key: cord- -fob lk m authors: fischbarg, j.; li, j.; cheung, m.; czegledy, f.; iserovich, p.; kuang, k. title: predictive evidence for a porin-type β-barrel fold in chip and other members of the mip family. a restricted-pore model common to water channels and facilitators date: journal: j membr biol doi: . /bf sha: doc_id: cord_uid: fob lk m water channels are the subject of much current attention, as they may be central for cell functions in a host of tissues. we have analyzed the possible fold of facilitators and water channels of the mip family based on structural predictions, on findings about the topology of chip , and on the biophysical characteristics of water channels. we developed predictions for the following proteins: mip , nod , glp, bib, γ-tip, fa-chip, chip k, wch-cd , and chip . we utilized kyte doolittle hydrophobicity, eisenberg's amphiphilicity, chou-fasman-prevelige propensities, and our own union algorithm. we found that hydrophobic amphiphilic segments likely to be transmembrane were consistently shorter than required for α-helical segments, but of the correct length for β-strands. turn propensity was high at frequent intervals, consistent with transmembrane β-strands. we propose that these proteins fold as porin-like -stranded antiparallel β-barrels. in water channels, from the size of molecules excluded, an extramembrane loop(s) would enter the pore and restrict it to a bottleneck with a width Å ⩽w ⩽ Å. a similar but more mobile loop(s) would act as gate and binding site for the facilitators of the mip family. protein water channels through membranes are currently the subject of much interest. based on their location and correspondence to: j. fischbarg distribution hasegawa et al., ; nielsen et al., ; zhang et al., a) they may be important for cell volume regulation, for epithelial transport of fluid in general, and for a host of other water movements that take place across cell layers in the lung, blood capillaries, ocular trabecular meshwork, and the like. in past decades, protein water channels were postulated to exist based on indirect but rather compelling evidence (hays & leaf, ; solomon, ; macey & farmer, ; parisi & bourguet, ; macey, ; verkman, ) . the strongest evidence for their existence came from the fact that such water channels appeared remarkably specific, permeable only to water and no other small hydrophilic solutes except for formamide (whittembury et al., ) . the recent sequencing, cloning, and functional expression of the channel-like integral membrane protein of kda (chip ) water channel (preston & agre, ; preston et al., ) enhanced the interest in this area, and prompted other laboratories to follow suit. the more recent clonings of several other water channels (fushimi et al., ; manrel et al., ; zhang et al., a; abrami et al., ) have moved the discussion to another level. water channels have been reasonably well characterized. hence, their known properties limit the possible three-dimensional foldings that might otherwise be proposed for their amino acid sequences. we undertook the current analysis to explore their possible structure based on predictive analysis, on initial findings about their topology, and on their characteristic permeability to water and other solutes. we propose that water channels and other members of the mip (major intrinsic protein of the lens) family of proteins may fold as porin-like [ -barrels. our proposal is meant to help future experimental tests of this and other possible folds for these proteins. the procedures we used for aligning and predicting protein secondary structures have been detailed before (fischbarg et al., (fischbarg et al., , . briefly, we used the bestfit and pileup routines of the gcg package (genetics computer group, version . ) to perform alignments. we predicted secondary structure using several algorithms: hydropathy, h; kyte-doolittle scale, (kyte & doolittle, ) ; amphiphilicity, g; (eisenberg, weiss & terwitliger, ) ; secondary structure propensities (prevelige & fasman, ) ; cfp algorithm, for chou-fasman-prevelige; the predictions from a robot that uses evolutionary information in conjunction with a database fed to a neural network (european molecular biology laboratory, heidelberg, germany) phd (profile network prediction heidelberg) unmanned server program phd (rost & sander, ) ; and our own union algorithm (fischbarg et al., ; fischbarg et al., ) that consists in adding hydrophobicity and amphiphilicity propensities, and subtracting cfp -point positiondependent turn propensities (pt) for given stretches of proteins. as in the papers above, we scaled h, g and pt values to - . -+ . , performed algebraic addition, and rescaled the resulting union propensities to - . -+ . . three-dimensional molecular modeling was done with the program hyperchem| (autodesk, sausalito, ca) on a computer, and with the program insightii on a silicon graphics platform. we aligned chip with rhodobacter capsulatus porin (por ), escherichia coli porin (por ), the reaction center l chain (rcl), colicin a (cola), and bacteriorhodopsin (br), and also aligned por with por . the alignments were run with standard gcg parameter values (gap weight = . , length weight = . ). the scores (data not shown) all fell in a "gray zone" (identity: to %; similarity: to %). it is well known that bacterial porins are evolutionarily quite distant among themselves, so in spite of the porins having the same secondary structure their sequences show little overall similarity (welte et al., ) , and therefore their alignment scores rarely exceed those above. the two porins examined here provided a good example. hence, a relationship between any two of these proteins can neither be affirmed nor refuted from such values; in particular, whether chip may fold as either a porin or a multihelical protein cannot be settled by the scores noted. figure la shows a multiple-sequence alignment of nine proteins of the mip family, namely: (a) the major intrinsic protein of the lens (mip ) (pisano & chepelinsky, ) ; (b) soybean nodulin (nod ) (sandal & marcker, ) ; (c) glycerol facilitator (glp) (muramatsu & mizuno, ) ; (d) drosophila big brain (bib) (rao, jan & jan, ) ; (e) the vacuolar membrane pro-tein ( -tip) (maurel et al., ); (f) the frog aquaporin (fa-chip) (abrami et al,, ) ; (g) the proximal tubule water channel (chip k) (zhang et al., a) ; (h) the collecting duct water channel (wch-cd ) (fushimi et al., ) ; and (i) the erythrocyte water channel (chip ) (preston & agre, ) . the similarity between some of these sequences has been pointed out before (chepelinsky, ) . however, the last two lines in the alignment of fig. l a add predictive information arising from the chip sequence. the last line shows the chip hydrophobicity profile (for a window of seven residues, and scaled ascendingly from to ). it is apparent that the regions with higher hydrophobicity, thus presumed to form either c~-helical or [ -strand structures, correspond to stretches of residues which are reasonably well conserved in the rest of the family. conversely, the gaps tend to coincide with regions of lower hydrophobicity, and thus presumably forming connecting loops. in line (i), the rectangles label those segments of chip for which we predict ~ -structure (see below). by and large, they correspond to stretches with relatively higher hydrophobicity and with very few gaps (only in out of the predicted strands). the patterns in fig it seems also noteworthy that some homologous segments in a same given region of the alignment are predicted as either oc or [ depending on the protein examined. this indicates to us that the prediction of a particular type of structure is perhaps less reliable than the prediction that there is secondary c~ or ~ structure in a given segment. assuming hence that there is either type of such structures in the segments delimited by phd, some of the segments appear too short to span the membrane as c~-helices (for which residues are presumed necessary), but have the correct length to span the membrane as [ -strands (usually - residues, but as few as residues may suffice (radding, ) ). as we have reported recently (fischbarg et al., ) , the conjunction of procedures we utilize gives reasonably accurate predictions of the global fold of those few membrane proteins that have been solved at high resolution so far. we give here an additional evaluation of our procedures in the bar diagram of fig. , which shows the correlation coefficients between predictive procedures and either existing structures, or the predicted structure of chip . for this purpose, we constructed an arbitrary step function (' 'structure") valued at . at existing (or predicted) (x or [ segments indistinctly, and at - . elsewhere. we verified that smoothing the predicted cfp and union profiles improved the correlation scores; we chose empirically to smooth the profiles with a fast fourier transform filter (span: seven points). as fig. shows, the union algorithm correlates better with the correct type of structure in both [ -barrel and (x-helical proteins (albeit by a narrow margin in the last case, and with understandably large errors given the minimal sample available). cfp overpredicts [ structures in the {;{-helical proteins, although here too the deviations may interfere with the actual picture. on the other hand, cfp correlates better with the structure in the ~ -barrels; the small error for cfp~ adds credence to it. for chip , union very narrowly favors [ over (x structures; however, cfp clearly selects global [ folding over (x. the difference in favor of the correlation of cfpp over cfp,~ appears too large to be attributed to overprediction. we applied to the proteins of the mip family the cfp, phd and union predictive algorithms. these results are shown in figs. a and b. for the union algorithm, we used a span of seven residues; we selected putative angle between residues of and degrees to search for regions with transmembrane (x-helix and [ -strand propensities, respectively. we also show the corresponding cfp (x and [ detailed propensities, and the marks for segments that exceed cfp standard thresholds. lastly, we show the phd predictions (already given partially in fig. lb) . we can discern no clear pattern to suggest a multi-(x-helical family. in fig. b , one can observe some three predicted long segments in the n-terminal half of the proteins. however, the pattern does not seem consistent: depending on which protein is examined, some of those segments break into smaller ones. even when the u,~ propensity is relatively high and maintained throughout the protein, as in mip , the cfp,~ and phd marks delimit a fair number of short segments. even more, in the same mip , the uc~ profile shows peaks corresponding to comparatively short segments; is the number of [ -strands expected from a porin fold. in fig. b , the cfpp marks and the phd predictions delimit consistent segments which are generally once more too short to span the membrane as o~-helices. the up profiles exhibit a periodicity consistent with a multistranded [ fold. such patterns are more readily apparent in the n-terminal half of glp, bib and ~-tip, and in the c-terminal half of the water channels. the close correlation of the cfpp and up profiles in the c-terminal half of chip , wch-cd and chip k appears also noteworthy. turn propensity information is implicit in the u profiles and is not shown in figs. a and b so as not to overload the graphs; still, there are frequent turn propensity markers, all the more so in relatively short proteins such as these. such high turn propensities are potential structure breakers, so their presence militates against the existence of relatively long, undisturbed segments of helical ((x or [ ) structures. for membrane proteins, this is consistent with the presence of numerous segments of the correct length to be transmembrane [ -strands. chip fig. presents in detail predictions for chip , along with marks for the nature of some residues, alignments, and mutation sites. the top panel shows hydrophobicity profiles with spans of and . in several instances, where the longer span finds one long hydrophobic segment, the shorter span contains a hint that such segments may be interrupted. the cf turn propensity plot and the marks for segments with suprathreshold turn propensity predict possible turns in regions for which h would predict a continuous segment. this panel also shows the prior prediction for chip by preston and agre ( ) ; the alphabetic nomenclature was used for predicted loops. immediately above are the marks for our current prediction for [ -strands. by comparing such marks to the turn propensity plot, it may be noted how in several cases the putative [ -strands coincide with segments relatively devoid of turn propensity, and are "nested" between two flanking turn peaks. the next two panels show cfp and union algorithms predicting (x and [ structures, respectively. both predict segments that tend to be too short to form transmembrane (x-helices. for comparison, our prediction for [ -strands is also given in each case. as can be seen, our predictions follow closely the pattern suggested by the cfpp and u~ profiles, especially in the c-terminal half. in the bottom panel, we compare how the predicted segments arising from several procedures are aligned. symbols at top and bottom mark the locations of charged (top) and aromatic (bottom) residues. the phd, cf(x, cf[ , and our own prediction marks are repeated here. the marks for por and por represent the segments of chip that align with the [ -strands segments of each respective porin. the x marks denote sites of recently performed mutations ) (labeled at the bottom). we first determined the approximate location of the structured segments, and then refined their lengths using a combination of approaches. one of them was a rule (see figs. and ) . stippled rectangles mark sites of mutations done in chip by preston et al. ( ) . the assignments of fig. were reached by dividing the chain in convenient stretches. mutations locate k inside the cells and n outside, hence an odd number of membrane crossings ought to exist for the segment between them. h has only one peak, but h has three. uc~ is unclear, but cfpc~, cfp[ and u[ mark three segments, and so does phd. in the porin alignments, por marks three segments and por two. from this picture, we extract a consensus of three segments. we mark their lengths selecting flanking aromatics for strands and , and choose the cfp[ mark to delimit strand . although this last maneuver places charged residues inside strand , this does not appear unwarranted since porins (not shown) have a fair number of charged residues in structured segments. segment - h shows one peak, h two, and there is a subthreshold turn at about residue . cfp(z, uo~, cfp[ and u[ mark two segments. por and por mark one and two segments, respectively. we note that the pair of cfp[ and u[ peaks appears well delimited, and opt for marking two [ -strands ( and ). segment - appears hydrophilic and with high turn propensity, so we resume at residue . h marks one segment, and h two. however, there are three turn marks in this stretch. cfpa and uc~ mark three segments; the pattern of cfp[ and u[ is consistent with three segments. the por alignment also shows three segments, so we opt for marking three [ -strands ( , and ). this leaves t in an inside loop, in contradiction with the outside placement deduced for it by mutagenesis . next comes a hydrophilic region ("loop c") which may have a short a-helical structure, judging from uc~ and cfpc~. we resume the analysis at residue . segment - h shows one peak, but h has two. cfpc~ marks two segments, and the uc~ peak is consistent with two segments. the cfp[ pattern is consistent with two peaks, and u[ marks two segments. both porin alignments mark two segments in this region. we mark two ~ -strands ( and ), which make the end of strand intracellular, back in register with the intracellular location deduced for r by mutagenesis . the next stretch up to residue appears a hydrophilic loop. c is in all likelihood extracellular, the site of action of external mercurial inhibitors on water permeation (van hoek & verkman, ; preston et al., ; zhang et al., a, b) . this requires an odd number of crossings for this segment. there is no clear evidence from any of the algorithms for more than one segment; the porin alignments also mark at most one segment. we mark one [ -strand ( ) and position it in the region where the u[ peak is highest and there is a limiting cfp[ mark. since there is evidence that the c-terminal end is intracellular, as evidenced by the k mutation , this segment is predicted to have an odd number of crossings. h shows one peak, h three. the cfpc~ and ucz patterns are unclear, pointing to between three and seven segments. on the other hand, cfp[ and u[ are remarkably well correlated with each other. cfp[ marks five segments, and u[ six. the por alignment marks six segments, and por four. we hence extract a consensus for five segments. to mark [ -strands - , we select the four regions for which the cfp[ and u[ peaks are in coincidence. strand is marked using the turn profile, the u[ peak centered on residue , and the por alignment mark for a [ -strand in that region. in closing this section, it seems worth noting the presence of stretches of residues with charges of opposite signs in close proximity (segments - , - , - , - , - , and - ). we discuss below i i i i i i i i i i i i i i i i i i i i , i i i i i . . of an epitope from the coronavirus e glycoprotein. *an earlier prediction (preston & agre, ) . how these may be related to electrostatic filtering to exclude electrolytes. the topology for chip suggested in fig. is given in more detail in fig. . we displayed graphically r. capsulatus porin (data not shown) and verified that replacement of individual porin residues in its [ -strands by the chip residues we predicted to be in the corresponding strands did not disturb the h-bonded patterns of the barrel. the predictions above for chip include a number of loops, so that one or more of them might enter the pore, restrict it as in porins (see below), and help confer chip its particular selectivity for water. kd) similar to that of chip . tim cannot be said to include a hydrated channel; the diameter of its -stranded [ -barrel is too small for the purpose and is filled with tightly packed side chains from the strands (data not shown). to form a channel, another ix-[ barrel protein would have to have either more numerous strands or a different sequence and fold; whether such a fold exists is not currently known. in terms of our proposal, an alternative explanation for the cd and ftir data is that the extramembrane loops may form some ix-helices. whether the amount of ix structure thus generated may be enough to correspond to the cd and ftir data is unclear. i ~ hmm e s c _q ] i s y ] m f l i d y t g c extra.ell uzar s c a i q v s f a i k y q r e i s a p v i v d a a i v l v_ g l r l i i g i g i v i d d i l c i t ~ y r s c- ~ a h f d l g ~ l l n i f d i c- p f w i s s t a s h l s n t s n ap r s la v l d dg a id / s r % [] [] ; r m [] since the structured transmembrane segments we predict are too short to be a-helices but have the correct length for [ -strands, we favor a [ -barrel fold for chip . we have previously employed prediction methods similar to those utilized here. in the first instance (fischbarg et al., ) , the u~ peaks identified the approximate location and length of the [ -strands in two porins. more recently, we have generated similar predictions for other membrane proteins (fischbarg et al., ) , including a short description of a putative multi-[ -fold for chip . as mentioned above, a prediction for the transmembrane and loop regions of chip has been advanced (preston & agre, ) by the researchers that cloned it. in that model, the transmembrane segments are all or more residues long, which is consistent with ix-helical structure. the topology of such prediction has been recently tested by vectorial proteolysis led to nonfunctional proteins, since these would have disturbed the channel fold. agre and colleagues themselves mentioned in their discussion ) that mutations in loops b and e are less tolerated than elsewhere in the chip molecule, and that such loops are candidate structures of the pore-forming domains of chip and other aquaporins. that would, of course, increase the number of transmembrane segments above the six originally proposed (preston & agre, ) . loop c deserves some consideration, since we predict it inside while the original proposal (preston & agre, ) has it outside. the evidence from protease digestion of chip plus an e insert at t locates such loop outside . however, this discrepancy may be only apparent; for instance, e may have somehow modified the protein fold so as to emerge on the outside. to be noted, aside from this aspect, the picture appears cogent so far. the width of the pore of the porin-like [ -barrel we propose is comparatively large. for instance, in r. capsulatus porin, the pore has an irregular ellipsoidal cross section with axes of some x a. if the entire width of such a pore would be available for diffusion, large solutes would traverse this channel. in the porin above, a loop enters the pore, remaining in contact with the barrel wall, and restricts the pore opening to a quasitriangular cross section of some a in height and a at the base. we are able to propose a barrel model for chip because it has loops that may restrict the size of the pore, otherwise the model could not be correct. there is evidence that water and formamide traverse a water channel (whittembury et al., ) but not urea (whittembury et al., ; van hoek & verkman, ) or compounds larger than it. if so, a steric restriction must exist, and must also be more pronounced than that for porin. from the molecular models illustrated in fig. , the minimum size a pore ought to have for the permeation of these molecules is: for water, . /~; for formamide, . ]k; and for urea, . a. hence, the loops inside the putative barrel ought to restrict the size of a bottleneck inside the channel to not less than and not more than a. as recently reviewed , chip does not allow the passage of electrolytes (h+ included). one explanation for this is that the molecule includes one or more electrostatic filters against electrolytes. such filters could result from the close juxtaposition of charged residues of opposite signs; indeed, some of the predicted loops contain such arrangements, as in segments - and - . for water by some of them, and for the facilitator properties uncovered so far for other ones. we propose that a restricted pore such as that found in porins would fit the description. figures , and exemplify this concept by displaying selected views of por . figure shows a cross section of por ; for clarity, only the backbone appears. as can be seen and as discussed above, one of the loops resides inside its pore and restricts its diameter. the shape of the loop hints at the possible presence of a pocket in its concave site, which might serve to bind substrates in the facilitators. figure shows a longitudinal section of a -a thick center slab of por (the ~ -strands in front and in back are not shown for clarity). as can be seen, the restriction created by the loop extends only to a central band rather than to the entire length of the pore. in addition, the loop that appears at the top right might represent an additional binding site or restriction. in fig. , the center of the loop has been removed, and the cpk spheres of the atoms lining the resulting pore are shown so as to give a view of the bottleneck in the pore profile. parenthetically, the model proposed here differs from one recently advanced by agre and colleagues in that in their case the channel would be circumscribed by two loops, while in the present one the channel is formed by the entire ~ -barrel translocation unit plus a single loop. as anticipated above, we propose that a fixed barrel framework and a disposition of the loops similar to that of fig. . as in fig. , except that the center of the loop is not shown and the channel-lining atoms are shown with their space-filling cpk spheres. porins could account for the selectivity and gating characteristics of m i p family proteins. as recently reviewed (cowan & rosenbusch, ) , " i n the past years detailed structures of a few m e m b r a n e proteins have so far revealed two folding patterns," meaning multi-a-helical and -barrel. it is, of course, still unclear whether additional patterns may emerge for integral m e m b r a n e proteins, but out of the two currently known patterns, the predictions we present favor a ~-barrel fold. clearly work remains ahead before a more definitive m o d e l for the proteins of the m i p family can be generated; we hope that our current proposal may provide a useful basis for further experimental tests of their characteristics. sequence and functional expression of an amphibian water channel, fa-chip: a new member of the chip family aquaporin chip: the archetypal molecular water channel the mip transmembrane channel family. in: handbook of membrane channels: molecular and cellular physiology. c. peracchia, editor folding pattern diversity of integral membrane proteins crystal structures explain functional properties of two e. coli porins the hydrophobic moment detects periodicity in protein hydrophobicity evidence that facilitative glucose transporters may fold as beta-barrels are most transporters and channels beta barrels? cloning and expression of apical membrane water channel of rat kidney collecting tubule tissuespecific expression of mrna encoding rat kidney water channel chip k by in situ hybridization studies on the movement of water through the isolated toad bladder and its modification by vasopressin molecular structure of the water channel through aquaporin chip. the hourglass model a simple method for displaying the hydropathic character of a protein transport of water and urea in red blood cells inhibition of water and solute permeability in human red cells the vacuolar membrane protein g-tip creates water specific channels in xenopus oocytes nucleotide sequence of the region encompassing the glpkf operon and its upstream region containing a bent dna sequence of escherichia coli chip water channels are localized in constitutively waterpermeable segments of the nephron the single-file hypothesis and the water channels induced by antidiuretic hormone genomic cloning, complete nucleotide sequence, and structure of the human gene encoding the major intrinsic protein (mip) of the lens isolation of the cdna for erythrocyte integral membrane protein of kilodaltons: member of an ancient channel family appearance of water channels in xenopus oocytes expressing red cell chip protein the mercurysensitive residue at cysteine in the chip water channel membrane topology of aquaporin chip. analysis of functional epitopescanning mutants by vectorial proteolysis in: prediction of protein structure and the principles of protein conformation proposed partial beta-structures for lac permease and the na+/h + antiporter which use similar transport and h + coupling mechanism similarity of the product of the drosophila neurogenic gene big brain to transmembrane channel proteins jury returns on structure prediction soybean nodulin is homologous to the major intrinsic protein of the bovine lens fiber membrane characterization of biological membranes by equivalent pores functional reconstitution of the isolated erythrocyte water channel chip secondary structure analysis of purified functional chip water channels by cd and frir spectroscopy water channels in cell membranes prediction of the general structure of ompf and phoe from the sequence and structure of porin from rhodobacter capsulatus. orientation of porin in the membrane contraluminal cell membrane water channels in pst exclude urea and acetamide but are formamide permeable cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to chip a point mutation at cysteine blocks the water permeability of rat kidney water channel chip k key: cord- - lozmf h authors: lion, marryanna; kosugi, yoshiko; takanashi, satoru; noguchi, shoji; itoh, masayuki; katsuyama, masanori; matsuo, naoko; shamsuddin, siti‐aisah title: evapotranspiration and water source of a tropical rainforest in peninsular malaysia date: - - journal: hydrol process doi: . /hyp. sha: doc_id: cord_uid: lozmf h to evaluate water use and the supporting water source of a tropical rainforest, a ‐year assessment of evapotranspiration (et) was conducted in pasoh forest reserve, a lowland dipterocarp forest in peninsular malaysia. the eddy covariance method and isotope signals of rain, plant, soil, and stream waters were used to determine forest water sources under different moisture conditions. four sampling events were conducted to collect soil and plant twig samples in wet, moderate, dry, and very dry conditions for the identification of isotopic signals. annual et from to was quite stable with an average of , ± mm, and a substantial daily et was observed even during drought periods, although some decline was observed, corresponding with volumetric soil water content. during the wet period, water for et was supplied from the surface soil layer between and . m, whereas in the dry period, approximately % to % was supplied from the deeper soil layer below . ‐m depth, originating from water precipitated several months previously at this forest. isotope signatures demonstrated that the water sources of the plants, soil, and stream were all different. water in plants was often different from soil water, probably because plant water came from a different source than water that was strongly bound to the soil particles. plants showed no preference for soil depth with their size, whereas the existence of storage water in the xylem was suggested. the evapotranspiration at this forest is balanced and maintained using most of the available water sources except for a proportion of rapid response run‐off. the response of tropical rainforests to environmental disturbance is of critical concern given the importance of their sustainability and potential to mitigate climate change. in these forests, changes in the amount and/or pattern of rainfall are perhaps the most important environmental disturbance. an understanding of fluctuations in evapotranspiration (et) under different moisture conditions is necessary to determine how tropical rainforests will respond to climate change. research to date has demonstrated that tropical rainforests maintain et even during the dry period. in pasoh forest reserve (fr), which is located in a dry zone of peninsular malaysia and receives the lowest yearly rainfall amount among adjacent south-eastern tropical rainforests, relatively stable et, which includes transpiration, interception evaporation, and soil evaporation, was observed even during the driest period, based on years of continuous eddy covariance (ec) measurement (kosugi, takanashi, tani, et al., ) . consequently, stable annual et rates ( , ± mm) were obtained despite the relatively small annual rainfall amount ( , ± mm, from to ) (kosugi, takanashi, tani, et al., ) compared to other southeast asian tropical rainforests (e.g., kume et al., ; vernimmen, bruijnzeel, romdoni, & proctor, ; noguchi, abdul rahim, & tani, ; kumagai et al., ) . no obvious decline in monthly et the english in this document has been checked by at least two professional editors, both native speakers of english. for a certificate, please see http://www. textcheck.com/certificate/blyfru variability was detected even during the driest month, although the amount of rainfall was much lower than et (kosugi, takanashi, tani, et al., ) . several other ec flux studies conducted in amazonian and southeast asian tropical rainforests have also consistently demonstrated this characteristic (e.g., costa et al., ; da rocha et al., ; kume et al., ; malhi et al., ) . in the amazonian rainforests, et did not drastically decrease and sometimes increased during the dry season (e.g., costa et al., ; da rocha et al., ) . the amazonian rainforest has distinct dry and wet periods, which is not the case in equatorial southeast asian rainforests, although dry and wet periods do exist as part of seasonal fluctuations, with considerable variability between years (kosugi et al., ; tani et al., ) . the stability of et in dry periods and in the dry season seen in tropical rainforests should be supported by stable water sources in the soil throughout the seasons. hydrogen and oxygen isotopes (δ o and δ h) provide a powerful tool for determining plant water resources under a number of environmental conditions (brooks, barnard, coulombe, & mcdonnell, ; dawson & ehleringer, ; eggermeyer et al., ; evaristo, jasechko, & mcdonnell, ; west et al., ; yang, wen, & sun, ) . using isotope indices, we can investigate whether soil water, streamflow, and plant transpiration are all sourced and mediated by the same well-mixed water reservoir originating in the soil (evaristo et al., ) . the depth of water uptake can also be estimated by measuring the isotopic composition of xylem water and soil water at different depths (ehleringer & dawson, ; yang et al., ) . many previous studies utilize only one of the dual stable isotopes of δ o (nie et al., ; querejeta, estrada-medina, allen, & jimenez-osornio, ) or δ h (filella & penuelas, ; jackson, cavelier, goldstein, meinzer, & holbrook, ) . however, the dual-isotope method (e.g., cramer, thorburn, & fraser, ; eggermeyer et al., ; evaristo et al., ; s. g. li et al., ; orlowski, frede, brüggemann, & breuer, ; rossatto, silva, villalobos-vega, sternberg, & franco, ; wang, song, han, zhang, & liu, ; west, hultine, burtch, & ehleringer, ) is a powerful tool for investigating the water source for et. the use of a dual-isotope approach could systematically help in the separation of distinct water pools in the ecosystem. these pools usually include water used by trees that does not contribute to streamflow or mobile water that is unrelated to the water used by trees (i.e., groundwater, streamflow, infiltration, and hill slope run-off; mcdonnell, ) . this approach can improve our understanding of the ecohydrological processes controlling water flows in soil-plantatmosphere continuums (berry et al., ) . our study of et and water sources in the pasoh fr comprised three objectives: (a) measure and calculate et using the ec method over a -year period ( - ); (b) determine spatial and temporal patterns of water uptake and provenance, using water budget methods combining et, precipitation, and soil moisture data; and (c) determine the provenance of water that is transpired at different times of the year by assessing the stable isotope signatures of water in precipitation, soils, plants, and streams. this information is expected to aid understanding of the likely impact of climate change on water demand and supply in tropical rainforests. the study was conducted in a lowland dipterocarp forest within the ha of pasoh fr (figure ) located at ° ′n, ° ′e at approximately to m above sea level. the soil in this area belongs to the local durian series, which is classified as an ultisol with a yellowish silt-clay layer ( - cm thick), overlaying a blocky indurated lateritic horizon ( - cm thick) on top of mottled white clay that overlays weathered shale down to a depth of - cm (leigh, ) . the a horizon was thin ( - cm) and consisted of brown silty loam, whereas deeper soils were bright yellowish or reddish brown and heavier (light to heavy clay; yoda, ) . lateritic gravel is abundant below cm of depth and increases with depth yoda, ) . the soil particle size distribution is characterized by low sand and high silt contents (yamashita et al., ) . the durian soil series at cm has . g cm − bulk density as well as . % moisture content at . bar and . % between . and bars. at the deeper soil layer ( cm), bulk density was . g cm − , and moisture content was . % ( . bar) and . % ( . - bar, universiti pertanian malaysia, ) . the underlying rocks at pasoh fr include shales and sandstones from the upper triassic age (gobbett, ; peh, ) . the textural details of this soil based on peh ( ) are listed in table . the maximum depth of tap roots was about m (niiyama et al., ) ; most of the fine roots were found at the a horizon (amir husni, ) . most fine roots that have a diameter of - mm were found at - cm soil depth, whereas fine roots with a diameter of - mm were in abundance at - -cm soil depth (based on figure in yamashita et al., ) . fine roots play an important role in absorbing water and nutrients and in dry matter production. the total fine root biomass down to -m depth was estimated at an average of . mg ha − using a pipe model approach and . mg ha − using a pit sampling method (niiyama et al., ) . the canopy height ranges between and m with emergent trees of~ m. rainfall distribution in pasoh fr is of short duration and high intensity, with a mean annual rainfall of , mm and mean annual air temperature of . °c ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (noguchi et al., (noguchi et al., , . streams in this area are typically shallow with relatively low baseflow (leigh, ) . direct run-off in response to rainfall is dominant, and the streams sometimes dry up during drier periods. continuous ec flux observations have been conducted at the microclimate observation tower since september (kosugi, takanashi, tani, et al., ; kosugi et al., ; takanashi et al., ) and showed very stable average monthly diurnal et courses. manokaran ( ) reported an annual interception value of mm ( . %) for pasoh fr. subsequently, tani et al. ( ) recorded an annual interception for pasoh fr from to of . mm ( . %). throughfall ranged between % and % in this forest (konishi et al., ) . in terms of run-off generation, a substantial amount of overland flow was measured using the trap method, indicating a close relationship with the amount of rainfall (leigh, ) . data were sampled at hz and sent to a data logger (cr- or cr- , campbell scientific, logan, ut). momentum, sensible heat (h, w m − ), and latent heat (ƛe, w m − ) fluxes were calculated as min averages. double rotation (mcmillen, ) was applied, assuming a zero mean vertical wind. the webb-pearman-leuning (webb, pearman, & leuning, ) correction for the effect of air density fluctuations was also applied, and linear trends in temperature and water vapour were retained. our ec measurements of et included transpiration, interception evaporation, and soil evaporation. we discarded all latent heat flux data recorded during and after rainfall; however, this gap was filled using available energy and sensible heat flux data measured using a three-dimensional ultrasonic anemometer, which can collect data during rainfall. a detailed description of these methods and instruments is provided by kosugi and colleagues (kosugi, takanashi, tani, et al., ; kosugi et al., ) . the energy budget correction was executed to overcome the energy imbalance problem described by takanashi et al. ( ) in estimating et. both sensible and latent heat fluxes are corrected using the bowen ratio to produce zero energy imbalance (kosugi, takanashi, tani, et al., ) . gaps were further filled using a second-order polynomial relationship between min λes after the energy budget correction method, and the available the energy released by changes in the air, vapour, and trunk storage was estimated using temperature and vapour pressure differences at the top of the tower based on ohtani et al. ( ) . air temperature and humidity at a height of m were observed using an hmp a or hmp c instrument (vaisala, vantaa, finland). an assmann psychrometer (sy- d, yoshino keiki, japan) was used to calibrate the vaisala sensors periodically. the vapour pressure deficit (vpd) of the air was calculated from these data. tipping-bucket rain gauges (ota keiki -t, japan) were used to measure rainfall at the top of the -m flux tower and at an observatory located m away from the tower. these data were compared and corrected with the rainfall measured with a storage gauge at the observatory. the volumetric soil water content (vswc) was measured using time domain reflectometry sensors (cs or cs , campbell scientific) at depths of . , . , and . m at three points around the tower logged at the -min intervals (noguchi et al., ) . this layer contains the majority of the fine roots. the mean rooting depth in a mixed dipterocarp forest has been identified as . m (baillie & mamit, ) . we used the daily average value of these nine sensors as a reference vswc for the surface layer between and . m. the time domain reflectometry sensors were calibrated using the standard procedure for calibrating capacitance sensors outlined by starr and paltineanu ( ) , which consists of nine steps (noguchi et al., ) . solar noon in this site peaks around : p.m. local time (kosugi, takanashi, tani, et al., ) . the antecedent precipitation index (api ) was tested and found to have a significant relationship with the vswc (noguchi et al., ) and was therefore used as a wetness index in this study area. the api is defined as ∑ i¼ p i =i, where p i is the daily precipitation (mm) and i is the number of preceding days (kosugi et al., ). . | isotope signatures of water in precipitation, stream, plants, and soil a storage-type rain gauge with a double-layered small-mouth inner glass bottle was installed at the observatory station to collect rainwater samples and was buried in the ground to prevent heating and evaporation. the storage gauge has a collar and is surrounded with a sponge to prevent splash-in from surrounding soil during rainfall. rainwater samples for isotope analysis were collected daily at : - : a.m. ; however, these samples were not taken during the rainfall. we collected eight samples when there had been no rainfall for more than a week. samples were filtered and transferred into two -ml polyethylene terephthalate bottles for stable isotope analysis. there was no rain over the course of these two sampling days. soil samples (n = ) were obtained using a hand auger at each depth, placed in -ml vials, sealed, and placed in a cool box to prevent evaporation. all plant and soil samples were frozen in a refrigerator (− to − °c) in the laboratory before water extraction. soil sampling was undertaken with care to prevent soil mixing between preceding layers and the current soil layer. auger holes were dredged several times before storage to ensure no material from the preceding layer was included in the current sample. tiny roots, stones, and leaf litter were separated from samples to prevent contamination. water extraction was conducted using a cryogenic vacuum distillation system (west, patrickson, & ehleringer, ) . cryogenic vacuum extraction is the most widely utilized method for plant and soil water extraction (ingraham & shadel, ; koeniger, marshall, link, & mulch, ; orlowski et al., ; vendramini & da s. l. sternberg, ; west et al., ) . we treated water extracted from plants with granulated activated charcoal to adsorb organic compounds that may influence the isotope contents of the samples (west et al., ) . the samples treated with granulated activated charcoal were kept for week before being transferred into measurement vials. all water samples, including precipitation, stream, plants, and soil, were filtered using a phobic polytetrafluoroethylene filter (with mm diameter and . -μm pore size) and transferred into -ml vials (c - g) in the laboratory for isotope analysis. a cavity ringdown spectrometer (l -i, picarro, ca) was used to analyse the isotope composition of the samples; this device had a specific analytical precision of . ‰ for δ o and . ‰ or less for δ h (katsuyama, ) . the delta (δ) the et reaches a ceiling at high vpd, whereas at low vswc during drier periods, a small decrease in et was observed with high vpd, indicating stomatal closure ( figure b ). comparing et with water budget in the soil provides some insight into the source of water for et (figures and ) . water was required in pasoh fr to accommodate et demand. a substantial amount of overland flow at the slope of this site was previously observed (leigh, ; peh, ) . however, some portion of such overland flow is assumed to infiltrate before reaching the stream. if this flow were taken into account, considerable water supply to the soil would be expected. nevertheless, the water supply would still be less than indicated by the antecedent precipitation values shown in this figure. figure examined the comparison between et demand and water budget in the surface soil layer to evaluate the source depth of water for et. the et demand was calculated as the cumulative budget of rainfall and et. if the budget was above , then the rainfall portion was not included. the soil water storage differential in soils at depths of - . m was calculated using the vswc, assuming uniform soil water content between and . m. if both decline slopes are similar, this indicates that most water was supplied from this surface layer, three rough groupings could be identified in the plots for plant water (table ) pasoh fr has typical monsoon-type rainfall variations (figure a,b) . it has significant interannual variations, with a bimodal pattern consisting of two distinct peaks; during march to may and october to december (marryanna et al., ) . using long-term average climate records, this area falls under the af (tropical rainforest climate) in the köppen-geiger climate classification scheme and is located within an equatorial region that hosts a fully humid climate (all months of the year are warm). this is the synoptic climate of southeast asia that is antecedent rainfall is also plotted to identify water sources southwest china, showed a smaller value ( , ± mm, z. li et al., costa et al., ; da rocha et al., ; kume et al., ; malhi et al., ) . nepstad et al. ( ) reported that some rainforests are able to extract soil water even in the dry season because of deep rooting depths. in pasoh fr, the rooting depth was concentrated in the a horizon (amir husni, ) but extended down to m (niiyama et al., ) . on the basis of energy imbalance filtering data, et was stable with a slight increase in the dry season in central amazonian rainforest sites, whereas in drier semideciduous sites, it showed a slight decrease during the dry season . this site-dependent difference was caused by differences in the increasing radiation energy and the stomatal behaviour during the dry season. et is mainly determined by atmospheric evaporative demand (e.g., carswell et al., ; da rocha et al., ; z. li et al., ) and stomatal control (e.g., cunningham, ) , the latter of which is influenced by the factors related to water supply, such as available water in the soil (e.g., burgess, adam, turner, & ong, ; rafael, todd, burgess, & nepstad, ) , rooting depth (e.g., z. li et al., ; nepstad et al., ) , and upward flow in the soil (e.g., da rocha et al., ; z. li et al., ) . in malaysian tropical rainforests located near the equator with indistinct dry periods, et was relatively constant (kumagai et al., ; kume et al., ; tani et al., ) . the variability of daily et at pasoh was dependent on available energy and vpd but was also moderately influenced by soil water content (figure a ,b, kosugi, takanashi, tani, et al., ) . ( ) could be the driving factor. the drought-tolerant tree and deep root system (hodnett, tomasella, marques, de, & oyama, ; nepstad et al., ) could also be the underlying factor. et decreased in the rainy season because the available energy and temperature decreased. it has been reported (kosugi, takanashi, tani, et al., ) that this forest used on average (±standard deviation) , ± mm year − for et during and . these values were slightly higher compared to those found in this study during and ( , ± mm). this is probably because of the drier conditions during these years. soil water content is a major control of hydrological processes such as et, the precipitation run-off response, energy transfer, and a climate predictor (e.g., betts, ball, beljaars, miller, & viterbo, ) . the soil water budget is a useful method for estimating total water loss from the soil caused by transpiration and soil evaporation (wilson, hanson, mulholland, baldocchi, & wullschleger, ) once soil water content has dropped below field capacity. the other benefit of the soil water budget is that it can provide insight into the relative contribution of various rooting depths to total transpiration sources (e.g., teskey & sheriff, ) . this forest needs at least four months of water storage to accommodate the et demand during extended dry periods ( figure ) . the typical clay soil in pasoh fr would delay water infiltration into the soil during rainfall and therefore contribute to overland flow. this means that the required water storage period should be longer than the analysis shown in figure . during dry periods, soil may experience excessive drying, and plants therefore need to use the previous water storage that is more strongly bound to soil particles. the slope of et demand (figure b) is an important indicator of the water change in the soil. when water supply from a specific soil layer is less than the et demand, the forest may obtain supplies from other sources. the analysis in figure shows that in pasoh fr, the forest mainly acquired water from the surface layer; however, during dry periods, insufficient water was available and water was acquired from deeper layers through the rooting system. the soil moisture content for soils at a depth of . m in this forest was reported to have an average field capacity of . %, a permanent wilting point of . %, and a saturation point of . % (peh, ) . in dry periods, the in pasoh fr was about m, and it was noted that fine roots were still found at -m depth (niiyama et al., ) . this finding supports our result. rainwater is partitioned into three components: (a) interception loss, (b) infiltration excess overland flow, and (c) water infiltration to soil. infiltrated water is divided between percolating water held at tension below field capacity (drainable pore space), plant-available water held at tension between field capacity and permanent wilting point (water available for plant et), and immobile residual water not participating in the hydrological cycle. our results (figures and ) strongly suggest that water sources for stream, plants, and soil at this forest, corresponding to infiltration excess overland flow + water infiltration to soil, plant-available water held at tension between field capacity and permanent wilting point, and immobile residual water not participating in the hydrological cycle, respectively, are separated. large fluctuations in isotope values in stream water corresponding with rainwater ( figure a ) indicate that the portion of new water runoff is large in this watershed. streams lose water flow during severe drought periods at this site. due to the rainfall characteristics in pasoh fr, which is intense and of short time duration (noguchi et al., ) , and the clay soils with low permeability, overland flow is often observed at the slope (leigh, ; peh, ) . our results are consistent with this. generally (e.g., clark & fritz, ) , the isotope values in stream water will lie close to the long-term weighted mean value for rainwater because stored, well-mixed groundwater maintains the streamflow. we need more intensive data of stream water to check this consistency and to obtain more precise partitioning of run-off pathway in this site. soil and plant water showed different characteristics from stream water and, additionally, differed from each other (figures and ) . only in the very dry period did plant and soil isotope values become closer, because plants used water from the same reservoir in the soil. clay soil typically has higher saturated and residual soil water content in the water retention curve, and hence a substantial portion of the water is strongly bound to soil particles and is not available to plants. this most likely explains the different water sources between soils and plants at the study site. interactions between the matrix and the gravitational potential lead to pores, with the largest diameter filling first and pores with the smallest diameter draining last and containing immobilised water compared to the larger pores (brooks et al., ) . evaporation from soil decreases rapidly with depth, and thus, plant roots are the primary cause of soil drying to below field capacity. additionally, water with the longest residence times in soil is more likely to be removed by plants and not delivered to the stream during dry seasons (brooks et al., ) . this situation was also observed in our study during the driest sampling period (june and march , figure a,b), when the isotope compositions of plant waters were similar to soil water and deviated from rainwater. this could indicate that plants are using water with the longest residence time and bound tightly to soil particles to supply their et demand during dry periods. we did not test how tightly the water was bound to the soil, and this could be a significant topic for future study. however, it can at least be inferred from figure that the vswc of soils from the study site was approximately . m m − in the driest period; thus, this value should be close to the residual soil water content. this means that the soil contained more than % water that was very tightly bound to soil particles, and thus inaccessible to plants. this amount of water would be included in the soil even in the wettest period, and we can conclude that this is one of the main reasons for the isotopic difference between water from soil and plants. the two only became similar in the very dry period. both the deviation in isotope values for plant water to the right side of the lmwl and the similarity in the range of fluctuation in the isotope values of plant water and soil water at the surface soil layer suggest that a substantial part of plant water was supplied from the surface soil layer. however, it should also be noted that the isotope values of plant water could not be explained entirely by surface soil water isotope values; this suggests plant water uptake also includes water in deeper soil layers. this analysis is consistent with the results of the water budget analysis shown in figure . it has been assumed that plants take up water according to their height (kim, park, & hwang, ) and rooting depth (meinzer, james, & goldstein, ) . water uptake and transport are associated with a hydraulic flow process that is controlled by resistance and hydraulic gradients (honert, ) . the overall resistance is determined by soil water potential, conducting vessels, transpiration rate, plant height, and gravity (kim et al., ) ; however, the results from this study do not necessarily reflect this trend. the isotope signals from trees of different height and species did not show any clear tendency of water uptake depth during the study period. from these results, we could not distinguish whether shorter or smaller trees use water from the shallow soil layer, whereas taller trees take water from the deeper soil layer. water uptake by co-occurring woody species showed that some species only take water from deep or shallow soil, whereas others use both layers (eggermeyer et al., ; west et al., ) . in tropical and subtropical forests, plants typically have shallow roots, meeting their water needs from the surface soil layer due to the plentiful rainfall (schenk & jackson, ) and soil properties would also be associated with variability in isotopic values (orlowski, pratt, & mcdonnell, ) . the comparison of different extraction techniques for different soil types (sandy and clay soils) identified that differences between the methods were small in sandy soil but large in clay soils (figueroa-johnson, tindall, & friedel, ; orlowski et al., ) . despite this, there are still many factors that may influence isotopic unfractionated water, and as a result, this issue remains poorly understood and there is a need for further studies (munksgaard, cheesman, wurster, cernusak, & bird, ; orlowski et al., ) . the centrifugation method to extract soil water, which can collect only capillary water with a certain limit of capillary pressure, could be used for this purpose. our study also found some classifications for plants in terms of water isotope characteristics. group showed isotope values close to that of soil water. this group consisted of an emergent tree (d. sublamellatus), two forest floor trees (m. lowii and b. parviflora), and a middle-sized tree (s. rugosum). group showed more negative isotope values that were out of the range of soil water, but still within the range of the antecedent rainwaters. all three trees in group were middle-sized trees (x. stipitatum, h. dictyoneurum, and d. malaccensis). the group tree (p. caput-medusae) was also middle sized and showed very stable and less negative isotope values during the whole observation period; however, these values differed from those of the deepest layer soil water. some group (x. stipitatum) and group (p. caputmedusae) trees have small stomatal conductance and maximum photosynthesis (kosugi, takanashi, yokoyama, et al., ) . these may be protective mechanisms allowing trees to retain more water in their xylem to prevent water loss, and the portion of storage water may be large. as the portion of newly absorbed water in xylem becomes smaller, the characteristics of isotope values would shift from that of group to group and finally to group . group may store more water inside the xylem, rendering these plants more drought resistant. all trees in groups and are medium-sized trees, which may indicate the origin of these characteristics. we found smaller stomatal conductance and photosynthesis in middle-sized trees at this site compared with emergent trees (kosugi, takanashi, yokoyama, et al., ) . these middle-sized trees might suffer from water stress and operate in a highly protective manner (kosugi, takanashi, matsuo, & abdul rahim, ). the utilization of released water from stored compartments near the canopy may play an important role in mitigating water stress in canopy leaves, thereby maintaining stomatal opening for photosynthesis. some previous studies have also shown that increased withdrawal of internally stored water occurs during water deficits (e.g., scholz et al., ) , whereas others have shown that stem-stored water may be used for buffering the daily water deficit even when soil water is abundant (goldstein et al., ; holbrook, ) . pasoh fr used most of the available water to maintain et particularly during dry periods in which there was no decrease of et ( figure d ). during dry periods, that is, when surface soil water was not available, the forest's root system was able to access water from greater soil depths to maintain et (nepstad et al., ) corresponding to the existence of a deep unsaturated soil layer in this forest. our sampling revealed no groundwater within m of the surface, including during the rainy season. streams in this area are typically shallow with relatively low baseflow (leigh, ) . direct run-off in response to rainfall is dominant, and the streams sometimes dry up during drier periods. this forest is mainly characterized by clay soil (yoda, ) , with low sand and high silt soil particle distributions (yamashita et al., ) . the physical traits of the clay soil likely influenced the water storage capacity of the watershed available for et. streamflow characteristics are typically determined by soil physical traits, topography and geography. in our study area, the uptake of almost all-available water by vegetation also influenced streamflow characteristics. in many tropical rainforests with rainfall amounts of~ , mm and having occasional dry period, the amount of run-off is not large, as illustrated by several reviews of hydrology in the humid tropical region (e.g., wohl et al., ) . rather, run-off is typically low and occasionally ephemeral during dry periods due to the large et demands of rainforest ecosystems. overall, although et during the observation years was lower than in previous studies, a stable pattern was observed. at least four months of water storage is needed to accommodate et needs during a very dry season. spatially, plants in pasoh fr typically obtained their water supply from the surface soil layer ( - . m) and sourced further water from deeper layers during the dry period. deep rooting and stomatal control are two well-known mechanisms that allow plants to cope with periods of high atmospheric demand and low water availability. isotope analysis results show that plants, soil, and stream have different sources of water in this forest. similarity in rainwater and stream water indicates the dominance of new water run-off. there are occasional isotopic differences between plants and soil water, probably because water source for plants is different from water strongly bounded to the soil. the source of water for this forest does not have a distinct pattern corresponding to soil depth and tree height. the results also suggest the existence and use of water storage 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water balance the hydrology of the humid tropics soil and belowground characteristics of pasoh forest reserve pasoh: ecology of a lowland rain forest in southeast asia seasonal variations in depth of water uptake for a subtropical coniferous plantation subjected to drought in an east asian monsoon region organic carbon, nitrogen and mineral nutrient stock in the soils of pasoh forest evapotranspiration and water source of a tropical rainforest in peninsular malaysia. hydrological processes key: cord- -od mf f authors: capodaglio, andrea g.; callegari, arianna title: online monitoring technologies for drinking water systems security date: journal: risk management of water supply and sanitation systems doi: . / - - - - _ sha: doc_id: cord_uid: od mf f more stringent constraints placed nowadays on water companies to provide high quality drinking water, increasing water resources scarcity in many areas of the planet, forcing water companies to work on marginal water bodies for supply, and the threat of hostile actions by political extremists and terrorist groups, that may willingly and deliberately cause contamination of an otherwise safe supply, are recent issues that have spurred demand for more efficient and comprehensive online water monitoring technologies. traditionally, quality parameters associated with drinking water provision were monitored using routine grab samples followed by laboratory analysis. this approach only allowed to capture small data sets, mostly unrepresentative of the true variance at the source, and allowed potentially important events to occur undetected. this paper examines state-of-the-art technologies for online monitoring of water quality in supply water systems, and reports some recent application examples. a safe and reliable supply of drinking water is nowadays an issue that bears strategic significance worldwide. in many areas of the globe the demand for high quality freshwater far exceeds the available supply, already; it is estimated, for example (who, ) that over million deaths each year are currently caused worldwide by pathogenic bacteria during the consumption of unsanitary water: it is easily foreseeable that in the future the issue of freshwater resource ______ * to whom correspondence should be addressed. andrea g. capodaglio, department of hydraulic & environmental engineering university of pavia, via ferrata , pavia, e-mail: italy capo@unipv.it and drinking water supply scarcity will continue to escalate, driven by population growth and economic development pressure. more stringent constraints placed nowadays on water companies to provide high quality drinking water, increasing water resources scarcity in many areas of the planet, forcing water companies to work on marginal water bodies for supply, and the threat of hostile actions by political extremists and terrorist groups, that may willingly and deliberately cause contamination of an otherwise safe supply, are recent issues that have spurred demand for more efficient and comprehensive online water monitoring technologies. micro-biological contamination of drinking water has an immediate effect on the emergence of infectious diseases. availability of micro-biologically safe drinking water is probably the most effective and economical way to ensure public health. in addition to "traditional" microbiological contamination by human waste, contamination of drinking water sources with pesticides and herbicides as part of the major contaminating factors is a growing problem worldwide. during the last decades, several studies revealed the presence of hazardous contaminants in wastewater effluents, being discharged in rivers and streams due to "normal" anthropic activities, including pesticides (Öllers et al., ) , natural and synthetic hormones (kolpin et al., ) , plasticizers, personal care products and pharmaceuticals compounds (daughton and ternes, ; jones et al., ) . a recent study estimated that there are , human pathogens including viruses and prions, bacteria and ricketssiae, fungi, protozoa and helminths; about % are considered to be emerging pathogens. their number continues to grow, as evidenced by the relatively recent emergence of severe acute respiratory syndrome (sars), caused by a novel coronavirus. in the past - years, on average at least one new pathogen has been identified each year, and many more have re-emerged after periods of inactivity or by appearing in areas where they were not previously reported (cunliffe, ) . pathogens identified in recent years include a range of organisms that can be transmitted directly from water or by water-related vectors. while a high proportion of the substantial burden of water-borne disease is caused by classical by pathogens such as salmonella typhi and vibrio cholerae, the spectrum of challenges is expanding (table ) . there are many reasons why water-borne pathogens emerge. in some cases this can be due to advancements in disease diagnosis and detection of the causative organisms: for example, it is likely that cryptosporidium caused diarrhoeal illness before it was diagnosed as a human pathogen in . in other cases true emergence can be caused by factors such as new environments, new technologies and changes in human behaviour. some pathogens have emerged for a combination of reasons. legionella was first detected in following the outbreak of legionnaires' disease in philadelphia. however, increased use of water-cooled air conditioning systems and spa pools has contributed to increased incidence of legionnaires' disease. as far as deliberate human hostile actions on urban water supplies, although none has been reported to date, in january, , the fbi circulated a reserved bulletin warning water industry managers that al-qaida terrorists may have been studying american dams and water-supply systems in preparation for new attacks (ionlife, ) . traditionally, quality parameters associated with drinking water provision were monitored using routine grab samples followed by laboratory analysis. this approach only allowed to capture small data sets, mostly unrepresentative of the true variance at the source, and allowed potentially important events to occur undetected. it is clear, that, in view of what just reported, this can no longer be considered a satisfactory procedure, and that online monitoring of water supplies, for a larger number of parameters than currently available, is quickly becoming an unavoidable choice. water utilities around the world are already using some form of online monitoring to warn of contamination of drinking water, in anticipation of yetto-be specified regulations: in the united states, turbidity is currently the only quality indicator for which there is a regulatory requirement for continuous online monitoring; in europe, the eu drinking water monitoring regulations (council directive / /ec) do not specifically require online measurements (awwa, ). online monitoring is usually defined as the unattended sampling, analysis and reporting of a parameter; it produces a sequence of data at much greater frequency than that permitted by manual (grab) sampling and it also allows real-time feedback for either process control, water quality characterization for operational or regulatory purposes, and alert/alarm purposes. online monitoring can be carried out at onsite as well as remote locations and will deliver measurements at intervals of seconds-to-minutes apart. clearly, online instrumentation must be placed at representative locations in the water system and must be periodically maintained by qualified technical personnel. monitoring requirements can be defined in relationship to: • source water quality: (a) variability, in space and time (very low for groundwater, low for lakes, high for rivers); (b) vulnerability (type and location of possible contaminating activity), time-of-travel of the contaminant to the intake, effectiveness of barriers, control options after an alarm • water treatment: process optimization options and response times, sampling frequency must allow adequate process control • distribution systems: minimization of deterioration of water quality over time and distance, early detection of cross-connections and water losses in addition, it must be considered that online monitors could have different sensitivity and selectivity according to the matrix and range of concentrations analyzed. a multibarrier approach to drinking water quality protection, such as those that are commonly used by facilities worldwide, is based on the concept that contaminants must be subject to as many points of control/treatment (barriers) as possible prior to the tap. the ideal location for control of contaminants is as close to the source as possible. a source water with low vulnerability is therefore characterized by few potential contaminant activities, transit time longer than that required for laboratory analysis, and the presence of multiple physical barriers between contaminating activities and point of intake. in a source water with moderate vulnerability, online monitoring of surrogate parameters (such as toc, doc, uv , ph and conductivity) may be considered to keep track of potential pollution. in a high vulnerability water source, online monitoring of chemical-physical-biological parameters (turbidity, ph, conductivity, redox, fish toxicity) and surrogate parameters in addition to specific indicators (e.g. vocs, phenols and specific toxicity tests) may be preferred. this is summarized in table . some parameters commonly used to monitor in online mode a supply source are listed in table . parameters used to monitor online water treatment are listed in table . both tables list also parameters for which online monitoring technology may not be widely available, nor field proved at the moment, but that may be useful for the given purpose (awwa, ) . the availability of real-time analytical information is one of the key benefits of online monitoring instrumentation: this information must however be conveyed to the appropriate user by means of a data collection and transmission system which is often referred to as scada, which consists of the individual online instruments, connected to programmable logic controllers (plcs) or remote telemetry units (rtus), that convert instrument outputs to the desired units, compare them to criteria set by the user, and generate signals for alarm or control to other process equipment. a host computer, that can be used to visualize or store data, or to further utilize them for specific purposes, almost always complements these systems. generally speaking, online monitoring instrumentation can be divided in: • physical monitors (turbidity, particles, color, conductivity, tds, hardness, alkalinity, acidity, streaming current, radioactivity, temperature, redox potential) • inorganic monitors (ph and do, disinfectants, such as chlorines and ozone, metals, fluoride, nutrients, cyanide) • organic monitors (carbon and hydrocarbons, uv adsorption, vocs, pesticides, disinfection by-products) • biological monitors (nonspecific, algae, protozoa, pathogens) • hydraulic monitors (flow, level and pressure) this paper will focus on the four former classes, discussing for each basic operating principles, and evaluation of the technology for online applications in the water distributing system. a wide array of technologies can be used for monitoring physical parameters, among them: light scattering/blocking (turbidity, particles, ss), light absorbance (color), electrochemical (conductivity, hardness, redox), electrophoretic (streaming current), chemical tritration (alkalinity, acidity, hardness) and other (radioactivity, temperature) (table ) . turbidity refers to the clarity of a water sample, and can be defined as an optical property that causes light to be scattered and absorbed by suspended particulate matter (inorganic or organic) in the sample. it is one of the most commonly monitored parameters in the water industry, and the one that is most amenable to online monitoring. a turbidimeter consists of a light source, a sample cell and a photodetector; commercial instruments differ in the type of light source and the number and location of photodetectors. color is caused by the absorption of visible light by dissolved/colloidal substances or small suspended particles and can be an indication of pollution by disinfection by-products, industrial pollutants and/or metals, although it does not, by itself, represent a health risk. color is in fact regulated as an aesthetic parameter only and is seldom monitored online, except in cases where the water source is subject to industrial effluents and/or storm runoff inputs. color is measured either visually or spectrophotometrically. dissolved organic/inorganic impurities (tds) are compounds of different nature and sources, and constitute the solid residue after all the water evaporates. tds must be determined gravimetrically in the laboratory, however, since these species tend to increase the electrical conductivity of the solution, conductivity is often used as a surrogate measure for tds, especially when in online mode. hardness is defined as the sum of divalent cations in a water sample, and is relevant in drinking water systems for aesthetic reasons and for corrosion concerns. alkalinity is defined as the acid-neutralizing capacity of a solution. both can be determined by chemical tritration (hardness with edta, alkalinity with an acid solution). inorganic monitors are used in online mode to detect influent and effluent water quality, and for treatment process control; the applicable technologies are listed in table . online monitoring of inorganic constituents is still in the early phase for many elements of interest to drinking water concerns. for metals, available technology is an adaptation to automatic operation mode of complex colorimetric methods developed for laboratory applications, and therefore turns out to be expensive and/or complex to operate, nor still suitable for installation in remote or unmanned sites. also, for many metals of interest in drinking water systems (as, cd, pb, hg, se, zn), online monitoring technologies do not exist at all. some promise for future application comes from developments in optode technology, coupled with miniaturized spectrophotometry. the technology for online monitoring of organic compounds includes toc analyzers, uv absorption and differential spectroscopy; it is much more developed than that for inorganics, for this reason, although neither eu nor us regulations require online monitoring of these substances, many drinking water utilities routinely use online organics monitoring to some degree. toc methods measure the carbon content of dissolved and particulate organic matter in the water, without giving information about the nature of this organic substances. table shows the different fractions of carbon measured by an organic carbon analyzer. toc measurement is defined by well established methods and occurs generally according to a four-step process: sample treatment (filtration), inorg. c removal (acidification to ph < ), oxidation (with uv, catalyzed uv, chemical oxidation, etc.) and co determination (ndir detectors, colorimetry, or conductivity methods). detection limits depend on the specific technique's operating temperature (usually > . mg/l for low temp., > mg/l for high temp.). most organic compounds found in water absorb uv radiation: using a uv spectrometer it is therefore possible to estimate the concentration of these compounds. originally, a uv light source with the single wave length of nm was used for such measures, however, in recent years, instrumentation reading the entire uv-vis spectrum ( - nm) has been developed and marketed (s-can, ) . uv absorption is a well-defined and commonly used methodology; evidence shows strong correlation between these measurements and organic carbon concentration measured with standard methods such as toc or others (figure ). in addition, it has been shown that other parameters can be measured indirectly by correlating their concentration values to uv absorption in the full spectrum ( figure ) ; several commonly sought organic compounds have typical absorption spectra that make their identification quite easy with appropriate instrumentation (figure ) . hydrocarbons in general are probably the main class of water pollutants found in surface and ground water (i.e., source water for drinking water systems). methods for online hydrocarbon detection include: fluorometry, reflectivity, light scattering and turbidity measurement, ultrasonic methods, electrical conductivity, spectroscopy, gas-phase detection (after volatilization), resistance-based sensors; some of these methods, however, give just an indication of the presence/absence of oil on the water surface. volatile organic compounds (vocs), including, among the others, aromatic compounds, halogenates and trihalometanes, are compounds that evaporate when exposed to air and can be of health concern when found in drinking water systems (trihalometanes are disinfection by-products -dbps -that can be precursors to the formation of carcinogens). their presence in drinking water can be a symptom of accidental pollution in the source water, of treatment failure/deficiency, or of incorrect disinfection procedures. current online monitoring technologies for vocs include purge-and-trap gaschromatography with flame ionization (fid), electron capture (ecd) or photoionization detectors or mass spectrometry (ms). detection limits for different substances vary according to the detector method. pesticides, including insecticides, fungicides and herbicides comprise triazines and phenlylurea compounds; they are monitored in drinking water systems in order to: detect accidental pollution in source waters, and check the effectiveness of treatment specifically designed to remove such substances. online monitoring of pesticides can be carried out using composite techniques, such as: • high pressure liquid chromatography (hplc)/diode array (da) detection, consists of extraction and enrichment, chromatographic separation and da detection. • gas chromatography (gc), consists of extraction and enrichment, gc separation and mass spectrometer (ms) detection. • liquid chromatography/mass spectrometry, consists in extraction and enrichment, lc separation and ms, thermospray, electrospray or particle beam detection. each technique is capable to optimally detect a group of compounds, for example, hplc/da can be used to analyse; atrazine, chlortoluron, cyanazine, desethylkatrazine, diuron, hexazinone, isoproruton, linuron, metazachlor, methabenzthiszuron, metobrorumon, metolachlor, metoxuron, monolinuron, sebutylazine, simazine and terbutylazine. in theory, any analytical laboratory method can be adapted for use as online measurement, provided that the requirements for consumables and manual intervention can be minimized: current online systems are often a "robotized" adaptation of known offline laboratory procedures, however, not always this solution is the most efficient one. a series of novel technologies, such as optochemical sensors, biosensors, and microbiological sensors are being tested for organics and hydrocarbon analysis. advances already in use include differential uv spectroscopy for dbps detection and microphase solid-phase extraction (spe) for the analysis of semivolatile organics (yongtao et al., ) . there are two basic types of biological monitors currently in use: those that use biological species as indicators of the presence of contaminants of concern (e.g. toxic chemicals), and those that screen for the presence of biological species of concern (e.g. nuisance algae, pathogens). in common us terminology, the term biomonitor usually indicates the former, and is in fact used as synonymous with toxicity monitor. in eu terminology, biomonitor refers generally to all types of biologically-based systems. at the present time, many existing biological monitors are quite new and can be considered experimental/unique applications. table shows an overview of the most common types of online biological monitors. sensitivity of test organism to individual compounds must be determined initially. online biological monitors are a very active area of r&d due to increasing regulatory and public demand pressures. while bacterial-based systems show great sensitivity and ease of operation, and development in this area will likely derive from improved fingerprinting of organisms and maintenance cost reduction, most advances can be expected from protozoan monitor technology, with techniques in uv absorption/scattering analysis that may soon allow automated detection of cryptosporidium and giardia. also, molecular techniques initially applied to the recognition of the genomic sequence of specific organisms in clinical applications (bej, ) , have also shown great potential for use in the detection of pathogens in water, and are producing extremely interesting results that could lead to widespread online use in the very next future (see also section ). chemical fingerprinting describes the use of a unique chemical signature, isotopic ratio, mineral species, or pattern analysis to identify different chemicals. optical fingerprinting by uv, vis, and nir absorption spectroscopy can be effectively achieved by low-cost and compact spectrometric devices, that can also be linked to an online diagnostic system, to directly identify some compounds (e.g. benzene) present in the water or to give an indication of the possibility of the presence of related compounds. in optical fingerprinting, a wide portion of the uv, vis and nir spectrum can be monitored simultaneously at high measurement frequency (minutes or fractions thereof); figure shows the spectral fingerprint of a typical municipal wastewater, in the range - nm, together with three other spectral readings from the same source that were recorded within min from the first. these show clearly different features, indicating a pronounced change in water quality. although this alone will not, in general, indicate the compound or compounds responsible for the change (they will need to be further investigated, if a definite answer is desired), but that can nevertheless trigger an alert to the system's operator, indicating deviation from routine conditions. some relevant applications taken from recent literature, are illustrated in the following sections, as examples of the current state-of-the-art in online monitoring technology. the river trent drains the large, mainly urban, catchment of midlands, with million residents, in the uk; although traditionally it has been considered too polluted to serve as a drinking water supply source, the river has undergone marked improvements in quality until, in , it was targeted as such. water is currently withdrawn from the river into bankside storage lakes, from where is taken to a purification plant. at this point, it became necessary to identify a suitable online monitoring strategy in order to protect this planned development (kirmeyer et al., ) . in order to protect water quality and provide operational data to the treatment plant operators, a list of parameters subject to online/laboratory monitoring has been established (table ). online monitors provide an alarm when a preset warning limit is exceeded, prompting immediate additional investigation. when a second, "action" preset limit is exceeded, water extraction into storage is automatically terminated. online monitors include traditional inorganic, organic and oil monitors, as well as an automated liquid chromatography (hplc) system for monitoring organics, originally developed for analysis of triazine and phenylurea in the rhine river, and uniquelly adapted to detect other acid herbicides and phenolic compounds known to be frequently present in the trent, at levels < . μg/l. vocs are detected by means of a purge-and-trap gc analyzer. maintenance requirements are relatively high (each unit requires - h of onsite attendance twice a week, in addition to the time required to review data, prepare buffers and standards, etc.). overall, in about years of operation, the system has recorded several incidents of parameter abnormality in the river trent, including ammonia toxicity events and contamination by various herbicides. it has been confirmed that online monitoring gives a far better definition of each recorded event than otherwise available, while spot samples analyzed at the laboratory in parallel have always been in good agreement with the online results. the llobregat river, near barcelona (spain) is characterized by a highly variable flowrate; to ensure sufficient supply during drought periods, dams have been built along its course. in addition, a continuous, automatic system with ten stations along its course has been put in place to monitor critical water quality parameters; these may vary depending on the monitoring location, in order to account for different needs and conditions. there are three types of stations: basic ( , upstream), complete ( , midstream) and special ( , mid-to-downstream); these are described in table . the objectives of the monitoring network are: • to provide warning of pollution incidents, ensuring safety of the water produced by the purification plants • to control instream water quality by monitoring changes in pollution indicators • to provide information for the operators of the water systems and planners through data access with a specifically created scada user interface in the course of several years of operation the automatic water quality monitoring network has recorded various incidents of pollution, including: • repeated occurrences of salinity discharges from potash mines, caused by accidental breakage of collectors or leaching due to rainfall • an event of chromium pollution in a tributary stream, due to industrial source discharges • multiple overflows of drainage and sewer systems causing variations of turbidity, toc, ammonia, uv adsorption and do • highly significant variations of physical and chemical parameters (mainly do, ph) due to photosynthetic activity . . on-line monitoring networks for drinking water security of karst water springs in geological karstic formations are the main source for drinking water supply for about % of the austrian population. water quality from these springs shows rapid, occasional instabilities for some parameters, caused by natural events, such as heavy rainfalls. rapid fluctuations of raw water quality, especially concerning turbidity, but also elevated concentrations of dissolved organic substances, and increased bacteria counts can occur in an unpredictable way during storm weather. furthermore, anthropogenic events like accidents and spills in proximity of the springs might also affect the quality of the raw water. the raw water quality is obviously one of the most important factors determining the final quality of the drinking water, in addition, rapid changes of source quality can limit the efficient usage of treatment procedures during drinking water production. as it is impossible to predict the impact of such events, it is vital to enhance drinking water security by monitoring the composition of the raw water continuously. main springs and important locations in the trunk mains had been monitored online since the late s. buildings equipped with power supply, pipe installations and special foundations had to be built for housing cabinet analysers, for monitoring spectral absorption coefficient (uv ), toc, turbidity, electrical conductivity and ph. in year , a novel submersible uv-vis-spectrometer, introduced by s::can messtechnik gmbh, capable of measuring light absorbance in the spectral range of nm to nm, and thus turbidity, organic carbons (e.g. toc, doc) and nitrate without any sample preparation were adopted. this new monitoring equipment provided new opportunities to the waterworks by allowing to monitor the totality of springs, even those without power supply, and by eliminating pumps, filters, membranes and reagents that were used by the old instrumentation, lowering substantially the overall cost of monitoring operations (weingartner and hosftaeder, ) . at present, vienna waterworks operates a s::can-based monitoring systems in order to monitor turbidity, sac , nitrate, toc, doc, temperature and electric conductivity at locations, since monitoring just one parameter would not meet the needs of efficient and safe drinking water supply, as different types of events cause different changes in the composition of the spring water. the results of these monitoring systems are transferred in real time to an early warning system that can be accessed from four central stations. this early warning system manages the raw water sources h a day. whenever current readings exceed limits that are specific to each parameter, the water of the spring of concern will not be used for drinking water production but drained away. oxidation of organic materials for drinking water disinfection is a commonly applied treatment step. during oxidation of natural organic substances in the source water, for example using ozone, large molecules, such as humic and fulvic acids, are cracked into smaller ones. a side-effect of this procedure is the increased availability of microbiologically assimilable organic carbon (aoc): whereas the original large molecules are not readily accessible to microorganisms, the smaller oxidation products can be digested and thus can stimulate bacterial regrowth in the distribution network. aoc is an important parameter especially in drinking water networks where no residual disinfectant is present in the water; its measurement is performed using cell cultures, which is a lengthy procedure. online uv/vis spectroscopy has proven itself as a tool that allows the collection of specific information on the removal efficiency for and subsequent concentrations of (organic) substances in water. the use of two on-line spectrometer instruments, placed before and after a treatment step, and the calculation of the differential spectrum between these two sites could open up a further area in water quality monitoring and process control as it allows calculation and prediction of water quality parameters previously unavailable (van der broeke et al., ) . two on-line spectrometers probes were installed in the pilot plant of amsterdam waterworks, weesperkarspel. in this pilot plant, the full treatment train of the waterworks is represented. the source, an artificial lake with high concentrations of natural organic matter, is sequentially treated in an ozone reactor (four bubble columns), pellet softening reactor, biological activated carbon (bac) filtration reactor and finally a slow sand filter. a parallel bac reactor was fed with water from the full scale treatment plant, which uses identical raw water, to be able to verify the effects of the changes in the ozone settings on water quality. the aoc calibration obtained in this way is a surrogate parameter, in the sense that the aoc concentrations in the water are far below the concentrations that can be distinguished using a uv/vis spectrophotometer without sample pre-concentration. the developed calibration, in combination with the on-line spectrometer probe, allows for on-line, in-situ measurement of dissolved ozone concentrations. the use of two instruments simultaneously, required to perform on-line differential measurements, allowed the prediction of the changes in aoc levels in individual treatment steps in real-time. the software ana::larm, for use with online uv spectrometers in a speciallydesigned configuration ( figure ), has been developed specifically for contaminant alarm systems based on thousands of spectra, and is successfully used in several applications in europe. the training of the alarm parameters can be done by the instrument automatically, or is done manually guided by the pc software. it allows for a very simple half-or fully automatic setup and configuration of up to eight spectral alarm parameters within a few minutes. it will react on any type of figure . configuration of online monitoring station for use with the software ana::larm organic contamination that provides an absorption signal in the uv range. the alarm sensitivity for many organic contaminants is between and ppb. at the same time it is most insensitive to any fluctuation of the matrix within the natural "normal" range, and thus keeps false alarms to an unmatched minimum. the approach and methods used are absolutely new and unique, and open a completely new perspective for water monitoring beyond the trending of "classical" concentration parameters (s-can, b) . a contaminant warning system (cws) as an integrated tool that employs in-situ sensors, supervisory control and data acquisition (scada) systems, and water quality event detection systems (eds) to continuously monitor network conditions and warn operations personnel of any potential contamination events, has been studied by the us environmental protection agency (epa) and sandia national laboratories (hasan, et al., ; grayman, et al., ) . the sensor component can be comprised of various water quality sensing platforms, including contaminant-specific sensors, or existing water quality sensors (e.g., ph, cl, electrical conductivity, etc.) as currently installed in many municipal water distribution systems to provide "surrogate" data to the cws's. it has in fact been proved by experiments conducted in laboratory and pipe test loop systems, that a majority of potential contaminants will change values of at least a surrogate parameter away from normal background levels (byer and carlson, ; cook et al., ; hall et al., ) . monitoring of surrogate parameters can therefore provide information on the presence of contaminants within a distribution system and this information can be transmitted to a central processing location through the scada system. the challenge is to analyze the surrogate parameter signals to accurately identify changes in water quality that are significantly beyond the range of the ambient variability of the background water quality. the recently developed the canary eds software (hart et al., ) is an open-source software platform that gathers water quality signal inputs from scada systems and processes the data using one or more event detection algorithms, using a number of statistical models to determine the probability of an anomalous water quality event occurring for a given time step and monitoring location within the distribution network (mckenna and hart, ). the canary system has not been used in real applications, yet, and offline case studies, based on real hystorical data, have evidentiated some problems in correctly pinpointing alarm causes, as changes in the network hydraulics (such as opening and closing valves, draining/filling of storage tanks and pump operations) can cause significant changes in water quality as waters of different ages and from different sources mix within the network. the existence of escherichia coli (e.coli) in drinking water is an important indicator of faecal pathogens and potential micro biological contamination. currently available detection methods for its presence in drinking water distribution networks are inadequate, for multiple reasons: first of all, current methods are very time consuming meaning that contamination will already have reached end-users before laboratory results are available; secondly, current sampling and analysis procedures lead to detection success rates of only % to maximum % (with optimized sample taking). research by scientists from the dutch kiwa shows that by using a network of on-line sensors, the success rate can be increased to % (koerkamp and van wijlen, ) . the core of this labonline system is based on a combination of a concentrator unit and a sensor system using disposable chips, which prevent the sensor system itself from cross-contaminations and guarantee reliable and high quality measurements in time. initially designed for e.coli, the system is theoretically capable of detecting a broad group of microbiological contaminants like bacteria and viruses. industrial production is scheduled for late . the same research group developed a sensor technology for pesticides based on a combination of an integrated optic chip, a biochemical transduction layer (micro)-fluidics, electronics and data acquisition and system control software, called optiqua mobilelab. the optiqua mobilelab sensor for the detection of pesticides will have the following characteristics: low cost per analysis, easy to use, detection of five pesticides (simazine, atrazine, glyphosat, ampa, bam ( , -dichlorbenzamide), detection of additional pesticides (available in later stages), on site detection, prompt analysis results (minutes), high resolution (e.g. detection limits . μg/l with a dynamic range of . - μg/l), robust, low maintenance system. knowledge of the sequence of microbial genomes has led to the development of molecular methods for detection of microbial pathogens in clinical specimens as well as water and other environmental samples. a wide array of molecular techniques has therefore been applied to the study of microbiological water quality issues. the application of molecular techniques, such as pcr (polymerase chain reaction) has generated a great deal of valuable information on the occurrence, diversity, and biology of pathogens in water (loge et al., ) . in addition, molecular methods demonstrate rapid detection and enhanced specificity compared to other analytical methods. a micro analysis system for water pathogen monitoring consists of a micro polymerase chain reaction (pcr) chip integrated with a continuous-flow microarray that is able to reduce the analysis time from about h to within several hours as compared with the existing epa approved methods was presented by yong ( ) . pathogen samples were successfully detected by the micro analysis system through dna amplification by the micro pcr chip followed by direct transfer of the amplicons to the microarray for detection. in addition to one species monitoring, the system shows potential in direct monitoring of a range of pathogens at the same time through pcr and different probes immobilized on microarrays. comprehensive two-dimensional gas chromatography also referred to as gcxgc, is an analytical technique in which all the eluted compounds from a first column are successively submitted to a new separation in a second column with different selectivity. contrary to gas chromatography (gc) which employs only one chromatographic column, gcxgc uses two chromatographic columns, coupled in series, with a modulator at their junction. developed in , due to its principle, gcxgc offers a much better capacity of separation and a better sensitivity than conventional gas chromatography. thanks to the sensitivity of gcxgc (three-to fivefold higher than gc) some compounds can be detected at the ng/l level. semard et al. ( ) applied this technique to the screening of wastewater and effluents samples. a large range of drugs (antidepressors, antibiotics, anticoagulants…), personal care products (sunscreens, antiseptics, cosmetics…) and carcinogen compounds were found in the raw waste water samples. in addition to the above mentioned micropollutants, a wide variety of nitrogen aliphatic and aromatic structures that could act as dbp (disinfection by-products) precursors, were also uncovered. this technology does not yet have the potential for direct online application. courtois et al. ( ) also propose a molecular technique, based on polymerase chain reactions to detect pathogens. to improve pcr diagnostics for routine analysis purposes, they focus on the processing of the sample, which is crucial for the robustness and the overall performance of the method. their objectives in sample preparation are to increase the concentration of the target organisms to the practical operating range of a given pcr assay; and to produce a purified dna extract that would be representative of the initial water sample and would be free of pcr-inhibitory substances. this can be achieved by means of a twostep ultrafiltration (uf) procedure by using protoptype hollow fiber uf cartridge, and a commercial uf centrifugal concentrator. this paper has overviewed existing instrumentation applicable to water supply online monitoring, and examined a few state-of-the-art application examples. it is clear that technological development in this field is very rapid, and that astonishing advances are anticipated in several areas (fingerprinting, optochemical sensors, biosensors, molecular techniques). software applications, together with new generation sensors, are also contributing to the identification of otherwise difficultly monitored parameters. in spite of the high technology being developed, monitoring costs are bound to become a lesser and lesser part of a water utility budget due to the fact that automation and technological simplification will abate the human cost factor (maintenance and other labour forms) and reduce significantly the complexity of procedures (with those, of reagent requirements, etc.). proper interpretation and use of the growing mass of water quality data that will become available through new technologies will allow better management of water resources, and water treatment and distribution facilities. online monitoring for drinking water utilities molecular based methods for the detection of microbial pathogens in the environment real-time detection of intentional chemical contamination in decision support system for water distribution system monitoring for homeland security universal concentration for large volumes of water samples before molecular detection of waterborne pathogens emerging enteric and potentially waterborne pathogens in the region ternes ( ) pharmaceuticals and personal care products in the environment: agents of subtle change? design of early warning and predictive source water monitoring systems on-line water quality parameters as indicators of distribution system contamination canary: a water quality event detection algorithm development tool safeguarding the security of public water supplies using early warning systems: a brief review case studies of online monitoring systems next generation sensors for detection of pesticides and e.coli in water pharmaceuticals, hormones, and other organic wastewater contaminants in u.s. streams, - : a national reconnaissance how safe is our water? the threat of terrorism human pharmaceuticals in the aquatic environment pcr detection of specific pathogens in water: a risk-based analysis on-line identification of adverse water quality events from monitoring of surrogate data: canary software simultaneous quantification of neutral and acidic pharmaceuticals and pesticides at the low-ng/l level in surface waters use of on-line uv/vis-spectrometry in the measurement of dissolved ozone and aoc concentrations in drinking water treatment on-line monitoring networks for drinking water security of karst water. all about karst and water, vienna. who ( ) emerging issues in water and infectious disease. world health organization ) micro analysis system for water pathogen monitoring spe twin-pal. online in situ monitoring of semivolatile organic compounds in water key: cord- -tmcm kxn authors: nakamura, shingo; ishihara, masayuki; sato, yoko; takayama, tomohiro; hiruma, sumiyo; ando, naoko; fukuda, koichi; murakami, kaoru; yokoe, hidetaka title: concentrated bioshell calcium oxide (biscao) water kills pathogenic microbes: characterization and activity date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: tmcm kxn bioshell calcium oxide (biscao) exhibits deodorizing properties and broad microbicidal activity. in this study, we examined possible utility of biscao water for that purpose. biscao water was prepared by adding wt% biscao to clean water and gently collecting the supernatant in a bottle. the same volume of clean water was gently poured onto the biscao precipitate and the supernatant was gently collected in a bottle; this process was repeated fifty times. the produced biscao water contained nanoparticles (about – nm) composed of smaller nanoparticles ( – nm), and was colorless and transparent, with a ph > . . in vitro assays demonstrated that biscao water eliminated more than . % of influenza a (h n ) and feline calicivirus, escherichia coli such as nbrc and o- :h , pseudomonas aeruginosa, salmonella, and staphylococcus aureus within min. we compared biscao water with the other microbicidal reagents such as ethanol, biscao, bisca(oh)( ) suspensions, povidone iodine, naclo, biscao dispersion and colloidal dispersion with respect to deodorization activity and microbicidal efficacy. the results showed that biscao water was a potent reagent with excellent deodorization and disinfection activities against pathogenic bacteria and viruses (including both enveloped and nonenveloped viruses). calcium oxide (cao) and calcium hydroxide (ca(oh) ) produced from limestone are readily available and important inorganic compounds used in various industries as adsorbents, toxic-waste remediation agents, and alkalization agents. however, both reagents contain harmful impurities, and cao is especially dangerous, because it easily generates high heat in response to hydration [ , ] . although scallop shells have been used as food additives and are a readily available source of cao and ca(oh) , most are discarded as industrial waste. these wasted shells accumulate on the shores of harvesting districts in japan, causing serious problems such as offensive odors and soil pollution due to release of harmful materials from the shells [ ] . the harmful materials in scallop shells can be removed by heating at a high temperature, and by grinding and sieving. most commercially available spraying biscao water, which is colorless and transparent with a ph > . , on a smooth metal or plastic surface, followed by drying, provides a white powder coating. scanning electron microscope (sem) images of dried powders of biscao water are shown in figure a ,b, and cryo-sem [ , ] images of biscao water are shown in figure c ,d. after drying biscao water, the biscao particles become larger ( - µm) compared to those in biscao water. the microparticles were connected with each other. nanoscale cao particles ( - nm) in biscao water aggregate to generate larger biscao particles ( - nm). furthermore, when the dried powder was suspended into clean water, the powder was insoluble and ph in the supernatant was below . , suggesting the microparticles were caco produced by interaction of ca + and co . molecules , , of spraying biscao water, which is colorless and transparent with a ph > . , on a smooth metal or plastic surface, followed by drying, provides a white powder coating. scanning electron microscope (sem) images of dried powders of biscao water are shown in figure -a,b, and cryo-sem [ , ] images of biscao water are shown in figure -c,d. after drying biscao water, the biscao particles become larger ( - μm) compared to those in biscao water. the microparticles were connected with each other. nanoscale cao particles ( - nm) in biscao water aggregate to generate larger biscao particles ( - nm). furthermore, when the dried powder was suspended into clean water, the powder was insoluble and ph in the supernatant was below . , suggesting the microparticles were caco produced by interaction of ca + and co . scanning electron microscopy (sem) images of drying biscao water and cryo-sem images of biscao water. the particle surface structure of drying biscao water at -fold magnification (a) and at , -fold magnification (b) was observed with sem images taken with a field-resolved scanning electron microscope. cryo-sem observations were performed on biscao water at -fold magnification (c) and , -fold magnification (d). arrows indicate nanoparticles comprising assemblies of smaller nanoparticles. bactericidal activities of biscao against escherichia coli (e. coli) nbrc , e. coli o- :h , p. aeruginosa, salmonella, and staphylococcus aureus (s. aureus) were evaluated by the japan food research laboratory (jfrl; tokyo, japan) ( table ). the levels (colony forming units; cfu) of e. coli, p. aeruginosa, and salmonella were reduced to below the detection limit within min, % of e. coli o- :h was eliminated after min, and % of s. aureus (a gram positive bacterium) was eliminated after min and reduced to below the detection limit after min. scanning electron microscopy (sem) images of drying biscao water and cryo-sem images of biscao water. the particle surface structure of drying biscao water at -fold magnification (a) and at , -fold magnification (b) was observed with sem images taken with a field-resolved scanning electron microscope. cryo-sem observations were performed on biscao water at -fold magnification (c) and , -fold magnification (d). arrows indicate nanoparticles comprising assemblies of smaller nanoparticles. bactericidal activities of biscao against escherichia coli (e. coli) nbrc , e. coli o- :h , p. aeruginosa, salmonella, and staphylococcus aureus (s. aureus) were evaluated by the japan food research laboratory (jfrl; tokyo, japan) ( table ). the levels (colony forming units; cfu) of e. coli, p. aeruginosa, and salmonella were reduced to below the detection limit within min, % of e. coli o- :h was eliminated after min, and % of s. aureus (a gram positive bacterium) was eliminated after min and reduced to below the detection limit after min. the virucidal activities of biscao water against influenza a (h n ), an enveloped virus, and feline calicivirus, a nonenveloped virus, were evaluated using the fifty-percent tissue culture infectious dose (tcid ) method [ , ] , which was performed by jfrl (table ) . feline calicivirus treated with biscao water was reduced to below the detection limit within min, and influenza a was reduced from . to . (tcid /ml) at min compared with the control, and then reduced to below the detection limit after min. we evaluated and compared the deodorization efficacy of biscao water, biscao and bisca(oh) suspensions, and biscao dispersion and colloidal dispersion using tainted pork meat as a malodorous material. tainted pork meat was mixed with each deodorant, then placed on petri dishes and sealed in plastic bags for h. the odor intensity was then measured using a handheld odor meter. all deodorants tested exhibited concentration-dependent deodorization effects. biscao water had the highest deodorization efficacy at each concentration tested; bisca(oh) suspensions were less efficient. biscao suspension, dispersion, and colloidal dispersion exhibited intermediate efficacies ( figure ). efficacies of biscao water, suspension, dispersion, and colloidal dispersions for deodorizing contaminated minced pork. each deodorant was added to tainted pork meat on petri dishes, then sealed in plastic bags for h. the odor intensity was measured using a handheld odor meter. the experiments were repeated four times. we investigated the microbicidal efficacy of biscao water, suspension, dispersion, and colloidal dispersion against a contaminated suspension comprising normal bacterial flora, compared to other antiseptics/disinfectants. equal volumes of each disinfectant and the contaminated suspension were mixed well and incubated at room temperature for min, then the number of colony forming units (cfu/ml) per sample was determined. during incubation of bathtub water with % dulbecco's modified eagle's medium (dmem) + bovine serum albumin (bsa) ( . wt%) at °c for h, the tc and cf values increased from ± cfu/ml and ± cfu/ml to . ± . (× ) cfu/ml and . ± . (× ) cfu/ml, respectively. the cfu/ml for tc and cf following treatment with undiluted (final -fold diluted) and -fold diluted (final -fold diluted) biscao water, and . and . wt% (final . wt% and . wt%) of biscao dispersion and colloidal dispersion were below the detection limit, whereas low counts of tc and cf remained viable following treatment with . wt% of biscao water, dispersion, and colloidal dispersion ( figure ). in contrast, some tc and cf remained viable following treatment with undiluted ethanol, -fold diluted, and -fold diluted (final -fold diluted, -fold diluted, and -fold diluted) ethanol, and . , . , and . wt% (final . , . , and . wt%) povidone iodine. the microbicidal activities of biscao and bisca(oh) (suspension) and naclo against tc and cf were intermediate between that of biscao water and povidone iodine, and no cfu were detectable following treatment with high concentrations of . wt% (final . wt%). plating and counting were performed as a set of technical replicates (n = ). efficacies of biscao water, suspension, dispersion, and colloidal dispersions for deodorizing contaminated minced pork. each deodorant was added to tainted pork meat on petri dishes, then sealed in plastic bags for h. the odor intensity was measured using a handheld odor meter. the experiments were repeated four times. we investigated the microbicidal efficacy of biscao water, suspension, dispersion, and colloidal dispersion against a contaminated suspension comprising normal bacterial flora, compared to other antiseptics/disinfectants. equal volumes of each disinfectant and the contaminated suspension were mixed well and incubated at room temperature for min, then the number of colony forming units (cfu/ml) per sample was determined. during incubation of bathtub water with % dulbecco's modified eagle's medium (dmem) + bovine serum albumin (bsa) ( . wt%) at • c for h, the tc and cf values increased from ± cfu/ml and ± cfu/ml to . ± . (× ) cfu/ml and . ± . (× ) cfu/ml, respectively. the cfu/ml for tc and cf following treatment with undiluted (final -fold diluted) and -fold diluted (final -fold diluted) biscao water, and . and . wt% (final . wt% and . wt%) of biscao dispersion and colloidal dispersion were below the detection limit, whereas low counts of tc and cf remained viable following treatment with . wt% of biscao water, dispersion, and colloidal dispersion ( figure ). in contrast, some tc and cf remained viable following treatment with undiluted ethanol, -fold diluted, and -fold diluted (final -fold diluted, -fold diluted, and -fold diluted) ethanol, and . , . , and . wt% (final . , . , and . wt%) povidone iodine. the microbicidal activities of biscao and bisca(oh) (suspension) and naclo against tc and cf were intermediate between that of biscao water and povidone iodine, and no cfu were detectable following treatment with high concentrations of . wt% (final . wt%). plating and counting were performed as a set of technical replicates (n = ). the number of colony forming units (cfu/ml) per sample was determined after disinfecting contaminated wood pieces with tc values of . ± . (× ) and cf values of . ± . (× ). the cfu/ml following treatment with undiluted and -fold diluted biscao water and . and . wt% of naclo, biscao dispersion, and colloidal dispersion were below the detection limit for both tc and cf, whereas low counts of tc and cf remained viable following treatment with -fold diluted biscao water and . wt% of naclo, biscao dispersion, and colloidal dispersion ( figure ). low counts of tc and cf remained viable following treatment with undiluted, -fold diluted, and -fold diluted ethanol; . , . , and . wt% bisca(oh) ; and povidone iodine. biscao (suspension) had a microbicidal activity against tc and cf intermediate between that of biscao water and povidone iodine: the tc and cf counts were below the detection limit following treatment with a high concentration of . wt% biscao (suspension). the number of colony forming units (cfu/ml) per sample was determined after disinfecting contaminated wood pieces with tc values of . ± . (× ) and cf values of . ± . (× ). the cfu/ml following treatment with undiluted and -fold diluted biscao water and . and . wt% of naclo, biscao dispersion, and colloidal dispersion were below the detection limit for both tc and cf, whereas low counts of tc and cf remained viable following treatment with -fold diluted biscao water and . wt% of naclo, biscao dispersion, and colloidal dispersion ( figure ). low counts of tc and cf remained viable following treatment with undiluted, -fold diluted, and -fold diluted ethanol; . , . , and . wt% bisca(oh) ; and povidone iodine. biscao (suspension) had a microbicidal activity against tc and cf intermediate between that of biscao water and povidone iodine: the tc and cf counts were below the detection limit following treatment with a high concentration of . wt% biscao (suspension). a suspension ( μl) containing . ± . (× ) cfu/ml of tc and . ± . (× ) cfu/ml of cf was inoculated on nonwoven surgical masks and dried or left wet, then biscao water was sprayed on some of the masks and air dried. the tc and cf of sprayed and unsprayed masks were about (× ) and , and (× ) and (× ), respectively ( figure ) . thus, dry white-powdercoated nonwoven surgical masks had strong antimicrobial activity. furthermore, the spraying of biscao water on the contaminated surface of a surgical mask resulted in almost total disinfection. no change in quality was observed in the surgical mask after spraying biscao ten times. this result suggested that surgical masks may be reused multiple times following disinfection with biscao a suspension ( µl) containing . ± . (× ) cfu/ml of tc and . ± . (× ) cfu/ml of cf was inoculated on nonwoven surgical masks and dried or left wet, then biscao water was sprayed on some of the masks and air dried. the tc and cf of sprayed and unsprayed masks were about (× ) and , and (× ) and (× ), respectively ( figure ) . thus, dry white-powder-coated nonwoven surgical masks had strong antimicrobial activity. furthermore, the spraying of biscao water on the contaminated surface of a surgical mask resulted in almost total disinfection. no change in quality was observed in the surgical mask after spraying biscao ten times. this result suggested that surgical masks may be reused multiple times following disinfection with biscao water. scallop shell is composed of caco and is converted to cao when heated above °c. according to the manufacturer, biscao is prepared by heating shell powder at °c for h to obtain over . % cao, grinding using a dry grinder, followed by cooling in a vacuum chamber and vacuum packing. the produced fine cao powder, biscao, has an average particle diameter of about μm [ ] . both biscao and bisca(oh) are poorly water-soluble under alkaline conditions. the generated precipitates in suspensions of high concentrations of biscao and bisca(oh) can result in a significant loss of cao and the plugging of spray nozzles. a methodology is therefore needed to prepare biscao and bisca(oh) dispersions without precipitates. we previously reported that the addition of phosphate compounds such as phosphoric acid (h po ), trisodium phosphate (na po ), na hpo , or sodium dihydrogen phosphate (nah po ) to biscao or bisca(oh) suspensions results in the formation of dispersions [ ] . furthermore, biscao and bisca(oh) colloidal dispersions can be prepared by mixing with na-polypo or na-tripo as flocculent agents. two layers quickly form a supernatant, and flocculants/precipitates composed of polymeric colloidal calcium phosphate [ ] . the present study showed that biscao water (ph > . ), in addition to biscao dispersion and colloidal dispersion, has microbicidal activity against various pathogenic bacteria, including s. aureus (a gram-positive bacterium) ( table ) and viruses such as influenza a (h n ) as an enveloped virus and feline calicivirus as a nonenveloped virus (table ) . biscao water, which is colorless and transparent with a ph > . , was sprayed and dried on smooth metal or plastic surfaces and provided a white powder coating. scanning electron microscopy (sem) images of drying biscao water showed microparticles ( - μm) connected with each other (figure -a,b) . cryo-sem observations indicated that cao nanoparticles ( - nm) aggregated to generate larger biscao particles ( - nm) in biscao water (figure -c,d) . we examined the deodorizing and bactericidal activities of biscao water, dispersion, and scallop shell is composed of caco and is converted to cao when heated above • c. according to the manufacturer, biscao is prepared by heating shell powder at • c for h to obtain over . % cao, grinding using a dry grinder, followed by cooling in a vacuum chamber and vacuum packing. the produced fine cao powder, biscao, has an average particle diameter of about µm [ ] . both biscao and bisca(oh) are poorly water-soluble under alkaline conditions. the generated precipitates in suspensions of high concentrations of biscao and bisca(oh) can result in a significant loss of cao and the plugging of spray nozzles. a methodology is therefore needed to prepare biscao and bisca(oh) dispersions without precipitates. we previously reported that the addition of phosphate compounds such as phosphoric acid (h po ), trisodium phosphate (na po ), na hpo , or sodium dihydrogen phosphate (nah po ) to biscao or bisca(oh) suspensions results in the formation of dispersions [ ] . furthermore, biscao and bisca(oh) colloidal dispersions can be prepared by mixing with na-polypo or na-tripo as flocculent agents. two layers quickly form a supernatant, and flocculants/precipitates composed of polymeric colloidal calcium phosphate [ ] . the present study showed that biscao water (ph > . ), in addition to biscao dispersion and colloidal dispersion, has microbicidal activity against various pathogenic bacteria, including s. aureus (a gram-positive bacterium) ( table ) and viruses such as influenza a (h n ) as an enveloped virus and feline calicivirus as a nonenveloped virus (table ) . biscao water, which is colorless and transparent with a ph > . , was sprayed and dried on smooth metal or plastic surfaces and provided a white powder coating. scanning electron microscopy (sem) images of drying biscao water showed microparticles ( - µm) connected with each other (figure a,b) . cryo-sem observations indicated that cao nanoparticles ( - nm) aggregated to generate larger biscao particles ( - nm) in biscao water (figure c,d) . we examined the deodorizing and bactericidal activities of biscao water, dispersion, and colloidal dispersion against tc and cf under various conditions such as particle size and concentration. biscao water, dispersion, and colloidal dispersion containing smaller particles and at higher concentration showed higher deodorization and microbicidal activities against tc and cf, probably due to higher brunauer-emmett-teller (bet)-specific surface areas (data not shown). we anticipate that differences in the bet-specific surface areas of biscao water, dispersion, and colloidal dispersion influenced their deodorizing and microbicidal activities. cryo-sem showed that the nanoparticles in biscao dispersion, colloidal dispersion, and biscao water were - nm [ ] , - nm [ ] , and - nm in diameter, respectively. the hydration of cao generates a strong base and is the primary mechanism for the deodorization and microbicidal activities of biscao dispersion. the cao content of biscao is much higher than that of bisca(oh) , and this suggested that biscao water, suspension, dispersion, and colloidal dispersion showed higher deodorizing and microbicidal activities than bisca(oh) because of the higher ph. however, our preliminary study showed that biscao exhibited higher activity than naoh solution at the same ph (data not shown). this suggests that alkalinity alone is not responsible for the deodorizing and microbicidal property of biscao. rather, we suggest that the microbicidal action is due to the reducing activity of biscao, and that the high disinfection activity of biscao is due to the oh− concentration of the thin water layer formed around biscao particles being higher than in the bulk solvent [ , ] . furthermore, active radical species generated from magnesium oxide and biscao may also contribute to strong disinfection activity [ , ] , as supported by a multiparameter flow cytometry study conducted by hewitt et al. [ ] . although high ph is certainly the main contributor to the deodorizing and microbicidal activity of biscao, active radical species generated from biscao may be an alternative microbicidal factor. the recent worldwide epidemic of coronavirus disease (covid- ) due to a newly discovered coronavirus is causing a crisis [ , ] . the world health organization (who) recommends "to ensure that environmental cleaning and disinfection procedures are followed consistently and correctly. thoroughly cleaning environmental surfaces with water and detergent and applying commonly used hospital-level disinfectants such as naclo are effective and sufficient procedures." [ ] . some antiseptics/disinfectants, such as ethanol and naclo, show significant activity towards sars-cov- by breaking the envelope of virus. however, they are cytotoxic to cellular and organic components, and high concentrations are required for antiseptic/disinfection activity [ ] [ ] [ ] . furthermore, chlorine-derived compounds are ineffective in the presence of organic materials [ , ] . therefore, antiseptics/disinfectants that can decrease the bacterial bioburden without harmful side effects and environmental disruption are essential for environmental hygiene and public health. the character of biscao water for having virucidal activity against an enveloped-type virus ( table ) may be valuable for the limitation of the spread of respiratory viruses such as covid- , although it is necessary to study additional microbicidal activity including coronavirus sars-cov- . recommendations for personal protective equipment (ppe), including face masks such as surgical masks or n respirators, are necessary for the protection of health-care personnel [ ] . recommendations on face masks vary across countries, and the use of masks increases substantially once local epidemics begin, including the use of masks in community settings. however, this increased use of face masks by the general public exacerbates the global supply shortage of face masks, sending prices soaring [ ] . in this study, spraying biscao water on the contaminated surface of a surgical mask resulted in almost complete elimination of the test microbes without changing the quality of the mask. there is little possibility that dry microparticle-aggregates blow up and/or suck in, since nanoscale cao particles in biscao water aggregate to generate larger biscao particles and the microparticles were composed of caco produced by interaction of ca + and co . this result suggested that face masks may be reused multiple times by sterilization with biscao water, which could contribute to their safe and economical reuse, rather than disposal after a single use. however, our preliminary experiments show that the potency of biscao water does not have a long-term effect on the spraying surface, therefore the spray may need to be performed both at the start of use and after use ( figure ). according to the manufacturer, scallop shell powders were heated at • c for h, then ground using a dry super grinder (nano jetmizer nj- -d; aishin nano technologies co. ltd., saitama, japan), followed by cooling in a vacuum chamber. this provided biscao dry powder with particle diameters of - µm (average µm). this was purchased from plus lab corp., kanagawa, japan. according to the manufacturer, the content of cao in this biscao preparation is . %. bisca(oh) was obtained from scallow, kohkin inst. co. ltd., tochigi, japan and had a dry-powder-particle diameter of - µm (average µm). the cao and ca(oh) contents were < % and > %, respectively. according to the manufacturer, biscao water was prepared by adding g of biscao to ml chilled clean water (< • c), gently mixing, and standing for min. the supernatant ( ml) was collected and transferred to a -l water tank. another chilled ml of clean water was gently poured and mixed onto the remaining biscao precipitate and the supernatant was collected in the tank. this process was repeated fifty times. the produced biscao water (total l) was colorless and transparent with a ph of about . , and the biscao water is now commercially available from plus lab corp. scanning electron microscope (sem) images of dry powder were obtained by osmium metal coating using a neo-osmium coater (neoc-stb; meiwafosis co., ltd., tokyo). the surface structure of each dry powder was observed using a field-resolved scanning electron microscope (jsm- f; jeol ltd. tokyo, japan). for cryo-sem, samples were frozen in liquid nitrogen, then knife-cut and observed using a jeol jsm f sem (jeol ltd., tokyo, japan) under vacuum conditions at − • c. the accelerating voltage was kv, and the detection signal was a backscattered electron image. we evaluated the bactericidal activities of biscao water towards e. coli (nbrc ), e. coli o- :h (atcc ), p. aeruginosa (nbrc ), salmonella (nbrc ), and s. aureus (nbrc ). each bacterial suspension was prepared in soybean-casein digest broth with lecithin and polysorbate (scdlp) liquid and agar medium. the assays were performed by the japan food research laboratory (jfrl; tokyo, japan). briefly, . ml of each bacterial suspension ( - cells/ml) was added to ml of biscao water. the mixture was stirred and then left at room temperature for , , and min to allow the bacteria to interact with biscao. at each time point, the mixture was ten-fold diluted with scdlp medium to terminate the interaction. the mixtures were subjected to ten-fold serial dilution with phosphate-buffered saline (pbs) onto scdlp agar plates ( . cm). the number of cfu was determined after incubating at • c for h. to evaluate the virucidal activities of biscao water, feline calicivirus (f- ; atcc vr- ) and human influenza a virus (h n ; atcc vr- ) were used and assayed using scdlp medium and the tcid method. the assays were performed by jfrl. briefly, viral suspension in pbs ( µl) was added to -ml biscao water. the mixture was stirred and then left at room temperature for , , and min to allow the virus to interact with biscao. at each time point, the mixture was ten-fold diluted with % fetal bovine serum containing dmem to terminate the interaction. the mixtures were subjected to two-fold serial dilution with pbs in a -well cell culture plate sown with crandell feline kidney (crfk) cells for feline calicivirus and madin-darby canine kidney (mdck) cells for influenza a virus. the antiviral activity of biscao water was estimated as the tcid ratio of the biscao-treated sample to the control (treated with clean water). the addition of . , . , and . g of biscao and bisca(oh) to ml of clean water, followed by rotary mixing, generated . , . , and . wt% biscao and bisca(oh) suspensions, respectively. next, . , . , and . wt% of na hpo (fujifilm wako pure chemical corp., osaka, japan) for biscao dispersion and na-polypo (fujifilm wako pure chemical corp.) for biscao colloidal dispersion were added to . , . , and . wt% biscao water suspension, respectively, then rotary mixed to prepare each biscao dispersion and colloidal dispersion. ethanol ( . %; fujifilm wako pure chemical corp.) was used and diluted with clean water. five grams of tainted pork meat was mixed with ml of each deodorant, then placed on petri dishes and sealed in plastic bags ( × cm) for h. the odor intensity was measured using a handheld odor meter (omx-srm; shinyei technology co. ltd., hyogo, japan). the microbicidal efficacy of biscao water against suspensions and wood pieces contaminated with high normal bacterial flora was evaluated and compared with the actions of ethanol, biscao and bisca(oh) suspensions, povidone iodine, naclo, biscao dispersion, and colloidal dispersion. various concentrations of povidone iodine and naclo were prepared by the dilution of -wt% isodine (meiji seika pharma co., ltd., tokyo, japan) and -wt% naclo (yoshida pharmaceutical corp., tokyo, japan) with clean water. the concentrations of naclo were confirmed as residual chlorine levels using clo (hclo and clo -) -selective test papers (high concentration, - ppm; low concentration, - ppm; kyoritu check laboratory corp., tokyo, japan). suspensions contaminated with normal bacterial flora were prepared by incubating bathtub water with % dmem + bsa ( . wt%) at • c for h [ , ] . ten milliliters of each disinfectant were added to ml of the contaminated suspension, mixed well, and incubated at room temperature for min. to prepare contaminated wood pieces, five wood pieces ( . cm × . cm × . cm) were added to ml of the contaminated suspension and incubated at • c for h, then rinsed with clean water [ , ] . for the disinfection assay of contaminated wood pieces, a wood piece was added to ml of each disinfectant, vortexed gently for min, and then tc and cf were released from the contaminated wood piece in ml of clean water by vigorous vortexing for min. to count the number of cfu, aliquots ( ml of each mixture) were gently poured into individual petri dishes containing prealiquoted portions of simple and easy dry medium for tc or cf (nissui pharmaceutical co., ltd., tokyo, japan) [ , , , ] , and the plates were incubated for h in a • c incubator (a ; ikuta sangyo co., ltd., ueda, nagano, japan). plating and counting were performed as a set of technical replicates (n = ). suspension contaminated with . ± . (× ) cfu/ml of tc and . ± . (× ) cfu/ml of cf ( µl total) was inoculated and dried for min at room temperature on a round area ( -cm diameter) of the surface of a surgical mask (surgical mask st; utsunomiya seisaku co. ltd. osaka, japan). "spray before use" was conducted by spraying about . ml of biscao water on the round area and drying for h at room temperature. "spray after use" was conducted by spraying about . ml of biscao water on the inoculated round area and drying for h at room temperature. the inoculated area of each mask was resected, and then tc and cf were released from the contaminated sections into ml of clean water by vigorous vortexing for min. to count the cfu, aliquots ( ml of each mixture) were gently poured into individual petri dishes containing prealiquoted portions of simple and easy dry medium for tc or cf, and the plates were incubated for h in a • c incubator. plating and counting were performed as a set of technical replicates (n = ). biscao water contains nanoparticles ( - nm) and is colorless and transparent, with a ph > . . biscao water completely eliminated various pathogenic bacteria within min and viruses within min in in vitro assays. furthermore, biscao water exhibited higher deodorization of tainted pork meat and higher microbicidal efficacy using suspensions contaminated with normal bacteria compared to ethanol, biscao and bisca(oh) suspensions, povidone iodine, naclo, biscao dispersion and colloidal dispersion. the results showed that biscao water has excellent deodorization and disinfection activities for pathogenic microbes, potentially for covid- measures. funding: this study was partially supported by jsps kakenhi (no. k for y.s.). the authors declare no conflict of interest, and the founding sponsor had no role in the design of the paper. an overview- a novel method to make regenerable core-shell calcium-based sorbants antimicrobial characteristics of heated scallop shell powder and its application antibacterial characteristics of heated scallop-shell nano-particles inactivation of avian influenza virus, newcastle disease virus and goose parvovirus using solution of nano-sized scallop shell powder sporicidal kinetics of baccillus subtilis spores by heated scallop shell powder comparison of antifungal activities of scallop shell, oyster shell and their pyrolyzed products disinfection treatment of heated scallop-shell powder on biofilm of escherichia coli atcc surrogated for e. coli o :h ability of heated acallop-shell powder to disinfect staphylococcus aureus biofilm heated scallop-shell powder treatment for deactivation and removal of listeria sp. biofilm formed at a low temperature comparison of various disinfectants on bactericidal activity under organic matter contaminated water skin cleansing technique with disinfectant using improved high-velocity steam-air micro mist jet spray chronic wounds and bacteria. clinical relevance, detection and therapy evidence-based practice: tap water cleansing of leg ulcers in the community a systematic review of wound cleansing for pressure ulcers stability of weak acidic hypochlorous acid solution with microbicidal activity effects of a low concentration hypochlorous acid nasal irrigation solution on bacteria, fungi, and virus disinfection by hypochlorous acid for pseudomonas aeruginosa-infected wounds in diabetic db/db mice development of antimicrobial biomaterials produced from chitin-nanofiber sheet/silver nanoparticle composites adsorption of silver nanoparticles onto different surface structures of chitin/chitosan and correlations with antimicrobial activities synthesis and application of silver nanoparticles (ag nps) for the prevention of infection in healthcare workers healing of pseudomonas aeruginosa-infected wounds in diabetic db/db mice by weakly acidic hypochlorous acid cleansing and silver nanoparticle/chitin nanofiber sheet covering bioshell calcium oxide (biscao) for cleansing and healing of pseudomonas aeruginosa-infected wounds in hairless rats cleaning technique using high-velocity steam-air micromist jet spray preparation and application of bioshell calcium oxide (biscao) nanoparticles-dispersions with bactericidal activity application of colloidal dispersions of bioshell calcium oxide (biscao) for disinfection persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents simple and environmentally friendly preparation and size control of silver nanoparticles using an inhomogeneous system with silver-containing glass powder an evaluation of the antibacterial action of ceramic powder slurries using multi-parameter flow cytometry human coronavirus: insights into environmental resistance and its influence on the development of new antiseptic strategies a toxicity index of skin and wound cleaners used on in vitro fibroblasts and keratinocytes in vitro toxicity of topical antimicrobial agents to human fibroblasts cytotoxicity of silver nanoparticle and chitin-nanofiber sheet composites caused by oxidative stress covid- coronavirus: recommended personal protective equipment for the orthopaedic and trauma surgeon rational use of face masks in the covid- pandemic key: cord- -yw jvf authors: li, w. d.; hou, j. l.; wang, w. q.; tang, x. m.; liu, c. l.; xing, d. title: effect of water deficit on biomass production and accumulation of secondary metabolites in roots of glycyrrhiza uralensis date: - - journal: russ j plant physiol doi: . /s sha: doc_id: cord_uid: yw jvf two-year-old seedlings of licorice plant (glycyrrhiza uralensis fisch) were exposed to three degrees of water deficit, namely weak ( – %), moderate ( – %), and strong ( – %) relative water content in soil, whereas control plants were grown in soil with – % water content. moderate and strong water deficit decreased the net photosynthetic rate, stomatal conductance, and biomass production. water use efficiency and the root-to-shoot ratio increased significantly in response to water deficit, indicating a high tolerance to drought. weak water deficit did not decrease root biomass production, but significantly increased the production of glycyrrhizic acid (by %) and liquiritin (by %) in the roots. therefore, a weak water deficit can increase the yield of root medical compounds without negative effect on root growth. licorice (glycyrrhiza uralensis fisch.) is a very popular medicinal plant, which roots contain glycyr rhizic acid and liquiritin mainly accumulated in the root and rhizome tissues [ , ] . recently, glycyrrhizic acid has been found to be highly active in inhibiting the replication of the severe acute respiratory syn drome (sars) associated virus and has been sug gested as a potential therapeutic agent for chronic hepatitis and acquired immunodeficiency syndrome (aids) [ ] . licorice plants appear to be highly drought tolerant, being a favorable plant to restore degraded desert, arid and semiarid ecosystems of northwest china [ ] . however, data on physiological processes, such as biomass production and secondary metabolite yield, in response to environmental condi tions are lacking [ ] . water deficit usually inhibits plant growth and pro ductivity by affecting gas exchange and especially pho tosynthesis [ , ] . water use efficiency (wue) can be traditionally defined as the ratio of net photosynthesis to transpiration over a period of seconds or minutes [ ] . the higher wue has been mentioned as a strategy this text was submitted by the authors in english. to improve crop performance under water limited conditions [ ] . however, in licorice plants photosyn thesis and biomass production as well as wue in response to water deficit were not studied. water deficit can induce the biosynthesis of some secondary metabolites [ ] [ ] [ ] , resulting in their accumulation in medicinal plants [ , , ] . for example, the concentration of rutin and chlorogenic acid increased with drought severity in tomato plants [ ] . although the responses of the metabolites to drought have been investigated in some medicinal plants [ , ] , no reference concerning the effect of various water deficit levels on their production by lic orice roots is available. the present study aims to determine the effect of water deficit on gas exchange, biomass and secondary metabolites production in licorice plants. it was hypothesized that a suitable water deficit, in addition to saving water, can also increase the amount of root secondary metabolites without negative effect on root growth. water treatments were carried out from may until the end of october in . four levels of soil rel ative water content (wc), - , - , - , and - %, represented control plants, weak, moderate, and strong water deficit, respectively. each pot was weighed and water was added to reach the target level at : p.m. every day. there were four replications per treatment arranged in a completely randomized block design. leaf gas exchange. the newly developed leaves from the middle part of the shoot were chosen for gas exchange measurement using a li portable pho water use efficiency (wue) was calculated as the ratio of the net photosynthetic rate to transpiration rate. biomass determination. biomass determination was carried out by the end of october. licorice plants were separated into the roots and shoots. dry weights were determined after drying for h at °c in an oven. root to shoot ratio = root dry weight: shoot dry weight. then the dried roots were used for glycyrrhizic acid and liquiritin analyses. glycyrrhizic acid and liquiritin analyses. glycyr rhizic acid and liquiritin were extracted as described in [ ] . dry roots were extracted with a tenfold volume of . % ammonia for min under ultrasonication ( w, khz). glycyrrhizic acid and liquiritin con centrations were determined with a hp high per formance liquid chromatography system (agilent technologies, united states) consisting of a g a pump, a g a degasser, and a g a autoinjector connected to a g b diode array detector (dad). the separation was performed on a dikma diamon sil tm c column ( mm × . mm × µm) with a mobile phase consisting of . % h po (solvent a) and acetonitrile (solvent b). the sample ( µl) was eluted with a gradient profile, and the column was maintained at °c [ , ] . statistical analysis. statistical treatment was per formed using a spss statistical package (version , spss, chicago, united states). the difference between the mean values of each treatment was deter mined using duncan's multiple range test and consid ered significant at p < . . the net photosynthetic rate, stomatal conduc tance, and transpiration rate decreased with increas ing water deficit (table ). compared to the control, - % wc had no effecton the net photosynthetic rate and stomatal conductance, but at - and - % wc, photosynthesis and transpiration were sig nificantly reduced. however, water deficit increased wue and the highest value was observed at the - % wc. dry weight of the plant and its organs decreased with increasing water deficit, but no effect was exerted at - % wc ( table ) . root dry weight decreased by . , . , and . % with increasing water deficit, while shoot dry weight decreased by . , . , and . %, respectively (table ). root to shoot ratio increased as water deficit progressed (table ) . the gain in the content of glycyrrhizic acid and liq uiritin in the roots under - % wc had the highest ( . and . %, respectively), followed by the mod erate treatment ( . and . %, respectively) as com pared to the control plants. there was no significant difference between the strong wc treatment and con trol (figs. a, b) . at - % wc, the amounts of glycyrrhizic acid and liquiritin in plant roots were the highest among the studied four levels of soil water con ditions: the gain increased by . and . %, respectively (figs. c, d) . at the lowest wc, glycyr rhizic acid amount in plant roots decreased, although no difference in liquiritin amount between these and control plants was found (figs. c, d ). it is well known that water deficit is one of the major factors limiting plant growth and yield [ , ] . in this study, licorice plants were able to grow and pro duce biomass even at - % wc (table ) , suggest ing that this plant can acclimate in response to unfa vorable environment and exhibit high drought resis tance. water deficit induces partial closing of stomata and both transpiration and photosynthesis decrease, thereby slightly increasing wue [ ] , especially at - % wc ( table ). the increase in the efficiency of water use under drought occurs at the expense of absolute biomass production [ ] . in order to dimin ish metabolism consumption and increase uptake of water under dry conditions, plants often decrease their growth rate and biomass production, and contribute more synthesized biomass to roots, so that they could maintain a higher root to shoot ratio ( table ) . parti tioning more assimilate to the underground parts and maintaining the higher root to shoot ratio may con tribute to enhanced water uptake [ ] . thus, these responses allow licorice plants to survive and even to continue to grow under conditions of water shortage, i.e., to develop drought tolerance. water deficit decreases plant photosynthesis and thereby reduces plant growth and biomass production ( tables , ). however, the dry weight of the roots decreased less than that of shoots (table ) . no signif icant difference was found between the dry weights of roots and the shoots at - and - % wc, while other treatments significantly decreased biomass pro duction (table ). our findings indicate that weak water deficit did not affect growth and biomass pro duction of licorice roots. it has been well documented that water deficit can also affect the production of secondary metabolites in some medicinal plants [ , , ] . biosynthesis of secondary metabolites is known to be affected by drought, indicating influence by environmental stim uli [ , ] . for example, the content of several alka loids increased in response to drought in tabernae montana pachysiphon [ ] . liu [ ] also found that camptothecin concentrations briefly rose when camp totheca acuminata seedlings experienced drought. glycyrrhizic acid and liquiritin, the major bioactive components of g. uralensis, are accumulated in the underground parts of licorice plants [ , ] . thus, soil wc plays a key role in their biosynthesis. weak water deficit significantly increased not only glycyrrhizic acid and liquiritin concentrations per gram dry weight (figs. a, b) , but also total amount of these com pounds in plant roots (figs. c, d) . the synthesis of secondary metabolites was stimulated under weak drought conditions. zhu et al. [ ] also found that mild water deficit significantly increased saikosaponin a and d contents in bupleurum chinense roots. our results confirm that weak water deficit can increase secondary metabolite contents and thereby increase the quality of medical row material. development of a monoclonal antibody based enzyme linked immu nosorbent assay for the analysis of glycyrrhizic acid separation of glycyrrhizic acid and liquiritin from glycyrrhiza uralensis fisch extract by three liquid phase extraction systems glycyrrhizin, an active component of liquorice roots, and replication of sars associated coronavirus effect of salt and drought stress on antioxidant enzymes activities and sod isoenzymes of liquorice (glycyrrhiza uralensis fisch), plant growth regul effect of low light intensity on growth and accumulation of secondary metabo lites in roots of glycyrrhiza uralensis fisch photosynthetic response to water stress and changes in metabolites in jasminum sambac, photo synthetica photosynthetic responses to chromosome doubling in relation to leaf anatomy in lonicera japonica subjected to water stress physiological and growth responses of populus davidiana ecotypes to different soil water contents impact of water supply on photosynthesis, water use and carbon isotope discrimination of sugar beet genotypes drought induced in vivo synthesis of camptothecin in camptotheca acuminata seedlings anti oxidant potential and indole alkaloid profile variations with water deficits along different parts of two variet ies of catharanthus roseus impact of fertilization on drought response in the medicinal herb bupleurum chinense dc growth and alkaloid contents in leaves of tabernaemontana pach ysiphon stapf (apocynaceae) as influenced by light intensity, water and nutrient supply drought stress in tomatoes: change in plant chemis try and potential nonlinear consequences for insect herbivores simultaneous determina tion of glycyrrhizic acid, liquiritin and fingerprint of licorice by rp hplc effects of drought stress and n supply on the growth, biomass partitioning and water use efficiency of sophora davi dii seedlings we would like to thank dr. e.s.j. harris (medical school, harvard university, united states) and dr. h. xu (institute of botany, chinese academy of sciences, china) for critical reviews of the manuscript.the work was supported by natural science foun dation of china (nos. , ). key: cord- - farxcc authors: koibuchi, yukio; sato, shinji title: numerical simulation of urban coastal zones date: journal: advanced monitoring and numerical analysis of coastal water and urban air environment doi: . / - - - - _ sha: doc_id: cord_uid: farxcc nan water quality is directly and indirectly influenced by a variety of flows. for example, since phytoplankton drifts passively according to water flows, it is strongly influenced by the distribution of the flow of the bay (lucas et al. ) . bay water motions also exert a strong influence on other water quality parameters. these kinds of influences are direct and obvious. the degree of influence naturally increases with the strength of the currents. in contrast, even if the flow is very small, it has some spatial patterns in common with outer bay directions. in this case, a nutrient load that is discharged at the head of the bay is transported to distant locations and, probably, the water exchange rate of the bay also increases. the nutrient loading that is permissible -i.e. which the bay can accept -will then increase due to the increase in the water exchange rate. for water retention rates, the strength of currents is not as important as spatial patterns. for example, tidal currents are dominant in urban coastal areas. however they are oscillatory. water particles in tidal currents move to the head of the bay during flood tides, but move back to the mouth of the bay during ebb tides. as a result, tidal currents do not substantially transport water particles. in contrast, density currents are clearly weaker than tidal currents, but they flow in one direction continuously and transport water particles more efficiently. as a result, they substantially effect water retention times and ecosystem characteristics. a water quality model for bays must therefore consist of a threedimensional circulation model and an ecosystem model that describes pelagic and benthic aspect of nutrients cycling. this section focuses on the physical modeling and the next section deals with water quality modeling. the final section discusses the application of these models to tokyo bay. many three-dimensional hydrodynamic models have been developed in the last decades, including pom (princeton ocean model; blumberg and mellor ) , ch d (curvilinear hydrodynamics in dimensions; johnson et al. ) and roms (regional ocean modeling system; maccready et al. ; li et al. ) . these models solve the navier-stokes equation with the forcing (the wind stress, coriolis force and buoyancy force) under adequate approximations that are called the hydrostatic and boussinesq approximations. hydrostatic approximation assumes that there is a perfect balance between pressure gradients and gravity: in other words, no acceleration occurs in a vertical direction. this is justified because the aspect ratio of urban coastal areas is extremely small, and hence the vertical motions are considered to be small and also to be further inhibited by gravitational forces under stable density stratification. this means that vertical acceleration is negligible and the fluid behaves as though it were under static equilibrium as far as vertical motion is concerned (prudman ) . density variations in urban coastal areas are also small (less than % or so), and so density can be considered to be constant, except when body forces resulting from the motion of a density stratified fluid in a gravitational field are concerned. this approximation is called the boussinesq approximation: in other words, changes in the mass or inertia of a fluid body due to the changes in its density are negligible, while the same changes in density are consequential when the gravitational field is present ( kantha and clayson ) . therefore, following boussinesq ( ) , this approximation justifies replacing r by a constant reference density r everywhere except in terms involving gravitational acceleration constant g. under such approximations, the governing equations are transformed as follows: here, t stands for time. u , v, and w are the velocity components in x , y , and z directions. the symbol r′ is the reference density and it is defined as r = r + r′ . the symbols a x , a y and a z are the eddy viscosities in x , y , and z directions. the symbol g is the acceleration due to gravity. all currents including tides, wind-driven currents, and density currents in urban coastal zones are strongly influenced by geometry and bathymetry, whereas these areas are rarely regular in shape. in particular, a coastline near an urban coastal area is more complex, due to reclamations and the constructions of harbors, than a natural one. in addition, the uniformity of the bathymetry further lessened as a result of dredging for vessel transport. a computational grid is required to accurately represent such complex geometry and bathymetry. for this reason, the selection of which grid system to use has varied along with the progress of modeling, although the governing equations are not basically different. for vertical coordinate systems (shown in fig. - ), cartesian ( z -coordinate vertical grid) and sigma-coordinate grids have been widely used. a cartesian grid is easy to understand, and shows the correspondence between program codes and governing equations. it is sometimes more accurate than sigmacoordinate grids, especially if the bathymetry of the bay is simple and mild. the sigma-coordinate system tends to have an error featuring the presence of steep-bottom topography. however, unless an excessively large number of vertical levels are employed, the cartesian grid fails to represent the bottom topography with satisfied accuracy. the sigma-coordinate system is convenient in the sense that it can essentially introduce a "flattening out" mechanism for variable bottoms at z = −h(x, y). the flow near the seabed is also calculated well. moreover, the sigmacoordinate system is easy to program since the number of vertical grids can be the same, and the setting of boundary conditions will be simple. the sigma-coordinate system has long been widely used in both meteorology and oceanography (phillips ; freeman et al. ) . after the incorporation of approximations, the governing equations in the sigma coordinate system are as follows: where, t stands for time. u , v, and s ⋅ are the velocity components in x , y, and z directions in the s ⋅ coordinate system. h is change in surface elevation, h is initial water depth, and h is the total water depth ( h=h + h ). f is the coriolis coefficient and p stands for pressure. r and r′ are the constant reference density and the deviation from it, and r = r + r′ . a h and a v are the horizontal and vertical eddy viscosity coefficients, respectively. g is acceleration due to gravity. recently, the stretched grid system (s-grid system) has also been popular. this grid system is an extension of sigma-coordinate system. generally, a sigma-coordinate grid divides the vertical coordinate into an equal number of points. the s-grid system has a higher resolution near the surface and bottom (haidvogel et al. ) . wind-stress and bottom friction are considered at surface and bottom boundary conditions, respectively. settings in boundary conditions are easy in sigma-coordinate systems since they use the same number of vertical grids. at lateral boundaries, normal velocities are set at zero, and a free slip condition is applied to the friction terms. at open boundary, the velocity gradient is set at zero. horizontal computational grids are also modified to fit topography. the simplest horizontal computational grid is the rectangular grid with fixed spacing. the rectangular grid is equivalent to the cartesian vertical grid. recently, curvilinear coordinate systems have been widely used. these systems allow greater flexibility than rectangular grid systems. fig. - is (from ming et al. ) an example of a horizontal curvilinear coordinate system. the chesapeake bay, like other bays, has a typical complex geometry, and thus a horizontal curvilinear coordinate system is advantageous (li et al. ) . this is extended to a nested grid system in which finer grids are used in regions to yield detailed information. in urban coastal areas, density difference plays an essential role for water quality and currents. one of the most important phenomena induced by the density effect is stratification. once stratification occurs in a coastal zone, surface water and bottom water are isolated. this process is very important when we discuss the distributions of pollutants from the land. stratification also enhances the increase of phytoplankton in the surface layer and oxygen depletion near the seabed. moreover, estuarine circulation is induced by density differences in the salty sea water and the river flow. fig. - shows a schematic diagram of estuarine circulation. river water runs through the urban area and runs off from the river mouth, spreading over the sea surface like a veil since river water has low density compared with saline sea water. to cancel the density difference between the river water and the saline water, a great deal of sea water is entrained into the river water flow. such a mixing process continues until the river water reaches the same density as the surrounding sea water, resulting in vertical circulation in the bays that is is several to ten times greater than the river flux (unoki ) . thus, estuarine circulation induces seaward currents on the surface and landward currents near the bottom. the speed of the currents is slow compared with the tidal currents, as explained previously. since estuarine circulations are in a fixed direction, its material transport is very effective over a long time scale in spite of the small magnitude of its velocity. estuarine circulation also plays an important role in the nutrient cycles of stratified bays. organic matters are deposited on the seabed after phytoplankton blooms or river runoffs. they are decomposed by bacteria in the seabed. these nutrients are supplied from the seabed under anoxic condition in summer. in order to include density effects in the numerical model, conservation equations for temperature and salinity are also included. then, to obtain a realistic prediction for vertical stratification, a turbulent closure model is employed (mellor and yamada ) . consequently, flows driven by various mechanisms -e.g. the gravitational, wind-driven, and topographically induced flows -can be reproduced within physical numerical models. diffusion equations for temperature and salinity in the sigma coordinate system are as follows: here, t and s stand for temperature and salinity, respectively. heat balance and moisture balance at surface are considered as surface boundary condition for temperature and salinity, respectively. c p and q stands for specific heat coefficient and net surface heat flux at the surface, respectively. r stands for river discharge. k h and k v are the horizontal and vertical eddy diffusion coefficients, respectively. the vertical mixing coefficients, v a and v k are obtained by appealing to the second order turbulent closure scheme of mellor and yamada ( ) , which characterizes turbulence by equations for the kinetic energy of turbulence, q , and turbulence macro scale l , according to: r r ( . ) wall proximity functions w is defined as follows: . ) mixing coefficients are given as: the stability functions s m , s h , and s q are analytically derived from algebraic relations. they are functionally dependent on , q and l . these relations are derived from closure hypotheses described by mellor ( ) and later summarized by mellor and yamada ( ) . a semi-implicit finite difference scheme has been adopted where equations are discretized explicitly in the horizontal direction and implicitly in the vertical direction. an arakawa c staggered grid has been used with the first order upwind scheme. the tri-diagonal formation of the momentum equation is utilized, and in combination with the mass conservation equation, an algebraic equation is obtained where the only unknown variable is the surface elevation h in implicit form. this algebraic equation is solved through the successive over relaxation (sor) method. the phenomena in urban coastal zone are not only physical but also biological or chemical, each of which relates to the other. the ecosystems and water quality of urban coastal zones are highly complicated. to deal with these complex systems, a water quality model is composed of a three-dimensional physical circulation model and an ecosystem model that describes pelagic and benthic aspect of nutrients cycling. the pelagic and benthic systems also have interactions with each other. in ecosystem models, each water quality variable is often called a compartment. various kinds of models are also proposed for ecosystem models (kremer and nixon ; fasham et al. ; chai et al. ; kishi et al. ) . some models, such as ce-qual-icm (cerco and cole ) each model is developed with basic aquatic compartments such as phytoplankton, zooplankton, and nutrients. some differences in the modeling of sediment, detritus, and the detailed modeling of phytoplankton exist, depending on the target ecosystems and the objectives of the study. as a result, no fully adaptive model applicable for all water bodies exists. if we were to make a model that could be adapted for all areas, its results would be too complex to discuss. it would not be so different from observing the real world. for example, ocean ecosystem models tend to focus only on pelagic systems. they tend to ignore benthic modeling, since the open ocean is deep enough to prevent the return of detritus to the seabed. meanwhile, ocean ecosystem models generally deal with some metals in order to represent the limiting factor of phytoplankton. these metals are fully abundant in urban coastal zones. however, they are often depleted during phytoplankton growth in the open ocean. on the other hand, the concentration of phytoplankton in coastal areas is highly variable both spatially and temporally as compared to the open sea. subsequent sedimentation of this bloom also constitutes a major input to benthic ecology (waite et al. ; matsukawa ; yamaguchi et al. ) . to represent these phenomena, ecosystem models of coastal zones usually cover benthic systems. ecosystem models solve conservation equations for relevant components with appropriate source and sink terms. this is the same as temperature and salinity modeling in physical models, as explained in sect. . . . for the sigma coordinate system, a mathematical formulation of the conservation of mass is written by: where c denotes concentration of the water quality variable and t is time. fluxes into and out of the target control volume are calculated by using physical model results. s(x,y,s,t) represents sources or sinks of the water quality variable due to internal production and the removal of the biogeochemical effect. it also represents the kinetic interactions of each compartment. w(x,y,s,t) represents the external inputs of the variable c . for example, phytoplankton constitutes the first level in the food chain of the pelagic ecosystem of bays. phytoplankton photosynthesizes by using sunlight and increases. at this time, the source term s of phytoplankton is increased depending on the amount of photosynthesis that takes place. phytoplankton is then decreased by the grazing of zooplankton. the source term s of zooplankton is increased along with this grazing, and the source term of phytoplankton is decreased. ecosystem models basically express the relationship of each compartment through mathematical expressions. these models provide a quantitative description of the influences of physical circulation on the biological and chemical processes of urban coastal zones. the ecosystem model introduced here was developed to simulate the nutrient budget of an urban coastal zone. it includes the temporal and spatial variations of phytoplankton, nutrients, detritus, and dissolved oxygen (do). in urban coastal zones, nutrients emitted from urban areas are not a limiting factor for phytoplankton growth. however, quantifying the nutrient budget is essential for analyzing and restoring the ecosystems of urban coastal zones. fig. . shows schematic interactions of a lower trophic ecosystem model which is used for tokyo bay (koibuchi et al. ) this model has state variables: phytoplankton (phy), zooplankton (zoo), nutrients (nh , no , po and si), labile detritus (ldon, ldop, ldosi) and refractory detritus (rdon, rdop, rdosi) for each nutrient, labile detritus carbon (ldoc), refractory detritus carbon (rdoc), dissolved organic carbon (doc), and dissolved oxygen (do), as well as sedimentation processed from particulate organic material. since the basic structure of the model follows the widely applied ce-qual-icm cole , ) , this section mainly focuses on our modifications of the ce-qual-icm model in the following description. this model deals with four phytoplankton groups. phyd is based on skeletonema costatum, which is a dominant phytoplankton species in tokyo bay. phyd represent a winter diatom group (such as eucampia ). phyr is a mixed summer assemblage consisting primarily of heterosigma akashiwo and thalassosira . phyz denotes the dinoflagellates. these four phytoplankton assemblages have different optimal levels of light for photosynthesis, maximum growth rates, optimal temperatures for growth, and half saturation constants for nutrient uptake. diatoms only use silica during growth. the time rate of change of phytoplankton due to biological activity and sink is given by: ( . ) where x = d , d , r, z , denote each phytoplankton assemblage. the phytoplankton growth rate m depends on temperature t , on photosynthetically available radiation i , and on the nutrient concentration of nitrogen, phosphorus, and silica : idealized nutrient cycling in tokyo bay's ecosystem according to the model of koibuchi et al. ( ) . cycling between the state variables: phytoplankton, zooplankton, nutrients (nitrogen, phosphorus, and silicate), labile detritus and refractory detritus for each nutrient, and dissolved oxygen, as well as sedimentation processed from particulate organic materials where m max (t) is the growth rate at ambient temperature, which relates m max , the maximum growth rate m max = m · . t (eppey ) , t opt , the optimal temperature of each plankton assemblage, and b and b are shaping coefficients, k no , k nh , k po and k si , which is the michaelis of the halfsaturation constant for each nutrient. i is exponentially decreasing with water depth z according to: . ) where i is shortwave radiation, and par is the fraction of light that is available for photosynthesis. k w , k chl , and k sal are the light attenuation coefficients for water, chlorophyll, and depth average salinity, respectively. suspended sediment reduces underwater light intensity and affects the growth of phytoplankton. the effect of suspended sediment concentration on light intensity should be simulated as its own compartment. however, the re-suspension rate of mixed mud and the available data on the concentration of sediment suspended in river water and on the seabed are very limited. therefore, we used observation results of salinity based on field observation data from and , as shown in fig. - . the function l(i ) represents the photosynthesis-light relationship (evans and parslow ) , the rate of phytoplankton grazing, g, which is a function of an ambient temperature: where k grz is the predation rate at °c. other phytoplankton loss terms are mortality, represented by the linear rate m p , where w px is the constant vertical sinking velocity for each phytoplankton. the growth rates of zooplankton are expressed as follows: here b is the assimilation efficiency of phytoplankton by zooplankton, and l bm and l e denote excretion due to basal metabolism and ingestion, while the remaining fraction is transferred to the detritus. m z is the loss coefficient of zooplankton mortality. the nutrient compartments have four principal forms for each nutrient (nitrogen, phosphorus, and silica): dissolved organic nutrients, labile and refractory particulate organic nutrients (lpon and rpon, respectively) , and dissolved inorganic nutrients. only the dissolved inorganic nutrients are utilized by phytoplankton for growth. nutrients are changed to these various organic and inorganic forms via respiration and predation. fig. . shows an example of nutrient cycles using phosphorus. dop, lpop, and rpop work as a pool of phosphorus. for example, certain labile compounds that are rapidly degraded, such as the sugars and amino acids in the particulate organic matter deposited on the sediment surface, decompose readily; others, such as cellulose, are more refractory, or resistant to decomposition. table . shows the distributions of each detritus form by each event, based on pett ( ) . ammonia and nitrate are utilized by phytoplankton for growth. ammonia is the preferred form of inorganic nitrogen for algal growth, but phytoplankton utilize nitrate when ammonia concentrations become depleted. nitrogen is returned from algal biomass to the various dissolved and particulate organic nitrogen pools through respiration and predatory grazing. the time rates for variations due to the biological processes of nitrate and ammonium are as follows. denitrification does not occur in the pelagic water systems in this model, but rather occurs in the anoxic sediment layer. as a result, if denitrification occurs in the sediment, nitrate is transferred by diffusion effect into the sediment. phosphorus kinetics is basically similar to nitrogen kinetics except for the denitrification and the alkaline phosphatase effects of the dop degradation processes. many phytoplankton can enhance alkaline phosphatase activity. this effect makes it possible for them to use phosphate from dop pools (fitzgerald and nelson ) . this effect is formulated in the following model: ( . ) where, r dop is the decomposition rate for dop, r dop-min is the minimum constant of dop decomposition(day − ), k po is a half saturation constant of phosphate uptake, and r dop-di is the acceleration effect of dop decomposition by diatoms. the kinetics of the silica is fundamentally the same as the kinetics of the phosphorus. only diatoms utilize silica during growth. silica is returned to the unavailable silica pool during respiration and predation. the sediment system is zoned in two layers (see fig. . ), an aerobic and an anoxic layer. organic carbon concentrations in the sediment are controlled by detritus burial velocity, the speed of labile and refrigerate organic carbon decomposition, and the rate constant for the diagenesis of particulate organic carbon. the thickness of the aerobic layer is calculated by oxygen diffusion when the amount of oxygen at the bottom layer of the pelagic system isn't zero. the nutrient model, which is a simplified version of the model, treats the nutrients ammonium, nitrate, phosphate, and silica and their exchanges with the pelagic system. silicate-dependent diatoms and non-silicate-dependent algae are distinguished. dissolved oxygen is an essential index for the water quality of an urban coastal zone. sources of do included in the model are reaeration at the sea surface, photosynthesis of phytoplankton, and do in inflows. the sink of do includes respiration of phytoplankton and zooplankton, oxidation of detritrial carbon (ldoc and rdoc), nitrification, and sediment oxygen demand. the time variation of do is formulated as follows: ( ) where, k oc is the oxygen to carbon ratio. k on is the oxygen to nitrogen ratio. k a and q a t- denote the reaeration rate at °c and the temperature coefficient for reaeration at the sea surface, respectively. k nh and q nh t- are the ammonia oxidation rate at °c and the temperature coefficient. k nit is the half saturation constant of ammonia oxidation. k rdoc and q rdoc t- are the rdoc mineralization rate at °c and the temperature coefficient for rdoc mineralization. k ldoc and q ldoc t- mark the ldoc mineralization rate at °c and the temperature coefficient for ldoc mineralization. k mldoc is the half saturation constant for ldoc mineralization. the concentration of do saturation is proportional to temperature and salinity. oxygen saturation value is calculated by using the following equation: where t is temperature and s is salinity. tokyo bay is located at the central part of the main island of japan. the inner bay, the north of which is km in length in its narrowest channel (fig. . ) along the main axis of the bay, connects to the pacific ocean. its average depth and width are m and km, respectively. tokyo bay is one of the most eutrophicated bays in japan. phytoplankton increase in the surface layer from late spring to early fall, and oxygen depletion and the formation of hydrogen sulfide occur on the sea bed. the sea-water color at the head of the bay sometimes becomes milky blue-green in late summer after a continuous north wind (koibuchi et al. ) . this phenomenon is called a blue tide. in the last decade, a variety of water quality observation equipment has been developed. this has made it easier to measure water quality than in the past. however, even with advanced technology, measuring the flux of nutrients is not easy. to quantify the nutrients budget, we applied our numerical model to tokyo bay. the computational domain was divided into km horizontal grids with vertical layers. computation was carried out from april , to march , , with time increments of s provided by the japan meteorological agency giving hourly meteorological data that included surface wind stress, precipitation, and solar radiation. at the open boundary, an observed tide level was obtained which can be downloaded from the japan oceanographic data center (jodc) of the japan coastal guard. don and dop was obtained at % of tn, tp based on the observation results of suzumura and ogawa ( ) at the open boundary. fig. - shows a temporal variation of the computed density at s . the simulation of water column density over the whole period (april -october ) agreed well with measured density. variations between simulated and observed values were generally less than . through the water column. time variation of density effectively reproduced observed results, including shortterm wind-induced variation, formation of stratification during summer, and mixing after continuous strong wind in the middle of october. calculation results also reproduced an upwelling event during the summer season. total chlorophyll-a concentrations in the surface water were reproduced relatively well by model simulations. the model captured the temporal increase in chlorophyll-a that were seen in the observation results, as denoted by arrows in fig. - . during these periods, phytoplankton increased more than m g/l. in tokyo bay, a red tide is defined as a chlorophyll a concentration of greater than m g/l. four different types of plankton assemblages do showed high variability compared with field measurements at the bottom, and was relatively higher than the field data from late september through october. oxygen-depleted water was made on the seabed, representing a basic trend for do variations (fig. - ) . the simulation of phosphate captured not only the observed increase in the surface layer during the summer season, but also inter-annual variability observed over the study period (fig. - ) . for example, phosphate concentrations increased from june to july due to phosphate release from sediment. simulation results represented this kind of trend based on the oxygen-depleted water. fig. - shows nitrate concentration. nitrate concentration in the surface layer fluctuated considerably during this period. nitrate levels doubled or tripled occasionally at the surface. the timing of the high nitrate concentrations and the low density in the surface layer coincided with increases in the river discharge. concentrations of nitrate were underestimated in bottom waters during summer. further study is needed to simulate denitrification processes in the sediment layer. fig. - shows the calculation results of an annual budget of nitrogen and phosphorus in tokyo bay. the annual budget is useful in understanding nutrient cycles. nitrogen is supplied to a considerable degree from rivers, since atmospheric nitrogen input is significant around urban areas. phytoplankton uptake the nitrogen and sink to bottom waters, where they are decomposed by heterotrophic processes which consume oxygen. at the head of the bay (between the line and line in fig. - ) , about % of nitrogen is sunk as detritus and % of it is lost into the atmosphere by denitrification. ammonia released from sediment reaches %. about % of the nitrogen load flows out from the bay. in contrast, atmospheric phosphorus input to the bay is negligible compared to the contribution of phosphorus from other sources. phosphate is released from sediment in the same amount as that discharged from the rivers, and it is transported to the head of the bay by estuarine circulation. as a result, the amount of phosphate in the inner bay remains high. in contrast, nitrogen is mainly supplied from the river mouth and transported quickly out of the bay. in conclusion, nitrogen and phosphorus showed important differences in the mechanisms by which they cycle in fig. - . summary of fluxes and process rates calculated in tokyo bay from january to january . units are given in ton/year for each element the bay. the regeneration of nutrients and their release from the sediment is an important source for phytoplankton growth and is equal to the contributions from the rivers. phosphorus in particular is largely retained within the system through recycling between sediment and water. these results denote the difficulty of improving the eutrophication of bays through the construction of sewage treatment plants alone. big cities have long been developed near waterfronts. even now, naval transport remains one of the most important transportation systems, especially for heavy industries and agriculture. today, many of the world's largest cities are located on coastal zones, and therefore vast quantities of human waste are discharged into near-shore zones (walker ) . fifty percent of the world's populations live within km of the sea. many people visit urban coastal zones for recreation and leisure, and we also consume seafood harvested from this area. as a result, effluents released into the water pose a risk of pathogen contamination and human disease. this risk is particularly heightened for waters that receive combined sewer overflows (csos) from urban cities where both sanitary and storm waters are conveyed in the same sewer system. to decrease the risk from introduced pathogens, monitoring that is both well designed and routine is essential. however, even though a surprising number of pathogens have been reported in the sea, measuring these pathogens is difficult and time consuming -not least because such pathogens typically exist in a "viable but non-culturable" (vbnc) state. in addition, the physical environments of urban coastal zones vary widely depending on time and location. their complicated geographical features border both inland and outer oceans, and so both inland and outer oceans affect them. for example, tidal currents, which are a dominant phenomenon in this area, oscillate according to diurnal periods. even if the emitted levels of pathogens were constant and we could monitor the levels of pathogen indicator organisms at the same place, they would fluctuate according to tidal periods. density stratification also changes with the tides. consequently, the frequent measurement of pathogens is needed to discuss the risk pathogens pose in urban coastal zones. however, this kind of frequent monitoring appears to be impossible. to solve this conundrum and achieve an assessment of pathogen risk, we developed a set of numerical models that expand upon the models developed in sect. . and that include a pathogens model coupled with a three-dimensional hydrodynamic model. section . . deals with the distributions of pathogens in urban coastal zones. the pathogens model is explained in greater detail in sect. . . . section . . deals with numerical experiments that help to understand the effects of appropriate countermeasures. figure - shows some typical density distributions patterns in urban coastal zones. the changing balance between tidal amplitudes and river discharge is responsible for the differences among these patterns. as tidal currents increase, the production of turbulent kinetic energy grows and can become the largest source of mixing in the shallow coastal waters. on the other hand, river-discharged water has a low density, creating a density difference between sea water and land-input water. in salt-wedge estuaries ( fig. - , top) , river water is discharged into a small tidal-range sea. the strength of the tidal currents decreases relative to the river flow. this creates a vertical stratification of density. as a result, river water distributes like a veil over the sea's surface and moves seaward. fig. - . cross-sectional view of the mixing patterns in urban coastal zones in contrast, bottom water moves to the river mouth and mixes with the river water. under such conditions, pathogens move on the surface of the sea, and further mixing with low-density fresh water is restricted by stratification. in partially mixed estuaries ( fig. - , middle) , the tidal force becomes a more effective mixing mechanism. fresh water and sea water are mixed by turbulent energy. as a result, pathogens that are emitted from sewer treatment plants are more mixed than those in the static salt-wedge estuaries. in well-mixed estuaries ( fig. - , bottom) , the mixing of salt and river waters becomes more complete due to the increased strength of tidal currents relative to river flow. here, the density difference is developed in a horizontal direction. as a result, pathogens are mixed in the water column and settle down on the sea bed, in turn contaminating estuarine waters during the spring tide or contaminating rainfall through re-suspension (pommepuy et al. ) . figure - shows distributions of pathogens under coastal environments. these pathogens encounter a wide range of stresses including uv rays (sinton et al. ) , temperature differences (matsumoto and omura ) , ph (solić and krstulović ) , salinity (omura et al. ) , and lack of nutrients. the pathogens are transported by currents and continue to become part of sedimentation and to be re-suspended in urban coastal zones (pommepuy et al. ) . the modeling of major pathogens of concern (including adenovirus, enterovirus, rotavirus, norovirus, and coronavirus) is not usually conducted owing to the difficulty of modeling and the lack of observational data in coastal environments. we modeled escherichia coliform (e. coli) by using experimental data in coastal sea water. this model consists of a three-dimensional hydrodynamic model and an e. coli model (onozawa et al. ) . the mathematical framework employed in the e. coli model takes the same approach that was explained in sect. . . . the mass balance of e. coli is expressed as follows: . ) where coli denotes concentrations of e. coli (cfu/ ml), and t is time. sink represents the sinking speed of e. coli . u i denotes flow speed for the calculation of the advection term. e i denotes the diffusion coefficients. sal denotes the salinity-dependent die-off rate ( ppt/day). sunlight is generally recognized to be one source by which bacteria are inactivated, due to uv damage to the bacterial cell (sinton et al. ) . however, this particular target area has high turbidity that rapidly absorbs uv rays at the sea's surface. as a result, this process has been ignored in this model. in this model, we can see the numerical simulations performed with two nested domains to fit the complex geography feature around the odaiba area . these nested grids make possible a representation of the stratification effect. a detailed configuration of the model is summarized in table - . the two computational domains cover the whole fig. - ) that includes both the salinity simulation and observation results. the simulation results show stratification, mixing, and an upwelling phenomenon, and include levels and timing. fig. - shows a comparison between observation results and a calculation for temperature and salinity in a fine grid scale (domain in fig. - ) . variations between the simulated and observed values were generally less than . °c and psu through the water column. the timing and periods of upwelling events were captured accurately. after rain fall, river discharge was increased remarkably. model results adequately represent precipitation variation events and their effects. figure - shows a comparison between modeled and measured e. coli at stn. . the current standard for acceptably safe beaches for swimming set by of the ministry of the environment of japan is a fecal coliform rate of coliforms unit per ml (cfu/ ml). this index of fecal coliform includes not only e. coli but also others. however, it is well known that the majority of the fecal coliform in this area comes from e. coli . therefore, we use a value of cfu/ ml e. coli as the standard for the safety of swimming in the sea. from the calculation results, we can see that durations when the standards are exceeded are very limited, and that most of the summer period falls below the standard for swimming. this result also denotes that the increasing rates of e. coli do not agree with levels of precipitation. even in small precipitations, e. coli significantly increased. understanding the effects of physical factors is important to understanding the fate and distributions of pathogens. such an understanding is in turn variations in levels of e. coli are directly correlated with the discharge from pumping stations, tidal currents, river discharges, and density distributions, as explained in sect. . . . as a result, the distributions of cso differ according to timing, even when the level of discharge is the same. we performed numerical experiments in order to evaluate the contributions of these different discharges and phenomena to cso distributions. the first numerical experiment was a nowcast simulation that calculated e. coli distributions under realistic conditions. the second experiment was a numerical experiment to estimate the effects of a waste-reservoir that was being constructed near the shibaura area. numerical experiments were also applied odaiba area, which is used as a bathing area. figure - shows temporal variations of precipitation and river discharges (top), as well as tide levels and e. coli concentrations discharged from three different areas. shibaura and sunamachi area are located at the upper bay location from the odaiba area. morigasaki has the largest area, but is located in the lower bay location from the odaiba area (see fig. in this spring tide period, tidal ranges can reach m. small precipitations were measured from august th to th. river discharge increased with precipitation, reaching m /s. the levels of e. coli increased rapidly after the rainfall event, due mainly to discharges from the sunamachi and chibaura areas. near the end of this period of increase, effluent from morigasaki also reached the odaiba area. fig. - shows the spatial distributions of e. coli from three different times. from this fig., the e. coli emitted from the morigasaki area can be seen to have been transported from the lower region of the bay to the odaiba area. this is because the small amount of river discharge resulted in a thin layer of low-density, highly concentrated e. coli on the surface of the sea, and tended to isolate the e. coli by preventing it from mixing with the water column. in contrast, fig. - shows a large precipitation case under the neap tide period. large amounts of precipitation produced a large river discharge that reached m /s. in this period, only the upper bay's csos arrived at the odaiba area. no contributions from the morigasaki area took place. in conclusion, the concentrations of e. coli vary widely according to space and time. the density distributions produced by the balance of fig. - . spatial distributions of e. coli at odaiba area tides and river discharges have very complex effects. e. coli concentrations reached maximum levels after small precipitation events, but did not increase so much under large precipitation events due to mixing. these kinds of results would be impossible to understand only from observation. the model successfully captured complex distributions of e. coli and helped our understanding of pathogens contaminations. to mitigate cso pollutions, the construction of storage tanks at three sites in tokyo has been planned by the tokyo metropolitan government. shibaura is the target area of this plan around the odaiba area. numerical numerical simulation was performed with and without the proposed storage tank, which has a capacity of , m . this storage tank can store csos after rainfall. to include the effect of continuous rain, we assumed that the csos stored in the tank could be purified within day. table . shows the calculation results for the mitigation effect of the storage tank. these numbers denote the dates when the standards for bathing in the sea (over cfu/ ml) were exceeded. fig. . shows the observation stations. from this table, stn. shows the largest decrease in csos among these five stations. this is because stn. is located closest to the pumping stations, and therefore would be most sensitive to the csos. before the construction of the cso storage tank, minimum bathing standards for cso levels were exceeded on days. after the construction of the storage tank, the duration of cso levels that exceeded safety standards decreased to days. there was an improvement of days. on the other hand, other stations only days, or in some cases, less than day. such differences could not be observed, especially in those stations that are located inside the odaiba area due to the enclosed feature of bathymetry. for example, over , storage tanks have been built in germany alone, and another , were planned during the s in germany. our plans for dealing with csos are not enough to mitigate the effects of csos completely. at the same time, these results show us the complexity of pathogens distributions and the importance of numerical modeling for this problem. numerical simulation is one of the most important tools for the management of water quality and ecosystems in urban coastal zones. we have developed a water quality model to simulate both nutrient cycles and pathogens distributions, and coupled it with a three-dimensional hydrodynamic model of urban coastal areas. to quantify the nutrients budget, a numerical model should include material cycles with phytoplankton, zooplankton, carbons, nutrients, and oxygen. we applied this model to the tokyo bay and simulated water column temperatures, salinity, and nutrient concentrations that were closely linked with field observations. this model successfully captured periods of timing, stratification events, and subsequent changes in bottom water oxygen and nutrients. our model results also indicated that there were clear differences between the material cycles of nitrogen and phosphorus inside the bay. the regeneration of nutrients and its release from sediment was found to be a source of phytoplankton growth on the same order of importance as contributions from rivers. in particular, phosphorus was found to have been largely retained within the system through recycling between sediment and water. we also developed a pathogen model that includes e. coli and is applied to the simulation of cso influences in urban coastal zones. these results indicate that, because of stratification, concentrations of e. coli significantly increase after even small precipitation events. from this study, the balance between tidal mixing and river waters can be seen to be significant. however, these are only two case studies; it remains necessary to simulate the structure and characteristics of cso distributions and their impact on urban coastal zone pollution. such simulations remain as future works to be undertaken. ministry of the environment a description of a three-dimensional coastal ocean circulation model phytoplankton kinetics in a subtrophical estuary: eutrophication three-dimensional eutrophication model of chesapeake bay user's guide to the ce-qual-icm: three-dimensional eutrophication model one dimensional ecosystem model of the equatorial pacific upwelling system part i: model development and silicon and nitrogen cycle temperature and phytoplankton growth in the sea a model of annual plankton cycles a nitrogen-based model of phytoplankton dynamics in the oceanic mixed layer extractive and enzymatic analysis for limiting or surplus phosphorus in algae a modified sigma equations; approach to the numerical modeling of great lake hydrodynamics model evaluation experiments in the north atlantic basin: simulations in nonlinear terrain-following coordinates verification of a three-dimensional hydrodynamic model of chesapeake bay small scale processes in geophysical fluid flows nemuro -a lower trophic level model for the north pacific marine ecosystem blue tide occurred in the west of tokyo bay in summer of study on budget and circulation of nitrogen and phosphorus in tokyo bay a coastal marine ecosystem: simulation and analysis simulations of chesapeake bay estuary: sensitivity to turbulence mixing parameterizations and comparison with observations processes governing phytoplankton blooms in estuaries. ii. the role of transport in global dynamics long-term isohaline salt balance in an estuary nitrogen budget in tokyo bay with special reference to the low sedimentation to supply ratio some factors affecting the survival of fecal indicator bacteria in sea water analytic prediction of the properties of stratified planetary surface layers development of a turbulence closure model for geophysical fluid problems viability and adaptability of e-coli. and enterococcus group to salt water with high concentration of sodium chloride numerical calculation of combined sewer overflow(cso) due to heavy rain around daiba in the head of tokyo bay kenetics of microbial mineralization of organic carbon from detrital skeletonema costatum cells a coordinate system having some special advantages for numerical forecasting enteric bacterial survival factors sunlight inactication of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters separate and combined effects of solar radiation, temperature, salinity, and ph on the survival of feacal coliforms in seawater characterization of dissolved organic phosphrous in coastal seawater using ultrafiltration and phosphoydrolytic enzymes relation between the transport of gravitational circulation and the river discharge in bays spring bloom sedimentation in a subarctic ecosystem the coastal zone seasonal changes of organic carbon and nitrogen production by phytoplankton in the estuary of river tamagawa key: cord- -drjfwcdg authors: shephard, roy j. title: building the infrastructure and regulations needed for public health and fitness date: - - journal: a history of health & fitness: implications for policy today doi: . / - - - - _ sha: doc_id: cord_uid: drjfwcdg . to recognize the importance to the maintenance of good health of adequate public health regulations and an infrastructure that provides clean water and appropriate waste management. . to see the lack of such amenities over many centuries, but the progressive development of public health bureaucracies dedicated to provision of an appropriate infrastructure for healthy cities, beginning during the victorian era. . to observe how responsibility for the provision of adequate housing for poorer city dwellers has been shared between government, benevolent entrepreneurs and charities. . to note the new challenges to public health presented by such current issues as the abuse of tobacco and mood-altering drugs, continuing toxic auto-emissions, the epidemic of hiv/aids, a decreased acceptance of mmr vaccinations, and the ready spread of infectious diseases by air travel. opportunities for the spread of communicable diseases have increased with the growth in size of cities. the success of urban living has depended in great part on governmental ability to maintain population health through the building of an adequate infrastructure to provide clean water and to dispose of waste, as well as the enactment of appropriate regulations to control the prevent the spread of infectious diseases. in this chapter, we will look at success in meeting these objectives in various communities from early history through the classical era, the arab world, mediaeval europe, the renaissance, and the enlightenment to the victorian era, concluding with some comments on current challenges to public health. in the hey-day of the persian empire, heat, cold, dirt, stench, old age and anxiety were all thought to contribute to ill-health. cyrus the great ( - bce) thus taught his soldiers not to urinate or spit into running water. dead matter was also carefully removed from water-courses, and the clothing of dead people was systematically burnt. during the mediaeval era, interest in public health was much more advanced in the islamic world than in northern and western europe. ali ibn-rabban ( - ce), a well-respected physician living on the south coast of the caspian sea, wrote in his seven-part medical work paradise of wisdom that: "no one should live in any country which does not have four things: a just government, useful medicaments, flowing water and an educated physician." in the eleventh century, the arabic biographer al-mussawir emphasized that the main duty of a monarch was the preservation of health and well-being in his subjects. thus, islamic legislation required physicians to pay regular visits to army units, prisons and people living in outlying areas. the practice of medicine was regulated through a religious office, the hisba, headed by an official called the muhtasib with some of the powers of a modern ombudsperson. one function of the muhtasib was to act as the city medical officer of health. he prevented people with elephantiasis from using the public baths, regulated the cleanliness of public places such as markets, and ensured that garbage collectors did not handle food. one interesting example of applied hygiene was the method used to determine an appropriate location for construction of the main hospital in baghdad (chap. ). the merits of various sites were compared by hanging up pieces of meat, and noting the location where decomposition proceeded the most slowly. the city of córdoba under moorish rule further illustrates the infrastructure typical of the arab world during the tenth century ce. among other facilities, the city boasted public baths. in northern and western europe, public health infra-structure such as aqueducts and sewers fell into disrepair following departure of the roman garrisons, and during the mediaeval era the sanitary conditions in most cities were appalling. positive developments were the development of quarantine procedures and the re-emergence of a few public baths. food inspectors were appointed, and some cities also introduced zoning regulations, requiring malodorous trades such as tanning to be undertaken outside the city walls. water supply and sewage disposal untreated waste was thrown directly into the rivers of london and paris, and travellers were advised: "wise men go over bridges, and fools go under them." in ce, king edward iii ordered the mayor of london to: "cause the human faeces and other filth lying in the streets and lanes in the city top be removed with all speed to places far distant, so that no greater cause of mortality may arise…" a fourteenth century ordinance prohibiting the emptying of latrines into a creek near london's city wall remained largely ignored, so that in the fifteenth century the stream was buried underground. substantial populations of hogs and cattle roamed the streets of many large cities, adding to the urban stench. uncontaminated water was a rarity, and a lack of refuse disposal encouraged rat infestations. infrequent bathing and unwashed woolen clothing led to a proliferation of fleas and other insect vectors of infection. during the mid-fourteenth century, two thirds of the european population was killed by the flea-borne bubonic plague (the "black death", - ce). many doctors deserted their patients during this epidemic, and others proposed preposterous remedies. guy de chauliac wrote: "so contagious was the disease…. that no one could see or approach the patient without taking the disease…for self-preservation, there was nothing better than to flee the region… to purge oneself with pills of aloes, to diminish the blood by phlebotomy and to purify the air by fire and to comfort the heart with senna and things of good odor and to soothe the humours with armenian bole and resist putrefaction by means of acid things." chauliac unwittingly kept the rats and fleas away from pope clement vi, by surrounding his bedside with charcoal burners. a few years following the black death, observant physicians hypothesized that ships arriving from overseas were contributing to the recurring epidemics of plague. at first, hostels for sick townsfolk and newly arrived visitors were set up outside the city, but this was not entirely effective in containing infection. thus in ce, a trentino ( days) of isolation on an uninhabited island was required at many european ports of entry. subsequently, the isolation period was extended to days, perhaps because of an ancient greek doctrine that a contagious disease became manifest within days. in britain, recently arriving travellers were quarantined on guardships, anchored in the thames estuary ( fig. . ). most of the roman baths in northern europe had been abandoned by the mediaeval era, in part because of the high cost of heating the bath water, and in part because the church considered public bathing as a common prelude to venal sins. the church also had concerns about reinforcing belief in the supposed healing powers of celtic water deities (chap. ). nevertheless, as prosperity increased in the latter part of the middle ages, public baths were built or reopened in various parts of europe. in britain, the king's bath was built over the sulis minerva temple in the city of bath, and paris had established public baths by the thirteenth century. in germany, the tradition of river bathing had persisted from celtic times, and a growing number of new public bath-houses were constructed during the th and th centuries. admission to a bath-house was expensive, and poorer germans considered the payment of "bath money" a great blessing. the full luxury package of a spa treatment included washing, scouring and slapping of the body with a sheaf of twigs, a steam bath, rubbing to induce perspiration, swatting the skin with wet rags, scratching, hair washing, cutting and combing, lavendering, and blood letting. unfortunately, some of the baths subsequently became the scene of debauchery, prostitution and infection, and by the sixteenth century, many were closed for fears of spreading syphilis, leprosy and plague ( fig. . ). personal hygiene substantial quantities of soap were traded during the mediaeval era, but this was used more for the washing of wool than for cleansing of the skin. monasteries boasted laundry rooms, and many women listed their trade as "laundry woman." however, the laundering of clothes was an infrequent luxury for poorer people, and indeed many had no spare set of clothing, so that fleas flourished in the poorer households. food inspection basic foodstuffs such as wine, beer, bread, meat, fish and salt were frequently adulterated in mediaeval times. to counter such abuses, several european governments appointed food inspectors. in britain, in , the assize of bread and ale regulated the price of these staples in relation to the price of corn. occasional renaissance scholars expressed some interest in health promotion. the english diplomat and scholar thomas elyot (c. - ) wrote a book entitle the "castell of health," summarizing the latest medical knowledge for those unfamiliar with greek, and the venetian nobleman luigi cornaro ( - ) wrote a book on the art of living a long life. santo santorio (chap. ) also sought to put hygiene on a mathematical basis. most of renaissance society showed little interest in public health or hygiene, as shown by the outbreak and management of the great plague. however, boards of public health were set up in some cities. two small advances in personal hygiene were the introduction of cotton clothing and a growing use of toothbrushes. diligent housewives adopted a few other simple changes in household management to preserve the health of their families, and cambridge university insisted on a direct control of its food supply, the great plague the london "plague" of ce was one in a series of european epidemics of bubonic plague dating back to the "black death." the great plague claimed at least , lives in central london, this being about a half of the population who had not fled from the city. indeed, the death count was probably underestimated, since publically appointed street monitors were open to bribery by those who did not wish to disclose that their house had become infected. samuel pepys commented that the prevalence of the disease was such that corpses could not removed during the hours of darkness ( fig. . ). people were confined to their homes if one family member was infected, thus virtually ensured the death of the entire household. two watchmen were posted at the doors of infected homes for days, at a cost of d per house per day, and the victims received a public stipend of d per day to pay for food, fuel and medicaments. believing that the disease was conveyed by miasmata, the college of physicians recommended using bonfires to displace the infected air. there was probably some incidental benefit from these fires, since the smoke tended to drive away the flea-ridden rats that were vectors of the disease. the epidemic was eventually checked by the great fire, which consumed both the rats and the plagueinfested slum dwellings. in europe, local boards of public health were established; they adopted various measures for the containment of epidemics and the provision of social support to the community. in some cases, they designated specific physicians to attend plague victims, and in florence, local doctors prepared a public information booklet that summarized current knowledge on plague prevention. a further responsibility of these boards was to deal with doctors who failed to report communicable diseases in wealthy patients. one roman doctor who was arrested for this offence was ordered to serve as resident physician at the local pest-house. outbreaks of the plague placed a severe financial stress upon some municipalities. in milan, extra funding was needed to hire physicians and grave-diggers, to pay for operating a quarantine "pest-house," and to reimburse the infected for two-thirds of the estimated value of their possessions, which were summarily burned. some municipalities set up immigration offices on mountain passes to control the arrival of infected travelers, and others restricted imports, exports, market trading, travel and funerals, although it was unclear how far these costly measures were successful in reducing the toll of disease and mortality. personal hygiene and household management one positive development during the renaissance was the introduction of washable cotton clothing and sheets. this greatly curtailed the spread of insect-borne diseases, particularly among those with sufficient wealth to own several changes of clothing. another innovation was popularization of the bristle toothbrush. this device had been invented by the chinese in the thirteenth century, but did not become popular in england until the late seventeenth century, beginning with the aristocracy. without necessarily knowing why, tudor housewives achieved some sterilization of their dairy equipment by scouring with salt and hot water, and then exposing utensils to bright sunlight. infestation of houses by fleas was also countered by sprinkling appropriate herbs beneath the rush mats that covered their floors. the renaissance saw further occasional attempts to control the quality of food, particularly for the wealthy. cambridge university insisted that the direct supervision of their refectories was important to preserving the health and well-being of their students. one of those promoting hygiene during the enlightenment was the physician james mackenzie, who in wrote a text on "the history of health and the art of preserving it." the enlightenment saw some improvement of health infra-structure many dwellings for the poorer citizens of europe.were now constructed of brick and boasted glass windows. and samuel johnson ( - ) was urging a pro-active response to the prevention of disease:"we must consider how many diseases proceed from our own laziness, intemperance or negligence… and beware of imputing to god, the consequences of luxury, riot and debauchery." the diderot encyclopédie, first published in , included a section on hygiene, which wss defined as: "the things which mankind uses or handles… and their influence on our constitution and organs." gottfried wilhelm leibniz ( - ) was perhaps the greatest enthusiast for public health during this era. he strove to establish a pattern of medical training that was oriented towards public health and preventive medicine rather than the treatment of disease. he reminded his colleagues that hippocrates had registered every successful cure, and he urged a similar meticulous recording of outcomes in order to provide a modern preventive medicine data-base. he proposed that standardized questionnaires should be developed to examine eating habits, and that careful mor-the enlightenment tality statistics should be collected so that findings could be correlated with the local climate, air conditions and the nature of the soil. a few other scientists such as hales (who improved the water supply for his village of teddington), and bernouilli (with authored a probability study demonstrating the merits of vaccination) were also interested in public health. but concern about the provision of clean drinking water, adequate treatment of sewage and garbage, and protection against communicable diseases remained the exception rather than the rule, with most countries making a poor showing on indices of population health. francis bacon published studies on the percolation, filtration, distillation and coagulation of water as early as . anton van leeuenhoek described the microscopic animalicules that he had seen in dutch drinking water in , and the french scientist joseph amy patented a water filter in . however, the quality of water in most large cities left much to be desired. philippe de la hire ( - ) mapped the area around paris, seeking to improve the water supply to versailles, probably as much to service the palace ornamental fountains as to provide clean drinking water in the town, and he built a massive aqueduct for this purpose (fig. . ) . he further suggested that householders should install a sand filter to purify the water collected from the roofs of their dwellings, although he noted that one alternative source of water, from underground aquifers, was rarely polluted. in , paisley, scotland became the first british city to establish a municipal water treatment plant. it used a sand filter that had been developed by robert thom. in , paris also constructed a large water treatment facility on the seine; here, river water was allowed to settle for hours, and was then passed through sponge pre-filters and main filters that contained sand and charcoal. despite these advances, the residents of broad st., in central london, faced a massive outbreak of cholera as late as , because they were drawing water from a shallow well that was located close to a cholera-contaminated cess-pit. too often, the city dwellers of the enlightenment continued to pass sewage into open gullies or cess-pits that were fig. . ruins of an aqueduct, built by philippe de la hire to improve the water supply to the city of versailles and its royal palace (source: http://en. wikipedia.org/wiki/ canal_de_l'eure) close to wells, and garbage was thrown directly onto the street. however, in , the conseil supérieur of new france ruled that in order to reduce infection, the houses in quebec city must have latrines, and that garbage must be carried to the river st. lawrence, rather than simply thrown out of the door. populatiion health during the enlightenment vital statistics provide simple objective indices of overall population health during the enlightenment. at birth, the average european could expect to live no more than years. a third to a half of the population died before reaching the age of years. those who survived to their mid-teens lived into their s or even their early s, and at the age of the aristocracy could expect to live a further - years; this was an improvement over the years of adult survival typical of the fourteenth century. survival prospects were much worse in north america than in europe during the enlightenment. many of the population succumbed to fevers, intestinal diseases, and, in the case of the african slaves, to harsh working conditions. a quarter of european immigrant children did not survive until their first birthday, and half of all marriages ended in the death of one partner before their seventh wedding anniversary. epidemics of beri-beri, smallpox, malaria and yellow fever wreaked havoc among early colonists. two of every three deaths were attributed to typhoid, dysentery or salt poisoning. in an attempt to reduce this terrible toll, newly arrived immigrants were initially isolated in "guest houses." replacement of contaminated water by wine, beer or cider, a reduced consumption of infected clams, and a scattering of the population to areas where there were copious fresh water springs reduced deaths from typhoid and dysentery, but progress in reducing overall mortality was slow. in early canada, dispersal of the population along the major rivers made major epidemics less likely than in the urban settlements of the united states, but isolation, accidents and harsh winters made canadian life expectancy worse than those in either europe or the u.s. only a small fraction of the population lived beyond years, and many of the children suffered from rickets and anaemia. typhus and smallpox were also recurrent problems. the victorian era was marked by growing government responsibility for the health of the public in large european cities. there was a gradual improvement in the quality of housing, and demographics showed a burgeoning birth rate. social reformers also succeeded in abolishing child labour and slavery from western society (chap. ). in this section, we will discuss the role of boards of health, continued deaths from poisoning, and improvements in housing conditions. major epidemics of influenza, cholera, typhus, typhoid fever and scarlet fever sparked a deep concern about population health in victorian england. in london, england, cholera killed , people in - and , in (chap. ). however, leaders of a new sanitary movement such as edwin chadwick ( - ) and thomas southwood smith ( - ) began to recognize that ill-health of the individual soon became ill-health of the population. they thus made urgent calls for the provision of clean drinking water, proper removal of refuse and sewage treatment. chadwick and smith sat as commissioners on london's general board of health that regulated the water supply and sewer connections for all new housing in the city, and provided adequate burial grounds for those who died. the quality of london's drinking water was rapidly upgraded, and money was spent on methods of preventing death during childbirth. the public health acts of and also established public baths and wash-houses, and by the s, health-conscious municipalities were building public swimming pools. in lower canada (quebec), a physician was appointed as health officer in , with the primary responsibility of monitoring the sick and starving people who were arriving on immigrant ships from europe. by , a strengthened five-member board of health was supervising quarantine arrangements on grosse isle, in the st. lawrence river near to quebec city. nevertheless, the number of immigrants was such that this holding facility was at times overwhelmed, and cholera periodically reached quebec and montreal, killing between - % of the population. in , people also died of typhus while they were quarantined at grosse isle. a central board of health for both upper and lower canada was created in . compulsory vaccination against smallpox was introduced in the early s. in the united states, organization of sanitary reform began rather later than in canada. the city of new york enacted the metropolitan health bill in , creating a -person board of health. immigrants were processed on ellis island, just outside new york city. the original wooden structure was quickly destroyed by a catastrophic fire, but a stone replacement building opened in . many immigrants spent only a few hours in the facility, but those with contagious disease were summarily denied admission to the united states. continued deaths from poisoning many victorians died from eating adulterated or diseased food. one report to the british privy council ( ) estimated that % of meat came from diseased cattle. flour was expensive, and bakers frequently adulterated it with chalk (to whiten it) and alum; often, the bakers also kneaded the mixture with their bare feet. an act prohibiting the adulteration of food was passed in , but its enforcement was an option for local authorities, so that it was not very effective. cooking was typically done in tin-lined copper pans; wealthier citizens replaced the pots when the tin had worn away, but the poor could not afford to do this, and in consequence they sometimes developed copper poisoning. other sources of poisoning in the victorian home were leaking gas pipes, lead used in white paint, and arsenic used to colour wallpapers. in the early nineteenth century, the sudden influx of country folk into the major cities of europe created hideous slums: "in big, once handsome houses, thirty or more people of all ages may inhabit a single room." housing gradually improved over the victorian era, as many workers accumulated sufficient funds to purchase modest but well-built homes. enlightened industrialists also constructed model housing estates for their employees. robert owen ( - ) organized a model community for his workers at the new lanark mills, in scotland, complete with a nursery school. he envisaged an even more ambitious employee housing project in new harmony, in, but this project failed within two years. the quaker chocolate manufacturer george cadbury ( - ) built a model village for his employees around his factory at bournville, near birmingham, and in the u.s. george pullman, the railway carriage czar, built a model town at pullman, il, in . charitable foundations such as the peabody trust began to replace the worst of london's slums with solidly-built if spartan apartments (fig. . ). the first peabody block, at spitalfields, included dwellings for the poor, shops complete with accommodation for the shopkeepers, and on the top floor baths and laundry facilities for a total cost of £ , . in the united states, building codes were improved during the victorian era, and a national housing association was founded in , under the aegis of the commission on the congestion of population in new york. there were also attempts to persuade philanthropists to build model tenements at low rents; buildings were bought, renovated, and then rented to relocated slum dwellers who were given "friendly instruction" on management of their new households. despite substantial progress in the delivery of public health, there remain a number of continuing challenges in the twenty-first century. current issues include the definitive control of the sales of tobacco and mood-altering drugs, the regulation of automotive emissions and other source of urban air pollution, management of the hiv/aids epidemic, concern over a growing reluctance to accept childhood vaccinations, and the management of infections spread by international air travel. in the edwardian era, cigarette manufacturers had promoted their wares as the cure for various respiratory conditions such as asthma and hay fever. but in , the american physician isaac adler pointed to a growing incidence of lung cancer, and he speculated that the abuse of tobacco and alcohol might be responsible. anti-smoking groups developed in germany following world war i, and a magazine (german tobacco opponents) was published from to . the nazi regime was opposed to smoking, with hitler declaring it a waste of money. in particular, women who smoked were considered as unsuitable to be german wives and mothers. during world war ii, the axis powers made much propaganda from the fact that hitler, franco and mussolini were non-smokers, whereas churchill, roosevelt and stalin were all heavy users of tobacco. evidence of the toxicity of tobacco steadily accumulated during the modern era. in , fritz linkint dresden demonstrated an increased prevalence of lung cancers in smokers. his research was confirmed in , with a case-control study by franz hermann muller of cologne. during the s, ernst wynder at the sloan-kettering institute in new york and richard peto and bradford hill at oxford university advanced even more compelling evidence that cigarettes were carcinogenic. hill concluded that consuming cigarettes per day increased the odds of dying from lung cancer as much as forty-fold. other damning evidence came from cellular pathology, animal experimentation and the demonstration of toxic chemicals in cigarette smoke. however, for a substantial part of the post-modern era, public health workers had to combat a deliberate campaign by the cigarette manufacturers to confuse and deceive the general public. the manufacturers were well aware of the damning facts by the early s, but their misleading propaganda was able to increase u.s. cigarette sales to a peak of billion units in . as late as , only a third of u.s. doctors considered smoking as "a major cause of cancer," and % of physicians were still smoking on a regular basis. beginning in the mid s, there was a dramatic decrease in the social acceptability of cigarette smoking, and growing restrictions were placed on public areas where smoking was permitted. this resulted from demonstrations that passive exposure to cigarette smoke gave rise to small but significant increases in the risks of chronic respiratory disease and asthma in childhood, and carcinoma of the lungs and cardiovascular disease in adults. public polls showed a growing acceptance of public health measures to control smoking in public spaces. cigarette manufactur-ers went to particularly great pains to obfuscate the risks of passive exposure to cigarette smoke, but adverse effects were clearly demonstrated during the s, not only by epidemiological research, but also by the exposure of volunteers to machine-generated cigarette smoke while they exercised in closed chambers. public health workers continue to face many challenges in reducing the sales of tobacco products, as manufacturers doggedly resist measures to reduce consumption through increased taxation, prohibition of sponsorships, and plain packaging. they constantly seek methods of creating new addicts, both through extensive advertising in third world countries and through such tactics as the marketing of electronic cigarettes. as recently as , cigarette smoking still accounted for . % of deaths world-wide. the toll from cigarettes is now compounded by the effects of mood-altering drugs. several countries (including canada) have abandoned attempts to prohibit the marketing of marijuana, with as yet no clear standards of dosages compatible with worker and road safety, and an ever growing segment of the north american population is becoming addicted to powerful opiates, with a high risk of deaths from overdoses. british columbia alone had deaths from opiate overdoses in , despite providing emergency workers with supplies of the antidote naloxone. the modern era saw a dramatic drop in the sulphurdioxide/large particulate smog associated with coal fires in many developed societies, but air pollution problems have continued from coal-fired power station and sautomotive emissions, particularly during thermal inversions. the exposure of cyclists and pedestrians to carbon monoxide was studied during the s. substantial concentrations of carbon monoxide were recorded on congested city streets, particularly if air movement was impeded by tall buildings, but any build-up of carboxyhaemoglobin in the blood stream was reversed quite quickly when the individual moved to a less polluted area. the only adverse clinical effect from carbon monoxide exposure was a somewhat earlier onset of angina if a person with coronary atherosclerosis exercised on a heavily polluted street. chamber experiments by steve horvath in santa barbara, ca, and larry folinsbee in toronto documented acceptable ceilings of exposures to the ozone that was formed by the action of sunlight upon the nitrogen oxides from vehicle and aircraft exhaust. the threshold concentration causing a minor disturbance of respiratory function in healthy exercisers was around . p.p.m., a level that was exceeded in some north american cities on heavily polluted days. to date, in many cities improved automotive emission controls have done little more than match the increase in vehicle registrations, and places such as paris and beijing have needed to forbid the access of drivers to the centre of cities on alternating days in order to reduce pollution levels. since ozone levels show a marked diurnal cycle, one immediate remedy for the active individual is to exercise at less heavily polluted times of the day (early morning or late at night). the ultimate solution to the problem of automotive exhaust probably lies in the replacement of gasoline-driven by electric or hydrogen-powered vehicles. the hiv/aids epidemic officially began in the u.s. in , when the centers for disease control reported a clustering of cases of pneumocystis pneumonia among homosexual men in los angeles. it was quickly realized that the condition was not limited to homosexual individuals, but was seen also in intravenous drug users, haemophiliacs and others receiving blood transfusions. thus, in august , the cdc coined the new term aids. a year later, luc montagnier and his associates at the pasteur institute in paris discovered the virus responsible for this disease. much effort has since been devoted not only to finding highly effective antiretroviral agents, but also in devising measures to reduce transmission of the disease. particular emphasis has been placed upon the wearing of condoms during sexual intercourse, in providing sterile needles for intravenous drug users through programmes of needle exchange and supervised injection sites, in closer control of blood banks and in ensuring sterility during drug injection treatments of tropical diseases. nevertheless, success in controlling the epidemic has as yet been only partial. in the u.s. the disease had already claimed , lives by ; a further million were living with the disease, and , fresh cases were diagnosed in that year. in rural africa, the situation remains even worse, with as many as a third of young adults currently infected. during the early part of the post-modern era, successful childhood vaccination campaigns brought the incidence of mumps, measles and rubella to a very low level in most developed countries, and the who set the year ce for the total elimination of measles and rubella from the european region. however, the percentage of children receiving vaccination has decreased in recent years, with parents weighing the low current risk of infections relative to the supposed dangers of developing meningo-encephalitis and autism. fears that vaccination would cause autism stemmed from a paper published by the british physician andrew wakefield, in . extensive research found no evidence to support his claims, and the british medical journal recently declared that the original article was fraudulent. further, the british general medical council found wakefield had been guilty of serious professional misconduct, and he was struck from the medical register. there have since been small outbreaks of measles consequent upon the decreased proportion of vaccinations in britain and in canada, and unfortunately many of the general public remain convinced that vaccination can cause autism. infectious diseases can now spread very rapidly, due to the ever-growing number of people who engage in global air travel. this problem is well exemplified by an epidemic of sars (severe acute respiratory syndrome). this began in mainland china in november of , and due to delayed reporting by the chinese authorities it spread rapidly around the world. the who issued a global health alert on april th . fortunately, application of rigid quarantine measures contained the epidemic, with relatively few deaths in north america, and by july th , the who was able to declare that the sars epidemic was over. many of the major epidemics of earlier eras were due largely to poor hygiene-a lack of clean water, poor sewage treatment, and an inadequate control of people who were already infected. although we often assume that these issues have now been resolved, it is important to recognize that in many third world countries supplies of clean water and adequate supplies of food are still lacking, with shortages often exacerbated by ethnic conflicts. the same issues of clean water, waste disposal and burial of the dead could still arise in wealthier countries today if there were to be an earthquake, a typhoon or a tsunami, and emergency services must be prepared to give the highest priority to an early re-establishment of the basic health infrastructure following any natural disaster. issues in the adulteration of food have now been largely overcome in developed society, but the current obesity epidemic underlines that problems still have to be resolved in terms of persuading food processors to avoid tactics designed to persuade consumers to overeat. for those who can afford housing, the modern single-family home is generally well-equiped to optimize the health of those who are living in it. massive tower blocks are less suited to a healthy and active life-style, particularly for families with young children. moreover, ever-increasing minimum specifications for housing, a growing world population and a lack of land is presenting public health agencies with the issue of a growing proportion of homeless individuals in many large cities. globalization is presenting new challenges to public health, not only with the rapid spread of infections, but also with the international enforcement of regulations on issues ranging from emission controls on cars to the quality of foods and medications. the ideal forum for developing appropriate preventive measures would seem the world health organisation, but unfortunately (as with many international bodies) its effectiveness is often limited by political considerations, including threats from some nations to slash funding unless criticism of their practices is shelved. . are the infrastructure constraints of an earlier era still compromising public health in third world countries? questions for discussion child against measles, mumps and rubella? what will be the likely new challenges to public health agencies over the next years? public health foundations: concepts and practices cigarette smoking: health effects and challenges for tobacco control plague and the poor in renaissance florence principles of water resources bathing in public in the roman world mission and method: the early nineteenth century french public health movement history of hygiene asian medical systems: a comparative study hygiene in the early medical tradition public health victorian medicine and popular culture private choices and public health. the aids epidemic in an economic perspective health, civilization and the state; a history of public health from ancient to modern times the nazi war on cancer housing in urban britain environmental policy and public health: air pollution, global climate change and wilderness a history of public health shephard rj. the risks of passive smoking on the mode of communication of cholera the new public health the establishment of a board of health for new york city in further reading some early societies had an infrastructure that provided clean water and the removal of sewage, but since this was usually available only to wealthy citizens, its impact upon the course of epidemics was limited. major cities such as london did not build a comprehensive infrastructure until the middle of the victorian era, when appropriate initiatives were taken by newly formed boards of public health. although the traditional concerns of public health have now been largely met in developed societies, new challenges are constantly arising. these include the control of tobacco products and mood-altering drugs, the reduction of automotive emissions and other forms of urban pollution, management of the hiv/aids epidemic, overcoming a growing reluctance to vaccinate infants, and countering the rapid spread of infections by air travel. key: cord- -p lijyu authors: rodriguez-proteau, rosita; grant, roberta l. title: toxicity evaluation and human health risk assessment of surface and ground water contaminated by recycled hazardous waste materials date: - - journal: water pollution doi: . /b sha: doc_id: cord_uid: p lijyu prior to the s, principles involving the fate and transport of hazardous chemicals from either hazardous waste spills or landfills into ground water and/or surface water were not fully understood. in addition, national guidance on proper waste disposal techniques was not well developed. as a result, there were many instances where hazardous waste was not disposed of properly, such as the love canal environmental pollution incident. this incident led to the passage of the resource conservation and recovery act (rcra) of . this act gave the united states environmental protection agency regulatory control of all stages of the hazardous waste management cycle. presently, numerous federal agencies provide guidance on methods and approaches used to evaluate potential health effects and assess risks from contaminated source media, i.e., soil, air, and water. these agencies also establish standards of exposure or health benchmark values in the different media, which are not expected to produce environmental or human health impacts. the risk assessment methodology is used by various regulatory agencies using the following steps: i) hazard identification; ii) dose-response (quantitative) assessment; iii) exposure assessment; iv) risk characterization. the overall objectives of risk assessment are to balance risks and benefits; to set target levels; to set priorities for program activities at regulatory agencies, industrial or commercial facilities, or environmental and consumer organizations; and to estimate residual risks and extent of risk reduction. the chapter will provide information on the concepts used in estimating risk and hazard due to exposure to ground and surface waters contaminated from the recycling of hazardous waste and/or hazardous waste materials for each of the steps in the risk assessment process. moreover, this chapter will provide examples of contaminated water exposure pathway calculations as well as provide information on current guidelines, databases, and resources such as current drinking water standards, health advisories, and ambient water quality criteria. finally, specific examples of contaminants released from recycled hazardous waste materials and case studies evaluating the human health effects due to contamination of ground and surface waters from recycled hazardous waste materials will be provided and discussed. after world war ii, industries began to produce a whole new generation of industrial and consumer goods made of synthetic organic chemicals such as plastics, solvents, detergents, and pesticides. industries profited enormously from the production and marketing of these products and consumers became accustomed to the convenience of synthetic products as well as cheap, convenient, throwaway packaging materials. as the industrial production of these products increased, so did the production, accumulation, and disposal of hazardous waste. prior to , facilities that handled and/or disposed of hazardous waste were not provided with detailed regulations and/or guidance on proper waste handling/ disposal techniques and, as a result, there were many instances where hazardous waste was improperly disposed. when chemicals are improperly disposed in the environment, abandoned hazardous waste sites are created that potentially affect human health and cost our society billions of dollars due to the high cost of not only evaluating human health and environmental impacts but also to performing site clean-ups. an example of one of the most well known incidents of improper disposal of hazardous waste was the love canal environmental pollution incident [ ] . this incident led to the passage of the resource conservation and recovery act (rcra) of . this act gave the united states environmental protection agency (usepa) regulatory control of all stages of the hazardous waste management cycle from the "cradle-to-grave:' beginning in the s, congress passed several other acts designed to protect human health and the environment ( table ) . based on the legislative directives in these acts, the usepa has issued numerous rules, regulations, and guidance documents that ensure that the use, disposal, processing, and handling of hazardous waste do not result in impacts to human health or the environment. state governments are authorized to implement these rules/regulations promulgated by the usepa, to permit facilities that handle hazardous waste in their states, and to create additional state rules and state regulations that apply to the operations of facilities in their specific state. the emphasis in recent years is to prevent pollution by recycling hazardous waste followed by proper disposal practices. rcra defines recyclable materials as "hazardous waste that are reclaimed to recover a usable product:' recycling is a broad term that applies to those who use, reuse, or reclaim waste to use as an ingredient to make a product and to use as an effective substitute for a commercial product. a material is reclaimed if it is processed to recover a useful by-product or forms the starting material for the systematic scientific approach of evaluating potential adverse health effects resulting from human exposure to hazardous agents or situations occur by the following steps: i) hazard identification; ii) dose-response (quantitative) assessment; iii) exposure assessment; iv) risk characterization [ ] . the overall objectives of risk assessment are to balance risks and benefits, to set target levels, to set priorities for program activities at regulatory agencies, industrial or commercial facilities, or environmental and consumer organizations, and to estimate residual risks and extent of risk reduction [ ] . diversity of risk assessment methodology helps ensure that all possible risk models and outcomes have been considered and minimize the potential for error [ ] . this section will provide information on the concepts used in estimating risk and hazard due to exposure to ground water and surface water contaminated from the recycling of hazardous waste and/or hazardous waste materials for each of the aforementioned steps. the first step in the risk assessment process is an evaluation of all human and animal data to determine what health effects occur after exposure to a chemical. well-conducted human studies are preferred, but occupational or accidental exposures to chemicals also provide useful information. however, in most cases, the results from animal studies are used as models to predict effects in humans since animal studies allow for controlled dose-response investigations and detailed, thorough toxicological analysis. some toxicants produce health effects immediately following exposure such as air pollutants that can produce eye irritation in individuals after a few minutes of exposure. other effects, such as organ damage due to metals and solvents, may not become manifested for months or years after first exposure. the time from the first exposure to the observation of a health effect is called the latent period. the length of this period is dependent on various factors such as the type of pathology induced by the compound/chemical of potential concern (copc), dose, dose rate as well as host characteristics such as age at first exposure, gender, race, species, and strain. other host factors that influence susceptibility to environmental exposures include genetic traits; preexisting diseases; behavioral traits such as smoking; coexisting exposures; and medication and vitamin supplementation [ ] . genetic studies include investigations of the effects of chemicals on the genes and chromosomes (genetic toxicology) and ecogenetics, a relatively new field, describes a host's genetic variation in predisposition and resistance to copc exposure [ ] . ecogenetics involves studies of specific exposures ranging from pharmaceuticals known as pharmacogenetics, pesticides, inhaled pollutants, foods, food additives, to allergic and sensitizing agents [ ] . moreover, induction of a health effect at the molecular level may occur after a single exposure, after repeated exposures, or after long-term continuous exposure. the length of the induction period may be a function of the same variables as the latent period. effective exposure time refers to the exposure time that occurred up to the point of induction [ ] . ineffective exposure is readily observed in dose-response curves as a saturation of response in the high dose range. an experimental study must follow the subjects beyond the length of the minimum latent period to observe all effects and cases associated with exposure. under ideal circumstances, a study will follow subjects for their lifetime. lifetime follow-up is common for animal studies but uncommon for epidemiology studies [ ] . qualitative assessment of hazard information should include a consideration of the consistency and concordance of the findings. such assessments should include a determination of the consistency of the toxicological findings across species and target organs, an evaluation of consistency across duplicate experimental conditions, and the adequacy of the experiments to detect the adverse endpoints of interest [ ] . for consideration of whether a copc is a carcinogen, qualitative assessment of animal or human evidence is done by many agencies, including the usepa and the international agency for research on cancer (iarc). similar evidence classifications are used for both animal and human evidence categories by both agencies. these evidence classifications are used for overall weight-of-evidence (woe) carcinogenicity classification schemes where the alphanumeric classification levels recommended by usepa [ ] are shown in table . usepa's woe carcinogenicity classification schemes were first recommended in the guidelines for carcinogen risk assessment (usepa, , hereafter " cancer table usepa's carcinogenicity classification scheme [ ] alphanumeric code evidence of noncarcinogenicity for humans; no evidence of carcinogenicity in adequate studies in at least two species or in both epidemiological and animal studies table weight-of-evidence classification scheme for qualitative assessment of chemical mixtures from mumtaz and durkin [ ] mechanistic understanding: i, ii, and iii i. direct and unambiguous mechanistic data ii. mechanistic data on related compounds iii. inadequate or ambiguous mechanistic data toxicologic significance: a, b, and c a. direct evidence of toxicologic significance of interaction b. probable evidence of a toxicologic significance based on related compounds c. unclear evidence of a toxicologic significance exposure modifiers: and . anticipated exposure duration and sequence . different exposure duration or sequence .a. in vivo data .b. in vitro data .b.i. anticipated route of exposure .b.ii. different route of exposure mixture is additive (=),greater than additive (> ), orless than additive ( <). guidelines") [ ] . however, the guidelines for carcinogen risk assessment, review draft (usepa, ,hereafter" draft cancer guidelines") [ ] recommend a woe narrative describing a summary of the key evidence for carcinogenicity. the draft cancer guidelines will serve as interim guidance until usepa issues final cancer guidelines [ ] . for evaluating chemical mixtures of noncarcinogens, mumtaz and durkin [ ] suggest the interaction data (i.e., independent joint action, similar joint action and synergistic action) and the qualitative and quantitative interaction matrix be taken into consideration when determining the hazard index. a qualitative woe scheme for evaluating chemical mixtures is shown in table . the woe takes into consideration the copc, data, reference doses/concentrations, and hazard index based on additivity [ ] . figure illustrates each of the chemical mixture's woe determination by a symbol indicating the direction of the interaction followed by the alphanumeric expression in table . the first two components are the major factors for ranking the quality of the mechanistic data to support the risk assessment. because toxicity studies must be evaluated to determine the quantitative dose-response relationship between the magnitude of exposure and the extent and severity of the adverse effect, a brief description of various toxicity tests will be provided. different methodologies are used to characterize doseresponse relationships, depending on whether or not the chemical has been identified as a carcinogen or noncarcinogen. carcinogens are assumed to pose some risk at any exposure level [ ] . four classes of toxicant-induced health effects include: i) cancer: genotoxic and nongenotoxic mechanisms; ii) hereditary effects: genotoxic mechanisms; iii) developmental effects: genatoxic or nongenotoxic mechanisms; iv) organ/tissue effects: nongenotoxic mechanisms [ ] . the evaluation of chemicals for acute toxicity is necessary for the protection of public health and the environment. acute toxicity is generally performed by the probable route of exposure in order to provide information on health hazards likely to arise from short-term exposure by that route (table ) [ ] . as shown in table , there are four categories ranging from i to iv based on increasing doses. generally, acute studies evaluate oral, dermal, inhalation, and eye and skin irritation as well as dermal sensitization. the acute inhalation studies are performed from one to seven days while the intermediate studies are performed from seven days to several months [ ] .an evaluation of acute toxicity data includes the relationship of the exposure to the copc and the incidence and severity of all abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects. an acute exposure is considered to be a one-time or short-term exposure with a duration of less than or equal to h. acute toxicity testing is conducted toxity evaluation and human health risk assessment of surface and ground water up to days of exposure and subacute testing for - days. testing periods for the evaluation of developmental effects is less than days since developmental toxicity can occur after short periods of exposure. sub chronic testing is typically conducted for days to year since subchronic exposures are considered to be multiple or continuous exposures occurring for approximately % of an experimental species lifetime. chronic exposures are assumed to be multiple exposures occurring over an extended period of time, or a significant fraction of the animal's or the individual's lifetime. to minimize the number of animals used and to take full account of their welfare, usepa recommends the use of data from structurally related substances or mixtures [ ] . review of existing toxicity information on chemical substances that are structurally related to the copc may provide enough information to make preliminary hazard evaluations that may reduce the need for testing. for example, if a chemical can be predicted to have corrosive potential based on structure-activity relationships (sars), dermal or eye irritation testing does not need to be performed in order to classify it as a corrosive agent. all the human carcinogens that have been identified have produced positive results in at least one animal model. in the absence of adequate human data, it is plausible to regard agents and/or mixtures for which sufficient evidence of carcinogenicity in animals exists to be a possible carcinogenic risk to humans [ ] . therefore, chemicals that cause tumors in animals are presumed to cause tumors in humans. in general, the most appropriate rodent bioassays are those that test the exposure pathways most relevant to human exposure pathways, i.e., inhalation, oral, dermal, etc. because it is feasible to combine bioassays together, it is desirable to tie these bioassays with mechanistic studies, biomarker studies, and genetic studies to understand the mechanism(s) of toxicity and/or carcinogenicity [ ] . a typical experimental design includes two different species, both genders, at least subjects per experimental group using near lifetime exposures. for dose-response purposes, a minimum of three dose levels should be used. the highest dose, typically the maximum tolerated dose, mtd, is based on the findings from a -day study to ensure that the test dose is adequate for the assessment of chronic toxicity and carcinogenic potential. the lowest dose level should produce no evidence of toxicity. in the oral studies, the animals are dosed with the copc on a -day per week basis for a period of at least months for mice and hamsters and months for rats [ ] . for dermal studies, animals are treated with the copc for at least h per day on a -day per week basis for a period. a minimum of h should be allowed for the skin to recover before the next dosing. the copc is applied uniformly over a shaved area that is approximately % of the total body surface area [ ] . the animals are evaluated for an increase in number of tumors, size of tumors, and number of rare tumors seen and/or expressed. even without toxicity, a high dose may trigger events different from those triggered by low-dose exposures. also, these bioassays can be evaluated for uncontrolled effects by comparing weight vs time and mortality vs time curves [ ] . if there is a divergence between the control group and the experimental group in the weight vs time curve, this indicates that there is a disruption of normal homeostasis due to high-level dosing. if there is a divergence in the mortality vs time curves, this indicates that there is an uncontrollable effect [ ] . the national toxicology program (ntp) criterion for classifying a chemical as a carcinogen is that it must be tumorigenic in at least one site in one sex of f rats or b c f mice. validation and application of short-term tests (stt) are important in risk assessment because these assays can be designed to provide information about mechanisms of effects. short-term toxicity experiments includes in vitro or short-term in vivo tests ranging from bacterial mutation assays to more elaborate in vivo short-term tests such as skin-painting studies in mice and altered rat liver foci assays. these studies determine if copcs are mutagenic, indicating they have the potential to be carcinogens as well. in general, stt are fast and inexpensive compared with the lifetime rodent cancer bioassays [ ] . positive results of stt have been used to predict potential carcinogenicity. common stt include the following: ames salmonella/microsome mutagenesis assay (sal); assays for chromosome aberration (abs); sister chromatid exchange induction (sce) in chinese hamster ovary cells; the mouse lymphoma l y cell mutagenesis assay (moly). there are several limitations to stt such as: stt cannot replace long-term rodent studies for the identification of carcinogens; the available tests do not detect all classes of copcs that are active in the carcinogenic process such as hormones; and negative results from stt cannot rule out carcinogenicity [ ] . the most convincing evidence for human risk is a well-conducted epidemiological study where an association between exposure to copc and a disease has been observed. these studies compare copc-exposed individuals vs non-copc-exposed individuals [ ] . the major types of epidemiology studies are cross-sectional studies, cohort studies, and case-control studies. cross-sectional studies survey groups of humans to identify risk factors and disease. these studies are not very useful for establishing a cause-and-effect relationship. cohort studies evaluate individuals on the basis of their exposure to the copc under investigation. these individuals are monitored for development of disease. prospective studies monitor individuals who initially are diseasefree to determine if they develop the disease over time. in case-control studies, subjects are selected on the basis of disease status and are matched accordingly. the exposure histories of the two groups are compared to determine key consistent features. thus, all case-control studies are retrospective studies [ ] . epidemiological findings are evaluated by the strength of association, consistency of observations, specificity, appropriateness of temporal relationship, dose responsiveness, biological plausibility and coherence, verification, and biological analogy [s].a disadvantage of epidemiological studies is an accurate measure of concentration or dose that the copc-exposed individuals receives is not available, so estimates must be employed to quantify the relationship between exposure and adverse effects. moreover, the control group is a major determinant of whether or not a statistically significant adverse effect can be detected. the various types of control groups are: regional general population; general population of a state; local general population; and workers in the same or a similar industry who are exposed to lower or zero levels of the toxicant under study [ ] . dose-response assessment is the fundamental basis of the quantitative relationship between exposure to an agent and the incidence of an adverse response. the procedures used to define the dose-response relationship for carcinogens and noncarcinogens differ. for carcinogens, a non-threshold, zero threshold, dose-response relationship is used when there are known or assumed risks of an adverse response at any dose above zero. non-threshold toxicants include hereditary disease toxicants, genotoxic carcinogens, and genotoxic developmental toxicants. for noncarcinogens, a threshold, nonzero threshold is used to evaluate toxicants that are known or assumed to produce no adverse effects below a certain dose or dose rate. threshold toxicants include nongenotoxic carcinogens, nongenotoxic developmental toxicants, and organ/ tissue toxicants [ ] . the two different approaches will be discussed separately in this section. the toxicity factors used to evaluate oral exposure and inhalation exposure are expressed in different units to account for the unique differences between these two routes of exposure. cancer slope factors (csfs), in units of (mg/kg/day)-t, and reference doses (rids), in units of mg/kg/day, are used to quantify the relationship between dose and effect for oral exposure whereas unit risk factors (urfs), in units of (jlg/m )-t, and reference concentrations (rfcs), in units of mg/m\ are used to describe the relationship between ambient air concentration and effect for inhalation exposure. the urf and rfc methodology accounts for the species-specific relationships of exposure concentration to deposited/delivered doses to the respiratory tract by employing animal-to-human dosimetric adjustments that are different than those employed for oral exposure. the interaction with the respiratory tract and ultimate disposition are considered as well as the physicochemical characteristics of the inhaled agent and whether the exposure is to particles or gases. most important is the type of toxicity observed since direct effects on the respiratory tract (i.e., portal of entry effects) must be considered as opposed to toxicity remote to the portal-of-entry [ ] . based on the differences between oral and inhalation exposure, route to route extrapolation of oral toxicity values to inhalation toxicity values may not be appropriate. please refer to appendix b of the soil screening guidance [ ] for a discussion of issues relating to route-to-route extrapolation. carcinogenic assessment assumes that exposure to any amount of a carcinogenic substance increases carcinogenic risk. thus, zero risk does not exist (a non-threshold response) because there is no carcinogen exposure concentration low enough that will not increase risk of cancer. a genotoxic carcinogen alters the information coded in dna; thus, it is reasonable to assume that these agents do not have a threshold so that a risk of cancer exists no matter how low the dose. there are three stages of genotoxic carcinogenesis: initiation, promotion, and progression. initiation refers to the induction of an irreversible change in dna caused by a mutagen. the initiator may be a direct-activating carcinogen or a carcinogenic metabolite. promotion refers to the possibly reversible replication of initiated cells to form a "benign" lesion. promoters are not genotoxic or carcinogenic but they enhance the tumorigenic response initiated by a primary or secondary carcinogen when administered at a later time. complete carcinogens have initiation and promotion properties [ ] . nongenotoxic carcinogenesis does not involve direct interaction of a carcinogen with dna. mechanisms of nongenotoxic carcinogenesis include an accelerated replication that may increase the frequency of spontaneous mutations or increase the susceptibility of dna damage. cancer may be secondary to organ toxicity and may occur only at high dose rates. moreover, many nongenotoxic cancer mechanisms are species-specific where the results from certain rodent species may not apply to human [ ] . several approaches and models are used to provide estimates of the upper limit on lifetime cancer risks per unit of dose or unit of ambient air concentration, i.e., the csf or the urf, respectively. the upper bound excess cancer risk estimates may be calculated using models such as the one-hit, weibull, logit, log-probit,or multistage models [ , ] . the linearized multistage model is considered to be one of the more conservative models and is typically used because the mechanism of cancer is not well understood and one model may not be more predictive than another one [ , ] . because the risk assessor generally needs to extrapolate beyond the region of the dose-response curve for which experimentally observed data are available, models derived from mechanistic assumptions involve the use of a mathematical equation to describe dose-response relationships that are consistent with biological mechanisms of response [ ] . "hit models" for cancer modeling assume that i) an infinite number of targets exist, ii) after a minimum of targets have been modified, the host will elicit a toxic response, iii) a critical target is altered if a sufficient number of hits occurs, and iv) the probability of a hit in the lowdose range is proportional to the dose of copc [ ] . the one-hit linear model is the simplest mechanistic model where only one hit or critical cellular interaction is required for cell function to be altered. multi-hit models describe hypothesized single-target multi-hit events as well as multi-target events in carcinogenesis. biologically based dose-response (bbdr) modeling reflects specific biological process [ ] . because a large number of subjects would be required to detect small responses at very low doses, several theoretical mathematical extrapolation models have been proposed for relating dose and response in the subexperimental dose range: tolerance distribution models, mechanistic models, and enhanced models. these mathematical models generally extrapolate low-dose carcinogenic risks to humans based on effects observed at the high doses in experimental animal studies. the linear interpolation model interpolates between the response observed at the lowest experimental dose and the origin. linear interpolation is recommended due to its conservatism, simplicity, and reliance because it is unlikely to underestimate the true-low dose risk [ ] . there is no universally agreed upon method for estimating an equivalent human dose from an animal study. however, several methods are currently being used to obtain an estimate of the equivalent human dose. the first method calculates an equivalent human dose from an animal study by scaling the animal dose rate for animal body weight. to derive an equivalent human dose from animal data, the draft cancer guidelines recommend adjusting the daily applied oral doses experienced over a lifetime in proportion to bw [ ] . for noncarcinogens, an uncertainty factor is employed to estimate the equivalent human dose from an animal study if pharmacokinetic data is not available. noncarcinogenic dose-response assessment utilizes a point of effects method which selects the highest dosage level tested in humans or animals at which no adverse effects were demonstrated and applies uncertainty factors or margins of safety to this dosage level to determine the level of exposure where no health effects will be observed, even for sensitive members of the population. also, benchmark dose modeling may be conducted if the experimental data are adequate. animal bioassay data are generally used for dose-response assessment; however, the risk assessor is normally interested in low environmental exposures of humans, which are generally below the experimentally observable range of responses seen in the animal assays. thus, low-dose extrapolation and animal-to-human risk extrapolation methods are required and constitute major aspects of dose-response assessment. human and animal dose rates are frequently reported in terms of the following abbreviations, which are defined below: loel lowest observed effect level in mglkg·day, which produces a statistically or biologically significant effect loael lowest observed adverse effect level in mg/kg·day, which produces a statistically or biologically significant adverse effect noel no observed effect level in mg/kg·day, which does not produce a statistically or biologically significant effect noael no observed adverse effect level in mg!kg·day, which does not produce a statistically or biologically significant adverse effect. key factors in determining which noael or loael to use in calculating a reference dose (rid) is exposure duration. as mentioned previously, acute animal studies are typically conducted for up to days, subacute studies for to days, and subchronic studies for days to year. chronic studies are conducted for a significant portion of the lifetime of the animal. animals may experience health effects during short-term exposure which may differ from effects observed after long-term exposure, so short-term animal studies less than days should not be used to develop chronic rids except for the development of interim rids or developmental rids. exceptionally high quality > day oral exposure studies may be used as a basis for developing an rid whereas the inhalation route is preferred for deriving a rfc [ ] . please note that the same approaches used to develop the rid are used to develop the rfc, the only difference being the route of exposure, animal-to-human dosimetric adjustments, and the units, (i.e., mg/m for the rfc vs mg/kg/day for the rid). the highest dose level that does not produce a significantly elevated increase in an adverse response is the noael. the noael from the critical study should be used for criteria development, i.e., the health effect that occurs at the lowest dose. however, if a noael is not available, then the loael can be used if a loael to noael uncertainty factor (uf) is applied. significance generally refers to both biological and statistical criteria and is dependent on the number of dose levels tested, the number of animals tested at each dose, and the background incidence of the adverse response in the control groups [ ] . noaels can be used as a basis for risk assessment calculations such as rids and acceptable daily intake values (adi). adi and rid values should be viewed as a conservative estimate of levels below which adverse affects would not be expected; exposures at doses greater than the adi or rid are associated with an increase probability (but not certainty) of adverse effects [ ] . who uses adi values for pesticides and food additives to define "the daily intake of chemical, which during an entire lifetime appears to be without appreciable risk on the basis of all known facts at that time" [ ] . in order to remove the value judgments implied by the words "acceptable" and "safety", the adi and safety factor (sf) terms have been replaced with the terms rid and up/modifying factors (mf), respectively. usepa publishes rids and rfcs in either iris or in the usepa's health effects assessment summary tables (heast) . rids and adi values (eqs. and , respectively) are typically calculated from noael values divided by the uf and/or mf: the uncertainty factor (uf) may range from to , depending on the nature and quality of the data and is determined by multiplying different ufs together to account for five areas of scientific uncertainty [ ] . the uf is primarily used to account for a potential difference between the animal's and human's sensitivity to a particular compound. the ufh and uf a accounts for possible intraand interspecies differences, respectively. as mentioned previously, an ufs is used to extrapolate from a subchronic duration study to a situation more relevant for chronic study and an ufl is used to extrapolate from a loael to a noael. an uf is used to account for inadequate numbers of animals, incomplete databases, or other experimental limitations. a modifying factor (mf} can be used to account for additional scientific uncertainties. in general, the magnitude of the individual ufs is assigned a value of one, three, or ten, depending on the quality of the studies used in developing the rid or rfc. this uf is reduced whenever there is experimental evidence of concordance between animal and human pharmacokinetics and when the mechanism of toxicity has been established. recently, benchmark dose modeling has been recommended by usepa instead of the noael approach. criticism of the noael approach exists because of its limitations, which include the following: i) the noael must be one of the experimental doses tested; ii) once the dose is identified, the remaining doses are irrelevant; iii) larger noaels may occur in experiments with few animals thereby resulting in larger rids; iv) the noael approach does not identify the actual responses at the noael and will vary based on experimental design. these limitations of the noael approach resulted in the benchmark dose (bmd) method [ ] . the dose-response is modeled and the lower confidence bound for a dose (bmdl) at a specified response level, benchmark response (bmr), is calculated [ ] . the bmdlx (with x representing the x percent bmr) is used as an alternative to the noael value for the rid calculations. thus, the calculation of the rid is shown in eq. ( }: advantages of the bmd approach includes: i) the ability to account for the full dose-response curve; ii) the inclusion of a measure of variability; iii) the use of responses within the experimental range; iv) the use of a consistent benchmark response level for rid calculations across studies [ ] . there are numerous informational databases or resources that provide risk assessors essential information. usepa publishes rids, rfcs, csfs, and urfs in the integrated risk information system (iris) or in the health effects assessment summary tables (heast). the information in iris followed by heast should be used preferentially before all other sources. a recent review of other available resources was published in a special volume of toxicology, vol , . articles by poore et al. [ ] and brinkhuis [ ] provide a thorough review of u.s. government databases such as usepa's iris at http://www.epa.gov/ iriswebp/iris/, national center for environmental assessment (ncea),atsdr's chemical-specific toxicology profiles and acute, subchronic, and chronic minimal risk levels (mrls ), and hazdat at http:/ /www.atsdr.cdc.gov/hazdat.html, among many other databases. the reviewers provide advise for effective search strategies as well as strategies for finding the appropriate toxicology information resources. exposure occurs when a human contacts a chemical or physical agent. exposure assessment examines a wide range of exposure parameters pertaining to the environmental scenarios of people who may be exposed to the agent under study. the information considered for the exposure assessment includes monitoring studies of chemical concentration in environmental media and/or food; modeling of environmental fate and transport of contaminants; and information on different activity patterns of different population subgroups. the principal pathways by which exposure occurs, the pattern of exposure, the determination of copc intake by each pathway, as well as the number of persons and whether there are sensitive subpopulations that need to be evaluated are also included in the evaluation. in this step, the assessor characterizes the exposure setting with respect to the general physical characteristics of the site, the site copcs, and the characteristics of the populations on or near the site. hazard identification/evaluation consists of sampling and analysis of soil, ground water, surface water, air, and other environmental media at contaminated sites. a common method used in screening substances at a site is by comparison with background levels in soil or ground/ surface water [ ] , determining if a chemical is detected or not and whether the detection limit for that chemical is less than reference concentrations as well as frequency of detection [ ] . once a list of copcs have been identified at the site, the availability of chemical characteristics such as struc-ture, solubility, stability, ph sensitivity, electrophilicity, and chemical reactivity and toxicity data are collected and evaluated to ascertain the nature of health effects associated with exposure to these chemicals. in many cases, toxicity information on chemicals is limited. knowing the copc's characteristics can represent important information for hazard identification [s] .also, sars are useful in assessing the relative toxicity of chemically related compounds. during this phase of exposure assessment, the major pathways by which the previously identified populations may be exposed are identified. therefore, locations of contaminated media, sources of release, fate and transport of copcs, pathways and exposure points, routes of exposure (i.e., ingestion of drinking water, dermal contact when showering) and location and activities of the potentially exposed population are explored. for example, the common on-site pathways evaluated when conducting a rcra remediation baseline risk assessment where unauthorized chemical releases have occurred includes direct contact with soil either by ingestion of soil and/ or inhalation of volatile chemicals or contaminated dust [ ] . the migration of chemicals off-site can occur via wind-blown dust and vapor emissions from soil, leaching of chemicals to ground water with subsequent movement off-site, and run-off surface water. these off-site chemicals can eventually accumulate in other transport media such that the copc ends up in vegetation crops, meat, milk, and fish that will eventually be consumed by humans. therefore, pathways, sources of release, locations of contaminated media, fate and transport of copcs, and location and activities of the potentially exposed population are explored. exposure points and routes of exposure (ingestion, inhalation) are identified for each exposure pathway. it is necessary to identify populations likely to receive especially high exposure and populations likely to be unusually sensitive to the chemical's effects. an example of possible point of exposures and exposure routes due to exposure to ground water or surface water (i.e., source medium) used for drinking water is shown in table . please note that all of these exposure path- volatilization from water air inhalation into enclosed space ways are typically not evaluated when doing a risk assessment on contaminated drinking water since the techniques and exposure parameters for evaluating these routes of exposure are not well developed. additional pathways to consider for surface water may include recreational exposures (i.e., swimming, boating), ingestion of contaminated fish, shellfish, etc., and dermal exposure to contaminated sediment. finally, an attempt should be made to develop a number of exposure scenarios. exposure scenarios are a combination of"exposure pathways" to which a single "receptor" may be subjected [ ] . for example, a residential adult or child receptor may be exposed to all the exposure routes in table (i.e., drinking water, showering/bathing, washing/cooking food, and volatilization from ground water or drinking water into an enclosed space). an industrial receptor may only be exposed through the drinking water pathway and volatilization from ground water into an enclosed space and not be exposed through showering/bathing or washing/ cooking, because these activities are not allowed at an industrial site. exposure scenarios are generally conservative and not intended to be entirely representative of actual scenarios at all sites. the scenarios allow for standardized and reproducible evaluation of risks across most sites and land use areas [ ] . conservatism allows for protection of potential receptors not directly evaluated such as special subpopulations and regionally specific land uses. the magnitude, frequency and duration of exposure for each pathway are next evaluated. for each potential exposure pathway, the chemical doses received by each exposure route needs to be calculated. because chemical concentrations can vary, many different studies might be required to get a complete picture of the chemical's distribution patterns within the environment. off-site sampling and analysis are preferred methods to determine the exposure concentrations in the environmental media at the point of exposure. because sampling data forms the foundation of a risk assessment, it is important that site investigation activities are designed and implemented with the overall goals of the risk assessment to be performed [ ] . for example, it is essential that appropriate analytical methods with proper quality assurance/quality control documentation be employed and that the analytical methods are sensitive enough to detect the copc at concentrations that are below health protective reference concentrations. after the sampling data is collected and evaluated, then statistical techniques may be used to calculate the representative concentration of copcs that will be contacted over the exposure area. different statistical techniques may be required for the determination of representative concentrations in ground water vs surface water [ ] . fate and transport models can be used to estimate current concentrations in media and/or at locations for which sampling was not conducted. in addition, an increase in future chemical concentrations in media that are currently contaminated or that may become contaminated can be predicted by fate and transport modeling. detailed discussions of these models are contained elsewhere in this book. each scenario described in the exposure assessment should be accompanied by an estimated exposure dose for each pathway. once the exposure pathway is determined, then the estimated risks and hazards from each exposure pathway can be characterized. exposure estimates for the oral pathway are expressed in terms of the mass of substance in contact with the body per unit body weight per unit time (i.e., intakes) whereas exposure estimates from inhalation pathways are expressed as mass of substance per unit volume (i.e., inhalation concentrations). the general equation for calculating intakes (mg/kg/day) is as follows [ ] : intake, the amount of chemical at the exchange boundary (mg/kg body weight-day) c copc concentration, average concentration contacted over the exposure period cr contact rate, the amount of contaminated medium contacted per unit time or event ef exposure frequency (days/year) ed exposure duration (years) bw body weight, the average body weight over the exposure period (kg) at averaging time or period over which exposure is averaged (days). each exposure pathway has slightly different variations of the above basic equation. please refer to appendix a for examples of equations used to calculate intakes for the major exposure pathway from ground and surface waters as well as examples of exposure parameters employed to calculate intakes: appendices a- and a- , ingestion of drinking water; appendices a- and a- , ingestion of contaminated fish tissue; appendices a- and a- , dermal contact with contaminated water; and appendix a- inhalation of volatiles from contaminated ground water or surface water. please refer to kasting and robinson [ ] and exposure to contaminants in drinking water [ ] for additional information on the various issues involved in the assessment of dermal exposure to water. the exposure parameters (e.g., cr, ef, ed, bw, and at) for each pathway are derived after an extensive literature review and statistical analysis [ ] . for example, information on water ingestion rates, body weights, and fish ingestion rates for adults, children, and pregnant women used to develop the national ambient water quality criteria were obtained from the following documents: exposure factors handbook [ ]; national health and nutrition examination survey (nhanes iii) [ ] ; and united states department of agriculture (usda) - continuing survey of food intakes [ ] . exposure parameters may represent central tendency or average values or maximum or near-maximum values [ ] . science policy decisions that consider the best available data and risk management judgments regarding the population to be protected are both used to choose appropriate exposure parameters. usepa emphasizes that exposure assessments should strive to achieve an overall dose estimate that represents a "reasonable maximum exposure (rme):' the intent of the rme is to estimate a conservative exposure scenario that is within the range of possible exposures yet well above the average case (above the h percentile of the actual distribution). however, estimates that are beyond the true distribution should be avoided. if near maximum or maximum values are chosen for each exposure parameter, then the combination of all maximum values for each exposure parameter would result in an unrealistic assessment of exposure. using probabilistic risk assessment, cullen demonstrated that if only two exposure parameters were chosen at maximum or near maximum values, and other parameters were chosen at medium values, than the risk and hazards estimates represented a rme (> % percentile level) [ ] . risk assessors should identify the most sensitive parameters and use maximum or near-maximum values for one or a few of those variables. central tendency or average values should be used for all other parameters [ ] . when central tendency and/or maximum values are chosen for exposure parameters used to calculate intake for an exposure pathway, single point estimates of risk and hazard are calculated (i.e., a deterministic technique). however, probabilistic techniques like monte carlo analysis can be employed to provide different percentile estimates of risk and hazard (i.e., soth percentile or th percentile estimates) as well as to characterize variability and uncertainty in the risk assessment. monte carlo simulation is a statistical technique by which a quantity is calculated repeatedly, using randomly selected values from the entire frequency distribution for an exposure parameter or multiple exposure parameters for each calculation. usepa recommends using computerized monte carlo simulations to provide probability distributions for dose and risk estimates by incorporating ranges for individual assumptions rather than a single dose or risk estimate [ ] . using better estimates for the distribution of contaminant levels is a major focus of recent risk assessment research. to obtain such estimates, several techniques, such as generating subjective uncertainty distributions and monte carlo composite analyses of parameter uncertainty, have been applied [ ] . these are approaches that can provide a reality check that is useful in generating more realistic exposure estimates [ ] . also, high-end exposure estimates (heees) and theoretical upper-bound estimates (tubes) are now recommended for specified populations as well as calculation of exposure for highly exposed individuals [ ] . heee represents an estimate of the exposure in the upper ninetieth percentile while tubes represent exposure levels that exceed exposures experienced by all individuals in the exposure distribution and assume limits for all exposure variables [ ] . please refer to the policy for use of probabilistic analysis in risk assessment at the usepa and guiding principles for monte carlo analysis at http:/ /www.epa.gov/ ncea/mcpolicy.htm [ ] . risk characterization, the last step in the risk assessment process, links the toxicity evaluation (hazard identification and dose-response assessment) to the exposure assessment. estimates of the upper-bound excess lifetime cancer risk and noncarcinogenic hazard for each pathway, each copc, and each receptor identified during the exposure assessment are calculated. another important component of risk characterization is the clear, transparent communication of risk and hazard estimates as well as an uncertainty analysis of those estimates to the risk manager. cancer risk is usually expressed as an estimated rate of excess cancers in a population exposed to a copc for a lifetime or portion of a lifetime [ ] . oral intakes are multiplied by the csf (eq. ), dermal intakes are multiplied by the csf adjusted for gi absorption (eq. ), and lifetime average inhalation concentrations are multiplied by the urf (eq. ) to obtain risk estimates. for evaluating the risk from oral exposure, the intakes from all ingestion pathways can be summed (i.e., ingestion of drinking water, ingestion of fish, etc.), then the total intake is multiplied by the csf, as follows: ( ) intakeoral the combined amount of copc from all oral pathways at the exchange boundary (mg/kg/day) (appendices a- to a- ) csf cancer slope factor (mg/kg/day)- • for evaluating dermal exposure, the dermally absorbed dose (dad) is calculated (appendices a- and a- ) and multiplied by an adjusted csf, csf dermal· the csf is typically derived based on oral dose-response relationships that are based on administered dose, whereas the dermal intake estimates are based on absorbed dose. therefore, if the csf is based on an administered dose, it should be adjusted for gastrointestinal absorption, if gastrointestinal absorption is significantly less than % (e.g., < %) [ ] . therefore, if an estimate of the gastrointestinal absorption fraction (absgi) is available for the compound and absgi is less than % [ , ] , then the oral dose-response factor, based on an administered dose, can be converted to an absorbed dose basis by dividing the csf by the absgr to form a csf derma!: where dad dermally absorbed dose (mg/kg/day) (appendices a- and a- ) ( ) csfdermal dermal cancer slope factor (mg/kg/day)- ; csfdermat=(csf/absg ). when absgr values are not available from bast and borges [ ] for a compound, then usepa region [ ] recommends the following defaults for absg : % for volatile organics; % for semi-volatile organics and nonvolatile organics; and % for inorganics. for evaluation of inhalation exposure, the lifetime average inhalation concentration is multiplied by the urf: where cinh concentration of copc at the exchange boundary (mg/m ) (appendix a- ) urf unit risk factor (jlg/m )- • to obtain a conservative total risk estimate, the risks for an individual copc from each pathway is summed and then the risks from all copcs are summed (eq. ): where ( ) rtotal sum of all risk estimates from all i h copcs from all pathways. however, usepa is still developing approaches to deal with the uncertainties associated with combining risk estimates of chemical mixtures across different routes of exposure (i.e., inhalation, oral, and dermal) since differences in the properties of the cells that line the surfaces of the air pathways and the lungs, the gastrointestinal tract and the skin may result in different intake patterns of chemical mixtures components depending on the route of exposure. another consideration in dealing with chemical mixtures is the chemicals in a mixture may partition to contact media differently [ ] .a risk estimate of x , x , or x is interpreted to mean that an individual has not more than, and likely less than, a in , , , in , , or in , chance, respectively, of developing cancer from the exposure being evaluated. the range of carcinogenic risks acceptable by the usepa is iq- to iq- • for chronic exposures to noncarcinogens, the intake of a copc is compared to the appropriate rid (i.e., oral rid or rid dermal) or rfc to form the hazard quotient (hq) [ ] . oral intakes are compared to the rid (eq. ), dermally absorbed doses (dads) are compared to the rid dermal (i.e., rid adjusted for gi absorption refer to the previous section for a discussion of procedures for adjusting toxicity factors for gi absorption) (eq. ), and inhalation intakes are compared to the rfc (eq. ) to obtained hazard quotients for each route of exposure: l intakeoral hqoral = rjd ( ) where intakeoral the combined amount of copc from all oral pathways at the exchange boundary (mg/kg/day) (appendices a- to a- ) rid oral reference dose (mg/kg/day) the total hazard index (hi) for an individual copc from all routes of exposure is the sum of the hqs from all applicable pathways (oral, dermal, or inhalation) (eq. ): hi; = l hq; ( ) where hii the sum of the hazard quotients from all relevant pathway for the i h co pc. in order to be conservative, a total hi can be calculated by summing the his from each individual copc. "if the overall hi value is less than one, public health risk is considered to be very low"; however, "if the hi value is equal to or greater than one, then the exposure assessment and hazard characterization should be investigated more thoroughly:' if the hi exceeds one, then the hazard estimates may be refined by grouping the copcs that affect the same target organ or have the same mechanism of action and adding only the his from similar-acting copcs [ , ] . ideally, chemicals would be grouped according to effect-specific toxicity criteria, information on chemicals exhibiting multiple effects would be available, and their exact mechanism of action would be known. instead, rids and rfcs are available for just one of the several possible endpoints of toxicity for a chemical and data are often limited to gross toxicological effects in an organ or an entire organ system. the list of these specific endpoints of toxicity is limited so it is best to consult a toxicologist during this step of the hazard evaluation [ ] . each copc and exposure pathway needs to be calculated to determine the actual risk. the hi provides a rough estimate of possible toxicity and requires careful interpretation [ ] . the hi does not account for the number of individuals who might be affected by exposure or the severity of the effects. usepa recommends that a hi of . be used for noncancer health effects. in the "real world", exposures generally involve complex mixtures of copcs. there are three basic actions for mixtures: i) independent joint action, which describes copcs that act independently and have different modes of action and are not expected to affect the toxicity of one another; ii) similar joint action or "dose addition;' which describes a mixture where the copcs produce similar but independent effects; iii) synergistic action, which the effective mixture cannot be assessed from the individual ingredients but depends on knowledge of their combined toxicity [ ] (table and fig. ). the total hi can exceed the target hazard level as a result of the presence of either one or more copcs with an hq exceeding the target hazard level or the summation of several copc-specific hqs that are each less than the target hazard level. it is important to mention that the numbers generated by risk assessors should not be viewed as either accurate measures or even predictors of rates of adverse health effects in human populations [ ] . the calculated estimates are routinely based on assumptions recognized as being conservative. thus, these numbers should be used as tools open to interpretation on a site-by-site basis. it is important for the risk manager to be informed of the uncertainties during the risk assessment process. significant limitations and uncertainties can exist throughout the entire risk assessment process; thus, it is important that a discussion of uncertainty accompanies risk assessment analysis so that the limitations of the quantitative results are taken into consideration. both qualitative and quantitative methods have been developed to analyze the uncertainty associated with risk assessment. a quantitative analysis may be conducted using either a sensitivity analysis or a probability analysis. listed below are the various reasons why uncertainty exists in a risk assessment analysis [ , ] : -deficient control groups -difference in smoking habits between an epidemiology study group and a risk group -differences or lack of consideration of pharmacokinetics and/or mechanism of toxicity between species -failure to diagnose or misdiagnosis the cause of mortality -inappropriate experimental study design -lack of knowledge regarding combined biological effects of exposure to multiple toxicants -limitation in data regarding nature and magnitude of levels in the environment -low-dose extrapolation from high-dose experimental conditions -reliance on mathematical models -toxicant interaction with another agent -use of animal studies in the determination of risk for humans it is important to make clear the distinction between risk assessors and risk management. risk assessors generate risk estimates but a risk manager considers these risk estimates, other scientific information, and integrates it into societal decisions [ ] . for example, risk managers consider data analysis, technical concerns, economic concerns, and social/political concerns in addition to comparing the risk estimates to an acceptable level set by federal or state health agencies [ ] . generally, trade-offs or compromises are made for the lowest possible risk and society's demand for jobs and economic growth. examples of questions that may be asked by risk managers are: "is a particularly deadly type of cancer in a narrow population worse or better than widespread effects of a non-lethal nature? can this decision be successfully defended in court?" in general, risk management decisions may be based more on political and economic factors than risk factors [ ] . risk assessment and risk management are an integral part of the contemporary regulatory scene. risk management refers to the selections and implementation of the most appropriate regulatory action based on: i) goals; ii) social and political factors; iii) available control technology; iv) costs and benefits; v) results of risk assessment; vi) acceptable risk; vii) acceptable number of cases [ ] . another aspect to consider is cumulative risk in either the risk assessment or risk management phase. cumulative risk evaluation considers all "involuntary" risk to which a receptor may be exposed by a variety of environmental risks such as: i) automobile exhaust emissions; ii) leaking underground storage tanks; iii) untreated sewage; iv) agricultural land runoff; v) industrial process air emissions; vi) conventional combustion-related air emissions [ ] . however, at the present time, definite guidance from usepa regarding the evaluation of cumulative risk is not available. a refined site-specific risk assessment takes into account the specific characteristics concerning the site, all relevant pathways a receptor is exposed to, and other site-specific information. this represents a "forward" calculation method where risk and hazard estimates are calculated. however, each state or usepa regional office may utilize slightly different exposure factors, exposure scenarios, target risk and hazard levels or different procedures to account for childhood exposure, cumulative risk, etc. it is a very time-and resource-intensive process, which involves numerous scientific policy decisions. however, risk and hazard estimates from a refined site-specific risk assessment typically provide more realistic estimates than a generic screening level risk assessment. in contrast, media-specific comparison values can be calculated based on a "backward" calculation method based on standardized equations, usepa toxicity values, standard exposure pathways or scenarios, default exposure factors, and conservative risk and hazard levels. usepa office of water has derived drinking water standards and health advisories to evaluate levels of contaminants in public drinking water supplies. to evaluate levels of contaminants in surface water, usepa publishes guidance documents [ ] as well as national recommended water quality criteria. state and tribal agencies then develop water quality standards for each water body in the state based on usepa guidance and the use designation for the individual water body. usepa must review the proposed state water quality standards before they become legally enforceable standards. if drinking water standards and/ or state and tribal water quality standards are available for the copcs present at the site, these standards generally must be used to evaluate human health impacts to groundwater and/or surface water, respectively. water quality standards apply to surface waters of the united states, including rivers, streams, lakes, oceans, estuaries, and wetlands. water quality standards consist, at a minimum, of three elements: ) the "designated beneficial use" or "uses" of a water body or segment of a water body; ) the water quality "criteria" necessary to protect the uses of that particular water body; ) an antidegradation policy. typical designated beneficial uses of water bodies include public water supply, propagation of fish and wildlife, recreation, agricultural water use, industrial water use and navigation. if information concerning copcs is not present in the drinking water and/or state and tribal water standards databases, or additional exposure pathways need to be included during the site assessment, then media-specific comparison values are available from the soil screening guidance [ ] , several usepa regional offices, and individual state governments (table ). these benchmark values may be used as a tool to perform initial site screenings or as initial cleanup goals, if applicable. the different media-specific comparison values are generic, but can be recalculated using more site-specific information and guidance provided on the applicable web addresses (table ). however, they usually do not consider all potential human health exposure pathways or consider ecological concerns. many of the databases listed in table also provide copc concentration in soil calculated with fate and transport models that are protective of ground water and surface water. if information concerning copcs is not present in these databases, or additional exposure pathways need to be included during the site assessment, then a detailed toxicity evaluation and risk assessment may need to be conducted based on state or other regulatory agency guidelines. usepa was granted authority to set drinking water standards by the safe drinking water act (sdwa) of . the sdwa has since been amended in and . the responsibility for implementing drinking water standards is delegated to states and tribes. usepa is responsible for identifying contaminants to regulate, establishing priorities for contaminants that are of the greatest concern, and then deriving national primary drinking water regulations. the sdwa is applicable to public water systems that provide water for human consumption through at least service connections or regularly serve at least individuals. the standards apply to the water delivered to any user of a public water system. the standards are not applicable to private wells, although state and local governments do set rules to protect users of these wells. owners are urged to test their wells annually for nitrate and coliform bacteria, to test their wells for other compounds if a problem is suspected, and to take precautions to ensure the protection and maintenance of their drinking water supplies. even though these drinking water standards do not apply to private wells, many states adopt them as ground water standards or use them to evaluate whether concentrations of contaminants in ground water are above a level of concern. the office of water establishes national primary drinking water standards, secondary drinking water regulations, as well as health advisories. the derivation of these standards, regulations, and health advisories are discussed below. the drinking water standards and health advisories tables may be reached from the office of science and technology ( ost) home page at http://www.epa.gov/ost. the tables are accessed under the ost programs heading on the ost home page. national primary drinking water standards are regulations the usepa sets to control the level of contaminants in the nation's drinking water. maximum contaminant level goals (mclgs) are the maximum level of a contaminant in drinking water at which no known or anticipated chronic adverse effect on the health of persons would occur, and which allows an adequate margin of safety. mclgs are non-enforceable public health goals. maximum contaminant levels (mcls) are enforceable standards that are set as close to mclgs as possible but take into consideration the availability of technology treatments and techniques as well as whether reliable analytical methods capable of detecting low concentrations of contaminants are available. the derivation of mclgs and mcls are discussed in the following sections. for noncarcinogens (not including microbial contaminants), the mclg is based on the rid. the definition and derivation of the rid has been discussed previously. the rid is first adjusted for an adult with body weight assumed to be kg and consuming l of water per day to produce the drinking water equivalent level (dwel): the dwel represents the concentration of a substance in drinking water that is not expected to cause any adverse noncarcinogenic health effects in humans over a lifetime of exposure and assumes the only exposure to the chemical comes from drinking water. however, exposure to the chemical can also occur through other pathways and routes of exposure. therefore, the mclg is calculated by reducing the dwel in proportion to the amount of exposure from drinking water relative to other sources (e.g., food, air). in the absence of actual exposure data, this relative source contribution (rsc) is generally assumed to be %. the final value is in mg/l and is generally rounded to one significant figure: mclg(mg!l) = dwel · rsc ( ) if the chemical is considered to be a class a orb carcinogen, then it is assumed that there is not a dose below which the chemical is considered safe. therefore, the mclg is set at zero. if a chemical is a class c carcinogen and scientific data provides information that there is a threshold below which carcinogenesis does not occur, then the mclg is set at a level above zero that is safe. prior to , the mclg for class c carcinogens was based on an rid approach that applied an additional uncertainty factor of to account for possible carcinogenic potential of the chemical. if there weren't any reported noncancer effects, then the mclg was based on a nominal lifetime excess cancer risk of - to - , if data were adequate. the office of water is now moving toward guidance contained in the draft cancer guidelines [ , ] which allows standards for nonlin-ear carcinogens to be derived based on low dose extrapolation and a mode of action approach. for microbial contaminants that may present public health risk, the mclg is set at zero because ingesting one protozoa, virus, or bacterium may cause adverse health effects. usepa is conducting studies to determine whether there is a safe level above zero for some microbial contaminants. so far, however, this has not been established. as mentioned previously, maximum contaminant levels (mcls) are enforceable standards that are set as close to mclgs as possible but take into consideration the availability of technology treatments and techniques as well as whether reliable analytical methods capable of detecting low concentrations of contaminants are available. if there is not a reliable analytical method, than a treatment technique (tt) is set rather than an mcl. a tt is an enforceable procedure or level of technological performance, which public water systems must follow to ensure control of a contaminant. in addition, mcls take into account an economic analysis to determine whether the benefits of enforcing the standard justify the costs. for group a and group b carcinogens, mcls are usually promulgated at the o- to o- risk level. secondary drinking water regulations are non-enforceable federal guidelines that take into account whether a chemical produces cosmetic effects such as tooth or skin discoloration or aesthetic effects such as affecting the taste, odor, or color of drinking water. because there are at least different contaminates (i.e., aluminum, chloride, copper, and fluoride) in drinking water that are not considered to be health threatening, secondary maximum contaminant levels (smcls) guidelines have been established for public water systems that voluntarily test the water. these secondary standards give the public water systems guidance on removing the contaminants. in most cases, the state health agencies and public water systems often monitor and treat their drinking water for secondary contaminants. in order to provide information and guidance concerning drinking water contaminants for which national regulations currently do not exist, the usepa health and ecological criteria division, office of water, in cooperation with the office of research and development prepares health advisories (ha). these detailed has are used to "estimate concentrations of the contaminant in drinking water that are not anticipated to cause any adverse noncarcinogenic health effects over specific exposure durations" [ ] . they include a margin of safety to protect sensitive members of the population (e.g., children, the elderly, and pregnant women). has are not legally enforceable in the united states, are only used for guidance by federal, state and local officials, and are subject to change as new information becomes available. included in the has is information on analytical and treatment technologies. has are provided for acute or shortterm effects as well as chronic effects. the one-day ha, the ten-day ha and the longer-term ha are based on the assumption that all exposures to the contaminant comes from drinking water whereas the lifetime ha takes into account other sources such as food, air, etc. the following types of has have been developed [ ] . one-day ha -the concentration of a chemical in drinking water that is not expected to cause any adverse noncarcinogenic effects for up to one day of exposure. a one-day ha is generally based on data from acute human or animal studies involving up to days of exposure. the protected individual is assumed to be a -kg child with an assumed volume of drinking water (di) of lingested/day. ten-day ha-the concentration of a chemical in drinking water that is not expected to cause any adverse noncarcinogenic effects for up to ten days of exposure. a ten -day ha is generally based on subacute animal studies involving - days of exposure. similarly to the one-day ha, the protected individual for the ten-day ha is assumed to be a -kg child with an assumed di of l l ingested/ day. longer-term ha-the concentration of a chemical in drinking water that is not expected to cause any adverse noncarcinogenic effects for up to approximately seven years ( % of an individual's lifetime) of exposure, with a margin of safety. a longer-term ha is generally based on subchronic animal studies involving days to year of exposure. the protected individual is assumed to be a -kg child with an assumed di of l ingested/day and a -kg adult with an assumed di of ingested/day. lifetime ha -the concentration of a chemical in drinking water that is not expected to cause any adverse noncarcinogenic effects for a lifetime of exposure. a lifetime ha is generally based on chronic or subchronic animal studies. the protected individual is assumed to be a -kg adult with an assumed di of l ingested/day. a dwel is calculated and multiplied by a rsc of % to account for exposure to drinking water as well as other sources (food, air, etc.). therefore, the lifetime ha is derived similarly to the mclg. the following general formula is used to derive the one-day, ten-day, and the longer-term has and the dwel: health advisories for the assessment of carcinogenic risk ( ) if a contaminant is recognized as a human or probable human carcinogen (groups a or b), a carcinogenic slope factor (csf) is derived based on techniques discussed above. the slope factor is then used to determine the concentrations of the chemical in drinking water that are associated with theoretical upper-bound excess lifetime cancer risks of w- , w-s, or w- • the following formula is used to calculate the concentration predicted to contribute an incremental risk level (rl) of - , w- , or - : cvw(mg.il) = ___ : : : . . _ where cow concentration in drinking water at desired rl (mg/l) rl desired risk level (lo- , - , or assumed body weight of adult human (kg) csf carcinogenic potency factor for humans (mg/kglday}- assumed water consumption of an adult human (l!day). ( ) if a dwel was calculated for a class a, b, or c carcinogen based on an rid study, (i.e., noncarcinogen), then the carcinogenic risk associated with lifetime exposure to the dwel can be calculated to assist the risk manager for comparison in assessing the overall risks. the theoretical upper-bound cancer risk associated with lifetime exposure to the dwel is calculated as follows: lid· csf risk = dwel · ---- kg ( ) toxity evaluation and human health risk assessment of surface and ground water usepa is required by the clean water act of to develop, publish, and revise ambient water quality criteria (awqc). the awqc "involves the calculation of the maximum water concentration for a pollutant that ensures drinking water and/or fish ingestion exposures will not result in human intake of that pollutant (i.e., the water quality criteria level) in amounts that exceed a specified level based upon the toxicological endpoint of concern" [ ] . in october , usepa issued new guidelines [ ] that replaced the awqc national guidelines [ ] . the awqs guidelines incorporated significant scientific advances in the following key areas: cancer risk assessment ( cancer guidelines [ ] vs the draft cancer guidelines) [ , ] ; risk assessments for class c carcinogens using nonlinear low-dose extrapolation; non-cancer risk assessments (benchmark dose approach and categorical regression); exposure assessments (consideration of non-water sources of exposures); bioaccumulation in fish (bioaccumulation factors, bafs, are recommended for all compounds to calculate concentration in fish tissue). in addition, the procedures for deriving awqc under the cwa were made more consistent to the procedures for deriving mclg by the sdwa. this section will discuss guidelines from the methodology for deriving ambient water quality criteria for the protection of human health, hereafter referred to as the awqc methodology guidance [ ] , accessible at http:/ /www.epa.gov/ost/ humanhealth/method/index.html. state and tribal environmental agencies are responsible for developing ambient water quality standards (awqs) for each water body in the state based on guidance provided by usepa [ ] and the uses that water bodies have been designated for (i.e., drinking water supply, recreation, or fish protection, etc.). these designated uses are a part of the water quality standards, provide a regulatory goal for the water body and define the level of protection assigned to it. the watershed assessment, tracking&environmental results database (waters), accessible at http:/ /www.epa.gov/waters/ provides information on the water body designation for each individual state and tribe. the exposure pathways typically evaluated for awqc are direct ingestion of drinking water obtained from that water body and the consumption of fish/ shellfish obtained from that water body. when an awqc is set, anticipated exposures from other sources of exposure (e.g., food, air) are taken into account for noncarcinogenic effects, or carcinogenic effects evaluated by the margin of exposure (moe) approach (i.e., class c carcinogens, using the woe cancer guideline terminology). the amount of exposure attributed to each source compared to total exposure is called the relative source contribution (rsc) for that source. the rsc is typically set at % but if a site-specific assessment is conducted for a particular water body and it can be demonstrated that other sources of exposures are not likely to occur, then the rsc can be set as high as %. an exposure decision tree approach is described in the methodology guidance to assist in calculating a site-specific rsc for a water body [ ] . the allowable dose (typically, the rid) is then allocated via the rsc approach to ensure that the criterion is protective enough, given the other anticipated sources of exposure: where awqc ambient water quality criterion (mg/l) rid reference dose for non-cancer effects (mg/kg-day) rsc relative source contribution factor to account for non-water sources of exposure -may be either a percentage (multiplied) or amount subtracted, depending on whether multiple criteria are relevant to the chemical bw human body weight ( default= kg for adults) di drinking water intake (default= l/day for adults) fli fish intake at trophic level (tl) i (i= , , and ) (defaults for total in-take= . kg/day for general adult population and sport anglers, and . kg/day for subsistence fishers). trophic level breakouts for the general adult population and sport anglers are: tl = . kg/day; tl = . kg/day; and tl = . kg/day bafi bioaccumulation factor at trophic level i (i= , , and ), lipid normalized (l/kg). the following equation is used for deriving awqc for chemicals evaluated with a nonlinear low-dose extrapolation (margin of exposure) based on guidance in the draft cancer guidelines: where pod point of departure for carcinogens based on a nonlinear low-dose extrapolation (mg/kglday), usually a loael, noael, or ledlo uf uncertainty factor for carcinogens based on a nonlinear low-dose extrapolation (unitless). for carcinogens, only two water sources (i.e., drinking water and fish ingestion) are considered when awqc are derived. awqc for carcinogens are determined with respect to the incremental lifetime risk posed by a substance's presence in water, and is not being set with regard to an individual's total risk from all sources of exposure [ ] . the cancer guidelines are the basis for iris risk numbers that were used to derive the current awqc, except for a few compounds developed using the revised cancer guidelines [ , ] . each new assessment applying the principles of the draft cancer guidelines [ , ] will be subject to peer review before being used as the basis of revised, updated awqc. the cancer-based awqc was calculated using the risk specific dose (rsd) and other input parameters listed below. the rsd and awqc for carcinogens was calculated for the specific targeted lifetime cancer risk (i.e., iq- , iq- , iq- ), using the following two equations: where rsd risk specific dose (mg/kg/day) target cancer risk iq- , iq- , iq- (lifetime incremental risk) csf cancer slope factor (mg/kg-day)- ( ) ( ) exposure parameters based on a site-specific or regional basis can be substituted to reflect regional or local conditions and/or specific populations of concern. these include the relative source contribution, fish consumption rate, baf (including factors used to derive bafs such as concentration of particulate organic carbon applicable to the awqc (kg/l) or concentration of dissolved organic carbon applicable to the awqc (kg/l), percent lipid of fish consumed by target population, and species representative of given trophic levels. states and tribes are encouraged to make adjustments using the information and instructions provided in the awqc methodology guidance [ ] . the national water quality standards database (wqsdb) at the web address, http:/ /www.epa.gov/wqsdatabase/, provides access to several wqs reports that provide information about designated uses, water body names, state numeric water quality standards, and epa recommended numeric water quality crite-ria. the wqsdb allows users the ability to compare wqs information across the nation using standard reports. some states and tribes use an incidental ingestion value (ii) instead of di value when the water body is used for recreational purposes and not as a source of drinking water. however, an ii value is not used to develop national awqc. the default value for ii is . l!day and is assumed to occur from swimming and other activities. the fish intake value is assumed to remain the same. besides protection of human health, awqc are developed based on other criteria such as organoleptic effects; aquatic life protection, sediment quality protection, nutrient criteria, microbial pathogens, biocriteria, excessive sedimentation, flow alterations, and wildlife criteria. for example, the national recommended water quality criteria table (http:/ /www.epa.gov/ost/standards/wqcriteria.html) lists freshwater or saltwater criteria maximum concentration (cmc) criteria values that are the acute limits for the priority pollutant for the protection of aquatic life in freshwater or saltwater. the freshwater or saltwater criterion continuous concentration (ccc) criteria value is the chronic limit for the priority pollutant for the protection of aquatic life in freshwater or saltwater, respectively. the table also includes criteria for organoleptic effects for pollutants developed to prevent undesirable taste and/or odor imparted by them to ambient water. in some cases, a water quality criterion based on organoleptic effects or aquatic life protection would be more stringent than a criterion based on toxicologic endpoints. information and links to guidance documents relating to these subjects may be reached from the office of water, water quality criteria and standards program page at http://www.epa.gov/waterscience/standards/. as more knowledge is gained about the waste generated and disposed in landfllls by our society, there is great concern about the toxic effects that this waste has on our environment as well as animal and human health. over the past decade, there have been numerous attempts to recycle various waste products generated by our society. this section will review some of the recent literature on recycled hazardous waste materials. recycled concrete pavement as aggregate for the construction of highways can produce effluent with a high ph that can enter the underwater drains [ ] . when portland cement is recycled, the concrete consists of limestones and minerals where - % is lime ( cao ), silica (si }, alumina (al }, and iron oxide (fe ). ca(oh} is formed sparingly in water and the saturated solution has a ph of . at oc. the ph of the water effluent in underdrains is approximately - . at this ph, the cac precipitates out and forms deposits on the screen [ ] . the deposition on the screens produces clogging and scales to form in the underdrain; thereby, causing vegetative kill around the outlet. in addition to recycled concrete, rubber is recycled for asphalt pavements. recycling of rubber allows a means of disposal of scrap tires and reduces the quantity of construction materials for the asphalt. asphalt pavement contains hot mix asphalt with and without crumb rubber modifier. the use of rubber tires reduces the weight of the asphalt and provides good drainage media as well as extending the life of the asphalt [ ] . while there is an apparent benefit for recycling rubber, it has been found by the minnesota pollution control agency that leaching can occur from the use of waste tires in sub grade roadbeds into the run -off water. in acidic conditions, leaching of barium, cadmium, lead, chromium, selenium, and zinc occurred from the asphalt while in basic conditions, there was leaching of polynuclear aromatic hydrocarbons. thus, the recommended allowable levels (rals) may be exceeded for drinking water standards in areas where there is recycled rubber in the asphalt. paper or wood itself does not contain any hazardous chemicals unless the paper undergoes recycling. the recycling process requires de-inking of waste paper prior to recovery of the fiber generating a sludge that contains particles of ink and fibers too short to be converted to a finished paper product [ ] . the de-inking chemicals such as sulfur, chlorine, cadmium, and fluorine are present in the sludge generated. a sludge is any solid, semi-solid, or liquid waste generated from a municipal, commercial, or industrial wastewater treatment plant, water supply treatment plant, or air pollution control facility exclusive of treated effluent from a wastewater treatment plant [ ] . thus, hazardous waste can be generated from the recycling paper process. a commodity used by numerous industries is plastic. because of the enormous amount of plastic disposed by consumers on a daily basis, it has become a common recycled item at many facilities. some metal sites will recycle the plastic insulation generated by their facility. the recycled plastic from such a facility generally includes metals such as lead, copper, manganese, and zinc, as well as dioxins, polychlorinated naphthalene, and polychlorinated biphenols. a leachate, contaminated run -off water, from the plastic "fluff" is formed during the recycling process. the leachate runs off into the water drains carrying hazardous chemical residue into soil and ground water. the plastic fluff is generally recycled on site into tiles, cushions, traffic cones, fenders, and highway barriers. the non-recyclable material and contaminated soil is generally taken to an off-site landfill. another common way to recycle plastic is to use the "sink-float" process where paper, fiber, and metal can be separated from the plastics and then recycled. the "sink-float" process uses water where the heavy items sink and the light items float [ ] . it has been demonstrated that recycled plastics can be used as construction material as an alternative to lumber. this product is made from used bottles collected at curbside for recycling. the recycled plastics undergo sorting to remove unpigmented polyethylene milk/water jugs and polyethylene terephthalate carbonated beverage bottles. the leftover plastic material is referred to as curbside tailings (ct). ct consists of approximately % polyolefin (polyethylene and polypropylene) with the remaining percentages made of polyethylene terephthalate, polystyrene, polyvinyl chloride, and other plastics [ ] . the ct product has reasonable strength compared to wood. we is et al. ( ) evaluated three ct recycled plastic formulations in fiddler crabs, snail, and algae [ ] . it was found that limb regeneration of the fiddler crabs was accelerated with all three formulations but had no effect on fertilized eggs or larval developments formulations. there was a significant reduction in the sperm fertilization success rate [ ] . furthermore, all three ct plastic formulation did not have an affect on the survival rate of snails or other algal species. the presence of metals in sludge and wastewater is a current problem. for instance, the use of sludge as a fertilizer of agricultural land generally receives cadmium (cd++) from aerial deposition and phosphatic fertilizers. cd++ is considered a hazardous chemical and has been shown to produce toxicity of the lung and kidney and to be carcinogenic in rats [ ] . the highest concentration of cd++ is found in tobacco, lettuce, spinach, and other leafy products/vegetables. using crop uptake data from field trials, it is possible to relate potential human dietary intake of cd++ on which hazard depends, to soil concentrations of cadmium [ ] . transfer via farm animals to meat and dairy products for human consumption is thought to be minimal even allowing for some direct ingestion of sludge-treated soil by the animals. background soil contains . to . mg cd++ /kg where % of cadmium is found in raw sewage that is converted to sludge. after the formation of sludge, % of the % cd++ is removed primarily by sedimentation. in order for cd++ uptake in roots to occur, cd++ must be in its soluble form adjacent to the root membrane for some finite period [ ] . generally, a decrease in ph in soil will enhance the solubility of cd++, which will increase the crop uptake of cd++. in , who/usepa agreed that the maximum acceptable daily uptake of cd++ was f.lg/day. where -lg cd++jday over a -year period would be necessary to produce toxicity to the kidney. farm animals fed fodder crops grown on sludge-treated soil will absorb ~ % of the cd++ ingested [ ] . in addition to recycling cd++, lead (pb )-edta wastewater also undergoes recycling. edta a chelating agent used in the soil washing process for the decontamination of pb contaminated soil. kim et al. [ ] outlines a method to recycle pb-edta wastewater by substituting the pb-edta complex with feh ions at a low ph followed by precipitation of pb ions with phosphate or sulfate ions. feh ions-edta will precipitate at a high ph with naoh. the recycled edta solution can be recycled several times without losing its extractive power [ ] . recycling computers can be extremely hazardous if not properly disposed. there are many parts of the computer that are toxic. to begin with, the cathode ray tube (crt) glass may be classified as a hazardous waste due to its high pb concentration. the liquid crystal display (lcd), which contains benzene material for the liquid crystal, is also considered hazardous. in addition, the mercury switch, mercury relay, lithium battery, ni-h battery, ni-cd battery, and polychlorinated biphenyl (pcb) capacitor are all hazardous materials. because of this, taiwan has recently established guidelines for the proper disposal of computer and/or computer parts [ ] . the nine guidelines are: i) landfill or incineration of scrap computers shall be avoided; ii) the phosphorescent coatings which have been applied to the glass panel of crt must be removed; iii) all the batteries (li, ni-cd,ni-h) must be removed by non-destructive means; iv) all the pcb capacitors which have a diameter greater than em and a height larger than em must be removed; v) all the mercury containing parts must be removed; vi) crt must be ventilated before stored inside a building; vii) the high-ph content funnel glass of the crt must be properly treated; viii) the lcd of notebook computer must be removed by non-destructive means; and lastly, ix) plastic that contains the flameretardant, bromine, shall be treated properly. hopefully, this model can be used in other countries where computer waste is becoming a major issue of environmental concern. organic solvents have many applications in the industry such as formulation of products, thinning of products prior to use or cleaning of materials by removal of contaminants. during this application, solvent emission and waste solvent generation occur. most organic solvents are known to have adverse effects on both human health and the environment. solvents may affect the body through inhalation and skin contact and lead to either acute or chronic poisoning [ ] . the effects of acute poisoning include narcosis, irritation of throat, eyes, or skin, dermatitis, and even death and the effects of chronic poisoning include damage to blood, lung, kidney, and gastrointestinal system and/or nervous system. in addition, many solvents are inflammable in nature. waste management of organic solvents includes: source reduction, recycling, treatment, and disposal [ ] . case studies indicate that dry cleaning facilities use perchloroethylene (perc) in which workers around the cleaning machines are subject to high health risks; thus, vapor recovery systems are used to reduce the perc emissions especially from older machines [ ] . riess et al. [ ] evaluated the recyclability of flame-retarded polymers that contain brominated flame-retardants from televisions (tv) and personal computers (pcs) obtained from a recycling company. the flame-retardants identified in the tv were: % high-impact polystyrene, % acrylonitrile butadiene styrene, % polystyrene, and % polyphenyleneoxide polystyrene. the flame retardants found in pcs were: % acrylonitrile butadiene styrene, % polyphenyleneoxide polystyrene, % high-impact polystyrene, % polystyrene, and % polyvinyl chloride. recycling may be practical if % new material is added to mixture [ ] . the denver potable reuse pilot project began in to recycle wastewater effluent to achieve potable water quality as well as being economically competitive with conventional technology. moreover, this project sponsored the first large-scale risk assessment studies using experimental animals [ ] . after ten years, this pilot project was converted to a demonstration treatment plant to address many of the technical and non-technical issues. the objectives of the "reuse demonstration project were (i) to establish end product safety, (ii) to demonstrate the reliability of the process, (iii) to generate public awareness, (iv) to generate regulatory agency acceptance, and (v) to provide data for a largescale implementation" [ ] . however, insuring end-product water safety proved to be difficult to demonstrate because the health standards established for drinking water were not intended to apply to treated waters. thus, additional criteria were used to prove that the effluent was suitable for human consumption. below were the criteria used in this project: -the product was compared with the national primary and secondary drinking water regulations values -the product was compared with federal or state regulated parameters -effluent levels were compared with the levels suggested to be hazardous -concentrations of product in the water were compared to denver's current drinking water criteria or other "acceptable" water supplies in the u.s. and/or worldwide -whole-animal studies (i.e., chronic toxicity, oncogenicity, and reproductive tests) were conducted using denver's current drinking water as a comparison standard the denver project used two dosage groups per water sample: reclaimed water from the demonstration plant with reverse osmosis treatment (rot) and denver drinking water from the foothills water treatment plant (dwt). ro and dw were administered to fischer rats and b c f mice at dosages at least times the amount found in the original water samples. ultrafiltration water treatment samples (uft) were only administered to rats at the high dose (soox) and distilled water was used as the control in both the rats and mice studies. in addition to the chronic toxicity studies, reproductive toxicity studies were performed to identify potential adverse effects on reproductive performance, intrauterine development, and growth and development of the offspring. the teratology phase will identify potential embryotoxicity and teratogenicity. administration of ro, uft, and dw water at times the amount found in the original water samples for weeks in rats did not result in any toxicologic or carcinogenic toxicity [ ] . the survival rate was slightly higher among the female rats ( %- %) compared to the male rats ( %- %). there were a variety of neoplasms seen in all treatment groups ( table ) . the "c" cell tumors in the thyroids were not considered treatment related because these neoplasms were within the anticipated ranges for the age and strain of the rat. similar results were seen with the mouse chronic studies where there was no toxicity or carcinogenicity seen after weeks of high dose treatment and the survival rate was identical to the rats. the organs most affected by the treatment were the hematopoietic system, liver, lung, and pituitary gland [ ] . the remarkable finding of the reproductive studies was "the absence of treatment- related effects on reproductive performance, growth, mating capacity, survival of offspring, or fetal development in any of the treatment groups" [ ] . the denver project met the outlined objectives at the start of the project and all three of the toxicity studies demonstrated that concentrations times the original amount seen in sample water did not cause any notable toxicity. thus, secondary wastewater can be recycled into safe drinking water for human consumption. chemical mixtures have always been an issue of concern to address/assess the toxicity to the environment and to humans. an interagency agreement between atsdr and the ntp resulted in participation in a public health service (phs) activity related to the superfund act (cercla comprehensive environmental response, compensation and liability act) [ ] . yang was the lead scientist at the national institute of environmental health sciences (niehs)/ntp for the development of the "superfund toxicology program". particular focus centered on chemical mixtures of environmental concern, especially groundwater contaminants derived from hazardous waste disposal and agricultural activities. yang states that obtaining a "representative" sample is practically impossible [ ] . a core sample from one location of a site will definitely be different from a core sample from a different location of a site. also, a core sample taken from the exact location at different times of day and/or different days will be different because weather, activity at the site, and composition of the waste can change and degrade or synthesize new compounds. thus, yang proposed a strategy to study chemical mixtures [ ] : . study chemical mixtures between binary and complex mixtures to avoid duplication of earlier studies that evaluated the two extremes . study chemically defined mixtures to make determination and mechanistic studies manageable . study chemical mixtures related to groundwater contamination because groundwater contamination is among the most critical environmental issue . study chemical mixtures at environmentally realistic concentrations to access the potential health effects of environmental pollution of long-term, low-level exposure . study chemical mixtures with potential for life-time exposure. a chemical mixture of groundwater contaminants from hazardous waste sites and agricultural activities were created. this formulation mixture contained -chemicals that simulated groundwater contamination as shown in table . the concentrations selected represent the average survey values of hazardous waste disposal sites representing all usepa regions. even though such a mixture may never exist in reality, new insights may be gained to elucidate potential health effects from laboratory animals to human. for most of the end-points examined in this study, the results were negative. the negative results of this study were significant because various mixtures were tested at -to -fold or several orders of magnitude higher than potential human exposure levels [ ] . insights gained from yang's project were: i) the effects will be subtle and marginal; ii) toxicologic interactions are possible at the environmentally realistic levels of exposure; iii) toxic responses may be from unconventional toxicologic end-point (immunosuppression, myelotoxicity); iv) possibility of subclinical residual effects may become more interactive with subsequent insults from chemical, physical, and/or biological agents; and v) negative results do not in-toxity evaluation and human health risk assessment of surface and ground water dicate safety for humans because the studies were done on rodents. subsequent work on this mixture at low doses increased the acute toxicity of high doses of known hepatic and renal toxicants [ ] . recently, niehs has begun to focus on simpler mixtures of chemicals that share common mechanisms of action rather than complex mixtures. over the past several decades, much effort has been made to establish national guidance on proper waste handling disposal techniques such that there are many local, state, national and federal agencies that provide guidelines to protect the surface and ground waters for humans. these guidelines also provide methods and approaches used to evaluate potential health effects and assess risks from contaminated source media, (i.e., soil, air, and water) as well as establish standards of exposure or health benchmark values in the different media, which are not expected to produce environmental or human health impacts. the use of the risk assessment methodology by various regulatory agencies using the following steps: i) hazard identification; ii) dose-response assessment; iii) exposure assessment; and iv) risk characterization balances the risks and benefits and sets the "acceptable" target levels of exposure to ground water and surface water. • for noncarcinogenic effects at=ed; for carcinogenic effects at= years. b exhibit - of the interim dermal guidance document [ ) . different regulatory or state agencies may recommend different exposure parameters based on scientific policy or risk management decisions. waste management guide. the bureau of national affairs industrial waste recycling in: jessup dh (ed) waste management guide. laws, issues, and solutions. the bureau of national affairs revised rcra inspection manual oswer directive quantitative risk assessment for environmental and occupational health casarett and doull's toxicology: the basic science of poisons the emerging field of ecogenetics guidelines for carcinogen risk assessment fr guidelines for carcinogen risk assessment. review draft. office of research and development a weight -of-evidence scheme for assessing interactions in chemical mixtures approaches and challenges in risk assessments of chemical mixtures. in: yang rsh (ed) toxicology of chemical mixtures health effect test guidelines: acute toxicity testing. us epa, office of prevention, pesticides, and toxic substances chlorethoxyfos-review of a repeated exposure inhalation study and evaluation of that study by the hazard identification assessment review committee. us epa, office of prevention, pesticides, and toxic substances biologic markers in risk assessment for environmental carcinogens health effect test guidelines: combined chronic toxicity/carcinogencity. us epa, office of prevention, pesticides, and toxic substances methods for derivation of inhalation reference concentrations and application of inhalation dosimetry. us epa, office of research and development soil screening guidance: technical background document. us epa, office of waste and emergency response health advisories of drinking water contaminants. us epa, office of water and health advisories assessment and management of chemical risks, vol risk assessment in the remediation of hazardous waste sites methodology for deriving ambient water quality criteria for the protection of human health issues in qualitative and quantitative risk analysis for developmental toxicology toxicology information resources at the environmental protection agency risk assessment guidance for superfund, voll. human health evaluation manual (part a) assessment protocol for hazardous waste combustion facilities can we assign an upper limit to skin permeability? international life science institute (ilsi) ( ) exposure to contaminants in drinking water, estimating uptake through the skin and by inhalation memorandum on body weight estimates based on nhanes iii data, including data tables and graphs. analysis conducted and prepared by westat, under epa contract number -c- - usda ( ) - continuing survey of food intakes by individuals and - diet and health knowledge survey measures of compounding conservatism in probabilistic risk assessment guiding principles for monte carlo analysis epa/ /r- / risk assessment forum chemical risk assessment numbers: what should they mean to engineers? risk assessment guidance for superfund, voll. human health evaluation manual (parte, supplemental guidance for dermal risk assessment derivation of toxicity values for dermal exposure supplemental guidance to ragss. region iv bulletins. human health risk assessment waste management division supplementary guidance for conducting health risk assessment of chemical mixtures guidelines for the health risk assessment of chemical mixtures the toxicity of poisons applied jointly a practical guide to understanding, managing, and reviewing environmental risk assessment reports addendum:region risk management -draft human health risk assessment protocol for hazardous waste combustion facilities epa year guidance document. contract number -w - guidelines and methodology used in the preparation of health effect assessment chapters of the consent decree water criteria documents implementing the food quality protection act. us epa, office of prevention, pesticides, and toxic substance remediation of hazardous effluent emitted from beneath newly constructed road systems and clogging of underdrain systems assessment of water pollutants from asphalt pavement containing recycled rubber in rhode island the rhode island department of transportation waste-to-energy plant for paper industry sludges disposal: technical-economic study superfund at work: hazardous waste cleanup efforts nationwide. us epa, solid waste and emergency response toxicity of construction materials in the marine environment: a comparison of chromated-copper-arenate-treated wood and recycled plastic the control of the heavy metals health hazard in the reclamation of wastewater sludge as agricultural fertilizer cadmium -a complex environmental problem. part ii recycling of lead-contaminated edta wastewater management of scrap computer recycling in taiwan management, disposal and recycling of waste industrial organic solvents in hong kong analysis of flame retarded polymers and recycling materials chemosphere health effect studies on recycled drinking water from secondary wastewater. in: yang rsh ( ed) toxicology of chemical mixtures toxicology of chemical mixtures derived from hazardous waste sites or application of pesticides and fertilizers. in: yang rsh (ed) toxicology of chemical mixtures toxicology studies of a chemical mixture of groundwater contaminants: hepatic and renal assessment, response to carbon tetrachloride challenge, and influence of treatment-induced water restriction texas natural resource conservation commission ( ) texas risk reduction program rule review draft addendum to the methodology for assessing health risks associated with indirect exposure to combustor emissions estimating exposure to dioxin-like compounds review draft development of human health-based and ecologically-based exit criteria for the hazardous waste identification project. office of solid waste i and ii cw · ir · ef · ed intake=--------- bw a a number of studies has shown that an age-adjusted approach should be used to calculate intakes for children for carcinogens to take into account the difference in ingestion rates, body weights, and exposure duration for children from to years old and others from to years [ ] . b the exposure parameters were taken from the texas risk reduction program rule [ ] and are provided as examples only. different regulatory or state agencies may recommend different exposure parameters based on scientific policy or risk management decisions. [ ] . ' use only when a rid is based on health effects in children [ ] . d the office of water is in the process of preparing an exposure assessment technical support document in which an age-adjusted approach will be used to calculate fish intakes for children for carcinogens to take into account the difference in ingestion rates, body weights, and exposure duration for children from to years old and others from to years [ ] . for copcs whose log k w< . for copcs whose log kow> . cf = cw · baf for dioxins, furans, and polychlorinated biphenyls cf = csed · bsaf cf =chemical concentration in fish (mg/kg), fresh weight (fw) cw =chemical concentration in water (mg/l) bcf =bioconcentration factor (l/kg fw)h baf =bioaccumulation factor (l/kg fw)b c,ed =chemical concentration in sediment (mg/kg) bsaf =biota-sediment accumulation factor (unitless)<• please refer to reference [ ] for a detailed discussion of procedures used to calculate chemical concentration in fish. different regulatory or state agencies may recommend different procedures based on scientific policy or risk management decisions [ , ] . b please refer to appendix a- of reference [ ] for bcf, baf, and bsaf values and procedures for calculating these values. also, please refer to [ , ] . c bsafs are used to account for the transfer of copcs from the bottom sediment to the lipid in fish [ , [ ] [ ] [ ] . organic compounds non-steady state" not applicable for inorganics cinh =the concentration of copc at the exchange boundary (mglm ) cw =chemical concentration in water (mg/l) vf =volatilization factor [(mglm )/(mg!l-h )]• ef =exposure frequency (days/year)h ed =exposure duration (years)h at =averaging time in years (period over which exposure is averaged)h a specific fate and transport models are used to derive volatilization factors to quantify the transfer of volatile copcs from ground water into an enclosed space, from ground and surface waters into ambient air, etc. these fate and transport models are discussed elsewhere in this book. b the exposure parameters for ef, ed, and at from appendix a - can be used for the residential adult, residential child, and commercial/industrial worker for some pathways, but site-specific exposure parameters may need to be developed for other pathways. key: cord- -savdgy u authors: bosch, albert; pintó, rosa m.; abad, f. xavier title: survival and transport of enteric viruses in the environment date: journal: viruses in foods doi: . / - - - _ sha: doc_id: cord_uid: savdgy u nan environmental virology may be defined as the study of viruses that can be transmitted through various environments (water, sewage, soil, air, or surfaces) or food and persist enough in these vehicles to represent a health threat. a wide variety of different viruses, representing most of the families of animal viruses, can be present in human and animal fecal wastes and urine. especially important are a variety of nonenveloped human and animal enteric pathogenic viruses that can enter the environment through the discharge of waste materials from infected individuals; contaminate food products and drinking and recreational waters; and be transmitted back to susceptible individuals to continue the cycle of infection (table . ). it is estimated that billions of cases of gastrointestinal illness occur annually worldwide (parashar et al., ; oh et al., ) . a good deal of these diarrheal cases are to some extent the result of fecal contamination of the environment (cabelli et al., ; koopman et al., ; fattal and shuval, ; moore et al., ) while outbreaks of hepatitis a and e are associated with water, shellfish, and crops (melnick, ; reid and robinson, ; halliday et al., ; bosch et al., bosch et al., , . the significance to human health of many of the non-human animal viruses present in environmental samples is less well understood and remains uncertain or unknown for many of them. it is remarkable, however, that zoonotic viruses infecting humans continue to be discovered or appear to reemerge as important human pathogens. one example of an emerging disease is severe acute respiratory syndrome, or sars, reported in november (ksiazek et al., ) . the primary mode of transmission of the sars coronavirus appears to be direct mucous membrane contact with infectious respiratory droplets and/or through exposure to fomites. several coronaviruses are known to spread by the fecal-oral route, but there is no current evidence that this mode of transmission plays a key role in the transmission of sars, although there is a considerable shedding of the virus in stools (tsang, ) . as a scientific discipline, environmental virology was born after a large hepatitis outbreak occurred in new delhi between december and january . the origin of the outbreak, which was attributed to hepatitis a at the time but now confirmed to be hepatitis e, was the contamination by sewage, from to weeks prior to the epidemic, of the jumna river, the source of water for the treatment plant. alum and chlorine treatment prevented bacterial infections, but , cases of hepatitis occurred among the population. as a consequence of this outbreak, studies in water and environmental virology began with efforts to detect poliovirus in water around years ago. since that time, other enteric viruses responsible for gastroenteritis and hepatitis have replaced enteroviruses as the main target for detection in the environment, although the near eradication of poliomyelitis from the globe calls for exhaustive studies on the occurrence of wild-type and vaccinal-type polioviruses in environmental samples. viruses are shed in extremely high numbers in the feces of infected individuals; patients suffering from diarrhea or hepatitis may excrete from to virus particles per gram of stool (farthing, ) . furthermore, a single episode of vomit of a patient with norovirus gastroenteritis may contain around particles (cheesbrough et al., ) . ingestion of sewagecontaminated water or food is the main route of infection with human enteric viruses, although the role of inanimate surfaces serving as vehicles for virus infection must not be underestimated. viruses with a viremic phase, such as the hepatitis viruses, may also be parenterally transmitted, although these days it is considered to be a much less frequent mode of transmission. a poorly understood aspect in the epidemiology of several enteric viruses is the role of animal viruses in human disease. nucleotide sequence analysis of some human enteric viruses has indicated a high degree of sequence similarity with animal strains. notably, hepatitis e virus-related sequences have been detected in pigs (meng et al., ; van der poel et al., ; banks et al., ) and birds (huang et al., ) . the threat of zoonotic infections may be either through direct transmission, suspected for hepatitis e virus (hev; reyes, ) and caliciviruses (humphrey et al., ) , or through incidental coinfection of a host with animal and human viruses, resulting in the mixing of genes and generation of novel variants (recombination/reassortment; unicomb et al., ) . recombination has been demonstrated as a mechanism for rapid expansion of diversity for noroviruses and rotaviruses, but it is likely to be a common feature of the rna viruses involved (jiang et al., ; unicomb et al., ) . viruses related to the human rotaviruses, astroviruses, noroviruses, sapoviruses, and hev circulate in several animal species, providing a huge reservoir for virus diversity (shirai et al., ; meng et al., ; van der poel et al., ; huang et al., ) . in the water environment, the fate of microbial enteric pathogens may take several potential routes ( fig. . ). mankind is exposed to waterborne enteric virus infections through shellfish grown in contaminated waters, contaminated drinking water, food crops grown in land irrigated with wastewater and/or fertilized with sewage, and, to a lesser extent, sewage-polluted recreational waters (tables . and . ). studies have documented the presence of enteric viruses in raw and treated drinking water (keswick et al., ) , and they are also frequently isolated from freshwater environments (table . ). however, epidemiological proof of human infection caused by these viruses as a result of water consumption is scarce. water-system deficiencies that caused or contributed to these outbreaks may be categorized under five major headings: (a) use of contaminated, untreated surface water; (b) use of contaminated, untreated groundwater; (c) inadequate or interrupted treatment; (d) distribution network problems; and (e) miscellaneous. pathogenic viruses are routinely introduced into the environment through the discharge of treated and untreated wastes, as current treatment practices are unable to provide virus-free wastewater effluents. virus concentrations of , to , pfu/l are commonly reported in raw sewage and may be greatly reduced during treatment; however an average of to pfu/l are normally found in effluents from wastewater treatment plants . sewage sludge, a by-product of wastewater treatment, is a complex mixture of solids of biological and mineral origin that is removed from wastewater in sewage treatment plants. the sewage may undergo primary treatment (physical sedimentation or settling); secondary treatment (primary sedimentation plus high-rate biological processes, such as trickling filter/ activated sludge); secondary treatment plus disinfection (chlorination, peracetic acid, uv or ozone); tertiary treatment (advanced wastewater treatment, including primary sedimentation, secondary treatment plus, for example, coagulation-sand filtration, uv, microfiltration); tertiary treatment plus disinfection; and lagooning (low-rate biological treatment). in any case, the type of treatment will determine the concentration of pathogens in a wastewater effluent and the relative risk of its disposal. an overview of the fate of enteric viruses in coastal environments is depicted in figure . . domestic sewage (in the form of raw sewage, treated effluent or sewage sludge) may be disposed of directly in the marine environment by coastal outfalls or by dumping from barges. in any case, viruses readily adsorb onto the abundant suspended solids present in the sewage and are discharged solid-associated into the marine environment ( fig. . a ). whereas viruses associated with small-size (< µm) particulate material tend to float in the water column (table . ), viruses adsorbed onto large/medium (> µm) particles readily settle down in the bottom sediment (table . ). viruses accumulate in the loose fluffy top layer of the compact bottom sediment ( fig. . b) and are thereby protected from inactivation by natural or artificial processes sobsey et al., ) . sediments in coastal seawaters act as reservoirs from which viruses may be subsequently resuspended by several natural or artificial phenomena. shellfish (fig. . c), being filter feeders, tend to concentrate viruses and bacteria in their edible tissues, and concentrations of these microorganisms in shellfish may be much higher than in the surrounding water. shellfish grown in and harvested from waters a . b o s c h e t a l . receiving urban contaminants ( fig. . d) have been implicated in outbreaks of viral diseases, notably viral hepatitis and gastroenteritis (halliday et al., ; le guyader et al., ; christensen et al., ; bosch et al., ; kingsley et al., ) . many of these outbreaks were related to water or shellfish meeting legal standards based on bacteriological criteria. this evidence supports the recommendation of monitoring shellfish and their overlying waters for viral contamination including the adoption of guidelines including virus standards. the possibility nowadays to detect the presence of human enteric viruses in different types of water samples and foodstuff, in particular shellfish samples, should be a valuable tool in the prevention of waterborne and food-borne diseases. unfortunately, in most outbreaks, virus detection is not attempted until after the outbreak and hence no prophylactic measures can be undertaken to decrease the severity of the outbreak. methods for the detection of viruses in food are discussed elsewhere in this book. the basic steps in virological analysis of water are sampling, concentration, decontamination/removal of inhibitors, and specific virus detection. sample concentration is a particularly critical step because the viruses may be present in such low numbers that concentration of the water samples is indispensable to reduce the volume to be assayed to a few milliliters or even microliters. in relatively nonpolluted waters, the virus levels are likely to be so low that optimally hundreds, or even thousands, of liters should be sampled to increase the probability of virus detection. a good concentration method should fulfill several criteria: it should be technically simple, fast, provide high virus recoveries, be adequate for a wide range of enteric viruses, provide a small volume of concentrate, and be inexpensive. table . shows a broad selection of currently available and widely employed procedures; some of them require large equipment. details on virus concentration procedures have been published elsewhere (american public health association, ; environmental protection agency, ) . all available concentration methodologies have important limitations, and their virus concentration efficiency depends, in part, on the quality of the sampled water. basically, all available procedures have been evaluated using samples spiked with known viruses. it is known that the recovery efficiency recorded with experimentally contaminated water dramatically decrease when the method is applied in actual field trials. additionally, none of the existing concentration procedures has been tested with all of the medically important virus groups; normally, a few specific enteric viruses have been employed to conduct the evaluation trials. however, several virus concentration methods have been used successfully to recover naturally occurring enteric viruses in water (finance et al., ; gerba and goyal, ; and gerba, ; environmental protection agency, ; lewis and metcalf, ; henshilwood et al., ) . most of the procedures for concentrating and extracting viruses make use of the properties of the viral proteinaceous macromolecules. certain protein structures confer on viruses in an aquatic environment the properties of a hydrophilic colloid of an amphoteric nature whose electric charge varies according to the ph and the ionic force of the environment. viruses can therefore be adsorbed onto and then detach themselves from different substrates that are positively or negatively charged depending on their ph. methods based on the adsorption of viruses from the sampled water onto a suitable solid surface from which they may subsequently be eluted into a much smaller volume are preferred for use with large-volume samples. different types of filters have been evaluated for the recuperation of aquatic viruses, in the form of flat membranes or cartridges. cartridge-type filters have the advantage to allow filtration of large volumes of moderately turbid water within a relatively short time. their chemical composition, and porosity vary enormously. a whole range of "negatively" or "positively" charged filters now exist. their efficiency depends on the type of water being tested and the presence of interfering substances such as detergents, suspended solid matter, or organic matter, which can affect the adsorption of viruses on these filters (sobsey and glass, ; hickey, , gilgen et al., ) . the disadvantage of the negatively charged membranes or cartridges (farrah et al., ) is that the water sample must be pretreated prior to concentration. this includes acidification of water and addition of salts to the water sample to facilitate virus adsorption because electronegative filters do not adsorb viruses well under ambient water conditions . the necessity of this pretreatment step limits the on-location use of this method to a certain extent, although automatic injection systems do exist for treating several hundred liters of water. virus concentration with electropositive filters may be performed on location at ambient conditions and without any prior amendment of the sample, which make this procedure most suited for in-field studies, provided that the sample ph is lower than . (sobsey and jones, ) . glass powder (sarrette et al., ; schwartzbrod and lucena, ; gajardo et al., ) or glass fiber (vilaginès et al., ) have also been satisfactorily used in different laboratories as adsorbent materials for virus concentration. viruses in eluate volumes too large to be conveniently and economically assayed directly for viruses, such as those obtained from processing large volumes of water through cartridge or large disk filters, can be reconcentrated by several methods. obviously, the recovery of small quantities of viruses from natural waters is dependent not only on the efficacy of primary concentration from the original large volume but also on the reconcentration of the primary eluate to a smaller volume. methods such as aluminum hydroxide adsorption-precipitation (american public health association, ), polyethylene glycol hydroextraction lewis and metcalf, ) , organic flocculation (katzenelson et al., ) , and ammonium sulfate precipitation (shields and farrah, ; bosch et al., b) that are impractical for processing large fluid volumes are suitable for second-step concentration procedures. alternatively, viruses can be sedimented depending on their molecular weight using ultracentrifugation (steinman, ; mehnert et al., ) . freeze-drying of samples pintó et al., ) and rehydration in a smaller volume provides a procedure for both virus concentration and removal of pcr inhibitors. ultrafiltration (divizia et al., b) can utilize size exclusion rather than adsorption and (or) elution to concentrate viruses can provide consistent recoveries of different viruses under widely varying water conditions. because an evaluation of the presence of viruses in sediment provides an additional insight into long-term water-quality conditions, several methods for the detection of viruses have been developed. these methods consist of virus elution from the solid materials followed by concentration of the eluted viruses. viruses are usually eluted from the sediments by using alkaline buffers (gerba et al., ; bosch et al., a; jofre et al., ) or chaotropic agents (wait and sobsey, ; lewis and metcalf, ; jofre et al., ) . procedures such as organic flocculation (wait and sobsey, ) , ammonium sulfate precipitation , polyethylene precipitation (lewis and metcalf, ) , or ultrafiltration (gerba et al., ) are commonly employed to concentrate viruses from the eluate. diseases associated with soil have been categorized according to the origin of the etiological agent as follows (weissman et al., ; santamaría and toranzos, ) : (a) soil-associated diseases that are caused by opportunistic or emerging pathogens belonging to the normal soil microbiota; (b) soilrelated diseases that result in intoxication from the ingestion of food contaminated with entero-or neurotoxins; (c) soil-based diseases caused by pathogens indigenous to soil; and (d) soil-borne diseases caused by enteric pathogens that get into soil by means of human or animal excreta. in this latter category are included viruses transmitted through the fecal-oral route. the transport of viruses through soil to groundwater and then to the community has been a topic of great concern. many epidemics of infectious diseases have been attributed to the consumption of contaminated groundwater, casting soil as a vector and source of important human disease agents. there is a concern about a possible increase in soil-borne diseases in human population, given the land disposal practices of sewage and sewage sludge. in developing countries, untreated domestic wastewater is used in agricultural irrigation, presenting a high risk to farm workers and to consumers of food products irrigated with wastewater (strauss, ) . in spite of the clear public health implications of the occurrence and survival of viruses in the soil compartment, studies on the fate of viruses in soil are scarce due to the complexity of the methodologies for virus extraction from soil. the most relevant factors controlling virus transport through soil are soil type, water saturation state, ph, conductivity of the percolating water, and soluble organic matter (table . ). the type of soil has a great influence on the level of viral transport. fine-textured soils tend to absorb viruses more readily than coarsely textured soils. as a general rule, sandy soils are relatively poor adsorbents of enteric viruses, whereas soils with clay content of % to % are excellent adsorbents (sobsey et al., ) . in consequence, viral adsorption increases with increasing clay mineral content (gerba et al., ) . the high adsorptive properties of a clay soil will prevent virus transport to another matrix, such as groundwater, whereas coarse soil will not. microbial movement in soils is also greatly dependent on the water saturation state. when the soil is saturated, all pores are filled with water, which allows faster virus transport through the soil because virus contact with the soil has been diminished. when the flow is unsaturated, the viruses are in closer contact with the soil, thus promoting virus adsorption to the soil (santamaría and toranzos, ) . considered soil ph as the single most important factor influencing viral adsorption, although the combined effect of organic matter and clay content, and cation-exchange capacity, could surpass the sole soil ph effect. at ambient conditions, viruses are usually negatively charged, thus being attracted to and entrapped by positively charged material in soil (sobsey et al., ) . in neutral and alkaline soil situations, viruses will not bind to any particulate matter and will be allowed to move freely in soil. there are, however, many exceptions to these general rules. virus absorption to soil is also affected by cation concentration. cations favor virus adsorption to soil by reducing their repulsive forces. sewage wastes provide an environment that enhances virus retention to soil, while this retention would be low in distilled water. as a matter of fact, distilled water may actually lead to the elution of viruses from soils, favoring virus mobilization and transport through soil. on the other hand, soluble organic matter will compete with the virus for soil adsorption sites. likewise, humic and fulvic acids will also compete with the virus and will reduce the level of adsorption of viruses to the soil (sobsey and hickey, ) . persistence is the term of choice to describe the capacity of a given virus to retain its infectivity in a given scenario. however, some authors unfamiliar with environmental virology claim that this term is confusing because it also describes the ability of certain viruses to produce infections in which, contrary to what applies in acute infections, a degree of equilibrium is established between the virus and the host (i.e., a cell or a whole animal). other authors avoid the use of the term survival to describe the natural persistence of virus infectivity, based on the ambiguity of the "live" condition of viruses. keeping in mind that a virus will be able to maintain its infectious status provided that all the virion components remain unaltered, the term stability may also be properly employed in this context. one critical question in environmental virology is whether or not viruses can persist long enough, and in high enough concentrations in the environment, to cause disease in individuals who are in contact with polluted recreational water, soil, or fomites, or who consume contaminated water or seafood. because viruses outside their hosts are inert particles, their chances of transmission from host to host are greatly dependent on the degree of their robustness, which allows them to remain infectious during the various conditions they may encounter in the environment. numerous physical, chemical, and biological factors influence virus persistence in the environment (table . ). some of the primary factors affecting the survival of viruses in liquid environmental matrices or media are temperature, ionic strength, chemical constituents, microbial antagonism, the sorption status of the virus, and the type of virus. considerable differences have been observed in the survival of viruses in different types of environmental samples. different behaviors and inactivation rates have been observed not only among viruses of different families and genera, but also among viruses of the same family, genus, and even among similar types or strains of virus (block, ) . among the chemical constituents of liquid or semisolid (feces, human night soil, biosolids, animal manures, etc.) environmental matrices, the amount and type of organic matter and specific antiviral chemicals (such as ammonia at elevated ph levels) play a role in virus stability. of the physical factors influencing virus persistence in liquid media, temperature, sunlight, and virus association with solids are among the most important. soil moisture, temperature, sunlight, and other soil characteristics may influence the persistence of viruses in soil. on inanimate surfaces, the most important factors that affect virus stability are the type of virus and surface, relative humidity, moisture content, temperature, composition of the suspending medium, light exposure, and presence of antiviral chemical or biological agents. most of these factors are also relevant for the ability of viruses to persist in aerosolized droplets, together with the moisture content and the size of the aerosol particles, and the air quality. some enteric virus infections follow a seasonal pattern, whereas others fail to do so. in regions with temperate climates, infections due to enteroviruses generally reach a peak in summer and early fall (moore, ) . on the contrary, rotavirus, norovirus, and astrovirus infections occur mainly during the cooler months (mcnulty, ; mounts et al., ; guix et al., ) , although seasonal and nonseasonal distributions of rotavirus in sewage have been described (hejkal et al., ; bosch et al., c) . on the other hand, cases of hepatitis a do not show a clear seasonal pattern (lemon, ) , whereas enteric adenovirus infections are reported to peak in midsummer (wadell et al., ) . these data suggest that temperature, and probably relative humidity, may be meaningful in the seasonal distribution of outbreaks of certain human enteric viruses (enright, ) , due to the influence of these factors on virus persistence. understanding environmental virus stability, and elucidating the factors that affect it, may shed some light on the potential public health risk associated with these environmental pollutants and at the same time provide tools to interrupt the chain of fecal-oral virus transmission. in this chapter, only studies involving the persistence of enteric viruses in the absence of any deliberately applied inactivation process are reviewed. neither work on virus disinfection nor studies conducted with potential indicators, such as bacteriophages, are considered because they will be discussed in other chapters. most studies to determine the potential of viruses to persist in environmental settings have been performed by artificially adding a known amount of infectious virus to a given sample, determining the reduction in the infectious titer after subjecting the spiked sample to designated conditions, and applying statistical procedures to determine the significance of virus decay. obviously, this implies the use of virus strains that may be propagated in cell cultures and enumerated through quantal infectivity assays (e.g., plaque assays), thus greatly restricting the range of viruses that are able to be included in these studies. molecular detection approaches such as pcr or rt-pcr are normally employed for fastidious virus analysis. however, they are unable to differentiate between infectious and noninfectious particles (kopecka et al., ; american public health association, ) and are, therefore, unsuitable for virus persistence studies, even when quantitative real-time procedures are employed. although reports on the presence of norovirus sequences in bottled mineral water raised a lot of concern (beuret et al., (beuret et al., , , many authors have shown the lack of correlation between virus persistence and molecular detection of virus genomes. it now seems obvious that infectious particles are degraded more rapidly than virus genomes. most enteric viruses of public health concern consist of rna genomes. in studies employing rt-pcr, it has been shown that poliovirus genomic rna is not stable in nonsterilized water (tsai et al., ) . although free dna is fairly stable, it is unlikely that a free single-stranded rna genome of noroviruses, astrovirus, poliovirus, or hepatitis a virus would remain stable without its protein coat in the environment. this presumption is less clear for the double-stranded rna genome of rotaviruses. nevertheless, it has been shown that altered nucleocapsids of noninfectious virions may still encapsidate a rt-pcr detectable single-stranded rna (gassilloud et al., ) . amplification of a piece of the virus genome is not indicative of the presence of the infectious agent. it can be assumed that even when different target sequences from unrelated parts of the genome are detected by molecular amplification, there is still no indication of the presence of unaltered capsid with functional surface residues involved in receptor recognition and cell attachment. from a strictly theoretical point of view, the use of an antigen-capture pcr assay, involving virus binding with a conformationally dependent monoclonal antibody and amplification of different unrelated genomic targets, could provide a fair estimation on the stability of a nonculturable virus. however, this approach requires a lot of experimentation before it can be considered adequate to be applied in virus persistence studies. in the meantime, infectious surrogates are usually employed to generate data on unculturable virus survival; for example, feline calicivirus has been used to mimic norovirus behavior (thurston-enriquez et al., a , b . another promising approach to increase the likelihood of detecting intact and potentially infectious viruses in cell cultures is to pretreat the virions with proteolytic enzymes and nucleases prior to nucleic acid extraction, amplification, and detection, thereby eliminating the detection of free nucleic acids or nucleic acids associated with damaged, inactivated virions (nuanualsuwan and cliver, ) . some health significant enteric viruses, such as rotavirus, astrovirus, and enteric adenovirus, replicate poorly in cell cultures; yet their persistence may be evaluated by integrated cell culture rt-pcr assays reynolds et al., ; abad et al., ; reynolds et al., ) . for this purpose, cells supporting the propagation of a wide variety of enteric viruses, such as caco- (colonic carcinoma) or plc/prf/ cells (human liver hepatoma), are used for an in vivo amplification step prior to molecular amplification (grabow et al., ; pintó et al., ) . it should be recognized, however, that most of the studies on virus persistence in the environment were performed under laboratory conditions and that data obtained from these studies may not truly represent their behavior under actual field conditions. the survival of viruses in environmental waters has been extensively reviewed (bitton, ; kapucinski and mitchell, ; block, ; bosch, ) . as previously mentioned, the most relevant factors affecting virus survival in the water environment are temperature (akin et al., ; raphael et al., ; bosch et al., ) , virus association with solids (gerba and schaiberger, ; la belle et al., ; rao et al., ; sobsey et al., ) , exposure to uv (bitton et al., ; bitton, ) , and the presence of microbial flora (gunderson et al., ; fujioka et al., ; toranzo et al., ; ward et al., ; gironés et al., gironés et al., , . the effect of temperature on viral persistence in water may be due to several mechanisms including protein denaturation, rna damage, and influence on microbial or enzymatic activity (dimmock, ; melnick and gerba, ; deng and cliver, ) . early studies pointed to damage to virion proteins as the primary target for viral inactivation at high temperatures, although damage to both protein and rna occurs at all temperatures (dimmock, ) . even though all viruses persist better at lower temperatures than at higher temperatures, some viral strains, such as hepatitis a virus and parvovirus, do exhibit higher thermal resistance than other viruses. as mentioned earlier in this chapter, virus adsorption to particulate material increases the persistence of enteric viruses in the water environment (gerba and schaiberger, ; la belle et al., ; rao et al., ; sobsey et al., ) , although differences have been observed among study locations (la belle et al., ) . the increased virus survival in the presence of sediment has important implications in the marine environment, because fecal contamination of coastal areas results in contamination of shellfish harvesting areas, accumulation of solid-associated viruses in sediments with sediments acting as virus reservoirs, and finally accumulation of viruses in shellfish. additionally, virus uptake by molluskan bivalves is enhanced by the presence of particulate material (landry et al., ) . although self-purification processes are reported to be more pronounced in seawater than in river water (matossian and garabedian, ; gironés et al., ) , the effect of salinity on virus stability is variable. thus, many studies have reported enhanced removal of virus infectivity in saline solution compared with distilled water (dimmock, ; salo and cliver, ) , whereas others report no significant effect of salinity on virus persistence (lo et al., ; fujioka et al., ) . in any case, the self-purification capacity of water is finite. several observations demonstrate the potential involvement of native aquatic microorganisms in the inactivation of viruses, particularly in marine habitats. however, data on the successful isolation of microorganisms with virucidal properties are scarce (fujioka et al., ; girones et al., ; bosch et al., ) . additionally, the ability of bacteria to inactivate viruses is usually lost while subculturing the microorganisms in the laboratory (gunderson et al. ; katzenelson ) , although in a few studies, such bacteria could be subcultured for more than year without losing their antiviral activity (girones et al., ; bosch et al., ) . in some studies, the virucidal agents in the tested waters could not be separated from the microorganisms (shuval et al., ; denis et al., ; fujioka et al., ; ward et al., ; gironés et al., ) , whereas in others the virucidal activity could be separated from the bacteria (matossian and garabedian, ; o'brien and newman ; toranzo et al., ; bosch et al., ) . the antiviral activity seems to be based on proteolytic bacterial enzymes that inactivate virus particles in water by cleavage of viral proteins, thus exposing the viral rna to nuclease digestion (toranzo et al., ; gironés et al., . it seems reasonable to assume that environmental factors and the compositional makeup of a given type of water may be substantially different from one geographical location to another, which implies that different data of virus persistence are produced when the same viral strain is suspended in water sampled from different sites . furthermore, it is highly likely that natural waters, particularly in the marine environment, contain a variety of potential antiviral factors, and that the antiviral action observed is generally the expression of the most dominant factor(s) present in any given water source. as has been mentioned earlier, soil pollution with human wastes may greatly contribute to groundwater contamination. because of the increasing empha-sis placed on land application as a means of organic waste disposal, it appears relevant to evaluate the persistence of human pathogens in soil. viruses in moisture-saturated soils may remain infectious for long periods of time, even at ambient temperatures of °c, in which the soil would be microbially active. if soil moisture drops under %, dramatic losses in virus infectivity are observed regardless of soil temperature or the medium in which viruses are applied (yeager and o'brien, ) . for example, enteric viruses survive for - days in an air-dried soil as compared with - days in soils with % moisture content. one of the pathways for virus removal from warm soils is through evaporation, which would account for the loss of viral pathogens from dry soils. the rate of evaporation is directly related to temperature and relative humidity. under constant moisture of % or greater, the main factors controlling the inactivation of viruses appear to be not only soil temperature but also soil texture. the survival of viruses is enhanced by a combination of low soil temperature and sufficient moisture (bitton, ) .as temperature increases, the virus inactivation rates also increase significantly (yeager and o'brien, ; straub et al., ) . at °c and with constant moisture, viruses are able to persist for days, whereas at °c no viruses persist after days. certain soil characteristics also influence virus survival. for example, virus persistence has been reported to decrease as a function of increasing soil ph and resin-extractable phosphorus (hurst et al., ) . increase in exchangeable aluminum, on the other hand, increased virus survival. the relative levels of clay and humic acids may also enhance virus survival (bitton and gerba, ) . viruses survive better in an adsorbed state than in suspension. virus adsorption to clay materials through electrostatic interactions is speculated to protect viral genome against nucleases or other antagonistic factors in soil (bitton and gerba, ) .additionally, clay contributes to virus survival by retaining minimum amounts of water, even in dry soils. this water provides the moisture required for virion stability. on the other hand, poorly absorbent sandy soils can increase their viral retention in the presence of divalent cations (mg + , ca + ) but not monovalent (na + ) or trivalent (fe + ) cations (lefler and kott, ) . clay loam soils generally afford more protection to viruses than sandy soils. however, in rapidly drying soils, virus persistence may decrease more deeply in clay soils than in sandy soils, due to the water-holding capacity of soil. clay soils can hold more water than sandy soils, but when water is evaporating from both soils, the clay soils, because of their mineral content, will retain the remaining water more tightly than sandy soils at the same moisture content, making them less apt for biological activity (straub et al., ) . the presence of indigenous microorganisms is deleterious to virus survival, although this effect is not observed at low temperature; at °c poliovirus remains stable through days (hurst et al., ) . indigenous soil aerobic microorganisms significantly reduce virus persistence, while indigenous anaerobes do not (hurst, ) . in a study involving a variety of soils and poliovirus, echovirus and hav suspended in groundwater, second-ary sewage effluent or primary sewage treatment effluent, hav was usually more persistent than poliovirus and echovirus; the % reduction times for hav were normally greater than weeks (sobsey et al., ) . this indicates that hav is an extremely stable agent, capable of persisting for more than months in soil, and hence it poses a health threat. the ultraviolet component of sunlight is destructive to viruses (bitton, ) . the uv has been shown to inactivate viruses at the surface of the soil but as the viruses move deeper in the soil column, it plays a minor role in inactivating viruses. disposable diapers may contribute to soil contamination with human pathogens. a field survey of virus inactivation in diapers buried in landfills for at least years showed complete inactivation (huber et al., ) . in laboratory conditions, hav and poliovirus experimentally seeded in disposable diapers showed . and log reduction, respectively, after days at °c, in aerobic conditions . quantitative interpretations (carrington et al., a (carrington et al., , b of existing data on poliovirus and cytopathic enterovirus decay rates in sludge-amended soil (tierney et al., ; hurst et al., ) indicated that, at the prevailing summer temperatures ( - °c), the decimal reduction rates were between . and . days, whereas at the winter temperatures ( - °c), it was days. carrington et al. ( a) analyzed data reported by straub et al. ( ) and found that decimal reduction time for poliovirus at winter temperature of °c and moisture levels of - % was days as compared with . days at summer temperatures of - °c at moisture levels of - %. most studies on virus persistence in soil have been performed in north american soil types and autochthonous climatic conditions. it has been pointed out that in other parts of the world, where mean soil temperature seldom exceeds °c at cm depth in summer and about °c in winter, viral decay rates would be slow or with decimal reduction times from days to more than days (carrington et al., b; rzezutka and cook, ) . however, the same authors suggest that cultivation of soil after sludge application would encourage virus decay by enhancing evaporation. outbreaks of acute gastroenteritis and hepatitis are a matter of concern in institutions such as daycare centers, hospitals, nurseries, schools, and military quarters. many of these outbreaks have been suspected to be caused by vehicular transmission of agents through contaminated environmental surfaces (ryder et al., ; halvorsrud and orstavick, ; rocchi et al., ; sattar et al., ; butz et al., ; green et al., ) . as has been mentioned earlier, stools from patients with diarrhea or hepatitis contain a very high number of the causative virus, and a single vomiting episode of an individual suffering from norovirus gastroenteritis may expel × virus particles, all of which are able to contaminate fomites (cheesbrough et al., ; green et al., green et al., , . it has been demonstrated that human enteric viruses are able to survive on several types of materials commonly found in institutions and domestic environments long enough to represent a source for secondary transmission of disease (hendley et al., ; sattar et al., sattar et al., , ansari et al., ; mbithi et al., ; abad et al., abad et al., , . the stability of health-significant human enteric viruses has been investigated on various nonporous (aluminum, china, glazed tile, plastic, latex, stainless steel, and polystyrene) and porous (cloth, different types of papers and cotton cloth) surfaces (sattar et al., ; abad et al., abad et al., , . as a general conclusion, when dried on environmental fomites, hepatitis a virus and rotavirus are more resistant to inactivation than enteric adenovirus, astrovirus, and poliovirus. the higher stability of hav in comparison with poliovirus, both of which belong to the picornaviridae family, is due to the inherently more stable molecular structure of hav capsid, concordant with the special codon usage described for this virus (sánchez et al., ) . in fact, it appears undeniable that poliovirus, which has been extensively employed as a model to elucidate enteric virus behavior in many scenarios, may fail to provide an adequate indication of the persistence of other human enteric viruses, such as hav, astrovirus, or rotavirus, dried on fomites (sobsey et al., ; mbithi et al., ; abad et al., abad et al., , . the resistance to desiccation appears to be of major significance in determining the ability of a virus strain to survive on fomites. a pronounced loss in virus titer at this stage dramatically reduces the chances of subsequent virus persistence. on the contrary, viruses involved in outbreaks probably transmitted through fecally contaminated environmental surfaces (i.e., hav, rotavirus, or astrovirus) show little decay at the desiccation step. on the contrary, hav and hrv, which have been involved in outbreaks probably transmitted through fecally contaminated environmental surfaces, show little decay at the desiccation step (mahl and sadler, ; keswick et al., ; sattar et al., ; sobsey et al., ; abad et al., abad et al., , . in spite of the experimental data on virus persistence on environmental surfaces, it is generally very difficult to determine whether, and to what extent, fomites play a role in the spread of infectious agents. keswick et al. ( ) have suggested that the prevalence of asymptomatic infections in daycare facilities may make contaminated surfaces in these environments a reservoir of infection for previously uninfected inmate children and their family contacts. as mentioned previously, there is a considerable shedding of the sars coronavirus in stools, where it remains stable at room temperature for several days (tsang, ) . although epidemiological evidence suggests that the major mode of transmission for sars coronavirus is by close personal contact with an infected individual, contact with environmental surfaces contaminated with respiratory secretions or other body fluids may also play a role in transmission (tsang, ) . in addition, sars coronavirus has been detected in a variety of environmental surfaces, such as the toilet and floor in the aparment of an infected individual and the walls and rooftop of a building with multiple cases (tsang, ) . hands are frequently in contact with environmental surfaces, and the potential for transfer of virus between surfaces and hands has been studied (hendley et al., ; ansari et al., ; mbithi et al., ) . it was ascertained in these studies that rotavirus and hepatitis a virus could retain infectivity for several hours on skin and could be transferred in an infectious state from fingertips to other surfaces and vice versa. enteric virus transfer between hands was apparently influenced by moisture. moisture would mediate suspension of virus particles and facilitate their movement between touching surfaces; drying would reduce this effect. laboratory studies have shown that viruses persist better in the environment at high relative humidity and at low temperatures (moe and shirley, ; sattar et al., ; sobsey et al., ; abad et al., ) . however, data on the effect of relative humidity on enteric virus survival is contradictory. these reported differences, particularly affecting rotavirus persistence, are difficult to explain but may be due to differences in the methodologies between these studies. temperature substantially affects the survival of feline calicivirus, an infectious surrogate for human norovirus, which is able to persist for long periods of time dried onto glass coverslips with log reductions of . after months and weeks, at °c and room temperature, respectively (doultree et al., ) . the authors suggested that the effect of temperature on feline calicivirus stability may reflect the greater prevalence of norovirus infections in cooler seasons (lopman et al., ) . because the fecal-oral route is the common means of enteric virus transmission, it seems reasonable to evaluate the effect of fecal material on the persistence of virus on fecally contaminated fomites. again, data on the protective effect of feces on viruses are contradictory; fecal matter appears to affect the survival of enteric viruses in opposite ways, depending on the type of surface and the virus strain (keswick et al., ; sobsey et al., ; abad et al., ) . aerosols are an important means of virus transmission in humans. various authors have reported the isolation of enteric viruses from aerosols produced by sludge-treatment plants (fannin et al., ; fattal et al., ; pfirrmann and bossche, ; alvarez et al., ) . the presence of microorganisms in aerosols generated from wastewater-treatment processes or in treated wastewater for agricultural irrigation is a potential danger to human health (teltsch et al., ; alvarez et al., ) . in hospitals, aerosolization of vomit was reported to be of major importance in the transmission of norovirus infection during outbreaks, while cleaning vomit or feces from patients did not significantly increase the risk of developing gastroenteritis (chadwick and mccann, ) . members of the caliciviridae family have been reported to be fairly stable in aerosols (donaldson and ferris, ) . the most important factors affecting the stability of viruses in the aerosol state are temperature, ph, relative humidity, moisture content, size of the aerosol particle, composition of the suspending medium, sunlight exposure, air quality, and virus type. the basis of virus inactivation in aerosols is poorly understood, although mechanisms for bacteriophage inactivation in aerosols have been proposed (trouwborst et al., ) . at high relative humidity, surface alteration of the virion has been reported, whereas at low relative humidity virus inactivation appears to be mediated by the removal of structural water molecules. relative humidity seems to confer a protective effect on aerosolized nonenveloped virus particles. thus, poliovirus was more stable in aerosol at °c at high relative humidity than at low relative humidity (hemes et al., ; harper, ) . picornavirus infectious rna may be detected at all humidity levels, suggesting that virus inactivation is caused by virion capsid damage (akers and hatch, ) . high relative humidity and low temperature enhance the persistence of bovine rotavirus in aerosols (moe and harper, ; ijaz et al., ) , although simian rotavirus sa survival in aerosols seems to be the best at intermediate relative humidity levels (sattar et al., ) . in any case, human, simian, and calf rotavirus strains may be detected in aerosols after as long as days (moe and harper, ; sattar et al., ; ijaz et al., ) , although discrepancies, probably due to methodological differences, are found among these studies. aerosolized adenovirus particles also show increased persistence at high relative humidity and low temperature (miller and artenstein, ; elazhary and derbyshire, ) . contrarily to nonenveloped viruses, viruses with an outer lipid envelope seem to be more stable at lower relative humidity (hemmes et al., ) . after days at °c and % relative humidity, infectious human coronavirus particles could be recovered in aerosols (ijaz et al., ) . virus infectivity in aerosols is also affected by solutes in the suspending media used for aerosolization. addition of salts and proteins in the suspending media provides a protective effect against dehydration and thermal inactivation of aerosolized picornaviruses (mcgeady et al., ; reagan et al., ) and may also influence the rehydration rate during sample rehumidification prior to the infectivity assay (benbough, ) . outbreaks of viral infection attributed to the consumption of contaminated soft fruit, salad vegetables, and other foods are increasingly reported (mead et al., ; lopman et al., ) . a recent example is an outbreak of hepatitis a virus in western pennsylvania in late , which affected more than people and resulted in three fatalities (mmwr, ) . the incident involved green onions imported from mexico and added to the restaurant's homemade salsa. those green onions were stored in a single container for up to days in the ice used for shipping them. some of the uncooked green onions were used in the restaurant's mild salsa that was prepared in large batches and stored for up to days. if the shipment ice was contaminated, prolonged exposure combined with the relatively long storage of salsa may account for why so many patrons became infected. green onions, which are multilayered and can retain soil particles that could harbor fecal contaminants, were probably contaminated during harvesting and packing. alternatively, hav-contaminated water used for irrigation, processing, and storage may have been the source of contamination. the high environmental persistence of hav makes any of these scenarios possible. data on the potential of enteric viruses to persist between the preparation of food and its consumption are required to ascertain the risk of virus transmission through food. this information is also important for the development of treatments applied to food in order to inactivate contaminant viruses. disinfection practices for food are reviewed in another chapter. examples of studies on virus persistence in food are depicted in table . . studies have shown that viruses remain infectious for several days or weeks on vegetable crops irrigated with contaminated sewage effluent or sludge (tierney et al., ; ward and irving, ) . several enteroviruses have been reported to survive during commercial and household storage for periods of up to weeks on vegetables irrigated with contaminated effluent (larkin et al., ; ward and irving, ) . the factors that affect virus survival in the environment, especially on fomites, are also relevant for the fate of viruses in food products. among them, temperature has a great influence on virus stability in food as in any other suspending matrix; the higher the temperature, the more pronounced the virus decay. natural or added constituents of food may influence the rate of virus inactivation by temperature (cliver and riemann, ) . for instance, salt used in pickling sausage batter has been shown to protect viruses from thermal inactivation (grausgruber, ) , whereas acidity often enhances the virucidal effect of temperature (cliver et al., ; salo and cliver, ) . additionally, viruses appear to resist thermal inactivation during cooking when fat levels are high (filippi and banwart, ) . furthermore, some ingredients may have a direct virucidal effect, as has been elucidated for free, unsaturated fatty acids with enveloped viruses (kohn et al., ) . naturally occurring substances in fruit juices have been reported to bear a reversible inactivating effect on enteroviruses, attributed to plant polyphenols such as tannins speirs, , ; cliver and kostenbader, ) . although the presence of fecal material and high relative humidity strongly enhances virus persistence (konowalchuk and speirs, ) , the effect of modified atmosphere packaging does not appear to be significant on virus persistence (bidawid et al., ) . as is the case with fomites, a rapid and marked decline in virus titer on crops/vegetables is attributed to drying/desiccation (larkin et al., ; tierney et al., ; ward and irving, ) combined with the action of sunlight and temperature (kott and fishelson, ) . direct sunlight irradiation (particularly its uv component) by itself is able to induce a pronounced reduction in virus numbers in food (badawy et al., ) . further work is required to develop robust and reliable quantitative methods to recover and detect health significant viruses in environmental and food samples. these procedures should also be adequate for newly recognized emerging pathogens of concern, as well as for non-human viruses capable of zoonotically infecting humans and having greater potential to cause human infection and illness. simple standardized diagnostic procedures for selected pathogens are needed to establish specific virological guidelines in selected food products, notably shellfish or food imports from regions with endemic infections. molecular characterization of agents responsible for waterborne and food-borne outbreaks will provide relevant information on the prevalence of infections among the population, which may be important in the development and/or efficacy of vaccines. the long pursued objective of the eradication of poliomyelitis will require comprehensive surveys on the occurrence of wild-type and vaccinal-type poliovirus in environmental samples that may represent potential reservoirs and vehicles of transmission. another important issue in environmental studies is microbial source tracking, which is imperative for the maintenance of microbiological quality and safety of water systems used for drinking, recreation, and in seafood harvesting, because contamination of these systems can represent high risks to human health and significant economic losses due to closure of beaches and shellfish harvesting areas. as mentioned earlier and discussed elsewhere in this book, bacteriophages and other microorganisms of fecal flora have been proposed as models of virus behavior. however, from the strictly structural point of view, there is no better surrogate of an actual virus pathogen to track their behavior in the environment than a noninfectious virus-like particle (vlp) of the same virus. recombinant vlps of health-significant viruses as norovirus and rotavirus have been employed to investigate the influence of electrostatic interactions in the filtration of norovirus in quartz sand and rotavirus behavior under disinfection conditions, respectively (redman et al., ; caballero et al., ) . as model systems, recombinant tracers are perfectly adequate for field studies of microbial tracking, as they may be produced in extremely high numbers (several milligram amounts). additionally, their noninfectious nature, due to the lack of a nucleic acid, makes them suitable for use in scenarios where the use of actual pathogenic viruses is not prudent; for example, drinking-water treatment plants, shellfish growing waters, or selected food products. survival of enteric viruses on environmental fomites astrovirus survival in drinking water potential role of fomites in the vehicular transmission of human astroviruses survival of a picornavirus and its infectious ribonucleic acid after aerosolization enteric viruses in ground and surface waters: a review of their occurrence and survival pcr for bioaerosol monitoring: sensitivity and environmental interference standard methods for the examination of water and wastewater viral water contamination as the cause of aseptic meningitis outbreak in belarus. cent waterborne outbreaks in sweden: causes and etiology rotavirus survival on human hands and transfer of infectious virus to animate and nonporous inanimate surfaces a possible virus aetiology in outbreaks of food poisoning from cockles comparative survival of enteric viruses and coliphage on sewage irrigated grass human and porcine hepatitis e virus strains the effect of relative humidity on the survival of airborne semliki forest virus norwalk-like virus sequences detected by reverse transcription-polymerase chain reaction in mineral waters imported into or bottled in switzerland norwalk-like virus sequences in mineral waters: one-year monitoring of three brands survival of hepatitis a virus on modified atmosphere-packaged (map) lettuce. food microbiol sewage pollution of marine waters: the risks of viral infection groundwater pollution microbiology: the emerging issues effect of solar radiation on poliovirus: preliminary experiments norwalk virus: a major cause of epidemic gastroenteritis viruses in environmental waters the survival of enteric viruses in the water environment human enteric viruses in the environment survey of viral pollution in besos river occurrence of enterovirus on marine sediment along the coast of barcelona (spain) detection of human rotavirus in sewage through two concentration procedures non-seasonal distribution of rotavirus in barcelona raw sewage waterborne viruses associated with a hepatitis outbreak the survival of human enteric viruses in seawater human enteric viruses in coquina clams associated with a large hepatitis a outbreak an outbreak of hepatitis a associated with recreational lake water prevalence of rotavirus on high-risk fomites in day-care facilities rotavirus virus-like particles as surrogates in environmental persistence and inactivation studies public health and water quality significance of viral diseases transmitted by drinking water and recreational water swimming-associated gastroenteritis and water quality review of the scientific evidence relating to the controls on the agricultural use of sewage sludge. part -the evidence underlying the department of the environment code of practice for agricultural use of sludge and the sludge (use in agriculture) regulations review of the scientific evidence relating to the controls on the agricultural use of sewage sludge. part -evidence since relevant to controls on the agricultural use of sewage sludge small round structured viruses: airborne transmission and hospital control transmission of a small round structured virus by vomiting during a hospital outbreak of gastroenteritis possible prolonged environmental survival of small round structured viruses human enteric viruses in oysters causing a large outbreak of human food borne infection in / significance of water and the environment in the transmission of virus disease antiviral effectiveness of grape juice foodborne diseases stability of viruses in low moisture foods surface water supplies and health outbreaks in drinking-water systems, - the survival of hepatitis a virus in fresh produce pharyngoconjunctival fever caused by adenovirus type : report of a swimming pool related outbreak with recovery of virus from pool water detection of animal viruses in coastal seawater and sediments persistence of inoculated hepatitis a virus in mixed human and animal wastes survie dans l'eau de mer de souches de virus a adn et differences between the thermal inactivation of picornaviruses at high and low temperatures isolation of wild type hepatitis a virus from the environment ultrafiltration: an efficient second step for hepatitis a virus and poliovirus concentration the survival of some airborne animal viruses in relation to relative humidity inactivation of feline calicivirus, a norwalk virus surrogate effect of temperature, relative humidity and medium on the aerosol stability of infectious bovine rhinotracheitis virus the epidemiology of paralytic poliomyelitis in hawaii usepa manual methods for virology effect of activated sludge wastewater treatment plant on ambient air densities of aerosols containing bacteria and viruses concentration of viruses from large volumes of tap water using pleated membrane filters comparison between adsortion of poliovirus and rotavirus by aluminum hydroxide and activated sludge flocs viruses and the gut epidemiological research on the relationship between microbial quality of coastal seawater and rotavirus induced gastroenteritis among bathers on the mediterranean israeli beaches survival of bacterial indicator organisms and enteric viruses in the mediterranean coastal waters of tel-aviv viral antibodies in agricultural populations exposed to aerosols from wastewater irrigation during a viral disease outbreak effect of the fat content of ground beef on the heat inactivation of poliovirus viral pollution of seawater at barcelona adenovirus type epidemic associated with intermittent chlorination of a swimming pool survival of human enteroviruses in the hawaiian ocean environment: evidence for virus inactivating microorganisms adsorption-elution with negatively and positively-charged glass powder for the concentration of hepatitis a virus from water genotyping of rotaviruses isolated from sewage presence of viral genomes in mineral water: a sufficient condition to assume infectious risk? effect of particulates on virus survival in seawater methods in environmental virology development of a quantitative method for detecting enteroviruses in estuarine sediments quantitative assessment of the adsorptive behavior of viruses to soils three-step isolation method for sensitive detection of enterovirus, rotavirus, hepatitis a virus, and small round structured viruses in water samples epidemic of gastroenteritis caused by oysters contaminated with small round structured viruses natural inactivation of enteric viruses in seawater isolation of marine bacteria with antiviral properties an epidemic of cockles associated hepatitis a in singapore comparative adsorption of human enteroviruses, simian rotavirus, and selected bacteriophages to soils viradel method for detection of rotavirus from seawater human enteroviruses in oysters and their overlaying waters detection of adenovirus types and by means of the plc/prf/ human liver cell line investigations of the inactivation of infectious swine paralysis virus in scalded sausages survival of hepatitis a virus (hav), poliovirus and f-specific coliphages in disposable and landfill leachates the role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay molecular epidemiology of astrovirus infection in characterization of a marine bacterium associated with virus inactivating capacity an epidemic of hepatitis a attributable to the ingestion of raw clams in shanghai an epidemic of rotavirus-associated gastroenteritis in a nursing home for the elderly airborne microorganisms: survival tests with four viruses seasonal occurrence of rotavirus in sewage virus survival as a seasonal factor in influenza and poliomyelitis transmission of rhinovirus colds by self-inoculation monitoring the marine environment for small round structured viruses (srsvs): a new approach to combating the transmission of these viruses by molluscan shellfish a community waterborne gastroenteritis outbreak: evidence for rotavirus as the agent heterogeneity and seroprevalence of a newly identified avian hepatitis e virus from chickens in the united states study of persistence of enteric viruses in land filled disposable diapers recherche des virus dans le milieu marin à partir d'échantillons de volumes différents an outbreak of calicivirus associated gastroenteritis in an elderly persons home: a possible zoonosis? waterborne outbreak of rotavirus diarrhoea in adults in china caused by a novel rotavirus influence of aerobic microorganisms upon virus survival in soil development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal effects of environmental variables and soil characteristics on virus survival in soil survival characteristics of airborne human coronavirus e comparison of the airborne survival of calf rotavirus and poliovirus type (sabin) aerosolized as a mixture characterization of a novel human calicivirus that may be a naturally occurring recombinant occurrence of bacteriophages infecting bacteroides fragilis and other viruses in polluted marine sediments gastroenteritis due to norwalk virus: an outbreak associated with a municipal water system processes controlling virus inactivation in coastal waters survival of viruses organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water detection of enteroviruses in treated drinking water survival and detection of rotaviruses on environmental surfaces in day care centers study of an epidemic of non a, non b hepatitis-possibility of another human hepatitis virus distinct from post-transfusion non a, non b type detection of both hepatitis a virus and norwalk-like virus in imported clams associated with food-borne illness unsaturated free fatty acids inactivate animal enveloped viruses survival of enteric viruses on fresh vegetables virus inactivation by grapes and wines antiviral effect of commercial juices and beverages norwalk virus enteric illness acquired by swimming exposure detection of naturally occurring enteroviruses in waters by reverse transcription, polymerase chain reaction and hybridization survival of enteroviruses on vegetables irrigated with chlorinated oxidation pond effluents a novel coronavirus associated with severe acute respiratory syndrome outbreak of viral gastroenteritis due to drinking water contaminated by norwalk-like viruses relationships between environmental factors, bacterial indicators and the occurrence of enteric viruses in estuarine sediments accumulation of sediment-associated viruses in shellfish persistence of virus on sewageirrigated vegetables recherche des virus et des salmonelles dans la loire par une méthode d'adsorption-élution sur filtres en microfibre de verre enteric virus behavior in sand dunes detection of hepatitis a virus, rotavirus, and enterovirus in naturally contaminated shellfish and sediment by reverse transcription-seminested pcr detection and analysis of a srsv strain in oysters implicated in an outbreak type a viral hepatitis-new developments in an old disease an outbreak of an enterovirus-like illness at a community wading pool: implications for public health inspection programs polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatits a virus and human rotavirus, from oysters, water and sediment samples the darwin outbreak of oyster associated viral gastroenteritis waterborne disease outbreaks - : a thirty-five-year perspective stability of human enteroviruses in estuarine and marine waters viral gastroenteritis outbreaks in europe identification of viruses isolated from sewage, river water and coastal seawater in barcelona virus survival on inanimate surfaces virucidal action of seawater effect of relative humidity and air temperature on survival of hepatitis a virus on environmental surfaces survival of hepatitis a virus on human hands and its transfer on contact with animate and inanimate surfaces survival of coxsackie virus b under diverse environmental conditions food-related illness and death in the united states detection of rotavirus in sewage and creek water: efficiency of the concentration method a water-borne urban epidemics of hepatitis, in: hepatitis frontiers the ecology of enteroviruses in natural waters a novel virus in swine is closely related to the human hepatitis e virus aerosol stability of three acute respiratory disease viruses surveillance for waterborne-disease outbreaks-united states hepatitis a outbreak associated with green onions at a restaurant the effect of relative humidity and temperature on the survival of bovine rotavirus in aerosol the effects of relative humidity and temperature on the survival of human rotavirus in feces enteroviral disease in the united states, - waterborne disease in the united states widespread outbreaks of clam-and oyster-associated gastroenteritis: role of norwalk virus transmission of viruses by drinking water cold weather seasonality of gastroenteritis associated with norwalklike viruses an australia-wide outbreak of gastroenteritis from oysters caused by norwalk virus infectious gastroenteritis in norfolk island and recovery of viruses from drinking water molecular analysis of poliovirus isolated from an aerosol generated by a waste water treatment plant pretreatment to avoid positive rt-pcr results with inactivated viruses inactivation of poliovirus and coxsackie viruses in surface water viral agents of acute gastroenteritis in german children: prevalence and molecular diversity epidemic hepatitis a from cockles hospitalizations associated with rotavirus diarrhea in the united states, through : surveillance based on the new icd- -cm rotavirus-specific diagnostic code occurrence of pathogenic microorganisms in the saint-lawrence river (canada) and comparison of health risks for populations using it as their source of drinking water occurrence and isolation of airborne human enteroviruses from waste disposal and utilization plants use of the colonic carcinoma cell line caco- for in vivo amplification and detection of enteric viruses detection of fastidious infectious enteric viruses in water astrovirus detection in wastewater samples waterborne non-a, non-b hepatitis environmental virology isolation of enteroviruses from water, suspended solids and sediments from galveston bay; survival of poliovirus and rotavirus adsorbed to sediments development of a method for concentration of rotavirus and its application to recovery of rotaviruses from estuarine waters long term survival of human rotavirus in raw and treated river water persistence of human rhinovirus infectivity under diverse environmental conditions filtration of recombinant norwalk virus particles and bacteriophage ms in quartz sand: importance of electrostatic interactions frozen raspberries and hepatitis a hepatitis e virus (hev): molecular biology and emerging epidemiology detection of infectious enteroviruses by an integrated cell culture-pcr procedure icc/pcr detection of enteroviruses and hepatitis a virus in environmental samples outbreaks of shellfish-associated enteric illness in the united-states: requisite for development of viral guidelines outbreak of rotavirus gastroenteritis among premature infants reovirus-like agent as a cause of nosocomial diarrhoea in infants survival of human enteric viruses in the environment and food effect of acid ph, salt and temperature on the infectivity and physical integrity of enteroviruses genome variability and capsid structural constraints of hepatitis a virus enteric pathogens and soil: a short review a new and simple method for recuperation of enterovirus from water effect of relative humidity on the airborne survival of rotavirus sa institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission survival of human rhinovirus type dried onto nonporous inanimate surfaces: effect of relative humidity and suspending medium spread of acute hemorrhagic conjunctivitis due to enterovirus : effect of air temperature and relative humidity on virus survival on fomites distribution of enteroviruses in sediments contiguous with a deep marine sewage outfall concentration des enterovirus dans les eaux par adsorption sur poudre de verre: proposition d'un apareillage simplifié. microbia concentration of viruses in beef extract by flocculation with ammonium sulphate coronavirus-, calicivirus-, and astrovirus-like particles associated with acute porcine gastroenteritis natural virus inactivation processes in seawater concentration of poliovirus from tap water using positively charged microporous filters influence of water quality on enteric virus concentration by microporous filter methods effect of humic and fulvic acid on poliovirus concentration from water by microporous filtration interactions and survival of enteric viruses in soil materials survival and persistence of hepatitis a virus in environmental samples survival and transport of hepatitis a virus in soils, groundwater and wastewater detection of rotavirus in sewage persistence of viruses in desert soils amended with anaerobically digested sewage sludge virus survival in sewage sludge amended desert soil health implications of excreta and wastewater use-hubei environmental sanitation study isolation and identification of pathogenic microorganisms at wastewater-irrigated fields: ratios in air and wastewater chlorine inactivation of adenovirus type and feline calicivirus inactivation of feline calicivirus and adenovirus type by uv radiation persistence of poliovirus in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent mechanism of poliovirus inactivation by cell-free filtrates of marine bacteria inactivation of some bacterial and animal viruses by exposure to liquid-air interfaces analysis of viral rna persistence in seawater by reverse transcriptase-pcr environmental issues. who global conference on severe acute respiratory syndrome (sars) evidence of high-frequency genomic reassortment of group a rotavirus strains in bangladesh: emergence of type g in hepatitis e virus sequences in swine related to sequences in humans, the survey of human enterovirus occurrence in fresh and marine surface waters on long-island round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water a large infantile gastroenteritis outbreak in albania caused by multiple emerging rotavirus genotypes enteric adenoviruses method for recovery of enteric viruses from estuarine sediments with caotropic agents virus levels in river waters virus survival on vegetables spray-irrigated with wastewater recovery of viruses from vegetable surfaces mechanism of inactivation of enteric viruses in fresh water an epidemic of gastroenteritis traced to a contaminated public water supply enterovirus inactivation in soil negatively charged filters farrah et al., positively charged filters sobsey and jones, ; gilgen et al., glass powder gajardo et al., sarrette et al., ; lucena, glass fiber vilaginès et al., organic flocculation katzenelson et al., ammonium sulfate precipitation bosch et al., b; farrah, polyethylene glycol hydroextraction farrah et al., ; lewis and metcalf, ultracentrifugation mehnert et al., ; steinman, lyophilization gajardo et al., pintó et al., ultrafiltration divizia et al., b key: cord- -yxttl gh authors: siegel, frederic r. title: progressive adaptation: the key to sustaining a growing global population date: - - journal: countering st century social-environmental threats to growing global populations doi: . / - - - - _ sha: doc_id: cord_uid: yxttl gh adaptation is an evolving long-term process during which a population of life forms adjusts to changes in its habitat and surrounding environments. adaptation by the global community as a unit is vital to cope with the effects of increasing populations, global warming/climate change, the chemical, biological, and physical impacts on life-sustaining ecosystems, and competition for life sustaining and economically important natural resources. the latter include water, food, energy, metal ores, industrial minerals, and wood. within this framework, it is necessary to adapt as well to changes in local and regional physical conditions brought on by natural and anthropogenic hazards, by health threats of epidemic or pandemic reach, by social conditions such as conflicts driven by religious and ethnic fanaticism, and by tribalism and clan ties. the principal problems with growing populations do not involve space although population density is a problem unto itself for reasons discussed in previous chapters. the main problems are how to nourish people with food and water. the chronic malnutrition that about billion people suffered from in is likely to grow in number in some regions due to global warming/climate change because humans cannot adapt to less food if they are already at subsistence rations. for example, the population in sub-saharan africa is million people. the population is projected to increase to about . billion in , an increase of about %. within the same time frame, the united nations estimates that acreage under maize cultivation in the region will decline by % because of heat and drought brought on by climate change. the loss of arable land for food production can be countered in sub-saharan africa if marker assisted hybridization of maize or maize genetically modified to withstand heat and drought come onto the seed market together with modified seeds for other food staples and if african nations that do not now accept gmo seeds do so in the future. if not, nations favored for food production by climate change will have a moral obligation to provide food staples to people in nations with declining food production at accessible costs based on their economies. it is clear that what happens in sub-saharan africa and other regions with declining cultivation acreage or that will bear other effects of climate change (e.g., drought, shifting rain patterns) will affect the rest of the worldwide community politically, economically, and socially. the earth's problems that associate with global warming/climate change will be further discussed in the last section of this chapter. water is the staff of life. it keeps the body hydrated and is necessary to grow food crops, hydrate food animals, and grow feed grains. chemically or biologically polluted water does not serve these ends. if ingested, contaminated water can result in sickness as discussed in chap. . water stokes industry and manufacturing as well, thus keeping economies in many countries contributing to a population's wellbeing by providing employment, goods, and services. ideally, these businesses contribute their fair share to a tax base that supports social needs (e.g., education, healthcare, maintenance of infrastructure). factory owners adapt and plan against water shortages by having a water recycling system in place but may also slow or stop production until operational water conditions return. citizens with a reliable water supply can adapt to periods of water shortage by limiting use according to mandates by government officials but still have water for basic daily needs. however, persons in nations with a chronic per capita water shortage may not have this option to serve their daily needs unless water is imported or new water sources are found (see chap. ). if imported water is not an option to meet immediate essential needs, an alternative adaptation for people (and animals) is to try to reach a location where water would be available to them. with growing populations, per capita water availability is greatly diminished (table . ), water shortages become endemic, and people are at risk of existing at subsistence levels or dying. most at risk from the lack of a basic water ration are pregnant women, infants, young children, and old people. water wars are a future possibility as nations battle for their peoples' survival unless political differences are set aside and projects are supported to develop and share water sources. in a welcome effort, jordan, israel, and the palestinian authority signed a memorandum of understanding in the world bank, december , with specific aims: ( ) produce millions of cubic meters of drinking water for a water-deficient region; ( ) pipe million cubic meters of water annually * km ( mi) from the red sea to the dead sea; ( ) build a desalination plant at aqaba that would supply water to aqaba and eilat; ( ) the israeli water utility would supply - million cubic meters of drinking water to the palestinian authority for the west bank population at a reduced cost; and ( ) there would be an inflow of water to slow and in the future perhaps abate and reverse the shrinking of the dead sea. funding for the estimated $ million, year project will come from the world bank, donor nations, and philanthropic groups. as the global population increases and more people in developing and less developed nations have more disposable income, there will be a growing draw on natural resources other than water and food to service their industrial, agricultural, and manufacturing needs and wants. competition can force economic wars among national and multinational corporations for the resources necessary to provide goods and services and thus drive up prices for resources. industries and manufacturing units that cannot compete economically for natural resources will shut down, thus contributing to unemployment and downturns in economies because of falling domestic demand. to keep order in the increasingly interdependent world economy, accommodation for shared natural resources (or substitutes for them) at affordable prices is the adaptationnecessary. this can be mandated by the world trade organization backed by other practical-minded international groups. another adverse effect of growing populations that is a national resource that can be lost at the expense of some countries to the benefit of others is brain power. this brain power has been cultivated at excellent universities in developing countries, often times at little or no cost to students (e.g., in medicine, science, engineering, economics, the arts) who attend and graduate in increasing numbers. where there are too many well-educated professionals but lack of employment opportunities for them in their fields of expertise, educated people have the option of relocating to another country that can nurture and use the expertise. many adapt to the employment problem by taking up this option. this may mean moving from a developing country to a developed country or from a less developed country to a developing or developed country. ultimately, this loss of citizens with special skills can hurt a country. to counter this brain drain or reverse it, a country can adapt by investing in its future to create programs and conditions that keep talented professionals home, or if they have emigrated, entice them to return. china and india are examples of countries that have successfully taken this tact. when there are increases in a population because of immigration, problems can ensue between immigrants and a general population. adaptation to diversity and the multicultural experiences it brings to a community is often not a comfortable change. the antipathy of some in a host country is based on slowness of the immigrants to learn the language and inability of host country citizens to understand what immigrants are saying among themselves. this makes citizens feel uneasy. some view immigrants as a threat to their own or a family member's employment or advancement. race difference is a factor that some cannot readily adapt to as is ethnicity with its traditions and customs unfamiliar to the general public. religion can be divisive if adherents to its beliefs engage in acts of hatred detrimental to the host country fueled by fundamentalists and zealots who interpret religious writings as giving them license to commit crimes or absolving them of the crimes. sadly, many citizens paint an entire religious community with the taint of the relatively few evildoers. adaptation to diversity is essential for our earth's citizenry with joint efforts by all to resolve worldwide issues (e.g., global warming/climate change) so as to become the keys to providing a sound future for coming generations. there has to be a shared attack on global threats, no matter what the language, race, ethnicity, or religious beliefs are, no matter social or economic status, no matter whether a threat affects less developed, developing, or developed countries. adaptation is a progressive process when dealing with natural hazards because as each type of natural hazard impacts global communities over time, lessons are learned from each one that give direction to the methods of adjustment. adaptation to living where hazards can be expected to strike and where populations continue to increase is dependent on what we learn from the study of past hazards. we can use this evaluation of measured and observed data to minimize the immediate effects and aftermaths of hazards and protect citizens from injury, death, and from damage or destruction of property or infrastructure when hazards strike in the future. in areas prone to earthquakes, we know that earthquakes do not kill and injure people but that collapsing buildings and infrastructure do. earthquakes are not predictable so that there is no adaptation by a timely evacuation to minimize deaths and injury. however, building structures to make them more earthquake resistant can save lives, reduce injuries, and protect property. thus, after a high-magnitude earthquake, forensic engineering teams come to assess the damage and determine where and why damage and destruction took place within the context of the magnitude of an earthquake, the type of motion it originated (shaking, jarring, rolling), its duration, the area it affected, and the geologic properties of rocks underlying structures' foundations. hazard assessment teams also evaluate other factors that contributed to additional damage such as ruptured gas lines that feed fires and ruptured water lines that inhibit fire control. the engineers establish how construction can be improved in the future in terms of construction techniques and materials to prevent the types of collapses and utility failures they investigated. municipalities revise building codes accordingly to direct reconstruction and future building projects. where possible, structures that withstood an earthquake with minor or no visible damage should be retrofitted to improve their resistance to the next "big one." with each event, we gain more data on how to better construct earthquake-resistant structures and alter building codes to more stringent specifications. in theory, this adaptationto an irregularly recurring global event is good, but in practice it is most applicable to nations with the economic resources for reconstruction according to revised building codes and where there is no corruption to allow a bypass of the code. the same can be stated for retrofitting to give more resistance to earthquakes to existing structures. many developed nations and nations rich in commodity exports (e.g., oil) have a moral obligation to donate funds, material, and expertise to help citizens in economically disadvantaged nations recover from a destructive earthquake. some commodity-rich and economically sound nations do not do so directly, whereas others, big and small, rally to help disaster victims. for example, immediately after megatyphoon haiyan devastated many regions in the central philippines in , israel sent medical doctors and nurses and field hospitals to help philippine citizens recover from the impacts of the typhoon. as discussed in an earlier chapter, volcanoes are predictable in terms of becoming active by emitting wisps of smoke, bulging on a slope, warming of the soil or nearby pond or lake waters, emitting increasing concentrations of gases, and showing increased low-frequency seismicity. however, this activity does not always result in an eruption. a marked increase in measurements and observations, especially the low-frequency seismic activity, suggests that an eruption is imminent. adaptation to living and working on or near a volcano means investing in equipment to monitor volcanic activity and listening to alerts from scientists monitoring its activity and being ready to evacuate by gathering important papers and precious mementos and prepared to load into transportation for evacuation to safe locations. governments adapt by charging geologists to map out areas considered as high-, moderate-, and low-hazard zones in the volcano environs. geologists do this by studying rocks deposited from past eruptions and assessments of the topography. municipalities then pass zoning regulations applicable to the hazard level. governments have adapted to repeated periodic flooding in areas by creating flood control systems described in chap. . dams hold water during times of heavy and/ or extended rainfall and release any overflow into channels that move water away from urban or rural population centers. levees increase the volume of water that can move through a channel, thereby keeping it from spreading into populated areas and cultivated farmland. for smaller waterways that flow through cities, municipalities may invest in deepening, widening, and straightening channels as well as erecting walls so that more water can flow through the area more rapidly without coming out of a channel. governments define zones on flood plains according to a recurrence interval of damaging floods (e.g., years) as being off limits for residential and factory/plant construction. as much as we plan to adjust to living in an area prone to flooding, there is always the possibility of a megaevent that can overcome in situ control systems. therefore, as described chap. , governments adapt to this possibility by installing flood prediction equipment in drainage basins to provide warning to those at risk from rising and sometimes raging waters. the warning gives people time to gather important documents and personal treasures and evacuate to safe areas. the apparent increase in the frequency and magnitude of storms and resulting flooding in recent years is thought by many weather scientists to be related to global warming and the increased amount of moisture in the atmosphere from warmer oceans that gathers in clouds and precipitates during storms. this will be discussed further in this chapter. adaptation to extreme weather events such as an extended period of drought, heat waves, and frigid weather means preparation to wait them out. some municipalities adapt to repeated, sometimes seasonal, times of short-term drought by storing a - month water supply in surface or underground reservoirs during periods of normal precipitation that can be tapped (conservatively) as needed. others may plan to move water via pipes or water tankers from where it is plentiful to where drought conditions exist. otherwise, to survive, people move as best they can to where they have access to water. in instances of years long drought, crops and livestock and other life forms may be lost. heat waves can kill. adaptation to heat wave conditions means that water has to be available to people to avoid dehydration. where possible, homes should have air-conditioning or fans to keep people comfortable and municipalities should have cooling centers to which people can go. personnel should check on senior citizens and escort them to cooling centers if necessary. clearly, economically advantaged nations have the resources to give support to citizens during natural hazards such as these. these nations, international organizations, and ngos have a moral obligation to help economically disadvantaged nations as is possible when hazard conditions such as these threaten populations. the most extreme of weather conditions that can injure and kill people and destroy housing and infrastructure are tropical storms that evolve into violent hurricanes (typhoons, monsoons) by increasing wind speeds and sucking up moisture (water) as they track across oceans toward land. when these storms make landfall, they drive storm surges that can wreak havoc onshore communities, and as they move inland precipitate heavy rains that cause life-threatening and destructive flooding. these violent storms are destructive to coastal populations and island nations and have regional reach inland as they move along paths until they finally spend their energy or move out to sea. on november , , the typhoon named haiyan, the strongest recorded typhoon ever to make landfall smashed into the central philippines killing more than , people, injuring about , , and displacing almost , people. there was a -m (* ft) storm surge driven by winds measured at over km/h ( mi/h) with gusts reaching km/h ( mi/h). the typhoon flattened the city of tacloban that was home to , residents, and there was major flooding inland. the weather alerts led to a government call for evacuation away from the predicted path of the storm, and about million people followed the evacuation warning, surely saving many lives. access to aid typhoon-ravaged areas was difficult, and there were shortages of water, food, and medical care for many evacuees for several days. the philippine central government and local officials were not prepared to deal with a storm of this magnitude but help started arriving from many nations worldwide. there was a post-event concern of attending to sanitation needs of survivors to prevent outbreaks of diseases such as cholera, typhoid fever, hepatitis, and dysentery. if the philippine government had adapted by adopting better policies with respect to response to high-category typhoons in addition to the call for evacuation, the impact of haiyan would have been ameliorated. one would hope that this deficiency would be dealt with to limit the effects of future like disasters. evacuation to prevent injury and death in coastal zones that could be struck by high winds, heavy sustained rains, and storm surges is dependent on weather bureau forecasts and warnings from police, firefighters, or other government-authorized personnel. homeowners adapt to hurricanes by securing roofing with additional nails or special fasteners as a retrofit precaution and by boarding up windows on structures before an incoming storm hits. governments have adapted to the onslaught of violent high-energy storms by constructing seawalls of varying designs and heights to protect population centers by damping the force of storm surges. in china, for example, a seawall . m (* ft) in height and that has been heightened in the past protects shanghai from the full damaging effects of high-category typhoons. as a result of rising sea level, the shanghai seawall and other that protect coastal cities from being flooded by surges from high-energy tropical storms will have to be heightened to afford a greater degree of protection to people and property. wildfires can be a natural hazard when ignited by a lightening strike. however, most wildfires are started by human carelessness such as tossing a lit cigarette on a forest floor or failing to completely extinguish a campfire, or by arsonists. one may adapt to living in an area with a history of wildfires in two ways, neither of which is practical or promises % protection. first would be to clear an area of vegetation in a -m ( ft) swath around a dwelling or site for building. second would be to build with nonflammable materials so that embers propelled during a wildfire could not ignite a structure. adaptation to the advance of a wildfire would be to heed warnings to evacuate carrying a prepared case with important documents and other items of personal value. to delay evacuation by going back to retrieve something from then home can be fatal as it was for two people in a recent (june, ) wildfire that destroyed almost homes in colorado springs, colorado, usa. when there is a hazard event coming that calls for evacuation, responsible and often economically advantaged governments have adapted to the threat by designating evacuation routes, by providing transportation for people who need it, by having evacuation centers stocked with water and food, cots and blankets, basic medical supplies and medical personnel, and by having phone service available for people that need it. in the case of a primary or triggered hazard that happens with little or no warning (e.g., an earthquake, a tsunami, a volcanic mud flow), search and rescue teams should be ready to move in soon after dangerous conditions ease and they can move with safety. there should be medical attention to treat injured survivors, and stations set up as soon as possible to provide water, food, and other essentials available to those that survived with little or no physical hurt. these first steps at adaptation are the keys to survival. recovery after a shock phase can be long and drawn out, depending in grand part on a nation's social and economic resources and physical and economic assistance from other nations, international institutions, and ngos. change on our earth's inhabitants global warming is a fact attested to by an overwhelming majority of the scientific community and unwaveringly supported by a february joint publication of the us national academy of sciences and the royal academy in the uk on the causes and evidence for global warming [ ] . as noted in earlier chapters, during the past century, measurements show that the earth has warmed by * . °c (* . °f). global warming is an ongoing process that is attributed in grand part to a slow but continuous and increasing buildup of greenhouse gases in the atmosphere. the greenhouse gas most associated with global warming is carbon dioxide (co ). a plot of the increase of co content in the atmosphere with time against the increase in global temperature shows an excellent correlation of one with the other. additional lesser contributors include methane (ch ), nitrous oxide (no ), and chlorofluorocarbons (cfcs). with the beginning of the industrial revolution and the increased use of coal as the principal energy source, the content of co in the atmosphere was parts per million ( . %). the combustion of coal and later oil (petroleum) and natural gas emits co to the atmosphere. initially, and for many years thereafter, the added greenhouse gases were taken up by vegetation for photosynthesis and was also absorbed by the oceans and other water bodies. this kept the atmosphere co close to the ppm pre-industrial level. however, with increased industrialization, the need for electrical power, and the use of internal combustion engines, the amount of co generated was greater than what could be absorbed by nature and the content of co in the atmosphere increased. during june , its concentration reached more than ppm, an increase of over % over the pre-industrial concentration (scripps institute of oceanography mauna loa measurement). the increasing co content, other greenhouse gases, aerosols, and particles acted as a media that admitted sunlight (heat energy) to the earth's surface but did not let all of the heat escape back into the atmosphere. this abets global warming. in the past two to three decades, the rush to industrialization in developing countries (e.g., china, india, and brazil) and their growing power needs and vehicular use has thwarted the implementation of international agreements to reduce emissions from coal-fired power plants, other industrial and manufacturing operations, and the transportation sector. a direct consequence of global warming is sea level rise (slr) caused by the progressive melting of icecaps and ice sheets in greenland, the arctic, and antarctica, and of mountain glaciers in the himalayas, the alps, the rocky mountains, and the andes. the * -cm (* in) sea level rise during the past century may see a rise of another * cm (* in)- m (* in) during this twenty-first century. one-third of the rise would be from the expansion of warmer sea water, one-third from icecap and ice sheet melt, and one-third from mountain glacier melt [ ] . in , other researchers used computer models on existing data and proposed that % of sea level rise between and was from glacial melt [ ] . following the same line of investigation, other scientists studied satellite data and ground measurements from alaska, the canadian arctic, greenland, the southern andes, the himalayas, and other high mountains of asia and estimated that glacier contributions to sea level rise from to was % and together with ice sheet melt explained % of slr [ ] . a publication in estimated that ocean thermal expansion - m deep and - m deep contributed up to % to sea level rise [ ] . these latter two estimations are in line with the ipcc prediction for melting ice and ocean thermal expansion contribution to the estimated rise of sea level by the end of the century [ ] . with a rise in sea level, marine waters encroach on land. as the rise continues, possibly at an increasing rate, it threatens habitation in lowlying islands, coastal villages and farmland in lowlying zones, and heavily populated cities worldwide settled on inshore terrain close to sea level (e.g., bangkok, ho chi minh city, jakarta, manila, miami, new york, boston, buenos aires, london, rotterdam). rising sea level and warming of ocean waters have other ramifications that affect coastal communities as well as inland areas. as explained in chap. , the warmer surface water releases more water vapor with heat energy into the atmosphere. when the water vapor molecules condense in clouds, heat energy is released. this energy gives more force to tropical storms as they form, track to shore, and move inland, or storms that move close to and along a coast. these storms may transition to hurricanes (typhoons, monsoons) with the violent winds that cause destruction, and heavy rainfall that triggers flooding if they move onto land. we recognize that rising sea level means that tropical storms that impact a coast with storm surges have a farther reach inland with their destructive energy that is more pronounced when the surge occurs at high tide. the surges also saturate farmland they reach with salt water that harms crops. they also carry salt water into fresh water marshes and ponds, thus disrupting ecosystems there. the increase in the number of these extreme weather events and the increase in violence and destruction they wreak on land compared with like weather events in the recent past (e.g., during the past years) strongly suggest that they are fueled to a significant degree by global warming. there are two possibilities for adapting to the effects of rising sea level on coastal urban centers, one impractical, the other very costly but doable. the impractical adaptation possibility is to move at-risk population centers inland, out of the reach of the destructive tropical storms. this does not lessen the threat of flooding. the move is possible in some cases where land is available, but such a move is not economically feasible. one practical but costly adaptation to mitigate encroachment from sea level rise and the effects of tropical storm surges is to surround cities at risk within place seawalls - m higher than recorded high tides or higher depending on historical records and contemporary published data. the walls can have a concave configuration so that surging waves lose energy when their lower parts hit and are curled back on themselves damping some wave energy or there can be a different configuration best for the site(s) to be protected. similarly, gates buried at strategic locations where there is ship access to consider can be built to be hydraulically driven so that they can rise from a near shore seabed site to mitigate the effects of storm surges. both techniques have been used at different global locations. we have read that climate change affects land-based agricultural production, both for crops and animal husbandry. the warming climate at higher mid-hemispheric latitudes and at higher altitudes does not favor the growth and normal yield and/or quality of many crops. depending upon the degree of climate change and the linked change(s) that may follow it, farmers can adapt in several ways to maintain or increase crop yield and nutrition value. for example, when warming starts diminishing the productivity of a traditional crop, farmers can sow crops that are known to grow well in warmer temperature and give a satisfactory economic benefit. however, new groups of weeds, pests, and diseases will migrate to the warmer growth environment and will have to be dealt with in order to protect the new crops. where the effect of global warming reduces water supply for rain-fed agriculture, for crops irrigated with surface waters, and for groundwater-irrigated crops when aquifer recharge does not balances discharge, agriculturalists can adapt in two ways. first is the use of a more efficient irrigation method that delivers water directly to a growing plant (e.g., drip or focused irrigation). this minimizes runoff and loss to evaporation. second and similar to what was mentioned earlier is to sow a crop that needs less water to thrive and that delivers a good yield, good-quality product. another result of global warming for some farmlands is a longer growing season. in this situation, growers can adapt by planting earlier and have the possibility of double cropping. they can also grow a cultivar that is later maturing and that gives a product that brings a good market price. however, switching to new crops in a warmer growth environment means that there will be an invasion of a new set of weeds, pests, and diseases to ward off. in any efficient operation, and as emphasized in earlier chapters, farmers adjust to a changing growth environment for a given cultivar by applying the optimum amounts of fertilizer and other agricultural chemicals as might be needed that nurture and protect it most effectively. this reduces agricultural costs and lessens runoff of these chemicals to ecosystems where they can be harmful. global warming can bring on abnormal weather extremes that affect agricultural productivity. in these cases, farmers have to plan ahead based on recent history of these conditions in their regions. drought, heat waves, and long-term rain or heavy rain in a short time present problems for both cultivars and food animals. periods of less than average precipitation may last months or years. depending on the amount of the deficit precipitation, adaptation can include storing water in reservoirs and cisterns during times of rainfall to be tapped during a drought to sustain food animals and crops during a short-term, not too severe drought. there is also the option of trucking in water to sustain livestock. long-term droughts when precipitation deficits are high take their toll on plants and animals to the detriment of agriculture in a region especially when accompanied by heat waves. they have caused recent disasters for crops and food animals on all continents less antarctica. farmers either wait out the "bad times," change the type of cropping they do, the livestock they tend to, or change careers. the adaptation from crops that have been grown successfully before the effects of global warming reduced yields and quality of a harvest, to those "same" crops that can grow successfully under the advancing warming changes just described generally means that hybridized species have to be developed and used as warming increases at a location and slowly tracks to higher latitudes and higher altitudes. thus, growers turn to plants that are created by hybridization as described in chap. : traditional methods and marker-assisted selection methods within the same species, and genetically engineered (-modified, -manipulated) methods using different species. hybridization is a slow process, sped up markedly by genetic engineering, a method that yields foodstuff not accepted by the european union and many nations outside the union, especially in africa. bred species are developed to carry one or more characteristics that favor crop resilience against the effects of climate change. these include resistance to disease, weeds, and pests, and tolerant of drought (water stress), heat, short-term inundation, and short-term saline exposure (see chap. ). hybrids have also been developed to give higher yields and more nutritious crops. thus far, research has been focused mainly on improving seed for world staples such as rice, maize (corn), wheat, sorghum, and soybean. there have been great successes where hybrid crops were agriculturalists' adaptation so that the possibility exists that we can feed the earth's growing populations and reduce chronic malnutrition. when this is coupled with the opening of additional arable acreage and the use of improved farming methods for seeding, watering, and harvesting, global food security can be strengthened for the existing world population and the future generations on earth. however, this will require economic and technical input by developed nations and international groups. without basic sustenance, people will have less resistance to diseases and there may be local or regional population crashes if diseases evolve into epidemics or pandemics that invade susceptible populations. warming of the open ocean water, enclosed aquaculture operations in ocean waters and on land water bodies has affected marine fisheries and marine and estuarine aquaculture that grow food fish and shellfish, and lakes that sustain fisheries. in marine fisheries worldwide (e.g., in the north atlantic, off the coast of peru, off the coast of the philippines), some food fish or fish captured for other purposes (e.g., to use in pet food, to use to make fertilizer) have migrated to cooler water in ecosystems with conditions conducive to their spawning and growth. in some cases, predators follow fish they prey upon that have migrated to cooler waters, but in other cases they find new prey to sustain them. in other situations, they may become prey for larger fish in an ecosystem. fishing fleets adapt by following the fish they hunt into cooler waters where ideally they capture the hunted species in quantities allotted them by national and international fishery governing body regulations. if the quota system is followed, this will allow recovery of fish populations and sustainable harvesting. aquaculture operations that provide important supplies of food fish worldwide can adapt to warming waters by raising food fish or shellfish that will grow and multiply under the changed range of day/night temperature conditions if the fish they are farming cannot survive in the warmer waters. aquaculturalists also have the option to move their facilities to cooler-temperature waters, but the economic feasibility of doing this has to be evaluated by a benefit to cost analysis. this analysis has to be for the time frame during which the cooler-ecosystem waters are estimated to remain stable within the framework of a time range against global warming/climate change. another adaptation is that food fish currently being raised can be genetically engineered to be resistant to a warmer growth environment without changing their nutrition yield, growth rate, and ability to reproduce. there are diseases that are global threats, others that put regions at risk, and yet others that menace smaller political divisions. humans adapt to the threat of sickness in a population or a sickness itself in several ways. scientists develop methods to eradicate a virus or bacterium health threat, or a chemical/radioactivity threat. failing this, health professionals act to control a disease, to slow or minimize its transmission, and to apply approved therapies and support research to find therapies to treat an illness if one is transmitted. the following discussion draws strongly on the disease fact sheets put out by the world health organization. vaccines provide a main line of defense against many diseases. smallpox has been eradicated on earth by vaccination. polio has all but been eradicated globally except for a few pockets of the disease in pakistan, afghanistan, and nigeria where, in some cases, religious fundamentalists have beaten and killed health workers tasked with giving the vaccine to children, and in other cases where parents have been warned by the zealots against allowing their children to be vaccinated. recently, polio cases were diagnosed mainly in somalia but also in kenya. this is attributed to the fact that by , , children in somalia have not received the vaccine and are at risk from this highly contagious disease. it is also attributed to crossborder migration of infected persons into kenya. both governments are stepping up their vaccination programs. there were cases of polio diagnosed in the rest of the world in . measles is a global disease that can be prevented by a vaccine that is safe and cost-effective. measles may soon reach the near-eradication stage. in and subsequent years, . million people, mainly children under years of age, died from measles. since , billion children were vaccinated, million in . by , % of the world's children received the measles vaccine, up from % in . from to , deaths from measles dropped to %, from , to , . when the vaccination rate reaches %, mainly in low-income countries, the world will have brought another disease close to elimination [ ] . seasonal influenza is a global viral illness that afflicts - million people. the sickness kills , - , people with severe symptoms annually. transmission of the virus takes place when an infected individual coughs or sneezes without covering his/her mouth and releases droplets that can be inhaled by someone up to a meter away. transmission can also be from hands carrying the virus. seasonal influenza affects all age groups, but children less than years old, people over , and those with complicating medical problems are most at risk. influenza is a disease to be controlled. the principal control is by safe and effective vaccines that can prevent - % of influenza cases in healthy adults. secondary controls are obvious for infected persons: cover the mouth when sneezing or coughing, and wash the hands frequently. the influenza vaccine is taken once annually. because strains of the influenza virus change from year to year, adaptation is needed. the adaptation is via a vaccine that is prepared with or strains that scientists determine will be most common during a coming season [ ] . other types of influenza and respiratory illnesses have the potential to cause an epidemic or pandemic. they include avian flu and its strains and swine flu if the strains develop the ability for person-to-person transmission after infection, and sars (severe acute respiratory syndrome) and middle east respiratory syndrome (mers) because there is human-to-human transmission of the sicknesses. to the present, the outbreaks of the animal influenza diseases have been contained by quarantining infected people during treatment and by culling flocks and herds, or if available, vaccination of healthy animals. the latter two respiratory illnesses are caused by the coronavirus, and infected people have been in isolation wards. for sars, an illness that broke out in and spread to countries, isolation of victims and treatment with antiviral drugs and steroids stopped the disease during . mers is a recent ( / ) illness that has been confined to jordan, saudi arabia, qatar, and the united arab emirates. the mers virus has been found in camels. infected persons are quarantined in hospitals, but an effective drug treatment is still being sought to complement the normal hospital care-afforded patients. hiv/aids is a global epidemic that killed million people in three decades since . worldwide, in , there were million people with hiv, mainly ( million or %) in sub-saharan africa and south/southeast asia. the illness is caused by the exchange of body fluids (semen, vaginal excretions, blood, breast milk) from an infected individual with an uninfected person. more than % of the cases of hiv are from heterosexual activity. there is no vaccine against hiv/aids, no cure for it, but there is a cocktail of medicines (antiretroviral treatment) that control viral replication and allow an infected person's immune system to strengthen. this keeps the illness at bay and afflicted people in general good health and productive in their communities. in , only . million (less than %) of those with hiv in low and middle economies received the antiretroviral treatment. this is changing as more hiv carriers have access to antiretroviral therapy and there are more donations from economically advantaged countries to support hiv stabilization and reduction programs. the number of new cases of hiv is not exploding because more than % of those infected are following protocols that reduce the transmission of the disease. the prevention of transmission methods include access to male and female condoms, blood screening before transfusions, and needle and syringe exchange programs for sterile injections by drug users. hiv testing and education programs and hiv treatment help prevent transmission because individuals in continuous treatment have a very low probability of passing on the disease. male circumcision reduces the infection in men by about %. there is still much progress to be made because there were . million new cases of hiv in , with . million of that total in sub-saharan africa. the hiv/aids is a global sickness that is slowly coming under control because of generous donations from governments and foundations in developed countries added to what low-and middle-income countries themselves provide to lower the prevalence and incidence of hiv in their populations [ ] . tuberculosis (tb) infected . million people globally in , killing . million persons. it is a bacterial disease that spreads among people when infected individuals cough, sneeze, or spit, releasing bacteria into the air where they can be inhaled by others a meter away. although tb occurs worldwide, developing countries carry the largest burden of cases and deaths ( %). the bulk of new cases are regional in asia ( %) with sub-saharan africa reporting a large share as well with , new cases per million inhabitants. there is no vaccination for tb, but the disease can be treated and cured. the treatment is a half-year course of four antimicrobial drugs that must be taken without fail and thus requires continual supervision by healthcare personnel. more than million people have been treated and cured of tb since and perhaps million lives saved by following the who stop tb strategy protocols including securing adequate, sustained financing, ensuring early reliable detection and diagnosis, and providing approved treatment with a secure effective drug supply. the number of people infected with tb is declining, and from to , the tb death rate dropped more than %. the success in dealing with tb is muted somewhat because a strain of the bacterium that causes tb has evolved to be multidrug resistant (mdr-tb). in , , cases of this variant were reported (of the . million cases worldwide), mainly from india, china, and the russian federation. these are treated with, but do not always respond to, the most effective anti-tb drugs. research into new drugs to deal with this problem is ongoing [ ] . there is the question of whether people visiting or immigrating from these countries should be screened before a host country issues them entry visas. regional illnesses threaten the health of s of millions of people mainly in tropical and subtropical areas and often affecting children. one of these, the guinea worm disease, is trending toward elimination, if not eradication. this is a parasitic disease caused when people swallow water contaminated with infected water fleas (microscopic copepods) carrying worm larva. the worms release, penetrate the intestines, and move through the body migrating under the skin until they emerge causing swelling and blistering. people infected with guinea worm disease cannot contribute to their communities for months. during the mid- s, there were . million cases mainly in african nations. but attention to where the sources were so that they could be avoided and treated, and assistance in generating clean water, were adaptations that brought the number of cases down to less than , in . the number of cases continued to decline and was reduced to in in four african countries: south sudan, chad, ethiopia, and mali. there is no vaccine against guinea worm disease. health officials adapt to counter this sickness in several ways. as noted above, access to clean drinking water is the best way to prevent infection. the prevention or transmission of the worms from infected individuals to healthy persons by proper treatment and hygiene and the use of the larvacide temephos to eliminate the parasite-infected water flea vector and other prevention protocols are important in the control and effort to eliminate/eradicate the disease [ ]. the (jimmy) carter institute, atlanta, georgia, usa, has been a principle force since in the fight to rid the world of guinea worm disease. in tropical and subtropical regions, there are three mosquito-vectored diseases that put millions of people at risk: yellow fever, malaria, and dengue fever. yellow fever is an endemic viral disease in tropical regions of africa and latin america with , cases reported annually that cause , deaths. there is no set treatment for afflicted people, but there is an adaptive preventive measure. a vaccine against yellow fever is available that is safe, affordable, and that gives lifelong immunity to the disease with one dose after - days for % of the people vaccinated. when there is the onset of a yellow fever outbreak where the population lacks vaccination protection, mosquito control is an essential first step in adaptation to prevent or slowdown transmission of the yellow fever virus. spraying insecticides to eliminate breeding sites and kill adult mosquitos is the control used during epidemics to make time for vaccination campaigns in a population and for immunity to take hold. there are limitations to the application of the yellow fever vaccine. first is that babies less than months of age should not be vaccinated or, during an epidemic babies less than - months of age should not receive the vaccine. second, pregnant women should not be vaccinated except when there is an outbreak of the disease. third, people with a strong allergy to egg protein or those with a marked immunodeficiency or with a thymus problem should not receive the vaccine [ ] . malaria is a parasitic disease caused by the bite of an infected mosquito. there is no vaccine against malaria, but one is undergoing a clinical trial in seven african nations with results expected in . a use or no use decision as a control method for malaria will be made in . promising results from an early-stage clinical trial of an unconventional vaccine prepared with live, weakened sporozoites of the malaria parasite were published in . plasmodium falciparum was given to healthy - year-old volunteers intravenously. the volunteers were grouped to receive - doses and subsequently exposed to bite by five mosquitoes carrying the parasite. none of the six that received five doses were infected with malaria. three of the that received four doses became infected, whereas of the that received lower doses became infected. of that received no vaccine, became infected. those that became infected were treated with malarial drugs and cured. clearly, higher dosages give protection against infection by malaria [ ] . more research and extensive clinical trials are necessary to determine how children respond to the vaccine with adjusted dosages and whether the results from earlystage trial are reproducible in larger volunteer populations. if the results of additional clinical trials go well, the hurdle of producing enough vaccine and adapting it to injection has to be faced. forty percent of the deaths from malaria are of african children in the democratic republic of congo and nigeria. in addition to sub-saharan africa, populations in asia (especially india and the greater mekong region) and latin america suffer from the disease. the effort to deal with the disease that is preventable and curable now centers on control and treatment to reduce the number of cases. in , the who reported that there were million cases and , deaths (with an uncertainty range of , - , ). in a report, researchers suggested that the number of deaths was understated and that their computer model gave a figure for almost double, , , deaths ( % uncertainty interval of , - , , ) [ ] . the who stood by its figure stating that much of the data in the cited study were based on verbal testimony of how people had died, not on laboratory diagnosis of samples. either figure represents too many deaths from the disease and have to be reduced. mosquito control is the adaptation that can reduce the transmission of the disease greatly. this includes personal protection by use of proper clothing and/or the application of mosquito repellent, the use of longlasting insecticidal (pyrethroids treated) nets to kill mosquitos and prevent nighttime bites, and indoor residual spraying (remains effective for months). those people infected can be treated with oral artemisinin monotherapy followed by a second drug. failure to complete the treatment as prescribed leaves parasites in a person's blood. no other antimalarial treatment is available so that parasite resistance could become a serious problem. for visitors to a malaria region, antimalarial drugs taken before, during, and after a trip can protect them from the disease. many countries in tropical and subtropical areas have used the above-cited strategies and others to work toward the elimination of malaria. malaria eradication is the goal of the who [ ] . dengue fever is a female mosquito-borne virus that infects people with an influenza-like disease in tropical and subtropical regions worldwide. the disease can kill if it evolves to severe dengue. it is endemic in latin america and asia where most cases now occur. since the s, the sickness has spread to more than countries putting about % of the world's population ( . billion people) at risk. dengue fever is especially endemic to urban/semi-urban environments. humans are the main carrier of the virus. after a mosquito bites an infected person, each subsequent bite by the infected mosquito creates another carrier. a mosquito can bite many people each time it feeds. in the americas alone, there were . billion cases of dengue fever reported in with , being severe dengue. there is no vaccination against dengue fever, but research continues to develop one. the main treatment for afflicted persons is to keep them hydrated. adaptation to deal with slowing or stopping the spread of dengue fever involves three main tracks in addition to spraying insecticide to kill mosquitos. the best control method to prevent the transmission of the virus is to deprive mosquitos of sites with shallow, standing water where they can lay eggs and multiply. control can be improved if communities cover and clean water storage containers regularly, and use proven insecticides on them as necessary. finally, individual protection such as the use of mosquito repellants and insecticide-impregnated bed nets can help reduce the incidence of dengue fever as it has with malaria [ ] . although controls are known, they are not always applied because of economics and other factors that prevent access to protection methods. the result is that the number of cases of dengue fever reported continues to grow globally. as populations increase in urban locations, the incidence of dengue fever can be expected to increase as well unless strict controls are enforced until a safe and cost-effective vaccine is developed. a positive aspect of the dengue fever problem is that recovery from one serotype of the virus gives immunity for life. however, there are four serotypes of the infectious virus so that recovery from one leaves a person susceptible to the others [ ] . chagas is another regional disease. it infects - million people annually, mostly in latin american countries. it is a parasitic illness that evolves after the bite of a blood-feeding triatomine bug, often on the face, where it defecates close by leaving parasite-bearing feces. parasites access the body when the feces are inadvertently smeared into the bite, the eyes, the mouth, or any skin lesion. the parasites circulate in the blood expressing their presence as a purplish swelling of one eyelid or as a skin lesion. there are several other symptoms as well in this acute stage of the illness, but these may be absent or mild. if diagnosed early during this stage, chagas disease is treatable. the parasite is killed with the medicines benznodazole and nifurtimox taken for months. there are limitations as to who can take these medicines (e.g., not by pregnant women or people with kidney or liver problems). the untreated sickness can cause cardiac alterations and digestive problems that show up - years after an untreated infection. chagas disease can be spread by blood transfusion and by organ transplant, making blood screening for the parasite essential before a procedure. it can also pass to a fetus from an infected woman. there is no vaccination against the illness so that control of the vector (triatomine sp.) is necessary. the controls adapted by many municipalities include insecticide spraying inside a home, the use of treated bed nets, and hygiene practices that protect food, its preparation, and its storage before eating it [ ] . the sickness may recur if control practices become lax. chagas disease is spreading as populations emigrate from latin america to northern countries. blood screening of visitors or immigrants from the countries where chagas is endemic may be necessary, and treatment followed by an infected individual before a host country issues an entrance visa. this would prevent the ingress and possible spread of chagas. outbreaks of diseases in town and cities is most often caused by bacterium-contaminated water or food and poor sanitation. sicknesses such as cholera, typhoid, and various other diarrhea types are examples of such diseases. they are all highly infectious if good hygiene practices are not followed. these diseases are endemic in many countries where populations do not have access to safe water and adequate sanitation. there are vaccinations for some of these sicknesses that may require more than one dose, but they may not be completely effective or long lasting and require revaccination at times specified by medical personnel (e.g., after - years). otherwise, infected persons can be treated with medicines such as oral rehydration pills or antibiotics. adaptation for prevention is easier called for than realistically available: washing hands with soap and clean water after visiting the toilet, and as noted above, access to safe water and good sanitation. given the millions of people infected by these bacterial diseases and the hundreds of thousand that die from them annually, generally in economically disadvantages countries, there should be an expanding global priority to eliminate the disease-causing conditions, and preparedness to combat an outbreak when it is reported. there are important factors to consider when adopting plans to halt or meliorate the effects of health threats to people in the near and extended future. one is the climate change-driven spread of tropical and subtropical diseases discussed earlier to newly warmer and moister higher-latitude and higher-altitude zones. another is the growth of populations mainly in tropical and subtropical regions in africa, asia, and latin america. together with this latter factor are the increasing populations and population densities in urban centers especially in the regions just cited. an additional factor to consider is whether there is accessibility to populations by healthcare workers or by people to healthcare clinics or hospitals, well-staffed and well-stocked with necessary pharmaceuticals. certainly, future planning has to include funding to support research to develop vaccines for diseases that do not have vaccination as an option against an illness (e. g., malaria, dengue fever) . in addition, improvement of vaccines that are available but that are not completely effective in terms of protection or the length of time they are effective should be a priority in pharmaceutical and biotechnology laboratories. scientists presented a fine review of the status of vaccine research from the design and development of vaccines to discussion of vaccines and infectious diseases (e.g., hiv, malaria, tuberculosis, pneumococcal disease, and influenza) [ ] . they also discuss vaccines against enteric infections and viral diseases of livestock as well as vaccines against non-infectious diseases (e.g., cancer) and against chronic noninfectious diseases. continued and repeated education classes on how to prevent the transmission of diseases and free supplies of materials that work to this end (e.g., insecticide-treated bed netting, male and female condoms) are essential to reducing the prevalence and incidence of diseases as are safe water and uncontaminated food. as new medicines or combinations of medicines are developed, tested, and found to be effective in controlling diseases, they become part of the protocol for either curing disease or controlling disease to reduce transmission while allowing persons to carry on with their lives. in these times of easy and rapid migration, one wonders whether screening of visitors or immigrants for diseases known to be endemic or active in the countries or regions from which they come should be required so as to prevent a carrier from infecting others and spreading a disease (e.g., chagas disease, cholera, tuberculosis). this was done at airports during the sars scare for people leaving or entering a country (e.g., china) and likely limited the transmission of the sars virus and spread of the disease. preparedness for a disease outbreak, response to an outbreak, and management of resources during and post-outbreak are the keys to adapting to health threats that could affect future generations. this means developing the capability to extend the reach of health services to regions where climate change brings warmer, moister conditions to higher-latitude and higher-altitude ecosystems that are now reached by disease vectors that have expanded into these formerly cooler and drier environments as a result of global warming. adapting to this reality and planning ahead makes it possible to deal with and stem an incipient outbreak of disease before it is transmitted and spread to the general population. this becomes essential when there is a future disease outbreak in large, dense populations in tropical and subtropical urban centers as well as those in regions warmed and humidified by climate change to subtropical and tropical settings. remember that urban populations worldwide, especially in africa, asia, and latin america, are where much of the global population growth will take place during the next few generations. under these conditions, diseases can spread rapidly in many ways. these include from bites of vectors, by respired droplets after an infected person coughs or sneezes, and by touching surfaces bearing viruses, bacteria, or parasites. diseases are also spread by ingestion of contaminated water and/or tainted food, and by other methods of infection transmission. disease transmission can be checked by rapid response teams with appropriate and sufficient supplies to treat (and perhaps places to quarantine) those in the infected population. lastly, it must be noted that there are many other diseases in addition to those cited previously for which prevention, treatment, and cures are research priorities in laboratories worldwide. in addition, there are addiction diseases that can trigger health problems in important segments of society. these include smoking (e.g., emphysema, lung cancer), alcoholism (e.g., cirrhosis of the liver), drugs (e.g., various psychological and physical ills), and overeating (obesity, diabetes, high blood pressure). adaptation to these health threats involves public education forums through various media outlets, counseling, and sponsored groups with their individual group meeting, and programs are assisting many in breaking from an addiction to the benefit of a healthier life. adaptation to meet the health challenges in the past, and in contemporary times has been a slow, progressive adventure with many successes but with much yet to be done. this is the planned path for the future: meet the challenges of societal health threats, resolve many, and keep researching to resolve others. a special ipcc report in examines in a general way adaptation to a changing climate as a risk management approach [ ] . it uses pre-planning to reduce exposure and vulnerability to extreme hazard events by preparing for them beforehand, responding to their impacts on people, structures, and infrastructure, and having in place recovery systems that can act when a danger condition eases. in this way, there will be an ability of populations to cope with future risks brought on by a changing force with which a hazard impacts a community, changes in the frequency of an occurrence, and extension of the spatial reach of its destructive power. much of this has been discussed in the chapters of the book you are reading. an understanding of what is being done now to adapt to the various problems society faces during the second decade of the twenty-first stimulates proposals of how to adapt to them as global conditions change in the future. to this end, the world bank commissioned a study on the effects global warming as it increased from . °c that exists on our planet now to what can be expected if the warming reached °c, a change that many scientists believe we can adapt to, and then reached °c as warming continues [ ] . the study centered on regions with high population growth and great susceptibility to be negatively impacted by climate changes: ( ) sub-saharan africa where food production is at risk: ( ) southeast asia where coastal zones and productivity are at risk; and ( ) south asia where there could be extremes of water scarcity and excess. the effects of higher temperatures from global warming and climate change included what has been discussed in previous chapters of this book: heat, drought, sea level rise, coastal zones, typhoons, flooding, river runoff, water availability, ecosystem shifts, crop yields, fishing, aquaculture, livestock, health and poverty, and tourism. projections such as those published in the world bank study give impetus to governments, international institutions, multinational companies, private foundations, and ngos to think now, to invest now, and to research now for adaptations that can be realized in good time and that will provide global citizenry with a good quality of life where needed. in this book, we have examined existing human populations and the problems they are experiencing in the second decade of the twenty-first century and have also considered growing populations globally and additional problems future generations will experience. we have discussed strategies on how to cope with manyfaceted threats to citizens. these include how to nourish those who need food and water, how to shelter people safely from natural and anthropogenic hazards, how to provide them with healthcare, education, and employment, and how to prepare them for the evolving global warming and the physical and biological dangers that ensue from climate change. given the present global conditions with about % of our earth's population suffering from malnutrition and more than % not having access to safe water, our capability of nourishing a billion and a half more people by is in question. also problematical is our capability to provide for an additional billion people years later, or a total of at least . billion people by the turn of the century, that is, if we reach those population figures or have population crashes such as from pandemics that can kill scores of millions if a disease is not immediately treatable, or an unlikely but possible nuclear conflagration that could do the same. less likely yet is an explosion of a small asteroid or comet in the atmosphere such as happened in a poorly inhabited area of siberia in . here, an exploding mass more than m in size knocked down millions of trees in an area greater than , km (close to mi ) with energy thought to be , times greater than the hiroshima atomic bomb. clearly, such an event could kill the population of a megacity if it were to occur. another question is whether national governments are economically strong enough and have the will to set priorities that adopt strategies to protect citizens from natural (e.g., earthquakes) and anthropogenic (e.g., pollution) hazards as well as from extreme weather conditions that are supported by global warming (pollution of the atmosphere) but are naturally occurring. countries can also improve social and economic conditions by investing in health care and education for their citizens in order to form a sound and knowledgeable cadre that would be attractive to investors interested in locating a development project that would provide employment. again, this is in question given limited national economic capabilities and the increasing numbers of people to be accommodated, especially in several developing and less developed countries in africa, asia, latin america, and the middle east. at this point, we must ask, "what is the carrying capacity of the earth?" have we reached it at billion given the billions who are today under served in developing and less developed countries? some scientists will answer yes, whereas others believe that advances in agriculture and technology can allow population expansion although to what point is not defined. can countries that are poisoning their environments do a turn around to save their citizens from grief? can they exert controls on operations that create unhealthy conditions that sicken people, lessen agricultural production, and otherwise disrupt local, regional, and global climate change: evidence and causes ( p) intergovernmental panel on climate change ( ) climate change (as four part report). part . the physical science basis mitigation of climate change past and future sea level change from the surface mass balance of glaciers a reconciled estimate of glacier contributions to sea level rise ocean thermal expansion and its contribution to sea level rise tuberculosis. fact sheet no. p . world health organization ( ) dracuncukiasis (guinea-worm disease) protection against malaria by intravenous immunization with a non-replicating sporozoite vaccine global mortality between and : a systematic analysis world health organization ( ) malaria. fact sheet no. p . world health organization ( ) dengue and severe dengue chagas disease (american trypanosomiasis). fact sheet no vaccines and global health ) ipcc special report summary for policy makers. managing the risks of extreme events and disasters to advance climate change adaptation turn down the heat: climate extremes regional impacts and the case for resilience. a report for the world bank prepared by potsdam institute for climate impact research and climate analytics key: cord- - g f authors: hurst, christon j.; benton, william h.; stetler, ronald e. title: detecting viruses in water date: - - journal: j am water works assoc doi: . /j. - . .tb .x sha: doc_id: cord_uid: g f various and divergent approaches that have been used to concentrate and assay viruses from tap water and environmental freshwaters are summarized and briefly explained. the basic principles behind the different methodologies and descriptions of the most recent developments are emphasized. comparisons help demonstrate the relative sensitivities of different concentration and assay techniques. viruses that replicate within and are subsequently shed from the gastrointestinal tract of animals are referred to as enteric viruses. these viruses are principally transmitted by the fecal-oral route, meaning that they cause illness after being ingested by an uninfected individual. the known human enteric viruses are listed in table . not all of these viruses, however, have been studied with regard to their presence in drinking water. although some of the viruses listed in table can also replicate in animals other than humans, their presence in raw waters is presumably related to human fecal pollution. the human enteric viruses are presumed to present the greatest concern with regard to waterborne viral disease. although raw waters may also contain related enteric viruses originating from wild and domesticated animals, most of these viruses are considered to have relatively limited pathogenicity for humans. infectious human enteric viruses can be found in surface freshwaters and groundwatersr- where they can originate from septic effluents and wastewater. -r viral contamination of surface water could also result from human recreational activities either in or around bodies of water." enteric viruses can survive improperly operated conventional water treatment trains and may then be found in potable water. - j.i".rr this cause for public health concern has resulted in the development of many methods for recovering both human enteric viruses and bacteriophages from water. bacteriophages (viruses that infect bacteria) have been studied in the hope that they may prove useful as indicators of the removal and september destruction of human enteric viruses during water treatment. the remainder of this article describes and categorizes the various viruses were concentrated from river water by directed adsorption onto a pleated membrane cartridge filter. techniques that have been used for detecting viruses in environmental freshwaters and in treated drinking waters. these techniques fall into two main categories. the first is virus concentration techniques, in which viruses recovered from large volumes of water are concentrated into smaller, more manageable volumes; the second is the viral assay procedures used to examine the concentrated samples. classification of techniques. five major categories of techniques are used for concentrating viruses contained in water samples (table ) . before examining thesedifferent concentration techniques, it is helpful to briefly mention the structure of what are termed classical viruses, a category that encompasses all of theviruses listed in table . classical viruses consist of a nucleic acid core, or genome, which provides their genetic information and is surrounded by a shell of proteins termed a capsid. in some types of viruses this capsid is, in turn, surrounded by a lipid bilayer membrane, or envelope. of the viruses listed in table , only coronaviruses possess such envelopes. nonenveloped viruses essentially present themselves to the environment as large protein structures. for this reason, studies like mizutani's,"" which addresses the adsorption of pro teins to glass surfaces, help explain the mechanics of virus concentration techniques. the only study found that addressed concentrating enveloped viruses from water samples used the herpes simplex virus and the measles virus,"" neither of which is considered enteric or otherwise of concern. with regard to the waterborne transmission of human illness. passiveadsorption. the first type of concentration technique mentioned in table is passive adsorption. this technique, originated by maccallum et als involves the use of pads consisting of either gauze alone or a combination of gauze and cotton batting for the purpose of absorbing a portion of the water sample into which the pads are placed. a small portion of the viruses suspended in the water sample are taken into these pads during the passive absorption process. any actual adsorption of viruses onto the surface of the gauze or cotton fibers is incidental. the pads may be used in either a stationary or flow-through configuration.r"."i viruses are recovered from the pads by expression of the absorbed fluid. additional fluids may be used in kneading the pads to help achieve recovery of any adsorbed viruses. fluids used for desorbing viruses from surfaces are termed eluants, and the process is termed elution. following exposure to an adsorbing surface, it is hoped that the fluid, or eluate, will contain any viruses that had been on the surface. the relatively low efficiency of gauze pads for concentrating viruses from water was demonstrated, however, by a study in which such pads were used for prefiltering a water sample to remove large particulate solids, before concentrating the viruses from the same water sample by passage through a layer of virus-adsorbing granular material.:{' directed adsorption involves the use of either filters or granular solids as surfaces onto which viruses are deliberately adsorbed and then ~~~~~~~~~~l~.~~. .~ .~ .~~ . , ~,~~-~~ ad. sorption is generally facilitated by modifying the water sample, the adsorbent material, or both. the adsorbent material can be modified either duringor after its manufacture. subsequent recovery of the adsorbed viruses can occur either by dissolving the filter material, if it is composed of alginate,"" or by exposing the adsorbent to a volume of eluant that is smaller than the original water sample and facilitates a reversal of the virus adsorption process. most techniques now used for concentrating viruses from large volumes of either tap water or environmental freshwaters belong to the category of directed adsorption. these techniques are discussed in greater detail later in this article. the general mechanisms involved in concentration methods of this type have been presented in publications by gartner, "' england, "" cookson, fifi mix, and sweet et viruses are adsorbed onto the surface of filter materials or granular solids, facilitated in many instances by pretreatment of the water. processes evaluated as pretreatments include removing soluble organics from the water using charged resins, adjustment of the water sample ph, and addition of salts of metal cations-particularly those that are multivalent such as aluminum and magnesium. efforts have also been made to pretreat the adsorbent material itself to enhance the efficiency of viral adsorption. examples of the latter approach include binding metal precipitates or charged polymers to the matrix of microporous filters. subsequent recovery of the bound viruses from the filter materials or granular solids used as adsorbents is achieved by exposing the adsorbents to an eluant. viruses are retained in the original water sample during a reduction of its volume achieved by pore size exclusion. this generally is accomplished by either recirculating the water under pressure through hollow fiber filters or over the surface of flat sheet filters in either recirculating or nonrecirculating systems. in some cases an eluant is subsequently passed through the same concentration unit to facilitate virus recovery. viruses are concentrated either by association with a generated precipitate or by selective partitioning of the water sample during processes that yield a polymeric phase separation, or they are retajned in the original water sample during a reduction in its volume through hydroextraction. ultrafiltration. ultrafiltration involves selectively removing small molecules from a water sample, including the water molecules themselves, and leaving behind large solutes or suspended particles such as the viruses. this technique uses filters that may be configured either as hollow fibers through which the water sample is passed or sheet filters against which the water is kept in motion by means of a recirculating pump or stirring apparatus. ". x.b the general mechanism used in this type of concentration technique has been discussed by mix.fi the filters may be pretreated before they are used on a water sample to suppress virus adsorption.@ alternatively, a second fluid, that either may or may not serve as an eluant, can be used to flush viruses from the surface of the filters after they have been used to concentrate a water sample. ultrafiltration is seldom used for directly concentrating viruses from water samples *secondary concentration is also frequently reterred to as second-step concentration or reconcentration. +may be supplemented with materials such as specific amino acids: other protein-derived products have ken evaluated as substitutes for beef extract (references for the latter include and ) $these types of rluants may include compounds such as alanine. edta. glycine. imidazole. lysine. and polysorbate . ." x. . . . . . several techniques fit into the category of direct physicochemical treatment for concentrating viruses from water samples. one general approach is to concentrate viruses through physical association with a flocculant precipitate that is induced by chemical supplementation of the water sample. the types of precipitates produced include aluminum hydroxide, aluminum phosphate, and calcium phosphate. . alternatively, proteinaceous materials may be added to the water sample and then precipitated by means of processes such as adding protamine sulfate or lowering the ph.~o viruses in the water are concentrated through association with these proteinaceous precipitates. polyethylene glycol has been used in two different ways for concentrating viruses from water samples. the first of these, two-phase separation, consists of adding sodium chloride, dextran sulfate, and polyethylene glycol to the water. ionic strength contributed by the sodium chloride causes polymeric incompatability, resulting in the formation of two distinct aqueous phases, one containing most of the dextran sulfate and the other containing most of the polyethylene glycol. the densities of these two phases differ sufficiently that they can then separate gravimetrically. viruses in the water sample undergo a partitioning during the generation and separation of these two phases and are largely concentrated into only one of them. ', . .@the second technique involves placing the water sample into a dialysis bag and packing the whole unit in solid polyethylene glycol. the polymer then concentrates the sample by drawing water out of the dialysis bag, a process called hydroextraction. . likeultrafiltration, thedifferent physicochemical concentration techniques are used seldom, if at all, for directly concentrating viruses from water samples much larger than l. the reason is that the time expended processing water samples by these techniques increases dramatically with sample size. both techniques are often used, however, in a secondary mode to reduce the volume of eluates generated during the processing of water samples by directed adsorption. this process is variously referred to as secondary concentration, second-step concentration, or reconcentration. affinity chromatography. affinity chromatography consists of passing an aqueous solution through a column of polysaccharide gel material bearing highly specific, purified antibodies that can selectively bind viruses or some other target antigen from the fluid. this binding process is reversible, and the bound antigens can subsequently be released by passing a smaller volume of a second aqueous solution through the column. the second solution differs from the first in terms of ionic strength and is formulated to reduce the favorability of antigen-antibody binding reactions. although this technique certainly merits mentioning, its use is limited with regard to recovering viruses from tap water or environmental freshwaters because it is costly and requires a large amount of preparation time and operational skill. in the literature, only one mention could be found of its use for recovering viruses from water samples.'s directed adsorption combined with secondaryconcentration. directed adsorption has generally replaced the other types of concentration techniques listed in table for use in recovering viruses from large volumes of water. the different solid adsorbents used successfully for this purpose are listed in table . these solids are divided into two groups, filter materials and granular solids. the types of filters that have been tested are yarn fiber that is wound around a hollow core to form a depth filtration cartridge,"'j ri sheet filter materials used as flat layers,l . "." . . . . ~- cartridges prepared as hollow tubesof filter material," , and cartridges composed of pleated sheets of filter material. jl viruses adsorb onto the filter matrix during the passage of viral-containing water samples through the filters. recovery of the adsorbed viruses is normally achieved by subsequently passing an eluant through the filters or by dissolving the filter if it is made of alginate. y granular solids are generally used in one of three modes: ( ) batch utilization by which thegranules are mixed into the water sample and then recovered using either filtration"" or magnetic attractionx" the latter requiring that the granules be magnetic; ( ) layers of granules either supported on or sandwiched between sheets of nonadsorbing flat filter material through which the water sample is passed;rrj or columns'",fi or fluidized bedsz of granules exposed to the water sample using a flow-through configuration. subsequent desorption of viruses occurs during exposure of the granules to an eluant using one of these same three modes. of the many different types and configurations of virus adsorbents, those now preferred for use in recovering viruses from large volumes of water are wound cartridge filters*' and pleated cartridge filter@ based on either glass fiber or nylon, including types that are positively charged, and columns of glass powder. also of interest is the use of sheet filter material that has been modified in situ either by a precipitation of metal hydroxides within the filter ma-trix " or by coating the filter with cationic researchandtechnology cultures of mammalian cells were prepared using a laminar-flow filtered air hood. polymersj the largest volumes of sample water that have been concentrated by means of cartridge filters and glass powder appear to be, respectively, , l"i and l.' in comparison, the technique of using a granular adsorbent sandwiched between layers of sheet filter material has been used for concentrating volumes of water as large as , l."x in the choice between filters and glass powder columns, the filters seem to offer greater advantage because they are easier to transport and operate in the field. this capability eliminates the need for transporting awkward high-volume water samples back to a laboratory. following the adsorption of viruses onto the filters, the filters can be transported, including postal shipment, back to the laboratory with little concern about harming either the filter or the adsorbed viruses.qo- elution of the filters and subsequent processing of the eluates can then be performed under the more manageable conditions that exist in the laboratory. theory of directed adsorption, elution, and reconcentration. the history of virus adsorption to filters dates back to a study published in by elford"" who determined that during passage of virus suspensions through collodion (nitrocellulose) filters, viruses were removed from the solution by adsorption onto the surface of the filter matrix as well as by simple sieving action. the adsorption of viruses onto filters, and presumably other solid adsorbents as well, is governed both by electrostatic interactions, which predominate at lower ph levels ( ) and by hydrophobic interactions, which predominate at higher ph levels (? )." -'j" the chemical composition of the filter matrix and the fluid in which theviruses are suspended influence the extent of attraction or repulsion between the viruses and the filter. other important factors that relate to virus adsorption onto filters include the chemical composition of the filter matrix, rwhe rateof water flow through the filter material," and the ratio of filter pore diameter to virus particle diameter."" these factors are important because if the distance between a virus particle and the filter matrix is toogreat (i.e., the pore size is too large) or the flow rate of the sample through the filter is too great, then the extent of attraction between the virus particle and filter matrix may be insufficient for adsorption to occur. cliver"" seems to deserve credit for first presenting the application of this adsorption phenomenon toconcentrating viruses from water samples. cliver also discovered that the adsorption of viruses to the membrane matrix was strongly inhibited by the presence of added proteins in the input virus .suspension. ".""."*he suggested that this inhibition resulted from a competition of the proteins and viruses for adsorption sites extant on the surface of the filter matrix."x this is indeed a reasonable conclusion in light of the structure of viruses, particularly those that are nonenveloped. other types of soluble organics, such as humic and fulvic acids, can also interfere with virus adsorption.zojzj . means of facilitating the adsorption of viruses onto solid matrixes include first removing dissolved organic materials from the water samples by passage through a resin colurnn" . ."" and adding salts to the virus suspension.loothese salts may includechlorides of sodium,loo magnesiurn,'*. ". .fin.s or, more effectively, aluminum. i~.~ .hj.r:~. virus adsorption can also be facilitated by adjusting the ph of a water sample, a process that can be performed either in a batching operation or in placing an inline injector ahead of the filter. jj". " a ph of approximately . seems preferable for use with negatively charged filters, j~ fl~x:~ versus the near neutrality (ph = ) used for the more positively charged filters.r",zx cliveralsodiscovered that theadsorption process was reversible and was able to recover viruses from filters by eluting them with a proteinaceous solution in the form of diluted blood serum.'" evidence collected from several studies suggests that eluantscan bedivided into at least two categories based on their mode of action.'",""~l~' the first category is proteinaceous materials, which simply compete with the proteins of the virus particles for binding sites on the adsorbent."" beef extract is now the most predominantly used eluant material in this category, although other proteinaceous products may prove to be suitable substitutes.~~~i"~l(':~ the second category consists of compounds that alter the favorability of adsorption."","".'"' these eluants include solutions that contain various active substances. among them are chaotropic agents like glycine or trichloroacetic acid,"'detergents,*~l~ and edta serving as a chelating agent for metal cations. " xvolumes of dechlorinated tap water simultaneously containing all three of the indicated viruses were adjusted to the desired ph ( . for type a and . for types band c) and then passed through a sterile .mm-diameter filter of the indicated type. adsorbed viruses were recovered by passing ml of the indicated eluant through the filter. calculation of viral recovery was based on the amount f virus present in the eluant. expressed as a percent of that contained in the water sample before filtration. values given represent the average of three independently conducted trials. saverage value for all three virus types precipitation.lo this mechanism differs from true coprecipitation, in which neither of the involved materials can precipitate on its own. a ph of approximately . appears optimal for recovering viruses from beef extract eluates by organic flocculation.~~~~l ~ with the exception of organic flocculation, the various techniques referenced in table for secondary concentration of eluates have been discussed earlier in this article. organic flocculation, introduced by katzenelson and his coworkers,"'" is performed by lowering the ph of the eluate, during which the proteinaceous material supplied by the beef extract spontaneously precipitates. viruses contained in the beef extract become associated with this precipitate, which can subsequently be collected by centrifugation and dissolved in a small amount of higher-ph buffer. the process by which virusesareconcentrated during this technique may result from a binding of the naturally precipitating material onto the surface of the viruses at higher ph levels where the material is in solution. this bound material would then cause the viruses to aggregate as the ph of the solution is lowered to induce comparison of techniques for concentrating viruses using membrane filters. the efficiencies of virus adsorption by different filter materials are compared in table . the efficiencies of adsorption in combination with subsequent elution are compared in table . the viruses f and +x are bacteriophages that infect escherichia coli. the procedures for their propagation and assay have been described by ward and mahler.l'o the virus designated pl is human poliovirus type , a human enteric virus. the procedure for propagation and assay of this virus has been described by benton and hurst."' conducted using water samples that simultaneously contained all three viruses. the viruses were then differentiated on the basis of selective assay procedures.l' these results demonstrate that the adsorption process is often far more efficient than the subsequent desorption. the use of these two processes in conjunction is often referred to as adsorption-elution. this process is sometimes called membrane chromatography when filters are used as the adsorbent.-'" although the incorporation of edta into filter eluants can increase their efficiency, as demonstrated by the results shown in table , a note of caution is important regarding its use for rotaviruses or reoviruses: edta can destroy the infectivity of these two virus groups by removing theouter shell layer of their protein capsids. virus assay techniques of the different filter types mentioned in tables and , type at is composed of glass fiber plus an epoxy resin binder, type b$ is composed of charged glass fiber, and type cg is composed of charged nylon. the type a material is considered electronegative, whereas the other two types of filter material are considered electropositive. the evaluations represented in these two tables were stages in the viral assay process. the various procedures that have been used for detecting viruses in concentrates prepared from environmental samples areoutlined in table exception of direct electron microscopy, each of thesecan be separated into three parts or stages. the first stage consists of the target material that is recognized by a given technique. in the case of electron microscopy, the target material is virus particles, termed virions, that appear intact. some assay techniques detect only infectious virions, a category that may differ from those virions recognizable by electron microscopy as visually intact but somehow damaged or deficient so that they lack infectiousness. still other techniques detect only either viral proteins or nucleic acids. the second stage of the various detection methods consists of target recognition; this is considered a nonvisual step. target recognition, in turn, facilitates the third stage of the detection methods, visualization of the target material. target visualization may be direct, as in observing virions by electron microscopy, or indirect, as in observing changes induced within host cells as a result of successful viral infection. indirect visualization may consist of detectingalterations in cellular morphology, including outright death, or the production of progeny viral proteins or nucleic acids. in general the infectivity of animal viruses contained in environmental samples is examined using cultures of animal cells that are prepared in the laboratory, rather than inoculating live animals. figure compares the relative sensitivities and required completion times for the assay techniques used in titrating viruses recovered from environmental samples. electron microscopy. the two different approaches for electron microscopy, direct and immune, are presented in figure in terms of the quantitation of physical viral particles. in contrast, all other viral assay techniques mentioned are represented in terms of their relative sensitivity for detecting viral infectious units. this distinction is important because for many different types of viruses, not all particles in a given preparation may be capable of producing an infection in host cells. for some types of bacteriophages, the ratio of viral particles to infectious units can be as little as between and . . for the various types of animal viruses, the ratioof viral particles to infectious units can range from . to more than .llz both categories of electron microscopy involve the examination of virus preparations after they have been negatively stained using solutions of electronopaque metals. in the case of immune electron microscopy, the viruses are incubated in preparations of diluted antibodies before they are negatively stained. the virions in the sample normally aggregate during this incubation if a serological identity exists between them and the antibodies. neither direct examination nor immune electron microscopy is practical for use with environmental samples because both require that extremely large numbers of viral particles be processed into volumes of, typically, less than ml before they are stained for observation. furthermore, bacterial viruses and other natural materials present in environmental concentrates could interfere with visualizing animal viruses by electron microscopy. cytopathogenicity and plaque assays. cytopathogenicity assays are based on the appearance of visibly observable, characteristically recognizable changes in the morphology of living host cells that result from the process of viral infection. such changes, termed "cytopathogenic effects," can be assessed by microscopic examination of the infected cells. plaque assays detect the production of focal areas of death within an infected culture of cells. detecting these areas of cell death is often facilitated by exposing the culture of cells to a vital stain such as neutral red. at modest concentrations neutral red does not interfere with cellular viability, yet it causes live cells to appear pink and dead cells to appear colorless. plaques, which represent the focal areas of cell death, are normally detected by visual examination of the cultures without the aid of a microscope. explanations of the procedures used in assaying virus preparations by either cytopathogenicity or plaque production, along with specific information on their use with environmental samples and comparisons of their relative effectiveness for this purpose, can be found in references and - . recent advances for these types of assays include the deliberate use of mixed cell types within single cultures that are to be inoculated with virus samples. another example is the treatment of cell cultures with nucleotideanalogs such as iododeoxyuridine before the introduction of sample material, both of which can increase assay sensitivity."' immunological assays. immunoassays are based on an identity binding between antibodies, used as "probes," and specific "target" antigens of interest to the observer. this identity binding is a natural recognition process and serves as an aid in determining the presence of the target antigens. in thecaseof viruses, such target antigens may be serologically distinctive portions of either proteins, glycoproteins, or glycolipids. depending on the laboratory protocol followed, these antigens may be suspended in aqueous solutions, trapped on filters, or associated with infected cells. the antibodies used as probes may first be "tagged" or "labeled" to facilitate subsequently identifying the presence of target antigens with which theantibodies bind. methods used for the taggingof antibodies include chemically incorporating radioactive isotopes into the molecular structure of the antibodies, which can then be detected by the isotope emissions. alternatively, the antibodies may be covalently linked tocompounds such as fluorescent dyes and functional enzymes. the dyes fluoresce when illuminated at the proper wavelength, normally in the near ultraviolet portion of the spectrum. when dealing with environmental samples, antibodies conjugated to fluorescent dyes are often used to detect the production of progeny viral proteins within individual cells of an infected host culture. this approach is termed cell immunofluorescence. during performance of the immunofluorescence assay, virally infected cultures of host cells are first exposed to a solution containing fluorescently tagged antibodies, termed probes, whose immunological reactivities are known. during this exposure the probe antibodies bind to the cells wherever the target viral antigen is present. the same cells are later examined using a specially adapted microscope that provides illumination at the proper wavelength for inducing the dye to fluoresce. the resulting fluo- immunological detection assays that use antibodies tagged with enzymes are broadly termed enzyme immunoassays. these enzyme-tagged antibodies can also be said to serve as probes. horseradish peroxidase is one example of the enzymes used as tags for this purpose. enzyme-tagged antibodies can be used in assays for the detection of viral antigens in solution, blotted onto filters, or associated with infected cells. evidence of the desired antigen-antibody binding reaction is subsequently provided by measuring a chemical reaction facilitated by the enzyme. very often the observed reaction involves conversion of a colorless substrate into a colored product. the use of enzyme-tagged antibodies for detecting antigens in solution is termed elisa, an acronym for enzymelinked immunosorbent assay. references and discuss the use of elisa to detect viral antigens present in environmental samples. one notable variation of the elisa technique for detecting viruses in environmental samples relies on first trapping the viral antigens onto filter paper, then performing the elisa test in situ on that filter.rz this approach can be termed a blot enzyme immunoassay. the use of enzyme immunoassays for detecting viral antigens directly associated with the infected cells of a host culture is similar to cell immunofluorescence and is, likewise, studied microscopically. in this article, this assay technique is referred to as a cell enzyme immunoassay. payment and trudel i"i. have provided examples of the use of cell enzyme immunoassays for detecting viruses in environmental samples and compared its sensitivity relative to cytopathogenicity assays. kurstak et aliz have prepared an excellent review on the use of enzyme immunoassays and other related procedures in virology. nucleic acid hybridization. nucleic acid hybridization assays are based on the use of homologous nucleic acid materials as probes to bind with, or hybridize to, specific target nucleic acids and thereby facilitate the detection of those target nucleic acids. hybridization, in this sense, is a physical pairing by means of hydrogen bonding between corresponding nucleotide sequences on those nucleic acid strands that respectively represent the probe and target. depending on the assay protocol, the target viral nucleic acid sequences may variously be freely suspended in an aqueous solution, bound on filter materials, or associated with virally infected cells. evidence of the desired hybridization (binding) reaction between the probe and targets may be provided by one of september rslatlve sa"*itl"lty in'ecllo"s unar assays, normally expressed in terms of infectious units per millilitre as a percent endpoint, were divided by a factor of to obtain endpoints expressed in terms of infectious units per millilitre. +adapted from reference several methods. the first of these involves the detection of emissions from radioactive isotopes incorporated within the probe nucleic acid material. , ~ a second, two-step method involves the use of probe nucleic acids that contain nucleotides tagged, or joined by covalent linkage, to markers such as biotin. in this case, the biotin moieties attached to the probe nucleic acids can, in turn, serve as binding sites for molecules of avidin, which are themselves covalently linked to an enzyme such as horseradish peroxidase. avidin is a natural compound that binds to biotin with high affinity. in this type of assay, the bound nucleic acid probe material is subsequently detected, as is done for enzyme immunoassays, by measuring the enzyme-mediated conversion of added substrate into products. the use of nucleic acid probes to directly detect viral genomic nucleic acid produced within infected cells is termed in situ nucleic acid hybridization. this process is conceptually similar to using antibodies as probes for detecting viral antigens associated with infected cells. the major difference is whether the target material consists of viral antigens or viral genomic nucleic acids. the use of nucleic acid probes for detecting viral nucleic acid that has been trapped on filter material is termed blot nucleic acid hybridization. a more detailed discussion on the use of nucleic acid probe hybridization assays for virus detection has been published by norval and bingham.i " comparison of viral assay methods. one important point to consider regarding the use of different assay techniques for identifying viruses in environmental samples, and particularly for viral particles recovered from samples subjected to drinking water treatment, is whether the assay result represents infectivity. none of the two types of electron microscopy procedures, the solution elisa, nor the blot styles of immunoassay and nucleic acid hybridization assay can, by themselves, reveal this most important piece of information. their results are instead qualitative or semiquantitative. the other methods compared in figure are directly usable for quantitating viral infectivity because they are based on detecting changes within host cells that are specifically caused by viral replication. the various changes measured are the disruption of normal cytomorphology (cytopathogenicity), overt cell death (plaque assay), and the production of either viral-specific antigens (immunofluorescence and enzyme immunoassay) or nucleic acid (in situ hybridization). the electron microscopy, solution elisa, or blotting techniques could be used for estimating the infectivity of viruses present in samples if a part of the sample is examined directly as collected and another part of the same sample is then inoculated into a host cell system supporting the production of viral products that could later be measured by the same type of assay. viruses are most suitably concentrated by directed adsorption onto the surface of either a filter material or granular solid. recovery from the surface is achieved during an elution treatment that reverses the adsorption process. as a result, the viruses are contained in a smaller volume of eluant fluid, which may or may not be further processed by a secondary concentration technique. the choice of a technique for quantitating viruses recovered by the concentration process often involves balancing the relative merits of detection sensitivity versus the time required for assay completion. this article presents information on the actual target materials detected by the various assay techniques and information on evaluating whether a particular type of assay procedure provides a direct measure of viral infectivity. variations of the nucleic acid hybridization assay may represent the optimum balance for viral assay techniques by simultaneously combining sensitivity, determination of viral infectivity, and rapidity with which the assay can be completed. cytopathogenicity, cell immunofluorescence, and in situ hybridization for the detection of indigenous adenoviruses present in concentrates produced from environmental samples are compared in table . these data indicate that the overall numbers of infectious viral units detected by either cytopathogenicity or immunofluorescence were approximately equivalent. the number of viruses detected by in situ hybridization was approximately percent greater than that revealed by the other two techniques. analysis of these results by means of the paired two-tailed t-test revealed that the differences in mean values between the titers obtained by cytopathogenicity and immunofluorescence were not significantly different (p = . ). the mean value of the titers obtained by in situ hybridization were, however, significantly different from those obtained by either cytopathogenicity (p = . ) or immunofluorescence (p = . ). this information suggests that in situ hybridization is superior toeither cytopathogenicity or immunofluorescence because of both its greater sensitivity (table ) and the speed with which the assay can be completed (figure ). . . this document has been reviewed and approved in accordance with us environmental protection agency policy. approval does not signify that the contents reflect the views or policies of the agency, and mention of trade names or products does not constitute endorsement or recommendation for use. . references . . a recent improvement in virus detection methodology, suitable for use in these types of viral assays, relies on the incorporation of selectively acting antiviral compounds such as guanidine into the medium used for maintaining cell culture viability. ' gmnidine suppresses enterovirus replication while not deleteriously affecting the replication of adenoviruses and thus imparts a measure of specificity into the assays. microbial., : ( this article, which reviews the subject of detecting viruses in water, encompasses two topics. the first topic consists of methods used for concentrating viruses from large volumes of water into smaller, more manageable volumes. the second topic consists of assay methods used for examining viruses contained in the concentrated samples. many of the references cited in this article contain flow diagrams that outline the techniques discussed. bull. acad. natl. med., : ( ) . occurrence of viruses in treated drinking water in the united states presence of enteric viruses in the drinking water of cochabamba ibolivia) comparison of adsorption-elution methods for concentration and detection of viruses in water evaluation of gauze pad method to recover viruses from water virus in water, i. a preliminary study on a flow-through gauze sampler for recovering virus from waters evaluation of methods for concentrating hepatitis a virus from drinking water concentration of poliovirus from tap water onto membrane filters with aluminum chloride at ambient ph levels concentration of poliovirus from tap water using positively charged microporous filters use of bituminous coal as an alternative technique for field concentration of waterboine viruses poliovirus concentration from tao water with electropositive adsorbent filters influence of water quality on enteric virus concentration by microporous filter methods simultaneous concentration of four enteroviruses from tap, waste. and natural waters evaluating adsorbent filter performance for enteri; virus concentrations in tap water concentration of viruses from water by using cellulose filters modified bv in situ precipitation of ferric and aluminum hydroxides concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater isolation of enteroviruses from water a new and simple method for recuperation of enterobiruses from wate detection d'enter- virus par concentration sur poudre de verre en lit fluidise a partir round-robin investigation of methods for the recovery of p&ovirus from drinking water conception d'une membrane filtrante a base d'alginate pour la concentration des virus hydriques virus isolations from sewage and from a stream receiving effluents of sewage treatment plants concentration of viruses from large volumes of tap water using-pleated membrane filters effects of humic materials on virus recovery from water effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration concentration of enteroviruses from large volumes of water simultaneous concentration of salmonella and enterovirus from surface water bv using micro-fiber glass filters rapid concentration of bacteriophages frbm aquatic habitats comparison of talc-celite and polyelectrolyte in virus recovery from sewage: development of technique and experiments with poliovirus (type , sabin)-contaminated multilitre samples use of talc-celite layers in the concentration of enteroviruses from large volumes of potable waters effects of bentonite clay solids on poliovirus concentration from water by microporous filter concentration of coliphage from water and sewage with charge-modified filter aid recent advances in the detection of human viruses in drinking water novel approach for modifying microporous filters for virus concentration from water concentration of enteroviruses on membrane filters enterovirus detection by membrane chromatography. transmission of viruses by the water route concentration of poliovirus in water by molecular filtration ein zweistufenverfahren zur virusanreicherungaus losungen mit geringem virustiter protamine precipitation of two reovirus particle types from polluted waters concentration of enteric viruses in water by hydro-extraction and two-phase separation. transmission of viruses by the water route detection of enteric viruses by concentration with polyethylene glvcol. transmission of viruses by the wilter route concentration of viruses on aluminum phosphate and aluminum hydroxide precipitates transmission i,f viruses by the water route the phase-separation method for the concentration and detection of viruses in water studies on the isolation and identification of hepatitis viruses in water protein adsorption on glass surfaces using porous glass the use of gauze swabs for the detection of poliomyelitis virus in sewers. poliomyelitis concentration of viruses from water by membrane chromatography membrane filters in virology a simple method for concentrating and detecting viruses in water comparative study of four microporous filters for concentrating viruses from drinking water developments in methods for the detection of viruses in large volumes of water concentration of bacteriophages from natural water virus detection in water by ultrafiltration retention and recovery of polioviruses on a soluble ultrafilter. transmission of viruses by the water route recovery of viruses from waste and other waters by chemical methods the chemistry of virus concentration by chemical methods mix, t.w. the physical chemistry of membrane-virus interaction recovery and removal of viruses from water-utilizing membrane techniques rapid concentration of bacteriophages from large volumes of freshwater: evaluation of positively charged, microporous filters detection of human rotavirus in sewage through two concentration procedures membrane filter evaluations using poliovirus concentration of virus from tap water at ambient salt and ph levels using positively charged filter media concentration of viruses in beef extract by flocculation with ammonium sulfate rotavirus concentration from raw water using positively charged filters a comparison of current methods of poliovirus concentration from tap water concentration of simian rotavirus sa- from tap water by membrane filtration and organic flocculation evaluation of procedures for recovery of viruses from water-i. concentration systems efficiency of several micro-fiber glass filto= fnr recovery of poliovirus from tap water concentration of heptatitis a virus recovery of small quantities of viruses from clean waters on cellulose nitrate membrane filters wound fiberglass depth filters as a less expensive approach for the concentration of viruses from water a portable device for concentrating bacteriophages from large volumes ot freshwater modifications of the tentative standard method for improved virus recovery efficiency modified membrane-filter procedure for concentration of enteroviruses from tap water effectiveness of the organic flocculation method in concentrating echovirus and coxsackirvirus a from water preformed magnesium hydroxide precipitate for second-step concentration of enteroviruses from drinking and surface waters detection of viruses in drinking water bv concentration on magnetic iron oxide concentration of seeded simian rotavirus sa- from potable waters by using talc-celite layers and hydroextraction adsorption of virusrs on magnetic particles. australian water resources council survie de virus enteriques adsorbes sur microfibre de verreau cours d'un transport postal. can. lour. microbial processing and transport of environmental virus samples a new series of graded collodion membranes suitable for general bacteriological use, especially in filterable virus studies influence of salts on electrostatic interactions between poliovirus and membrane filters effects of chaotropic and antichaotropic agents on elution of poliovirus adsorbed membrane filters virus interactions with membrane filters virus retention by membrane filters factors in the membrane filtration of enteroviruses humic acid interference with virus -recovery by electropositive microporous filters efficient filtration and sizingof viruses with membrane filters reduction of interferingcytotoxicity associated with wastewater sludge concentrates assayed for indigen&s enteric viruses induction of cytopathogenicity in mammalian cell lines challenged with culturable enteric viruses and its enhancement by -iododeoxyuridine evaluation of procedures for recovery of viruses from water-ii. detection systems influence of inoculum size, incubation temperature, and cell culture densityon virus detection in environmental samples improved method for the use of proportioning injectors to condition large volumes of water for virological analysis comparison of positively charged membrane filters and their [jse in concentrating bacteriophages in water comparison of commercial beef extracts and similar materials for recovering viruses from environmental samples second-step reconcentration of environmental samples by ammonium sulfate flocculation of beef extract organic flocculation: an efficient second-step concentration method for the detection of recovery of viruses from water by a modified flocculation procedure for second-step concentration immuno-peroxidasemethod with human immune serum globulin for broad-spectrum detection of cultivable human enteric viruses: application to enumeration of cultivable viruses in environmental samples hepatitis a virus concentration on cellulose membranes imoroved method for recovery of enteric viruses from wastewater sludges uptake of f through plant roots evaluation of mixed cell types and -iodo- ' deoxyuridine treatment upon plaque assav titers of human enteric viruses hybridization as methods for the detection of adenoviruses clinical and research application of an enterovirus group comparison of methods for rotavirus detection in water and results of a survey of jerusalem wastewater an a-elisa to detect heptatis a virus iii estuarine samples ljse of the nitrocellulose-enzvme immunosorbent assay for rapid, sensitive, and quantitative detection of human enteroviruses detection and quantitation of human enteric viruses in waste waters: increased sensitivity ijsing a human immune serum globulin-immunoperoxidase assay on ma- cells enzyme immunoassays and related procedures in diagnostic detection of hepatitis avirus by hybridization with ~single-stranded -rna probes detection of hepatitis a virus in seeded estuarine samules bv hybridization with cdna probes advances in the use of nucleic acid probes in diagnosis of viral diseases of man suppression of viral replication by guanidine: a comparison of human adenoviruses and enteroviruses key: cord- -istz iql authors: nan title: procedures to investigate waterborne illness date: - - journal: procedures to investigate waterborne illness doi: . / - - - - _ sha: doc_id: cord_uid: istz iql humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. not only safe water is limited to drinking water, but recreational water can also be a source for waterborne illness—both from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. safe water is not limited to drinking water, since recreational water and aerosolized water can also be sources for waterborne illness, from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. often when an outbreak is first suspected, the source is not clear, i.e., food, water, animal contact. investigation is usually needed to find the common source. in some outbreaks the food may first be identified as the source, such as with produce, but the ultimate source could be contaminated irrigation water. investigators have to keep an open mind until laboratory and/or epidemiologic evidence links cases to the primary source. although we frequently think of waterborne illness originating from a microbiological agent, we should be aware that water may also be contaminated by pesticides, fertilizers, and other chemicals which may enter through industrial discharge, agriculture runoff, or deliberate contamination. waterborne illness acquired from microorganisms may be classified as: • toxin-mediated infections caused by bacteria that produce enterotoxins or emetic toxins that affect water, glucose, and electrolyte transfer during their colonization and growth in the intestinal tract; • infections caused when microorganisms invade and multiply in the intestinal mucosa, eyes, ears, or respiratory tract, or contact the skin; • intoxications caused by ingestion of water containing poisonous chemicals or toxins produced by other microorganisms manifestations range from slight discomfort to acute illness to severe reactions that may terminate in death or chronic sequelae, depending on the nature of the causative agent, number of pathogenic microorganism or concentration of poisonous substances ingested, and host susceptibility and reaction. the public relies on public health regulators to investigate and mitigate waterborne illness. mitigation depends upon rapid detection of outbreaks and a thorough knowledge of the agents and factors responsible for waterborne illness. public health and law enforcement agency officials should always be alert to the rare possibility of an intentional contamination of water supplies by disgruntled employees or terrorists. the purposes of a waterborne illness investigation are to stop the outbreak or prevent further exposure by: • identifying illness associated with an exposure and verifying that the causative agent is waterborne • detecting all cases, the causative agent, and the place of exposure • determining the water source, mode of contamination, processes, or practices by which proliferation and/or survival of the etiological agent occurred • implementing emergency measures to control the spread of the outbreak • gathering information on the epidemiology of waterborne diseases and the etiology of the causative agents that can be used for education, training, and program planning, thereby impacting on the prevention of waterborne illness • determining if the outbreak under investigation is part of a larger outbreak by immediately reporting to state/provincial/national epidemiologists in the instance of a bottled water outbreak, halting of distribution and sale of product and recall of product, some of which may already be in consumers' homes, are necessary to prevent further illness. as epidemiologic data accumulate, information will indicate the source of the problem, whether a municipal water treatment plant, bottled water manufacturing plant, or recreational water exposure, and suggest methods for controlling and preventing waterborne illness. this information will guide administrators in making informed decisions to provide the highest degree of waterborne safety. a flowchart, sequence of events in investigating a typical outbreak of waterborne illness (fig. ) shows the sequential steps, as presented in this manual, in investigating a typical outbreak of waterborne illness and illustrates their relationships. a description of each step is presented in this manual. can be recognized at any point during the outbreak investigation. if intentional contamination is suspected follow your notification scheme in emergency response plans (this could include law enforcement, emergency management and other government agencies) the primary purpose of a waterborne disease surveillance system is to systematically gather accurate information on the occurrence of water-related illnesses in a community, thus allowing development of a rational approach for the detection, control and prevention of waterborne illness. other purposes are to (a) determine trends in the incidence of waterborne diseases, (b) characterize the epidemiology of waterborne diseases, (c) gather and disseminate information on waterborne diseases, and (d) develop a basis for evaluating control efforts. it may be useful to coordinate this system with, or integrate it into a foodborne disease surveillance system. however, while the procedures are quite similar from an epidemiologic viewpoint, they may differ with respect to personnel or agencies involved. an effective disease surveillance system is essential for detection of disease caused by either unintentional or intentional contamination of food. an effective waterborne disease surveillance system consists of: • early reports of enteric and other illnesses that may be related to water exposure or consumption • coordinated effort between local and state public health partners, water utility and water recreation staff • systematic organization and interpretation of data • timely investigation of identified outbreaks or clusters of illness • dissemination of outbreak reports and surveillance summaries to all appropriate stakeholders many types of reporting systems may already exist at the local or state/provincial level, and these should be incorporated into a waterborne disease surveillance program. these include (a) mandatory (or voluntary) laboratory-or physician-based reporting of specific infectious diseases, (b) national-based surveillance systems such as calicinet (cdc ) or nors (cdc in the us, (c) physician office, hospital emergency, and urgent-care clinic medical records, (d) public complaints made to health agencies and/or local water utilities, (e) school illness and absentee records, (f) absentee records of major employers, (g) water treatment records kept by water utilities (e.g., turbidity, disinfection levels, occurrence of coliforms), (h) increased sales of antidiarrheal drugs and anti-nausea medications, and (i) source water quality data kept by environmental agencies (e.g., departments of natural resources and geological survey agencies). another type of surveillance mechanism that may supplement or enhance existing reporting systems is a daily log of illness and water quality complaints. agency contemplates initiation or development of a waterborne illness surveillance program, give top priority to identification of appropriate financial, political, strategic, and administrative support. then, identify a key person to create, implement, and manage the system. this person takes responsibility for: • reviewing the types of reporting systems that already exist in your agency or in other agencies that could be incorporated into a waterborne illness surveillance system • identifying the types of information that cannot be obtained from existing reporting systems but that need to be collected or addressed by the waterborne illness surveillance system • identifying ways to merge or integrate the data collected by existing systems with data gathered in the waterborne illness surveillance system • identifying collaborating agencies and staff • develop a mechanism to communicate and update all stakeholders (may be by blast e-mail or periodic conference calls) • providing training in surveillance methods for agency staff and other partners to enhance cooperation • assembling materials that will be required during an outbreak investigation • evaluating the effectiveness of the system. develop procedures to seek and record complaints about waterborne illnesses, water supplies, and water recreational sites. for example, list the telephone number of the waterborne illness investigation unit prominently on local and state public health and water utility websites. to be most effective, have this number monitored / by staff or an answering service. if possible, the utilization of social media such as facebook or twitter should be considered and monitored as many large municipalities (including drinking water utilities) and recreational facilities have an internet presence. if your agency has social media accounts, consider using this vehicle to further disseminate information regarding waterborne illness clusters or outbreaks. identify medical care facilities and practitioners and seek their participation. direct educational activities, such as newsletters and talks at meetings, to stimulate participation in the program. encourage water treatment utilities and operators of recreational water sites to report suspected complaints of waterborne illness to the appropriate local agencies. also, encourage private and hospital laboratories to report isolations of parasitic agents (e.g., giardia, cryptosporidium), viruses (e.g., norovirus and hepatitis a virus), bacteria (e.g., e. coli (pathogenic), salmonella, shigella, vibrio cholerae), and other agents that may be waterborne. develop a protocol for notification and coordination with agencies that might cooperate in investigational activities, including -h-a-day, -days-a-week contacts. a comprehensive contact list should be constructed and updated at least twice a year as individuals may change. notify and coordinate with state/provincial or district agencies, national agencies that have surveillance and water regulatory responsibilities, and other national and international health agencies, as appropriate. for example, it may be useful to find out the level of participation within a certain jurisdiction in national-level outbreak surveillance programs such as nors (cdc, ) or other national surveillance system. delegate responsibility to a professionally trained person who is familiar with epidemiologic methods and with the principles of water treatment and recreational water protection. this person will (a) direct the surveillance program, (b) take charge if waterborne and enteric outbreaks are suspected, and (c) handle publicity during outbreaks. delegate responsibility to others who will carry out specific epidemiologic, laboratory and on-site investigations. if an intentional contamination event is suspected, local and national law enforcement agencies will likely become the lead agency responsible for the investigation. with this in mind, it is critical to identify appropriate individuals and include them in communication and any practice drills that may occur. if a relationship has been established prior to any event, the investigation may run more smoothly. enlist help from a team of epidemiologists, microbiologists, sanitarians/environmental health officers/public health inspectors, engineers, chemists, nurses, physicians, public information specialists, and other (e.g., toxicologists) as needed. free flow of information and coordination among those participating in waterborne disease surveillance and investigation are essential, particularly when several different agencies are involved. water-related complaints are equally likely to be directed at health departments or water utilities but also perhaps to different jurisdictions. therefore, it is essential that these complaints be registered by an agency and that the information is rapidly shared within and perhaps outside of a particular jurisdiction as part of an integrated surveillance system. whenever possible, share the information with participating parties by rapid means such as e-mail and by calling / contact phone numbers. if an intentional contamination event is suspected, contact emergency response and law enforcement for their early involvement. select staff members who will participate in the waterborne disease surveillance program on the basis of interest and ability. inform them of the objectives and protocol of the program. emphasize not only the value of disease surveillance, but also the value of monitoring water quality and treatment performance. if possible, provide printed learning material that can be referenced later. encourage the use of epidemiologic information and approaches in routine disease surveillance and prevention activities. develop their skills so that they can carry out their role effectively during an investigation, and teach them how to interpret data collected during investigations. conduct seminars routinely and during or after investigations to update staff and keep agency personnel informed. train office workers who will receive calls concerning waterborne illnesses to give appropriate instructions. those who participate in the investigation will learn from the experience and often are in a position to implement improvements after completion of the investigation. assemble and have readily available kits with forms and equipment as specified in table a (equipment useful for investigations). restock and maintain kits on a schedule recommended by, and in cooperation with, laboratory staff to ensure their stability and sterility. verify expiration dates, and use kits before this date or discard and reorder. assemble a reference library on waterborne illnesses, investigation techniques, and control measures from reference books, scientific journal articles, manuals, and reputable internet sources (e.g., www.cdc.gov, www.who.int/en/, www.hc-sc.gc.ca/ index-eng.php, www.gov.uk/topic/health-protection/infectious-diseases); make it available to the staff in an easy-to-access format. (see further reading for suggestions). organize a multiagency team with representatives from public health agencies, regulatory agencies, and water utilities and with local political officials to review and exercise existing emergency response plans in the event of a large scale waterborne disease outbreak or other disaster likely to result in waterborne illnesses. local public health agencies have the primary responsibility for the restoration and protection of health of a community following an outbreak event or other emergency. emergency operational procedures should include the following: • an emergency notification list that includes phone numbers and e-mail addresses of key persons/agencies that need to be informed about possible outbreaks and that should receive emergency press releases. every state/province has an emergency management agency and depending upon the scale of the event, it may be useful to coordinate efforts. • clear guidelines for household water consumption following an event. for example, boil-water advisories or instructions to drink only bottled water. statements should be reviewed to ensure current relevance and updated to reflect the most current knowledge. • a plan for dissemination of information to the public; select a coordination point to which all news media and outside agencies will be directed, and designate one person or one telephone number as the contact. (more than one contact person can create confusion). • alternative drinking water sources to be used in cases of emergency and plans for the distribution of this water, if necessary. these include alternative munici-pal systems, bottled water supplies, portable filtration and/or disinfection devices and home treatment units. (special attention should be given to backup supplies for hospitals, nursing homes and other places where lack of safe water would be immediately life-threatening). • identification of specialty laboratories at the state/provincial and national level that are capable of performing (and willing to perform) procedures not routinely done at local laboratories (e.g., large volume water sampling and processing for pathogenic parasites and viruses, serotyping, electron microscopic examination of stool samples, molecular and immunodiagnostic tests for pathogens in environmental and clinical samples). one or more of these tests may be necessary to identify the causative agents in an outbreak and confirm their transmission route. • a plan to exercise procedure with tabletop exercises involving all pertinent partners on a regular basis and implement any necessary adjustments based upon review of after-action findings. notification of a suspected outbreak is often received by health authorities from a laboratory report or a family physician and can be documented on a log such as form a (foodborne, waterborne, enteric illness complaint report). public health investigators will then interview cases and persons at risk who are well (controls) to make epidemiologic associations to find a common source. from here a hypothesis can be formed. further investigation will involve: • collecting clinical samples and water samples • conducting an on-site investigation at the facility alleged to be responsible to determine the mode of contamination or process failure, e.g., low disinfectant level • characterizing the etiologic agents by laboratory analysis using various typing schemes. dna profiling or pulsed-field gel electrophoresis (pfge), of isolates from clinical and water samples to "fingerprint" and link strains of the etiologic agent among cases and to the source prompt handling and referral of water-related complaints, rapid recognition of the problem, and prevention of further illnesses are the foundations of a successful investigation. complaints of water problems are as likely to be reported to a water utility as to a health department. communication is essential between these agencies. this first contact with the public is a vital aspect of an investigation of a potential outbreak and needs to begin by public health professionals as quickly as possible, usually within hours, sometimes by putting less urgent activities aside. as indicated earlier, any action respecting a potential deliberate contamination of water will generate a specific approach to further action. upon receiving a complaint or an alert about a water supply or water exposure or illness potentially attributed to these sources, record the information on form a. alerts may be initiated by reports from physicians, laboratories, or from hospital emergency rooms. alerts may also include an increase in a particular pfge pattern from clinical isolates. an investigation may be initiated to determine if there is a common water exposure among patients with the pfge pattern. the pattern may be compared with similar pfge patterns in pulsenet databases to determine if there are similar occurrences of the pattern in water and clinical isolates nationwide or internationally, e.g., for food that might have been contaminated with water, bottled water. the form provides information upon which to decide whether an incident should be investigated. form a is not difficult to fill out and can be completed by a public health professional, a trained water utility staff member, or trained office worker. assign a sequential number to each complaint. if additional space is needed to record information, use the reverse side or attach additional sheets. always ask the complainant to provide names of other persons at the event, or using the water supply or recreational water under suspicion, whether or not ill, and names of any other persons who are known to be ill with the same symptoms. follow up by contacting these additional persons. emphasize to the persons making alerts or complaints the need to retain a sample of the suspect water and to save clinical specimens (vomitus and stool) from ill persons using a clean utensil in a clean jar or plastic bag and to seal tightly and label clearly with the name of the person and date, and store in a refrigerator (do not freeze). also consider family members not ill for case-control studies. advise complainants to collect a liter (quart) of water immediately, preferably in sterile containers but otherwise in jars that have been boiled or in plastic bags, or if this is not feasible, in other clean containers. tell the complainant to save any ice cubes or refrigerated water, either in their present containers or in unused plastic bags, in the refrigerator or freezer (if already frozen) where they are normally kept. instruct the ill person to hold all clinical specimens and water samples until the health agency evaluates the epidemiological evidence and arranges, if necessary, to collect them. if it is determined that the specimen or sample is not necessary, notify the complainant and advise on proper disposal of the material. unfortunately, the specific etiologic agent cannot be identified in a large proportion of waterborne outbreaks because water samples and clinical specimens (a) were not collected in an appropriate time-frame (not early enough during illness), investigate outbreaks (b) are too old, (c) are too small in volume, especially for giardia and viruses which require liters, (d) have not been examined for the appropriate agent. contaminants may be in the water system for only a short time, and concentrations of toxic substances and numbers of microorganisms may decrease significantly because of dilution or disinfection. if there is a cluster of cases, monitor reports from physicians, complaints about water, or records of laboratory isolation of enteric pathogens that may suggest outbreaks of disease or contributory situations. collect clinical specimens and water as soon as practicable according to the section obtain clinical specimens in this book. extract key information (see* and † entries) from form a and enter it onto form b (foodborne, waterborne, enteric illness and complaint log). record time of onset of the first symptom or sign of illness, number of persons who became ill, predominant symptoms and signs, whether ice or water was ingested, and the name of the water supply or recreational water alleged to have caused the illness, and whether a physician had been consulted, and/or had taken feces or emesis samples, and/or prescribed antibiotics. also, enter on form b names of the places or common gatherings (other than home) at which the stricken persons ingested water during the weeks before onset of illness (see table for an example). enter a code for the water source (e.g., community, non-community, individual well, bottled, stream/ lake, vended, or untreated) . under "history of exposures" column indicate whether the afflicted person had recent domestic or international travel, attended a child care facility, or had recent contact with ill persons or animals. under "comment" column, enter notations of type of agent isolated, results of specimen tests, places where water was consumed during travel, names and locations of restaurants or other foodservice facilities, and other pertinent information including hospitalization, occupation, or place of employment. at this phase of the investigation it will probably not be known whether the illness is waterborne, foodborne, or person-toperson spread. this log can be kept either in hardcopy or in electronic format. see table (below) as an example of a log. interpretation of table . entry -get further details on the patient's symptoms and seek other cases. the report of foreign travel suggests an infection that may have been acquired outside the country. follow-up of such cases may identify an outbreak of international scope. if so, inform state/provincial and national authorities concerned with surveillance of waterborne disease about the situation. entry -possibly food associated; alert food safety officials. entry -initiate investigation; the two cases of conjunctivitis suggest the possibility of a common-source outbreak associated with the motel pool. entry -initiate investigation; cases indicate an outbreak that has a common time-place association. entry -this could be related to entry , because this person reported swimming in the same pool. exposure history: dt domestic travel (out of town, within country); it international travel; cc child care; ci contact with ill person outside household or visitor to household; a an exposure to ill animal; c contact with ill person within household responsible for the investigation, if this was not done when the surveillance protocol was established. delegate sufficient authority and provide resources to the head investigator so that the investigation tasks can be accomplished effectively and efficiently. inform all outbreak investigative team members that any findings are to be reported to this delegated authority. a list of all team members and additional contacts such as administrative contacts, sanitarians/environmental health officers/ public health inspectors, local and regional contacts, physicians, clinical laboratories, or other persons who may become involved in outbreak investigations should be assembled. before beginning the investigation, check the supply of forms and the availability of equipment suggested in table a (equipment useful for investigations) and obtain any needed materials or additional equipment. general resource materials describing signs and symptoms, incubation times, and specifics regarding specimen collection and appropriate kits to be used should be maintained and readily available to those processing the initial calls, which may help to formulate the initial hypothesis. if the alert or complaint suggests a possible outbreak, inform laboratory personnel of the type of outbreak and estimated quantity and arrival time of clinical specimens and water samples collected. this information will give laboratory managers time to prepare laboratory culture media, prepare reagents, and allocate personnel. at a minimum, the laboratory should have six to eight stool culture kits on hand or readily available, since in many cases, stool specimens must be collected within h of onset of illness to isolate and identify certain pathogens (e.g., campylobacter spp., and salmonella spp.). consult laboratory personnel about proper methods for collecting, preserving, and shipping environmental samples and clinical specimens if such information is needed. obtain appropriate specimen containers and sample submission (chain of custody forms) from them. once the investigation is underway, the proper clinical specimens should be collected as soon as possible before patients recover and become less likely to submit specimens. all suspected waterborne outbreaks should be examined and a determination made regarding the feasibility of conducting a thorough investigation even if the time to collect proper clinical specimens has passed. an ill person or family member, physician, hospital staff member, or operator of a water utility or recreational site may report suspected cases of waterborne illness. whatever the source of the report, verify the diagnosis by taking a thorough case history and, if possible, by reviewing clinical information and laboratory findings. (this analysis can be further substantiated by detecting suspected etiologic agents in water). verification of the diagnosis is done in consultation with medical professionals. when a complaint involves illness, complete forms c - (case history: clinical data and case history: food/water history and common sources) either at the time of initial notification, during a personal visit, or during a telephone call to the person reported to be ill. use this same detailed interview approach with every person who has been identified in the initial complaint or alert, even though some may not have been ill. be aware that potential cultural and language barriers can make interviews difficult. a different interviewer may be needed to accommodate these barriers. continue this until sufficient information is obtained to decide whether there is, indeed, an outbreak of waterborne illness. from persons who are at risk of illness but who remained well, also obtain water and -hour food histories, inquire about recreational water exposure in past weeks, and information about their activities in common with the ill persons. information from these persons is as important to make epidemiologic associations as it is from the cases. when it is apparent that an outbreak has occurred and a specific event has come under suspicion, substitute forms d - (case history summaries: clinical data and case history summaries: water/laboratory data) for form c. form d can be used initially in many routine waterborne illness outbreak investigations where it is obvious that a common-source outbreak has occurred or when all of the ill persons consumed water together (e.g., drank from the same public system, consumed ice at an event) or recreated at the same place (e.g., swam in the same lake or used the same hot tubs). this will simplify recording, because most affected persons will give similar information. at this time, notify the district, state, or provincial epidemiologist about the outbreak. if a specific pathogen (e.g., norovirus, e. coli o :h , cryptosporidium spp.) has been identified as the etiologic agent, consider developing a form for recording relevant information. many state/provincial or national public health agencies have standard forms tailored to specific pathogens. include signs and symptoms of the illness and other clinical information, the etiology of the agent, and usual methods of transmission. computer programs (e.g., epi info™) can aid in the design of such standard forms. upon contact with the affected person, identify yourself and your agency and explain the purpose of the visit or call. a professional attitude, appropriate attire, friendly manner, and confidence in discussing epidemiology and control of waterborne illnesses are essential for developing rapport with affected persons or their families and in projecting a good image of the investigating agency. keep in mind that you are not interviewing someone you inspect or regulate, but that you are providing a service to the affected person. exhibit genuine concern for persons affected and be sincere when requesting personal and confidential information. communicate a sense of the urgency of the investigation, and emphasize that their participation will make a positive contribution for the control and prevention of waterborne illness. parental consent must be obtained before interviewing children under years of age. in some locations, consent from the affected person's physician may also be required. after asking open-ended questions about the person's food exposures and illness history, follow up with more specific questions to fill in the details and better ensure a thorough recall. base your level of communication on a general impression of the person being interviewed, considering information about age, occupation, education, or socioeconomic status. tact is essential. use either form c or form d, as appropriate, as a guide. state questions so that the persons who are being interviewed will describe their illnesses and associated events in their own words. try not to suggest answers by the way you phrase questions. fill in form c - (if appropriate) and take additional notes during the interview. ask specific questions to clarify the patient's comments. think questions through before conducting the interview. realize that people are sometimes sensitive to questions about age, sex, special dietary habits, ethnic group, excreta disposal, and housing conditions. nevertheless, any or all information of this type can be relevant. word questions thoughtfully when discussing these characteristics and habits. such information can often be deduced from observations. if doubt remains, confirm your guesses by asking indirect questions. information on recent travel, gatherings, or visitors may provide a clue to common sources or events that would otherwise be difficult to pinpoint. review known allergies, recent immunizations, recent changes in the patient's medical status, and similar information. remember that the agents associated with waterborne disease can also be spread by other means such as consuming food, person-to-person, visiting child care centers, animal-to-person in petting zoos, through walk-in-spray fountains, and pools for young children. as persons describe their illnesses, check boxes next to appropriate symptoms or signs on form c . do not ask about all symptoms or signs listed; however, ask about those marked with an asterisk if the ill person does not mention them. if there are questions, explain symptoms to the patient in understandable terms. the symptoms and signs in the first two columns of form c are usually associated with poisoning or intoxication, although some occur during infections. those in the third, fourth, and fifth columns are usually associated with enteric infections, generalized infections, and localized infections, respectively. those in the last column are usually associated with disturbance of the central nervous system. diseases in any category will sometimes be characterized by a few symptoms and signs listed in the other columns, and not all signs and symptoms occur for any one ailment or for all persons reporting illness. if an illness seems to fall into one of these categories, mention other symptoms in the category and record the patient's response. whenever possible, use physician and hospital records to verify signs and symptoms reported by patients. clinical data may strengthen or dismiss the possibility of waterborne illness. before contacting a physician or a hospital, become familiar with laws and codes relating to medical records to ensure that you have legal access to these records. legal release forms may be necessary to obtain some records. do not distribute names of patients, their other personal identities (e.g., address, phone number), or their clinical information to unauthorized persons. the entries begin with the day of illness, followed by the previous days. if the illness, however, began early in the day or before any of the listed meals, modify the entries on the form so that the -hour history can be completed in the space pro-vided on the form. if the incubation period is days to a week in duration, use additional copies of form c and modify day or day before subtitles. signs and symptoms will sometimes give clues to the transmission route by indicating the organ systems affected. if the early and predominant symptoms are nausea and vomiting, ask about the most recently ingested water or beverage within the past h. in these situations, suspect high-acid water supplies, carbonated beverages and fruit drinks, because these tend to leach metallic ions from water pipes and containers. if diarrhea, chills, and fever predominate, be suspicious of water and beverages ingested - hours before onset of illness for salmonellosis, shigellosis, and norovirus related gastroenteritis. if the incubation period averages - weeks, consider typhoid fever, cryptosporidiosis or giardiasis. diseases with incubation periods exceeding weeks (e.g., hepatitis a and e, amebic dysentery, or schistosomiasis) can be handled as special cases for which longer histories would be sought. others, such as chronic lead and arsenic poisoning, have incubation periods of variable durations and onsets so gradual as to be indeterminable. see table b (illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents) for details on specific pathogens, table c (illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents), and table d (illnesses acquired by inhalation of microorganisms aerosolized from water. a classification by symptoms, incubation period, and type of agent). other microorganisms not listed in tables b, c, and d that can be potentially spread by water include the bacteria klebsiella pneumoniae, mycobacterium avium complex, acinetobacter calcoaceticus, elizabethkingia meningoseptica, stenotrophomonas maltophilia, pseudomonas putida, serratia marcescens, protozoa isospora, microsporidium, algae schizothrix calcicola. these microorganisms are less frequently identified with waterborne illness, but they may become opportunistic pathogens, particularly for highly susceptible and immunosuppressed persons. further investigation is needed to confirm their role in the spread of waterborne diseases. gather information about all sources of water to which the patient(s) may have been exposed weeks before onset of illness. the water supply and the event that precipitated the illness might not be obvious. persons often have difficulty recalling exposure to all water sources including; ice or water ingested; aerosols and recreational water contact. therefore, if the person does not remember specific exposures to water, ask about the water consumed in usual or routine daily habits and the amounts ingested; exposure to recreational waters; and unusual exposures or events attended during this interval. this may stimulate recall of away-from-home water consumption or contact that was unusual. ask about other risk factors for enteric illness, such as contact with young children and child care centers, animal contact, ingestion of raw foods of animal origin, and usual food preference habits. for persons who have been traveling, ask them where (both cities and rural areas) they have traveled during the incubation period of suspected agents. determine if they drank water from any taps or pumps in rural areas they visited. ask whether unheated (or untreated) tap water or beverages containing unheated (or untreated) water or ice was ingested at restaurants, in hotels or at events in the places they visited. also, ask whether they ingested bottled water and, if so, the brand name. find out whether they drank water from streams, ponds, springs, or other natural water sources. if they did, ask if they observed any abnormal condition of the water such as algal growth, high turbidity or discoloration. ask if domestic or wild animals had access to the water. if they have skin or eye infections or generalized infections, ask them to name all swimming pools, water slides, beaches, lakes, ponds, or other chlorinated and nonchlorinated water courses where they swam, waded or bathed during their trip. also ask them whether they used any hot tubs, spas, whirlpools, or similar devices. this information sometimes provides clues to common sources or to events that otherwise would be difficult to discover. record the information on form c . in a protracted outbreak, or when investigating an outbreak of a disease with a long incubation period, expect recall to be poor. in this situation, obtain from ill persons and others at risk a listing of their water, ice, and beverage preferences and amounts usually ingested, or their purchases of these items within the range of the incubation period of the suspected disease. as a guide, draw up a list of either water, ice, and beverages that are commonly consumed by the affected group or those waters, ice, and beverages previously identified as vehicles of the suspected disease under investigation. summarize data from all copies of forms c - on form d. form d allows rapid review of all exposed persons (ill or not ill) and serves as a basis for analyzing the data. diagnosis of most diseases can be confirmed only if etiologic agents are isolated and identified from specimens obtained from ill persons. get specimens from the ill persons to confirm an etiologic agent. • in large outbreaks, obtain fecal specimens from at least ten persons who manifest illness typical of the outbreak • in smaller outbreaks, obtain specimens from as many of those ill and those at risk as practicable, but from at least two, and preferably ten, ill persons • try to collect specimens before the patient takes any medication. if medication has already been taken, collect specimens anyway, and find out the kinds and amounts of medicine taken and the time that each dose was taken • also get control specimens from persons with similar exposure histories that did not become ill obtain clinical specimens at the time of the initial interview during acute illness or as soon as practicable thereafter. even though this is not always possible, take specimens even after recovery because etiologic agents may remain in low populations or concentrations. if a disease has already been diagnosed, collect specimens as listed in table b . if a disease has not yet been diagnosed, choose kinds of speci-mens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in the treatment of cases detected later. apart from the fact that people are more likely to cooperate while they are ill, some pathogens or poisonous substances remain in the intestinal tract for only a day or so after onset of illness. if the patient is reluctant to provide a fecal specimen explain that the specimen will be tested to identify the causative agent and compare it to any agent recovered from the water. if a disease has not yet been diagnosed, choose specimens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in treating cases detected later. some pathogens (e.g., salmonella, parasites) may be recovered for weeks after symptoms have abated. if applicable for the disease under investigation, take specimens even after recovery because some etiologic agents may remain in low numbers, and changes in serologic titers can be detected. before collecting specimens, review table e (guidelines for specimen collection) and, if necessary, get additional instructions from laboratory personnel and seek their advice on how to preserve the stool specimens if you cannot deliver them to the laboratory immediately. many public health agencies have special fecal specimen kits. demonstrate to the patient how to use the materials in the kit, how to complete the form in the kit and how to mail it if you are not going to pick it up. if mailing specimens, make sure that you are aware of the regulatory requirements that may apply to the transport of infectious material. stool specimen containers for intestinal parasite examination are not suitable for bacterial or viral examinations because they ordinarily contain a preservative, such as formalin or polyvinyl alcohol. if an inappropriate transport medium is used, a specimen can be rendered unsuitable for laboratory examination. feces. if the patient has diarrhea or is suspected of having had an enteric disease, obtain a stool specimen (preferred specimen) or a rectal swab. instruct patients to provide you with their own specimens by one of the following means. . if practicable, give the patient a stool specimen container with a wooden or plastic spoon or a tongue depressor. a clean container available in the home (e.g., a jar, or disposable container that can be sealed) and a clean plastic spoon or similar utensil can be used if laboratory containers are not available. . label the specimen container with the patient's name age/date of birth and date of collection. . collect the stool specimen by one of the following methods: (a) put sheets of plastic wrap or aluminum foil under the toilet seat and push them down slightly in the center, but not so far as to touch the water in the bowl. sheets of paper can be tacked on the rise of a latrine and pushed down to form a depression in which to catch feces. take care to ensure that toilet cleaning chemicals and other microorganisms in the toilet bowl do not contaminate the fecal specimen. after defecating, use a clean spoon or other utensil to transfer about g of feces into a specimen container or other clean container. (b) defecate directly into a large clean dry container or bedpan. use a clean spoon or other utensil to transfer about g or the size of a walnut of feces into a specimen container or other clean container. (c) scrape feces off a diaper with a clean spoon or other utensil to transfer about g of feces into a specimen container or other clean container. . collect fecal swabs by twisting the cotton-wrapped end of the swab into the stool obtained in one of the ways described above. follow instructions given in table e . if necessary, use fecal-soiled toilet paper or cloth diaper and twist a swab into the top of feces. take care to ensure that there is no carryover of toilet paper as they are impregnated with barium salts which are inhibitory to some fecal pathogens. dispose of excess fecal material into the toilet and carefully wrap all soiled articles (e.g., by placing them inside two plastic bags) and dispose of in domestic waste. check that the specimen container is tightly sealed and properly labeled and place into a clean outer plastic bag (special zip lock bags for clinical specimens, if available). store the specimen in a cool place, preferably at °c to await pick-up or despatch. do not freeze. feces from rectal swabs. collect rectal swabs by carefully inserting the swab approximately . cm ( in) beyond the anal sphincter. gently rotate the swab. fecal matter should be evident on the swab. vomitus. if the person is vomiting or subsequently does so, arrange to collect vomitus. tell the patient to vomit directly into a sterile specimen container or a plastic bag. otherwise, transfer some vomitus from a clean receptacle into the container with a clean spoon. refrigerate, but do not freeze, this specimen until it can be picked up or delivered to the laboratory. blood. take blood if a patient has a febrile infection or when infectious agents are suspected (see tables b, c, and d) . blood specimens are collected for: • bacterial culture • detection of antibodies to specific agents • detection of certain toxins before collecting specimens, get additional instructions from laboratory personnel and seek their advice. blood should be obtained by an appropriately trained and accredited person (check appropriate laws). collect blood during the acute phase of illness, as soon as the febrile patient is seen (within a week after onset of illness) and, if comparing of serologic titers, again within weeks (usually - weeks later) during the convalescent phase. draw ml of blood (from an adult) or ml (from a child) or - ml (from an infant). if possible, collect the blood from the same patients from which stool specimens were obtained if both specimens are to be examined. label tubes and vials at every step of serum transfer. do not freeze whole blood because the resultant hemolysis interferes with serologic reactions. collect into a sterile syringe or evacuated sterile tube that does not contain anticoagulants. if practicable, centrifuge the blood at , rpm for min; pour off the serum into small screw-cap vials and store at approximately − °c. if the serum cannot be separated immediately, rim the clot with a sterile applicator stick and refrigerate approximately °c to get maximum clot retraction if the specimen is to be stored unfrozen overnight. if centrifugation cannot be done, store the blood specimens in a refrigerator until a clot has formed, then remove the serum and transfer it with a pasteur pipette into an empty sterile tube. send only the serum for analysis urine. instruct patients to collect urine in the following manner. clean the area immediately around the urethral orifice with a paper pad that has been pre-moistened with % tincture of iodine or other appropriate antiseptic. then begin to urinate into a toilet and collect ml (about oz) of midstream urine into a sterile bottle. use either a second antiseptic-moistened pad or an alcohol-moistened cotton ball or tissue to clean any drops from the top or side of the bottle. other instructions. follow applicable instructions given in table e . before or immediately after collecting clinical specimens, use waterproof permanent markers to label each container with the patients name, complaint number, case identification number, specimen number, date and time of collection, tests requested, and other appropriate information. tightly seal all containers. clinical specimen collection report for each specimen. complete form e (clinical specimen collection report). the complaint number, case identification (id) number, and specimen number must be entered on each report so that laboratory results can later be correlated with other data. on form c record the type of specimen collected, and submit both the specimen and a copy of form e to the laboratory. send a copy of the laboratory report to the patient's physician or call if urgent. if the patient/case or other household member collected any water, ice, or beverage as instructed during initial contact, label containers with the complaint/outbreak and sample numbers. proceed as instructed in table f (general instructions for collecting water samples for microbiological analysis) and complete form f (water/ice collection report) and/or forms g -g as applicable. record conditions of collection as called for on the forms. if a hypothesis associates the illness with water, caution these persons not to use the water source unless the water is first boiled and to discard all previously prepared ice and water-containing beverages until notified otherwise. develop a working case definition to classify exposed persons as either cases or non-cases. start with the most specific symptoms (such as diarrhea and vomiting) rather than broader symptoms such as nausea or malaise. for example in an outbreak of gastroenteritis, a case might be defined as a person from whose stool a specific pathogen was isolated. it may be a person who was at risk and developed diarrhea within a specified period of time. diarrhea will have to be defined, perhaps as three or more loose, watery stools during a -hour period. in some cases, a particular pathogen responsible for the outbreak might have been identified from clinical specimens. a case definition, which is developed later in the investigation, might include either a person having specific signs and/or symptoms within a period of time or a person from whom a specific pathogen was isolated. the ultimate case definition has a tremendous impact on the investigator's ability to make illness and exposure associations and to calculate probability of these associations. sometimes the first symptom or sign provides a clue to developing a case definition. information in tables b, c, d, g, and h can be useful in making case definitions. compare newly identified cases with the definition to see whether each is part of the outbreak. classify cases into categories: • a confirmed case is a person with signs and symptoms that are clinically compatible with the disease under consideration and for which there is either (a) isolation of an etiologic agent from (or otherwise identified in) an appropriate specimen from the patient, or (b) serologic evidence of a fourfold or greater rise in convalescent antibody titer. a confirmed case must also have possible exposure to the etiologic agent within the incubation period of disease. see table e . criteria for confirmation of etiologic agent responsible for outbreaks of waterborne illnesses for definitions of confirmed cases for specific waterborne diseases: • a presumptive case is a person with signs and symptoms that are clinically compatible with the disease under consideration, and for which there is laboratory evidence of infection (e.g., an elevated antibody titer but less than a fourfold increase), but the etiologic agent was not found in specimens from patients or no specimens were collected. a presumptive case must also have possible exposure to the etiologic agent within the incubation period of disease. • a suspected case is a person with signs and symptoms that are clinically compatible with the disease under consideration and history of possible exposure, but laboratory evidence is absent, inconclusive or incomplete. • a secondary case is a person who became infected from contact with a primary (outbreak-associated) case or from a vehicle contaminated by a primary case. onset of illness for secondary cases typically is one or more incubation periods after the outbreak-associated cases. it is not essential, however, to classify cases into these categories. do so only if it aids in developing a final case definition or in making comparative analyses of data. consider doing analyses using case definitions of both confirmed and combined confirmed, presumptive, and highly suspect cases, and compare the results. make a preliminary evaluation of the data collected as soon as possible. if you decide that there is an outbreak, use the information you have to develop a hypothesis about the causal factors. an outbreak is an incident in which two or more persons have the same disease, have similar clinical features, or have the same pathogen (thus meeting the case definition), and there is a time, place, or person association among these persons. a waterborne outbreak is traceable to ingestion of contaminated water or ice or to contact with contaminated water. a single case of either chemical poisoning or a disease that can be definitely related to ingestion of drinking water or contact with water can be considered an incident of waterborne illness and warrants further investigation. waterborne methemoglobinemia in an infant who resides in a rural area having a high concentration of nitrates in well water is an example of a single case of waterborne illness due to ingestion. a rare diagnosis such as primary amebic meningoencephalitis in a person who swam in a body of freshwater and inadvertently ingested the ameba, naegleria fowleri, through the nose is an example of a single incident related to water contact. sometimes it will be obvious from an initial report that an outbreak of waterborne disease has occurred simply because of the number of persons displaying certain signs and symptoms at or near the same time. many complaints, however, involve illness in only one or a few persons. it is often difficult to decide whether ingestion or contact with a particular water source and onset of illness was associated or coincidental. certain diseases that are highly communicable (e.g., shigellosis and epidemic viral gastroenteritis) may result in secondary infections from person-to-person spread or from subsequently contaminated food or water. however, if complaints are received from several persons who are associated with ingesting water or contact with water at the same place, water is likely to be involved. routine review of the log pertaining to potential waterborne illnesses for similar complaints can often be useful in detecting time, place or person associations. an investigation may also proceed based upon the suspicion of an intentional contamination of a water source. a time association exists if the time of onset of similar illnesses is within a few hours or days of each other. place associations exist when persons have ingested water from a particular single source, have swum in, worked in or otherwise been exposed to the same water, have attended the same event, or reside in an area common to all. person associations indicate a shared personal characteristic, such as being of the same age group, sex, ethnic group, occupation, social group, or religion. waterborne illnesses transmitted by a community water supply usually afflict persons of both sexes and all ages throughout the community. non-community water sources, such as bottled water, ice, water from individual wells, or water from areas of recreation should also be considered when making associations. keep in mind that water can contaminate foods during washing or freshening, and it can contaminate utensils and vessels that are used to handle or store foods. water may therefore be a source of contamination of another vehicle. also, water can be ingested as aerosols generated by shower heads, whirlpools, hot tubs, fountains, cooling towers, and irrigation devices. once some of these associations become obvious, question other persons who could be at risk because of their time, place, or person associations with the ill persons. from time, place, or person associations that have been established or suggested by the investigation, formulate hypotheses to explain (a) the most likely type of illness, (b) the most likely vehicle involved, (c) where and the manner by which the vehicle became contaminated, and (d) other possible causal relationships. the section "collection and analysis of data" describes calculations that can aid in the formation of these hypotheses. test hypotheses by obtaining additional information to support or reject them. if the hypothesis includes food contamination, the instructions given in the manual, procedures to investigate foodborne illness, might be useful. guidelines for confirmation of waterborne outbreaks are presented in table g and guidelines for confirmation that water is responsible for illness are presented in table h . if there is strong evidence to support a hypothesis that the outbreak is waterborne, take precautionary actions. the choice of action is dictated by the (a) suspected causal agent, (b) size of the water source, (c) availability of alternate water sources, and (d) expected use of the water. on the basis of available information, estimate the population at risk and engage any public relations staff with your organization to help inform all persons potentially impacted. when dealing with a microbiological contaminant or agent, consider issuing a boil-water advisory with water treatment guidelines (e.g., heating water in a covered container to a rolling boil for at least min and keeping it covered until use). other options that can be explored include chlorinators that can be installed in individual and non-community systems. for community and non-community supplies in which chlorine is already used, increasing the chlorine dosage and opening hydrants and taps to draw the super-chlorinated water through the whole system might be an option. increasing chlorine is sometimes not effective because the chlorine contact time is too short or super-chlorinated water does not reach some parts of the system. furthermore, chlorination is ineffective against cryptosporidium oocysts and requires a long contact time to kill other human pathogens like hepatitis a virus and giardia. for suspected chemical contamination contact a specialist for further assessment and remedial strategies, such as activated charcoal filters. as a last resort, shut off the contaminated system until the source of contamination is found and controlled. be cautious when you take this drastic measure, because it may do greater harm than good by causing lack of water for hospitals, nursing homes, or for firefighters to extinguish fires. if the water is shut off or the treatment facility or distribution system disrupted (as in the case of floods or other disasters), consider means to distribute water from an alternative source to healthcare facilities and homes. if an illness could have resulted from water contact, close the offending water source, post warning signs around it, and patrol the area. where there is a swimming pool, hot tub, spa, fountain, or whirlpool, evaluate the recirculation system and its operation. it may be that increasing disinfectant concentration by super-disinfection could resolve the problem. where there may be chronic operational problems, evaluate ph, disinfectant concentration, and bacteriological laboratory records. choose your course of action, including consultation with appropriate professional experts, depending on the contributing factors existing at the time of investigation. verify the effectiveness of these actions (e.g., boil-water advisory, superchlorination, provision of alternate water source) to protect public health by monitoring illness levels in the population to determine if the outbreak terminates. if the outbreak continues unabated, consider the possibility of other transmission routes. also, verify the effectiveness of repairs to the water system, super-disinfection, and other actions by closely monitoring the quality of the water supply or recreational water to determine if laboratory reports indicate that the water is now safe for consumption or contact. if there is a public health threat, work with any available public relations staff to announce the outbreak in the mass media so that the public who consumed or was otherwise exposed to the implicated water can be alerted to take appropriate action including seeking medical consultation or treatment. provide only objective factual information about the outbreak. coordinate among the investigating agencies to assure that a consistent and accurate message is delivered. it is easy for agencies to miscommunicate before and during a water crisis (see box , false alarm; box , the walkerton outbreak; box , the flint water crisis). it is often preferable to have one spokesperson for all agencies. do not release preliminary information that has not been confirmed. the person giving information about an outbreak should be well informed about the etiologic agent being investigated and prepared to deal with questions. if the health hazard warrants a public warning at the hypothesis stage, tell the public why emergency measures are being invoked and that subsequent information may be cause to modify the action. as the investigation proceeds and the etiologic agent is confirmed and contributory factors are identified, consider terminating emergency measures, and give advice on specific control and preventive measures. attempt to reach all segments of the population at risk; this may require communication in multiple languages. route all news releases or statements to all persons involved in the investigation. in situations involving large outbreaks or highly virulent or toxigenic etiologic agents, set up an emergency hotline for the public to call to ask questions. this is likely to occur if there is an intentional contamination event where there is high publicity and public concern. train staff to handle these calls in a consistent manner so that the advice is the same who gives it. faulty information derived from poorly tested hypotheses can lead to severe political, legal or economic consequences. an example of this occurred in sydney, australia, in when an apparent water contamination event was publicized for the public to take precautionary actions. the false alarm was costly because of rebates to water customers, additional water testing, and for hiring extra staff, as well as a loss of confidence in the facility (see box , false alarm). they may then be disseminated by the mass media with inappropriate interpretations of the public health significance. furthermore, this information may be used as an unrealistic base for water programs or water regulations because of either misinterpretations or pressure from misinformed consumer-advocate groups. all involved parties should follow a written protocol for cross-agency communication and release of information to the public. unreasonable delays are unacceptable. test hypotheses by obtaining additional information to either confirm or refute their validity. do this by case-control or cohort studies, additional laboratory investigations, and on-site investigations (e.g., laboratory reports of water testing). sydney water (a new south wales state-owned corporation) supplies million liters of water each day to . million properties in sydney and its outlying areas. the city has a large and complex catchment with nine major dams and several storage reservoirs. about , km of water main, almost pumping stations and many tunnels deliver water from four main river systems. the water is filtered through eleven treatment plants. seven are owned by sydney water and four are privately owned. these plants provide % of sydney's drinking water and one plant, prospect, provides up to %. in , the quality of sydney's drinking water came under acute review when giardia and cryptosporidium were found in the city's main water supply at the warragamba dam. initially, low levels of these parasites were first detected in the water supply on july, but these were within the acceptable health limits. in days following, much higher levels were recorded, and on july the first "boil water" alert (in which residents were instructed to boil their tap water before use) was declared for the eastern central business district of sydney. however, by late on july high readings were found in samples at the prospect filtration plant, in a reservoir and at a location further down the system, and a "boil water" alert was issued for the south of sydney harbour, and on july a sydney-wide "boil water" alert was issued affecting most of sydney's residents. on august the warning was discontinued. however, high levels were again found on august (the second event), although it was believed that most organisms would likely be dead. more positive readings were found on august , although at lower levels. further contamination was identified on august and an extended boil water alert was again declared. this was progressively lifted suburb by suburb until further contamination was reported on september (the third event). a -week alert was then instituted, which was finally lifted on september . it was determined that the parasitic contamination was caused by low-quality surface water entering the dam. this contaminated source was attributed to moderate rainfall in july, followed by heavy rainfall in august and september which caused intermittent supplies of the raw water to enter the dam. despite high levels of cryptosporidium (up to > , oocysts) and giardia (up to > cysts) being recorded in july and august, , no increase in human cryptosporidiosis or giardiasis was detected in the exposed population. the incident was highly publicized and caused major a public alarm because the number of people affected, the on and off boil water alerts, and the fact that the filtration plant had been advertised as one of the best in the world. the economic and political repercussions were extensive. the cost of the crisis to sydney water was estimated at a$ million which included $ million paid in rebates to customers, $ million in lost revenue, water testing and staff costs and at least $ . million for damages claims. these costs exclude those relating to improvements to the system and infrastructure. the lack of cases of cryptosporidiosis, giardiasis or other water-related health problems led to suggestions that the parasites were either not an infectious type, or not as extensively distributed. an inquiry after the event revealed the publicity as an exaggeration of fact, with australian water technologies, part of sydney water, severely overestimating levels of cryptosporidium and giardia present in the water, with the recorded levels exposed to consumers as not harmful to human health. the handling of the crisis by state-owned sydney water was heavily criticized, causing the resignation of both the chairman and the managing director, and bringing up issues of private vs. public ownership and scientific uncertainty. the eventual consequence of the state inquiry was the establishment of the sydney catchment authority in to assume control of sydney's catchments and dams, while sydney water maintained responsibility for water treatment and distribution and for sewage collection, treatment and disposal. if an outbreak investigation requires resources beyond your agency's capacity, request assistance from other health professionals. it is desirable to have a team including, if feasible, an epidemiologist, an engineer, a microbiologist, a sanitarian/ environmental health office/public health inspector, a chemist, a physician and others, to undertake a detailed waterborne illness investigation. such personnel can usually be provided by local, state/provincial, or national agencies concerned with health, environment, or agriculture, depending on the expertise needed. for events suspected to arise from intentionally contaminated food, contact emergency response or law enforcement agencies. continue to search for and interview both ill persons who have had time, place, or person associations with the identified cases (see the section on "make time, place, and/or person associations"). review recently received complaints in the water-related complaint log (form b). contact other nearby health agencies, hospital emergency rooms, elderly care centers, and local physicians to discover other epidemiologically related cases. call previously contacted persons to see whether they know anyone else who has become ill or had a common association suggested by data in the log. the illness you are investigating may be part of a larger multijurisdictional outbreak, and therefore communicate with adjoining local and state agencies to learn if they are seeing similar illnesses. state or provincial public health agencies can check reportable disease records and state/provincial public health laboratories can start looking for clusters in isolates that they are characterizing. for outbreaks where intentional contamination of water is suspected or confirmed, public health and law enforcement agency officials may conduct the investigation jointly. if it becomes apparent that an outbreak is associated with a specific water supply (source) or recreational water or event, use form d for recording information. at this stage of the investigation, interviews can be expedited by reviewing the event itself to stimulate each person's recall. ask about specific symptoms and signs that are known to be common to the syndrome, as well as, time of ingestion or contact with water and onset of illness. mention each source of water to which the person may have been exposed, and ask each person (whether a case and well persons at risk) which of the water sources had been ingested or contacted. the number of persons to be interviewed depends on the number exposed and the proportion of them who are probably affected; if fewer than persons were at risk, try to interview all of them; if several hundred are involved, interview a representative sample. be sure to obtain clinical specimens from these cases and well persons at risk (controls). it is more difficult to obtain positive results if symptoms from persons have ceased. there may be situations where self-administrated questionnaires are sent to cases and persons at risk. use either form c or form d or modified versions for this purpose. after questionnaires have been completed, summarize the data on form d. also, identify and interview secondary cases if they become apparent. because no two waterborne disease outbreaks are identical, the order of the expanded investigation may not always follow the outlined sequence of procedures. some investigative steps can usually be done simultaneously by different investigators. additional procedures may also be required. the principles and techniques described will suffice for most investigations. modify forms, if necessary, to accommodate the type and amount of information to be collected. make on-site observations. prove or refute hypotheses developed during the epidemiological portion of the investigation. focus on sources and modes of contamination and ways contaminants could survive and pass through water treatment. as applicable, conduct an on-site investigation of source (lakes, streams, areas around groundwater, etc.), treatment facilities, distribution lines, cross connections, water reservoirs, places of recreational water contact and/or sites at which aerosols were generated. such an epidemiologically focused investigation is quite different from sanitary surveys done during routine evaluations of water source sites, treatment plants or recreational water facilities. not all drinking water (even municipal and bottled water) is disinfected; so, it is important to identify whether the water source is treated and if so, how. some treatments (filtration, reverse osmosis, membrane treatments, riverbank filtration, and others) may not be complemented with a disinfection step. sanitary survey information can provide information about potential sources of contamination in the area of a usually pristine water source. microbiological records of a water supplier, particularly if any total coliform positive samples were found by the system in the last months, may help identify a contamination pathway. if significant matters relating to water quality are observed or otherwise identified during the investigation, note them and communicate them to those responsible for the water system and to the proper authorities. do not lose the focus and objectivity of the investigation by confusing matters of quality and aesthetics with factors related to contamination by, and survival of, infectious and toxic agents. use the haccp-system, also known as systems analysis, way of thinking in your investigation. contact the person with the highest responsibility for the operation and maintenance for the implicated water source, water treatment facility, and/or distribution lines. identify the types of records that ought to be reviewed during the investigation and their likely source. do not forget that the responsible authorities also can have records (about water quality, if there has been a change of municipal water supply, industrial water pollution, wastewater pollution). they can be good sources of information about recent pipe breaks and other water system issues that could be related. in many cases they will be aware of the potential for contamination upstream of source water intakes. if applicable, obtain water distribution maps and recent water quality reports from appropriate departments. if you are not familiar with the community in which the investigation is to be done, obtain maps of the area to locate streams, lakes, water treatment facilities, and other community features that might have a bearing on the investigation. check if there are water protection areas and their rules. get plans and specifications on design of treatment facilities from consulting engineers or state agencies that approve these facilities. contact weather bureaus, airports, radio/television stations, or newspapers for information on heavy rainfall, flooding, extremely low temperatures, droughts, or other unusual weather conditions that preceded the outbreak, if this information is unknown to investigators. contact police or fire departments about traffic accidents, which can be the source of the outbreak. review all background data pertaining to the suspect water. as information is gathered, record it on applicable parts of form g. discuss with laboratory personnel that a field investigation will be made, and get their suggestions regarding samples and specimens that should be collected (see tables e and f) . confer with them about special analyses, media, and sampling procedures; make arrangements for rapid transport of samples to the laboratory. the samples must maybe be transported at the right temperature. pick up appropriate forms and sample collection equipment (preferably preassembled in a kit-see table a ). the laboratory can probably help assemble this kit. during the investigation, identify factors that contributed to contamination and survival of the etiologic agents and perhaps also to their growth or amplification or another cause of the outbreak. identified factors and situations that have contributed to waterborne disease outbreaks include those listed in table . focus the investigation on the potential situations listed in table , as applicable. remember that other possibilities can occur. describe circumstances that contributed to contamination and that permitted the etiologic agent to survive so that it reached drinking, agricultural, industrial, or recreational water. also describe circumstances that allowed pathogenic bacteria or algae to multiply in the water. write your findings down on the back of form g (illustration of contamination flow) or on a separate sheet. continually update the listing in table with newly available data. introduce yourself (who you are, where you come from, who ordered you there) to the owner, resident, or persons in charge and state your purpose, when you arrive at the place of the suspected contaminated water source. emphasize that your visit is to confirm or eliminate suspicion that this water was a source of illness. tell him or her that a complete epidemiologic study is in progress and that other possible sources (such as food) will be investigated as well as operations of this site. explain that your investigation is not to fix blame but to identify the cause of the outbreak. emphasize that the findings can yield benefits related to the ability to identify needed improvements, to educate staff and to provide public support. try to create an atmosphere of cooperation. maintain an open mind and try to answer all questions. if you can not answer a question, tell the person that you will come back with an answer. come back to the person within or weeks even if you do not have any new information. give the person your phone number and e-mail address and tell the person to contact you if the person has more information later. privately interview key persons responsible for operating or repairing water facilities. do not forget to interview persons from other work shifts. identify persons who were working there at the likely time of contamination and have since left and interview them as well. ask questions to determine the flow of water and operations from intake through distribution through plumbing systems. ask about any changes in operation, unusual events in the watershed or repairs to the water facilities. ask if you can check records, both in paper form and on the computer (monitoring system), analyses of results, and/or incident reports. plant personnel may not describe water treatment or installations exactly as they existed at the time that a mishap occurred. they may fear criticism or punitive action as a result of their possible role in the causation of the outbreak. their descriptions should be plausible and should account for possible sources and modes of contamination and indicate possibilities for survival of pathogens. if a description does not contain all the information desired, reword questions and continue the inquiry. confirm accounts by private interviews with others knowledgeable of water treatment or operation of the facility. be alert for inconsistencies among the accounts told by different persons. seek resolution of discrepancies in accounts by watching actual procedures as they are being carried out, by taking appropriate samples, or by conducting experiments. a communicative working relationship between the plant management and the investigator influences plant workers' attitudes toward the investigative team. consider the position, feelings, and concerns of the manager and staff; defensive reactions are normal on their part. diagram each phase of the water system or situation under study on form g (illustration of contamination flow). insert special symbols and notes for all sites that might be involved in introducing contamination to the water or where contaminants might have survived treatment. record other information gathered on the appropriate parts of forms g - . review and collate appropriate information on quality control and operational records from the water utility and from responsible agencies. as applicable, obtain information on quality of untreated surface or groundwater from a local, state/ provincial, or national pollution control or geological survey agency. also, seek water distribution maps, well logs, descriptions of geological conditions and indices of groundwater quality from them. for surface water supplies, obtain information on upstream discharges and unusual events that may have affected raw water quality. get data on finished water quality in the distribution system from a local, state/ provincial water surveillance or regulatory agency. water suppliers also frequently have records of raw and finished water quality. review data on quality control tests (e.g., ph, chlorine residual, chlorine demand, bacteriological and chemical tests, turbidity, jar test data, incident reports) that are available. obtain data on cross connection control programs and sewer repairs from the water supplier or other local agencies (e.g., building inspectors, sewage departments). review files for data concerning potential sources of contamination for individual or semipublic water supplies (e.g., diagrams of septic tank systems, sewer line locations, well logs, small individual wastewater plants, accidental industrial pollution of water, traffic accidents involving chemicals, salting of roads or sawmills). check if they have any haccp-systems or water safety plan and, if so, how they monitor their ccps (critical control points) and if they are implementing control measures. ask them about haccp, to see if they understand the system and if results are documented. check if the haccp-system is validated (should be documented) and that they are conducting internal audits. get information on all aspects of normal operations as well as unusual events or conditions to determine whether such events were coincidental with the time of suspected contamination as determined from the epidemic curve. also, consider the time it takes for a contaminant in the raw water or treatment plant to reach households in the affected community. ask responsible persons for this information. compare data on heterotrophic plate and total coliform counts of raw and finished water leaving the treatment facility and of water in distribution lines. also, compare data on chlorine residuals within the plant with that in distribution and check, if they have, that the uv-light is functioning. review other test data (e.g., turbidity and chemical analyses) that may indicate a problem situation. identify locations and dates of sample collection. take photos, if it is allowed, of things you suspect are not right. go back more than one incubation period of the disease under investigation. record this information on form g (record review of on-site investigations, and test results prior to and during outbreak). photocopy appropriate records for confirmation and subsequent review and attach them to the record review form. be alert for evidence of falsification of records. while reviewing records, watch for evidence of the following: • potential contamination of groundwater systems because of proximity to septic tank systems, latrines, animals manure or landfills, industrial contamination of the water supply, small sewage plants, especially old and nearly forgotten ones, and recent heavy rain • high heterotrophic plate or coliform counts, or counts that exceeded the average (median) or typical count • sudden changes in water quality or operating practices that suggest the possibility of contamination or treatment failure • high turbidity, unusual odor, color, or taste, or high coliform counts in raw water, which can indicate potential overloading of the normal treatment process • high levels of ammonium, nitrate and nitrite, which can indicate organic and inorganic contaminants • low chlorine residuals in treated water or higher-than-normal amounts of chlorine used, which can indicate either a high chlorine demand or a sudden high level of contamination • a sudden drop in amount of disinfectant used, possibly indicating failure or interruption of a disinfection process. no functioning alarm system • a sudden change in the amount of a chemical (e.g., alum or ferric sulfate) used, suggesting equipment disfunction or inadequate coagulation or flocculation and thus poor filtration • lack of treatment chemicals if a more corrosive water supply is used (see box , the flint water crisis) • pump failures, draining of distribution lines or reservoirs, or massive pumping to fight fires, which can produce low pressures that can cause contamination through cross connections or back siphonage • repairs to water mains, wells or pumps where contamination could have been introduced record this information on form g or other appropriate form in the g series. as applicable, investigate the water source, treatment facility, distribution and plumbing systems, sites where water was contacted, and sites at which microorganisms amplified and aerosols disseminated. use forms in the g series as guides while observing facilities, gathering data, making measurements and collecting samples. google or bing maps or other similar resources' views of the watershed can be very helpful in identifying potential sources of contamination that you will need to investigate further. these maps can also facilitate your own map and diagram making on form g . the water source may be surface or ground or in some cases a combination of the two. verify this by observations at the site and by talking to the property owner or persons responsible for operation or maintenance of water supply or recreational facilities, as applicable. examination of "weather events" such as heavy rainfall may indicate a potential for surface water contamination (see box , the walkerton outbreak). when a surface water is either suspected or implicated as the source of a contaminated supply, get information about the watershed concerning possible sites of contamination of the suspected etiologic agent. this includes, but is not limited to (a) land use, (b) sewage effluent from treatment plants and septic tanks, (c) industrial plants that may be discharging toxic waste, (d) mining wastes, (e) landfill leachates, (f) slaughterhouse discharge wastes, (g) animal feed lots, (h) both domestic and wild animals that use the source water for drinking, (i) sludge disposal from sewage treatment plants or septic tanks (e.g., land spreading or lagoons), (j) storm water discharge. if this information is not available from records or persons familiar with the site, visit the site and observe possible sources of contamination and pollution (e.g., while traveling by foot, vehicle, boat, or helicopter, as applicable). record this information on form g (source and mode of contamination of surface water). diagram the surface water and sites of contamination on form g . note type and location of sources of contamination and their distances from the water. visit groundwater sources. using form g (source and mode of contamination of groundwaters) as a guide, question owner or operator and inspect groundwater installations to ascertain character of the land and surface and subsurface soil and water. when a well or improved spring is under consideration as the source of the contaminated supply, observe its location relative to possible sites of contamination and to whether its construction allows contaminants to reach the water. determine locations of all sewage outflows or disposal sites (e.g., septic tanks and absorption lines, cesspools, privies, and other sewage disposal facilities), gradients, and distances from the well or spring. determine the type of soil at the site. if the soil is limestone or fissured rock or if there is a high ground or perched water table, pollution may travel many miles. in this case, the search for sources of contamination may have to be expanded for a considerable distance from the well or spring. ascertain whether there were heavy rains, heavy snow melts, or sudden discharge in may, , many people in walkerton, a small ontario, canada, community of about , people, began to simultaneously experience bloody diarrhea and other gastrointestinal infections. on may - , torrential rain had unknowingly contaminated the town's water system, but operators failed to check residual levels for a period of several days, allowing unchlorinated water to enter the distribution system. however, the privatelyowned walkerton public utilities commission insisted there was no problem with the water despite other laboratory tests showing evidence of e. coli contamination. illnesses began about may , with the first death occurring on may and the seventh and last on may . by may , however, many more cases had been diagnosed, the infectious agent determined to be e. coli o :h , and contaminated well water was confirmed as the source of the e. coli; all this allowed the region's medical officer of health to issue a boil water advisory, warning residents not to drink the tap water. two days later, laboratory results identified the presence of campylobacter and e. coli o :h and dna testing showed that the contaminating source was a cattle farm a short distance from a well used for the water supply. by the time the outbreak was over, > were ill and had died. the people who died directly from drinking the e. coli-contaminated water might have been saved if the walkerton public utilities commission had admitted to contaminated water sooner. those in charge of the water utilities at the commission had no formal training in their positions, retaining their jobs through three decades of on-the-job experience. they were later found to fail to use adequate doses of chlorine, fail to monitor chlorine residuals daily, make false entries about residuals in daily operating records, and misstate the locations at which microbiological samples were taken. regulations state that water suppliers are required to treat groundwater with chlorine to sufficiently neutralize contaminants and sustain a chlorine residual of . mg/l of water after min of contact. had utility operators adhered to the protocol, the disaster most likely would have been averted. the operators knew that these practices were unacceptable and contrary to ministry of environment guidelines and directives; they eventually admitted falsifying reports and were sentenced to short jail terms. the ontario government was also blamed for not regulating water quality and not enforcing the guidelines that had been in place. the water testing had been privatized in october . an enquiry found that the water supply, drawn from groundwater, became contaminated with the e. coli o :h strain from manure from cattle on a farm washing into a shallow water supply well after heavy rainfall. the risk of contamination from farm runoff into the adjacent water well had been known since . key recommendations from the enquiry included source water protection as part of a comprehensive multibarrier approach, the training and certification of operators, a quality management system for water suppliers, and more competent enforcement, which were incorporated into ontario new legislation. the bottom line of the enquiry was that officials and municipal water facilities operators and managers across north america need to recognize public waters are a most valued but vulnerable public resource. investment in keeping them safe and secure needs to be a community top priority. from dams that could have resulted in flooding within the duration of the incubation period of the disease under investigation. obtain information on the depth of the well in reference to the ground water table from the owner or by referring to any available well logs on public file or from local drillers. observe well construction and get information about depth of casing, depth and method of grouting, and whether there is an underground discharge. observe whether there is an impervious well platform and whether the pump or casing seal was subjected to flooding. illustrate the situation by showing location of the well in this event is considered a disaster, still unfolding, initiated from a political decision to save money, and ending up with acute and chronic illnesses and deaths to residents of a michigan city, as well as high system remediation and health-related costs to the taxpayer. on april , , flint, genesee county, michigan, switched its water supply from detroit's system to the flint river as a cost-saving measure for the struggling, majority-black city on the recommendation of the state-appointed emergency manager. flint agreed to separate from the detroit water and sewerage department and go with the karegnondi water authority, including the decision to pump flint river water. this was to be an interim measure until a new pipeline from lake huron was constructed in to serve the region. soon after the switch, residents begin to complain about the water's color, taste and odor, and to report rashes and concerns about bacteria. in august and september city officials issued boil-water advisories after coliform bacteria were detected in tap water. in october , the michigan department of environmental quality (mdeq) blamed the cold weather, aging pipes and a population decline. in the same month a general motors plant in flint (continued) reference to possible sites of contamination on form g . note distances between the well and contamination sites and elevations. determine whether any pumps were out of order or had been repaired during the interval of concern. if priming of the pump was done, find out the source of the water used. record this information on form g . test hypotheses of modes of contamination by conducting a dye test and/or sampling the water. (see appropriate sections of this manual for directions.) collect samples of water from these sites and submit them for analysis of the suspected etiologic agent or for any physical, bacterial, or chemical tests that will provide evidence of contamination or movement of the contaminants. (see procedures for collecting water samples) record these results on forms g or g and i (laboratory results summary). when appropriate, confirm hypotheses by a dye or other tracer test. (see section on this subject). determine the means by which the etiologic agent survived treatment or was otherwise not eliminated or inactivated. consider the treatment process as a series of barriers placed between contaminants and consumption of the treated water. the operation of each barrier should be optimized. review available data for each step in the treatment process. records of well-maintained and properly calibrated continuous monitoring equipment will be especially valuable. look for failures in the barriers, which could include (a) lack of disinfection, (b) inadequate concentration of disinfectant or contact time, (c) interruption of disinfection, (d) inadequate filtration, (e) lack of corrosion inhibitors, which may follow inadequate pre-filtration treatments. in in flint, michigan excessive levels of lead were found in drinking water from corrosion of water distribution pipes (see box , the flint water crisis). corrosion inhibitor had not been added. also, look for possible introduction of contaminants within the treatment process, such as in treatment chemicals. stopped using municipal water, saying it was rusting car parts. on january , , the city announced that flint's water contained a high level of trihalomethanes, a byproduct from increased disinfection by the city. though this is in violation of the safe drinking water act, officials told residents with normal immune systems that they have nothing to worry about. in january , detroit's water system offered to reconnect to flint, waiving a $ million connection fee but the offer was declined by the emergency manager. by february, state officials continued to play down any water problems saying that the water was not an imminent "threat to public health." on february , parts per billion (ppb) of lead were detected in drinking water at a flint home and the federal environmental protection agency (epa) was notified. the epa does not require action until levels reach parts per billion, but science indicates that there is no safe level for lead in potable water. officials from epa and mdeq discussed the lead level in the sample, and epa found that the state was testing the water in a way that could profoundly understate the lead levels. on march , , a second testing detected ppb of lead in flint drinking water. a consultant group hired by flint, reported that the city's water met state and federal standards, and it did not specifically report on any lead levels. in may, tests revealed high lead levels in two more homes in flint. in july, an epa administrator told flint's mayor that "it would be premature to draw any conclusions" based on a leaked internal epa memo regarding lead. however, in september, flint was asked to stop using the flint water supply or consider corrosion control for it, because it was causing lead to leach from the water pipes and children had high levels in their blood. state regulators insisted the water was safe. nevertheless, on october , the governor of michigan ordered the distribution of filters, the testing of water in schools, and the expansion of water and blood testing after a briefing on the lead problems with the mdeq and federal officials. at the same time, flint city officials urged residents to stop drinking water. on october , flint reconnected to detroit's water system, and residents were advised not to use unfiltered tap water for drinking, cooking or bathing. on october , the director of mdeq reported that his staff had used inappropriate federal protocol for corrosion control, and soon after, the governor announced that an independent advisory task force would review water use and testing in flint. on december flint added additional corrosion controls, and soon after an emergency was declared. at the end of december, the task force found that the mdeq was accountable for its lack of appropriate action, and the director resigned. on january , , the governor asked the national guard to distribute bottled water and filters in flint, and president obama declared a state of emergency in the city and surrounding county, allowing the federal emergency management agency to provide up to $ million in aid. three days earlier the crisis expanded to include legionnaires' disease, because of a spike in cases, including ten deaths, after the city started using river water. on january , the michigan department of health and human services stated it did not have enough information to conclude that the increase in cases was related to the ongoing flint water crisis, although the. head of michigan's communicable disease division had said three months earlier that the number of legionella cases at that time "likely represents the tip of the iceberg." as of february , the number of reported cases was close to . a flint hospital official was surprised that michigan and local health agencies did not inform the public about the legionnaires' outbreak in genesee county in - until january ; the hospital earlier had spent more than $ , on a water treatment system and bought bottled water for patients. the source of legionella is not known but it was likely in the flint river, and possibly extensive flushing of flint's colored water, which had undesirable odors and tastes, by residents may have caused chlorine residual in the pipes to be washed away, leaving the pipes susceptible to growth of the legionella; in addition, aerosols from the extensive flushing from turned-on faucets might have led to close contact between the residents and the pathogen. the investigation of the cause of the illnesses continues with criminal charges laid against michigan departmental employees. observe treatment processes from the water inlet to the finished water discharge. diagram on form g the treatment process; insert notations of hazardous situations that were observed. collect samples of water at the inlet, after each phase of treatment that may have functioned suboptimally or failed, and at the outlet. test the samples for pathogens that cause a syndrome characteristic of that being investigated, for indicator organisms and for physical and chemical characteristics of the water, as appropriate to the situation. evaluate effectiveness of the disinfection process and resulting residuals. determine the type of disinfectant (e.g., gaseous chlorine, hypochlorite, chlorine dioxide, chloramine, ozone, ultraviolet irradiation) used and whether the disinfection treatment was adequate for the volume of water treated. determine, by talking to water treatment plant employees and reviewing records of the plant or regulatory agency, whether there were any interruptions of disinfection during the two weeks prior to the first onset date. determine contact time between the point of addition of the disinfectant and the first point of use. measure the chlorine residual, ph and temperature of the water just before it leaves the plant. observe the condition, operation, and maintenance of disinfectant dispensing equipment. review plant records to identify any sudden changes in disinfectant demand that causes temporary depletion of disinfectant residuals and allows survival of pathogens. review maintenance records for disinfectant dispensing equipment and quality assurance records for online analyzers. record this information on form g a (disinfection failures that allowed survival of pathogens or toxic substances). calculate disinfectant rate applied and usage (see formulae in form a). for example, to calculate disinfectant rate, if the flow rate is , , gal/day and the dosage is lb/day: the destruction of pathogens is dependent on (a) type and condition of microorganisms present, (b) type of disinfectant used, (c) concentration of available chlorine or other disinfectant, (d) contact time, (e) water temperature, (f) ph, (g) degree of mixing, (h) presence of interfering substances (which may be related to turbidity). utilize treatment records that provide small scale time resolution, such as online monitoring data, to determine whether the process was stable during the time period in question. daily averages may provide evidence of massive failures, but will not provide information about whether consistent treatment was being provided. in general, the relative effectiveness of microorganisms' resistance to free chlorine, from high resistance to low resistance, is as follows: • protozoan oocysts (i.e., cryptosporidium) • protozoan cysts (i.e., giardia, entamoeba histolytica) • viruses (hepatitis a virus, poliovirus) • vegetative bacterial cells (shigella, escherichia coli) protozoan oocysts are highly resistant to chemical disinfectants, but not to physical means such as uv light or ozone (gas). microorganisms within each group and strains among the same species differ somewhat in resistance. the state of injury induced by environmental impacts and selection of resistant strains influence survival. aggregation of microorganisms and/or close association with debris shield them to various degrees from lethal effects of disinfectants and attachment to surfaces such as pipe walls to form biofilms that protect organisms from inactivation by disinfectants. a measurement of microbiological inactivation by disinfectants is the ct value (ct calc ), which is the product of the free residual disinfectant concentration (c) in mg/l that is determined before or at the first user (customer) and the corresponding disinfectant contact time (t) in minutes (i.e., c × t). refer to table i (ct . values for inactivation of giardia cysts at different concentrations of disinfectants, temperatures, and ph values) and table i (ct values for inactivation of viruses at ph - , at different temperatures with different disinfectants for comparing disinfectant efficiencies). make residual measurements during peak hourly flow. for comparisons of ct values between the indicated ph, temperature, and concentration values, use linear interpolation. (for example, for free chlorine, °c, concentration mg/l, ph . = [ − = ; / = ; + ] = ). if no interpolation is done, use the ct . value at the higher temperature, at the higher ph and higher concentration. a simple ct calculation, for example, using a disinfectant concentration (c) at the basin effluent of . mg/l and a detention time (t) of min, is as follows: use this calculation for comparing to values in table i or j. the calculated ct value should be higher than the value stated in the table for specific conditions of disinfection, temperature, ph, and concentration (residual). in this situation, if the temperature of the water was °c, the ph and the concentration of chlorine mg/l, a ct value of would be needed for a . reduction of giardia cysts. the ct value of . would have been inadequate to meet the criteria and could explain the survival of the pathogen under investigation. microbial inactivation efficiencies vary considerably among different disinfectants and are influenced by the characteristics of the water and water temperature. tables i and j show that, in general, ozone is more effective than chlorine dioxide, which is more effective than free chlorine, which is more effective than chloramines. also, in general, longer contact time increases the degree of inactivation, and higher water temperatures as well as lower ph values increase rates of inactivation. rapid mixing of the disinfectant with water increases disinfection efficiencies, whereas dissolved organic matter reacts with and consumes the disinfectant and forms products that have weak or no disinfection activity. certain inorganic compounds and particulate matter also react with disinfectants. the ct value must be determined sequentially whenever a disinfectant is added to water. contact time (t) is the duration in minutes for water to move from the point of application of the disinfectant or the previous point of residual disinfectant measurement to the point where residual disinfectant concentration (c) is measured. it is measured from the first point of disinfectant application and from all subsequent applications until or before the water reaches the first user. determine contact time in pipelines by dividing internal volume of the pipe by the maximal hourly flow rate through the pipe. determine the flow rate from (a) plant records, (b) continuous monitoring device readings, (c) measurements at hourly intervals, or (d) if this sort of information is unavailable, measurements at expected high flow periods. use tracer studies to determine contact time within mixing basins and storage reservoirs. these values represent only % effectiveness because of short circuiting. chlorine, fluoride, and rhodamine wt (but not b) are commonly used tracer chemicals. contact time is usually measured by a step-dose method, but a slug-dose method is used where chemical feed equipment is not available at the designated point of addition or where such equipment does not have the capacity to provide the necessary concentration. (see appropriate epa literature for procedures, and consider getting engineering expertise if these matters are too complex.) estimate whether pathogens had been inactivated. to do this, divide the ct calc value by a value (ct x% ) resulting in a certain percentage inactivation (e.g., . % [ -log] or ct . for giardia cysts and . % [ -log] or ct . for viruses). this gives an inactivation ratio. see table i for ct . values for giardia and table j for ct . values for viruses. following is a sample calculation for data in table i when water temperature is °c, ph in a clearwell (reservoir for storing filtered water) is . , time (t) (either calculated or measured by dye test) is min, and the disinfectant used is chlorine: the desired ct value for . % inactivation of giardia for ph at °c is between and depending on concentration of disinfectant. in this case, the disinfectant measured at the clearwell outlet is . mg/l. therefore, the result, , is larger than the value, , required in the table; hence, these disinfection concentration (c) and time (t) conditions should result in a . % or greater inactivation of giardia cysts. for free chlorine, a -log inactivation of giardia cysts provides greater than a -log inactivation of viruses. the following example, using the data in table i , demonstrates a means to calculate the increased disinfectant dosage needed for a plant during the transition from summer to winter, when the water temperature fell from to °c, chlorine dioxide was the disinfectant used and the t value (calculated or measured) is min. using table i in this situation, the plant should have increased the chlorine dioxide concentration from . mg/l to . mg/l to maintain the same efficiency of disinfection. if this had not been done, it may explain the survival of pathogens in the water supply. the sum of these ratios gives the total inactivation ratio, which should equal or more to provide effective disinfection. make calculations and record information on form g a. the following example shows the way this is done. chlorine is added to three basins. chlorine concentration, contact time, temperature and ph are measured at these locations and recorded as shown in table . data from table i is combined to do the calculation. the resulting sum exceeds . . this ensures that the plant met the recommended or required ct. regulations may require that community and non-community public water systems that use surface water or water under direct influence of surface water meet a criterion (e.g., minimum of . % [ -log] removal and/or inactivation of giardia cysts and a minimum of . % [ -log] removal and/or inactivation of viruses of fecal origin that are infectious to humans). removal and/or inactivation of microorganisms may be accomplished by either filtration plus disinfection or disinfection alone, depending on the water source. water systems using chlorine with ct values that attain minimal level or inactivation of giardia cysts will result in inactivation of . % ( -log) of viruses. evaluate the prefiltration processes (e.g., coagulation, flocculation and sedimentation). coagulation is a process that uses coagulant chemicals and mixing, by which colloidal and suspended materials are destabilized and aggregated into flocs. flocculation is the process that enhances agglutination of smaller floc particles into larger ones by stirring. sedimentation is the process by which solids are removed by gravity separation before filtration. observe whether these processes reduce turbidity. calculate detention (transit) time within the settling tank and seek information about frequency and method of cleaning the tank. for example, if an -ft-deep sedimentation basin has a volume of million gal, and the plant flow rate is million gal/day, detention time in the basin is: (in your country you may want to calculate rates based on metric measurements) several different types of filtration may be used in water treatment facilities. these are conventional, direct (both conventional and direct are referred to as "rapid" filtration), slow sand filtration, and diatomaceous earth filtration. conventional filtration consists of a series of processes including coagulation, flocculation, sedimentation, and filtration. direct filtration consists of a series of processes including coagulation and filtration but excluding sedimentation. slow sand filtration is a process involving passage of raw water through a bed of sand at low velocity (usually less than . m/h), utilizing both physical and biological means to remove particles and microorganisms. in diatomaceous earth filtration, water is passed through a precoat cake of diatomaceous earth filter medium while additional filter medium is continuously added to the feed water to maintain the permeability of the filter cake. if done properly, each filtration method results in substantial particulate removal. when rapid sand filters have a head loss of about - ft, they require back washing. filters are backwashed by reversing the flow of the filtered water back through the filter at a rate between and gal/min/ft of sand-bed area. sometimes water jets at the surface aid in loosening and removing deposited material on the sand. observe an actual backwash and look for indications of short-circuiting or areas of the filter material that seem agglomerated or resist being cleaned by the flowing water. if backwash water is not discharged to waste, evaluate where it is released. slow sand filters eventually become clogged. when this occurs, a scraper or flat shovel is used to remove the top layer of clogged sand, and new sand (equivalent to the depth removed by scraping) replaces the old. test the effectiveness of filtration for each filter unit by observing capacity and filtering area relative to volume and turbidity of the filtered water. also, review turbidity, headloss, and filter rate record. look for anomalies, especially in the few hours after a filter is returned to service, and before the filter is backwashed. review criteria that cause a backwash to be initiated, and establish if these criteria were followed during the time preceeding the outbreak. determine the source of backwash water and the frequency of back washing of filters from records and head gauge readings. check whether the water used to back wash or clean filters came from an untreated source and determine the fate of the backwash water. in the case of illness due to chemical substances, evaluate types of chemicals used and condition, operation and maintenance of chemical feeding equipment. consider sampling backwash water for pathogens under investigation. review plant records for results of monitoring and be alert for changes that suggest treatment failure. record this information on form g b (source of contamination and treatment failures that allowed survival of pathogens or toxic substances.) data in table k (estimated removal of giardia and viruses by various methods of filtration), give a summary of expected minimal removal of giardia and viruses in well operated filtration systems. values can be subtracted from ct values required for disinfection. although contamination is likely to be associated with raw incoming surface water, look for bypass connections where raw or partly treated water can get into treated water. also look for common walls that separate treated and untreated water. consider the possibility that a contaminant was introduced in any of the treatment chemicals themselves, or as an act of sabotage. determine whether any flooding has occurred during the interval of concern. check absentee records for possible enteric illness of the water treatment plant staff. such illness may reflect either sources of contamination or victims. record this information on form g b. at domestic locations, evaluate treatment devices (such as chlorinators, filtration units, softening equipment) as described above, but modified to fit the situation under investigation. record observations and measurements on forms g a and g b, as applicable. the water distribution system can be complex, with multiple entrance points for treated water and different pressure zones in which water can enter but not leave. water flows in the direction in which it is being "requested," so can flow in different directions in the same pipes from one hour to the next. contaminated water can enter a potable water supply from a non-potable water supply when the two are directly connected. such interconnections are referred to as cross connections. to evaluate such situations, trace lines of the treated supply from the point of treatment or entrance into a building to points of use and associated plumbing. look for any interconnections of other water supplies, such as wells, waste lines or holding tanks for water intended for fire control. if cross connections are found, look to see whether backflow prevention devices are inserted between the lines and, if so, whether they are functioning properly. also, look to see whether there is an air gap between the water inlet and vessel or tank. evaluate the arrangement and operation of check valves on connections between the two water systems. review inspection report for backflow prevention devices. contaminated water can also enter a treated supply by siphonage from a contaminated vessel or sewerage to the potable water line having negative pressure. this is referred to as back siphonage. examine all water vessels to see whether they contain submerged inlets or hoses connected to water faucets, and if so, whether properly functioning vacuum breakers are in place. without proper air gaps or properly functioning vacuum breakers, there is a possibility of siphonage of water from plumbing fixtures in upper stories to lower stories when line pressure is negative. this may occur when faucets on lower floors are opened after the water supply valve has been turned off for repairs or when the supply line has had a sudden loss of pressure, as can happen with nearby heavy use of water (e.g., to fight fires or irrigate) or when pressure lines are broken. measure water pressure on upper stories of buildings to determine whether negative pressure occurs. (pressure losses may be transient and of very short duration.) interview building managers and residents about whether there were (a) any repairs of water service during the past month, (b) fires that occurred nearby, or (c) other situations that could have caused negative pressure in the water line. also, if appropriate, review fire and utility department records for information about these situations. get dates of line repairs to see whether they correlate with the time of incubation periods of early cases. measure chlorine residual (of chlorinated water systems) and take samples for microbiological tests at several strategic locations in the distribution systems. perform calculation on comparison of disinfectant residual. if a toxic chemical poisoning is under investigation, talk to home owner, building manager or maintenance staff about whether pesticides or other toxic compounds were sprayed with equipment connected to a hose or a sprinkler system. furthermore, interview building managers and residents about whether there are persons residing there who either are or recently were ill with diarrhea. they may represent sources of the etiologic agent or may identify victims. interview those identified about the onset of their illness and symptoms and examine their plumbing systems. record information obtained during the investigation of distribution and plumbing systems, and record related calculations on form g (source and mode of contamination during distribution and at point-of-use). evaluate implicated waters used for swimming, water skiing, bathing, clothes washing by hand, or agricultural activities, in a manner similar to that described under the section on investigation of surface water source. if the potential site of contact was natural surface water, determine whether the water is likely to be infested by parasites and look for the presence of snails (swimmer's itch). for swimming pools, measure the water's ph, chlorine residual, water temperature, and turbidity, if applicable. also, review pool records for previous information on these characteristics. high turbidity in pools, hot tubs, and spas is a sign of either poor filtration or inadequate disinfection. evaluate whether the resulting water would adequately protect those who swam or waded in it or had any physical contact with it. evaluate filter and chlorination equipment as described for water treatment. backwash filters and collect a sample to get an indication of microorganisms present on the filter (thus obtaining historical information). this approach has been useful for identification of pseudomonas aeruginosa. look for the presence of slime on tub, whirlpool, slide and pool surfaces, and collect some of this material for analysis for p. aeruginosa. if the answer is not obvious, ask ill persons whether they had puncture injuries or wounds or scrapes while immersed in water. record this information on appropriate parts of form g (contamination source and survival of pathogens or toxic substances for recreational waters). collect samples of the water (see section on "collect water samples"), and test them for pathogens and/or indicator organisms, as applicable. the agents listed in table d can multiply in water and if such water is aerosolized, they can be transmitted to human beings via the respiratory route. highly susceptible persons (e.g., the elderly, smokers, immunosuppressed individuals) are the usual victims. look for possible sites where water may have been or is being disseminated as aerosols. consider (a) air conditioning cooling towers and evaporative towers, (b) hot water systems (heaters and tanks), (c) shower heads, (d) faucets with aerators, (e) mist machines used to freshen fruits and vegetables in markets, (f) humidifiers, (g) nebulizers/respiratory therapy equipment, (h) whirlpools and spas, (i) dental drills and cleaners, (j) cooling water apparatus for grinders, (k) splash from hoses, (l) water pressure line breaks, (m) decorative water features, (n) outside misters, (o) other aerosol-producing devices. sample water from all suspect sites for legionella or other waterborne agents that may cause illness when inhaled. it is not possible to recognize by visual inspection the potential for water to be contaminated with legionellae. warm temperatures, especially those between °c ( °f) and °c ( °f), are conducive to growth of legionellae. additionally, stagnant water allows time for legionellae to multiply, especially in dead-end lines, reservoirs and hot water tanks, and in water trapped in shower heads and faucet aerators. if it is deemed appropriate or necessary to sample for detection of legionella in the environment, collect water samples from suspect sources. it is important to use a lab with proven expertise in isolating and characterizing legionella, such as those labs in the u.s. certified under the environmental legionella isolation techniques evaluation (elite) program. the centers for disease control (cdc) have a convenient form for recording case histories (http:// www.cdc.gov/legionella/downloads/case-report-form.pdf). it is not appropriate to sample air for detection of legionella hazards. it may, however, be appropriate to use micromanometers or smoke to trace direction of air flow to determine route of dissemination. micromanometers measure pressure differences, and flow can be assumed to travel from high to low pressure areas. smoke moves from areas of higher pressure to areas of lower pressure and is extremely sensitive to air currents. observe direction and spread of smoke movement. record this information on form g (contamination source and sites of amplification and aerosolization of pathogens). prior to the collection of samples, investigators should consult with the testing laboratory that will be used, to receive specific laboratory sampling instructions and sampling kits. sampling protocols for potable and non-potable sources are dependent on the specific etiological agent and the related analytical procedures performed by the testing laboratory. collect samples promptly to test for possible etiologic agents and for microorganisms indicative of fecal contamination. contaminants in water are in a dynamic state; their presence and quantity differ with time and place. see table f (general instructions for collecting drinking water samples) for guidance on collecting and shipping samples for viral, bacterial, and parasitic analyses. samples for bacteriological tests can be collected in one of three ways: (a) by letting a stream of water flow into a container or by submersing a container into a volume of water, (b) by passing a large volume of water through a filter, (c) by putting moore swabs (see table a for description) or similar absorbent materials in surface water or drains for a few days (see table f ). use bottles that have been cleaned, rinsed, and sterilized, or use sterile plastic bags to collect and store samples for bacteriological examination. for a chlorinated water supply, or when in doubt about the presence of residual chlorine, use bottles containing mg/l sodium thiosulfate to combine with any free chlorine in the sample and prevent lethal effects of chlorine on microorganisms in the sample. this compound will not interfere if used for non-chlorinated water. when collecting water samples, first try to get "historical" samples that might give an indication of the condition of the water at the time it was ingested by those who became ill. obtain historical samples from water in bottles in refrigerators, toilet tanks, hot water tanks (for chemical analyses only), fire truck reservoirs, storage tanks, and taps at seldom-used and dead-end locations, and from ice in refrigerators and commercial ice plants. direct the laboratory to test historical samples for pathogenic organisms or toxic chemicals, as well as indicator organisms, because these samples have a chance of still containing the etiologic agent, whereas samples collected during the investigation several days or weeks after the event may be of water that has been flushed free of contamination or has been significantly diluted. take samples from to points throughout the distribution system. sample dead-end locations if they are found. do not neglect to obtain raw water samples even though treatment is provided. this is important, as it suggests possible sources of contamination and reflects the effectiveness of treatment. compare these test results with records of results on previous samples of raw or treated water. before drawing a sample from a water tap, make sure the tap is connected to the supply to be tested. do not collect samples (other than for legionella) from hose connections, sprays, or swivel faucets; uncouple these connections or choose different outlets. it is unnecessary to flame outlets, as this does not improve the quality of the sample. first, ensure your hands have been thoroughly washed then take a line sample by allowing the water to run to waste for - min. adjust the flow of water so that the thiosulfate will not wash out of the bottle or bag (do not overfill-most laboratory bottles indicate a maximum fill line). keep sample containers closed until the moment they are to be filled. hold the bottle near the base, fill to the "fill line" or within an inch of the top without rinsing, and immediately replace the stopper or cap and secure the hood, if attached. if a whirl-pak™-type plastic bag is used instead of a bottle, hold the base, rip off the perforated top, open the bag by pulling the side tabs apart, grasp the end wires, and place the bag under the flowing water. remove the bag before it is completely filled and squeeze most of the air out; fold over the top of the bag several times and secure by twisting the end wires. take a source or a distribution line sample by opening the tap fully and letting the water run to waste for sufficient time to empty the service line (or if in doubt, for min) and proceed as above. collect samples from open shallow wells and step wells by dropping a clean wide mouth container on a string or rope into the well. allow the container to sink below the water surface and then pull it out of the well. pour contents into a sample jar or bag. collect samples from rivers, streams, lakes, reservoirs, springs, toilet tanks, and non-pressurized storage tanks by holding a ml sample bottle near the bottom and plunging it neck down to a depth of cm ( in) below the surface; turn it right side up, and allow it to fill. don a plastic disposable glove when small vessels used for drinking are sampled in this manner. when collecting these samples, move the bottle in a sweeping, continuous, arc-shaped motion, counter to stream flow or in a direction away from the hand. collect samples at locations approximately one-quarter, one-half, and three-quarters the width of the stream or water course. special apparatus can be used for sampling at various depths. samples can then be taken by positioning large bottles on a rod or pole at the desired depth and location before pulling their stoppers with a wire, string or thin rod. samples of bottom sediments are sometimes useful for the detection of certain pathogens. collect surface scum or regions containing dense particulate colored material when seeking cyanobacteria (blue-green algae). collect slime, if present, when seeking pseudomonas. if large amounts of water are needed, seek assistance and obtain specialized sampling equipment from agencies responsible for water quality. if possible, avoid wading when sampling bodies of water because wading often stirs up bottom sediments. if this is the only way to get a sample, however, wade against any current (e.g., upstream in creek or river) and keep moving forward until sample taking is completed. piers or similar structures, or the front end of a drifting or slow moving boat, make good sampling stations. concentration of bacteria by the use of swabs, filters, or by absorption, is particularly important when waterborne pathogens are sought. to concentrate bacterial pathogens from flowing water (e.g., streams, lakes, sewer lines, or drains), suspend moore swabs (or non-medicated sanitary napkins or non-medicated tampons if moore swabs are unavailable) for - days. these can be held in place by wire just below the surface or at other depths. if rodents are about, put moore swabs in wire baskets. after the sampling period, either put swabs or pads into a plastic bag and pack in ice, or put the swabs or pads directly into an enrichment broth for the pathogen sought. take or send these to the laboratory promptly. concentration of microorganisms can be increased by filtration with a variety of filters (e.g., membrane filters, cartridge filters, or other filter media). when membrane filters are used for pathogenic bacteria recovery, pass at least l of water (relatively free of turbidity) through a sterile . μm membrane filter. for viral analysis, use virus-absorbing electropositive cartridge filter to concentrate l or more water (see table f ). keep filters cool (but not frozen) and ship to a reference laboratory for further processing. for giardia cysts and cryptosporidium detection, collect samples by passing at least l water through a cartridge filter (see table f ). for inorganic chemical analyses, use l polyethylene containers. these should be new, or acid-washed if previously used. collect the water without flushing the lines, preferably in the early morning before water is used. for trace metal analyses, preserve one sample with ml of high-grade nitric acid to a ph of or less. this is particularly important whenever it is suspected that metals may have leached from water pipes or vessels. for organic chemical analysis, use l glass containers with teflon-lined caps. clean and rinse the containers with a good quality laboratory solvent and heat at °c for min. rinse the cap thoroughly with distilled water. fill the container so that there is a minimum of air space. for physical analyses, collect at least l, or other amounts requested by the laboratory. collect ice aseptically in sterile plastic bags or jars. use sterile tongs to collect cubes; sterile spoons for collecting chipped or crushed ice; and sterile chisel, hammer, or pick to chip block ice. put block ice or large chips into plastic bags. if legionella is sought, sample water at sites of any source that may have been aerosolized and send to a lab with proven expertise in legionella isolation and characterization, such those in the cdc elite program. this includes cooling towers, evaporative condensers, water heaters and holding tanks, humidifiers, nebulizers, decorative fountains and whirlpool baths (see section on investigating sites where aerosols are disseminated for a more complete listing). turn off fans of condensers before sampling; if this is not possible, wear a respirator. use ml to l polyethylene bottles that have had sodium thiosulfate added if the water to be tested has been chlorinated. for each sample, don disposable plastic gloves and collect the sample by inverting the bottle and moving it in a continuous arc away from the hand. measure and record water temperature. handle samples as described in table f . rub swab over faucet aerators and shower heads if these are considered as sources of aerosols. break stick and allow tip to fall in a tube containing - ml sterile water (not saline). investigators are often requested to test air to demonstrate the presence of legionella in aerosols. although legionellosis is an airborne disease, legionellae are susceptible to low humidity and become non-viable on drying. therefore, air sampling is an ineffective and inefficient way of determining whether a legionella hazard exists, and it can thus be misleading. label each container with sample number, date, time of collection, and your name or initials. complete the water/ice sample collection report, form f, for the first sample. list additional samples with sample numbers and other pertinent information on the back of the form. in those situations where the laboratory needs additional information, attach the appropriate g series forms. send the original form f and list with samples to the laboratory; retain a copy for your files. inform the laboratory of the type and number of samples and specimens; also, consult with the laboratory on methods to preserve and transport samples, if necessary, and on time of their arrival. if legal proceedings are anticipated, deliver sample personally to the analyst, or seal the sample container in such a way that it cannot be opened without breaking the seal. note on form f the method by which the bottle was sealed. maintain a chain-of-custody log to document the handling of the sample, and have the log signed and dated each time it changes hands. consult with state/provincial regulatory agency on complying with legal requirements for chain-of-custody procedures. recipient should record on the form whether the sample was sealed when the laboratory received it. if analysis cannot be done on the day of collection, chill water samples rapidly and hold them at temperatures at or below °c ( °f), but do not freeze, because populations of bacteria such as escherichia coli and of parasites decrease during frozen storage. hold ice samples frozen; if this is not possible, keep the temperature below °c h ( °f). investigators should consult with the testing laboratory that will be used to receive specific laboratory sample packaging, labeling, and transportation instructions as protocols are dependent on specific transportation regulations (iafta, tdgr) within each jurisdiction. ensure each sample is uniquely identified and labeled (as per the receiving laboratories requirements). many laboratories include barcode labels along with the sample containers within the sample collection kits. ensure that the correct label is affixed onto the correct sample container and that this information is transferred to the shipping manifest accurately (chain of custody form). specimens should be packed and the packages labeled according to applicable regulations governing transport of hazardous materials. generally, the transport of samples of water and ice intended for laboratory analyses are packed and shipped in a manner to ensure the sample does not change from the time of sampling to the time received by the testing laboratory and shipped using the most expeditious means (e.g., personal delivery or overnight mail). typically samples of water or ice are packed with refrigerant (ice packs, dry ice, etc.) in insulated and sealed containers (see table f ). several measurements are routinely called for during on-site investigations. brief instructions are given for those that are commonly done; nevertheless, follow manufacturer's instructions if these are available. color comparison kits are available for testing for free, combined and total residual chlorine. the diethyl-p-phenylenediamine (dpd) test is an example (see table a ). check instrument calibration regularly. use dry reagents, because the liquid forms are unstable. chlorine comparators can be used to test for bromine by multiplying the result by the factor . and to test for iodine by multiplying the result by the factor . . measure temperature. measure water temperature by immersing the sensing end of either thermocouples, transistors, or thermometers into the water. sometimes measurements need to be made at various depths; use thermocouples with wire leads of sufficient length for this purpose. calibrate temperature measuring devices periodically. measure ph. calibrate the ph meter as recommended by the manufacturer with at least two standard buffers (e.g., ph . or . ) and compensate for temperature, if the meter does not do it automatically, before each series of tests. remove a sample of water to be tested and immerse the ph electrode into the sample; record the reading. ph can also be measured by color comparators that employ color indicator solutions or discs. (ranges of ph color indicator solutions are bromophenol blue, . - . ; bromocresol green, . - . ; methyl red, . - . ; bromocresol purple, . - . ; bromothymol blue, . - . ; phenol red, . - . ; cresol red, . - . ; thymol blue, . - . ; and phenolphthalein, . - . .) in this case, water containing more than mg/l chlorine in any form must be dechlorinated with sodium thiosulfate before the ph indicator solution is added to prevent decolorization of the indicator. always report temperature at which the ph is measured. measure turbidity. nephelometric turbidity unit (ntu) is the usual standard unit, but other turbidity measurements (such as particle counts) are used. the ntu requires a nephelometer, which measures the amount of light scattered predominantly at right angles and absorbed by suspended particles (e.g., clay, silt, finely divided organic matter, inorganic matter, soluble colored organic compounds, and microscopic organisms) in the water sample. calibrate turbidimeters with a standard reference suspension. make turbidity measures on the day samples are taken. vigorously shake samples, wait until all air bubbles have disappeared, and then pour sample into turbidimeter tube. read directly from scale on instrument or from an appropriate calibration scale. pump chemical smoke into the air at the exit of the device suspected of releasing aerosols. observe the direction and spread of the smoke. otherwise, measure pressure differentials with a micromanometer. measure other attributes of water. follow instructions given by manufacturers or in standard reference books (see further reading). use fluorescein dye, lithium or other tracers in appropriate soils to determine the means by which contamination from sewage, industrial wastes, or other sites of pollution reached the water supply. fluorescein dye is particularly helpful in evaluating flow of contamination through fissured rock, limestone, gravel, and certain other soils. this dye is not readily absorbed or discolored by passage through these soils or sand, as are many other dyes, but it is discolored by peaty formations or highly acid (ph < . ) soils. make a concentrated fluorescein dye solution by mixing g of fluorescein powder into a liter of water. usually, / to l of this solution are sufficient for the test for up to , l of water. fluorescein dye is also available in liquid and tablet form. one tablet will dye approximately l (~ us gal). pour the calculated amount of fluorescein solution or put a sufficient number of fluorescein tablets into a receptacle at a point of potential pollution. usually this point will be located within yards and at a higher elevation than the water source under study. cesspools, latrines, distribution boxes, sink holes, borings, septic tanks, drains, manholes, toilets and plumbing fixtures are typical places to introduce the dye. if dye is poured into a plumbing fixture or dry hole or boring, add water to wash it down. the amount of dye to use varies with the distance the dye must travel, the expected time of the journey, the size of the aquifer or water channel, and the nature of the soil. take samples of the water when the dye is introduced into the test hole or fixture and then hourly for up to h to detect arrival and departure of fluorescein. if no dye is observed, repeat the test with twice the amount of dye. whenever possible, use a fluorescent light or fluorometer to analyze water samples for evidence of fluorescein. a fluorometer can be set up and calibrated, and a continuous recording can be made. this meter can detect fluorescein in concentrations of μg/l (ppb). fluorescein dye will temporarily color water, which discourages use of the water until the dye is sufficiently degraded or diluted. alternate tracers can be used if specific ion meters are available. the dye stains all it touches. methanol is a good solvent for the dye, and hypochlorite solutions decolorize it; both can aid in removing stains. abrasive soaps are useful for cleaning stained skin; fluorescein-stained clothing should be washed separately. appearance of dye in a water supply is conclusive evidence of seepage from the site where the dye was introduced. failure to detect dye, however, is not conclusive evidence that seepage did not or would not occur if more dye had been added or if weather conditions or subsurface flow had been different at the time of the test than during the outbreak event. illustrate source and direction of contaminated water flow as indicated by the dye test on form g . take photographs of sources of contamination and evidence of staining of the ground at the site or dye-stained color of the water. in situations where a single source of contamination is obvious or where multiple sources are readily apparent, dye studies serve little purpose. drinking water, however, is not the only source of water that may contribute to outbreaks. other sources of water that can contribute to outbreaks include water not intended for drinking, recreational water and water used in agriculture during harvesting and packaging. legionnaires disease is the pneumonia caused by the inhalation of contaminated water aerosol containing the bacteria legionella, with legionella pneumophila being responsible for % of all infections. it is also a common cause of healthcare associated pneumonia. legionella can replicate within free-living amebae in water, allowing it to resist low levels of chlorine used in water distribution systems. risk of infection is more common in warm and humid weather, when water droplets are able to drift further due to higher absolute humidity. fifty percent of all legionella outbreaks have been traced to cooling towers with l. pneumophila serogroup responsible for all cooling tower outbreaks. all aerosol generating devices, however, can be potential sources of legionella. some other sources of aerosolization that may have contributed to or be associated with outbreaks include: whirlpool displays, building's air conditioning systems, water spray fountains, public bath houses, vegetable misting systems in grocery stores, evaporative condensers, showerheads, humidifiers, air scrubbers, car washes, ornamental and decorative fountains, potting soil, respiratory therapy equipment, dental units, road asphalt paving machines, car windshield washer fluid and car air-conditioning systems. in the investigation of a legionella outbreak, (see box , the flint water crisis, which describes a likely legionella outbreak from a commercial water source) due to the varied sources, there is a need to use a broad investigative questionnaire and the collection of environmental data. environmental factors such as dry bulb temperature, relative humidity and wind rose data can provide information regarding drift evaporation, deposition (settling) and the size of the affected zone. although aerosol drift can carry legionella up to mi ( km), the risk of infection is usually highest within ft ( m) of the source. there are also air dispersion models that can be used to determine drift zone and the use of human activity mapping in the identification of potential sources. in general, e. coli and norovirus are the most common pathogens responsible for recreational waterborne outbreaks associated with non-treated water such as beaches and lakes. cryptosporidium, which is resistant to chlorination, is the most common pathogen resulting in outbreaks in treated water venues such as swimming pools and water spray parks. it should be noted that e. coli, the indicator of choice of recreational water samples, is not indicative for the presence of norovirus and giardia, cryptosporidium. e. coli can also be "naturalized" and have been found to survive and multiply in beach sand. beach water sampling results therefore may provide false positive or false negative results and may not be the best indicator for the presence or absence of pathogens. recreational waterborne outbreaks are not just traced to the ingestion of contaminated water (table c . illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents). hot tub rash, or pseudomonas dermatitis/folliculitis commonly occurs in public hot tubs or spas such as those found in hotels. the rash is often a result of skin infection from the bacteria pseudomonas aeruginosa colonizing in the hair follicles after exposure to contaminated water. pseudomonas aeruginosa is an opportunistic pathogen that can survive within the biofilm on the tub surface or within the piping system. outbreaks can occur when there is a heavy bather load resulting in an increase in chlorine demand, which in turn reduces the effectiveness of the disinfectant to control the population of pseudomonas. blue-green algae or cyanobacteria bloom can occur in warm, slow-moving or still water. when conditions are favorable, mostly during hot summer weather, cyanobacteria populations may increase dramatically, resulting in a "bloom" as they rise to the surfaces of lakes and ponds. they resemble thick pea soup and are often blue-green in color. although blooms can occur naturally, water bodies which have been enriched with plant nutrients from municipal, industrial, and agricultural sources are particularly susceptible. some cyanobacterial species may contain various toxins, some are known to attack the liver (hepatotoxins) or the nervous system (neurotoxins); others simply irritate the skin. health effects from cyanotoxin exposure may include dermatologic, gastrointestinal, respiratory and neurologic signs and symptoms (table b . illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents). water can also be an indirect cause of foodborne outbreaks by providing a media for the survival, transportation and the introduction of pathogens into food products. water used during production, including irrigation, pesticides and fertilizers application and washing, frost protection, harvesting, has long been recognized by food safety scientists as one of plausible and probable sources of the contamination of fresh fruits and vegetables. there have been many outbreaks from produce traced to pathogens being introduced by contaminated irrigation water. although harvested products are sometimes washed with chlorine solution, pathogens may still survive the process through internalization. e. coli o :h may migrate to internal locations in plant tissue and be protected from the action of sanitizing agents by virtue of its inaccessibility. experiments have also demonstrated that e. coli o :h can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant. however, this claim has been refuted by others. salmonella and e. coli can also adhere to the surface of plants, and enter through stomata, stem and bud scars and breaks in the plant surface caused by harvesting and processing. water containing bacteria can be drawn into the produce if it is immersed in or sprayed with water that is colder than the produce itself. e. coli o :h may also use its flagella to penetrate the plant cell walls and attached to the inside of the plant. once attached, it may be able to grow and colonize the surface of the plant. the concerns are not just with bacteria. the present of norovirus in the hydroponic water can result in internalization via roots and dissemination to the shoots and leaves of the hydroponically grown lettuce. irrigation water may be contaminated from runoff from nearby domesticated animals and their lagoons, feedlots, ranches into rivers; from feral/domestic animals with direct access to creeks, ditches, rivers, ponds; from sewage flows into waterways and contaminated wells. in some parts of the world sewage contaminated water is preferred for irrigation despite a potential risk of transporting enteric pathogens, since it carries nutrients (n and p) for the plants. there is sufficient information to conclude that the application method of irrigation water to fresh produce can have an effect on the microbiological risks associated with the crop. in general, keeping water away from the edible parts of ready-to-eat crops that are consumed without cooking can result in a lowered risk of a foodborne outbreak. the least to more risky methods for irrigations for microbial contamination are: subsurface irrigation (buried soak hoses) < drip irrigation < indoor flood irrigation (hydroponics) < outdoors flood irrigation (water-filled furrows) < overhead irrigation (sprinklers). an outbreak of illness arising from exposure to water demands immediate epidemiological investigation to assess the situation, gather, evaluate, and analyze all relevant information, with the goal of ( ) halting further spread of this illness, and ( ) predicting, preventing, and/or attenuating future outbreaks. this twofold mandate of epidemiology is usually described as "surveillance and containment." at the commencement of an investigation, the unknowns usually outnumber the known facts. there is no substitute for prompt, thorough, and careful collection of interview data from ill and well persons who ingested or contacted the suspect water, attended a common event, or who were part of a group of persons where illness occurred. careful analysis of these data, particularly with reference to common patterns of "time," "place," and the characteristics of the persons involved, can often eliminate many vehicles, agents, and pathways quite early in the investigation, and focus on the remaining possible vehicles, routes, and agents. later, laboratory results may confirm the agent, the specific pathology, the route taken by the infection or toxic agent, and indicate what is needed to stop the spread, but early epidemiology can often be invaluable in predicting the outcome and taking preventive steps to contain the problem before the lab results are available. lessons can be learned from most outbreak investigations and are invaluable for increasing our understanding of these pathologies, and preventing their future occurrence. an outbreak is defined as either an unusually large occurrence of an expected illness at that time of year in that place, or the occurrence of a type of illness that does not usually appear at that season and location. the "time" factor should be studied immediately by plotting the onset time of each case on a time-based grid, to create the epidemic curve. although any number of cases can be involved, the minimum number for an "outbreak" to be declared is two associated cases, with special exceptions such as naegleria fowleri where, because of the severity and the possibility that cases may have been missed, a single case constitutes an "outbreak." although the epidemic curve is usually measured in hours or days, protracted exposure to agents in water may mean apparent sporadic cases linked to a common source over months or years. if an "outbreak" is suspected by a sudden increase of cases, determining who is to be categorized as a "case" is not necessarily a simple process. many people notoriously fail to report enteric illness for many reasons: embarrassment, lack of time, no clear idea which agency should be notified, mild self-treatable symptoms, or simply because they prefer not to make a fuss. they may therefore be incorrectly classified at least initially as "nonill." consider also that - % of the general population will have experienced some form of "upset stomach" in the last hours, regardless of exposure to the suspect item, and they may be incorrectly classified, at least initially, as "ill." to reduce the "false negatives" and "false positives" that are expected with self-reporting, the investigator needs to establish a working case-definition. a careful case definition categorizes people as "case" or "control" with the best accuracy possible within the time constraints and resources available. a case definition could be considered "too sensitive" if it classifies as a "case" a person who experiences: "… at least one episode of stomach cramps, nausea, vomiting, or diarrhea in the last hours." this would confuse subsequent analysis, and produce more false positives. similarly, a case definition could be considered "too specific" if it classifies as "not-ill" a person who had experienced only three episodes of diarrhea or vomiting, because they failed to satisfy a case definition requiring "…at least four episodes of vomiting or diarrhea in the last hours." should this last individual, having been declared as not fitting the case definition, be taken into the "notill" group, the error and subsequent analysis is confounded even further. a reasonable case definition therefore attempts to reduce both types of errors, and will depend upon the early indications of what the etiology may be. in the instance of a suspected salmonellosis, a case could be defined as "a person who was in good health before attending the event on monday may rd, and who experienced two or more of the following symptoms anytime up to midnight, sunday may th.: nausea, vomiting, stomach-cramps, diarrhea, headache, or fever." note that a case definition should include a place of exposure if known, a timeframe during which symptoms may have been experienced (salmonellosis has a range from to h. usually - h), and the additional footnote that the individual was not already symptomatic before the suspected "exposure." calculate the percentage of ill persons who manifest each symptom by dividing the number of persons reporting the given symptom by the number of cases ( for the example, table ) and multiplying the quotient by . the distribution of symptoms can be used to identify the most likely pathogen, and aids in requests to the laboratory for microbiological assays of samples and specimens. other symptoms (e.g., prostration, lethargy, weakness) may be included if deemed appropriate or helpful, but the six symptoms in table should always be included. headache, for instance, is associated with many viral infections (e.g., norovirus, rotavirus), but much less so with bacterial infections. fever is usually associated with an invasive bacterial infection (such as salmonellosis or campylobacteriosis), and is not usually seen in outbreaks of simple enteritis (such as with cholera). this information helps to determine whether the outbreak was caused by an agent that produced intoxication, an enteric infection, or generalized illness. in the example given, a predominantly diarrheal syndrome without much fever or headache tends to eliminate some of the viral infections (norovirus or rotavirus) or the host-adaptive/invasive serotypes of salmonella (e.g., s. dublin or s. choleraesuis). median onset time calculations may further reduce possible candidate etiologies. in historical investigations, or where no laboratory confirmation is possible, the symptom profile and onset times can sometimes predict the etiology of the outbreak within reasonable certainty. an epidemic curve (also called an onset curve or onset distribution) is a graphic illustration called a histogram that shows the distribution of the time of onset of first symptoms for all cases that are associated with the disease outbreak. paper printed with square "grid" lines will allow the investigator while on site to represent each case as a single "block." the horizontal axis is the sequence of intervals of time and date. the unit of time that defines the width of each interval depends on the characteristics of the illness under investigation. for example, intervals of days or weeks are appropriate for diseases with long incubation periods, such as cryptosporidiosis or hepatitis a. intervals of a day or half-day are appropriate for outbreaks of enterohemorrhagic e. coli strains or shigellosis, while single-hour, -h, or -h intervals will be more suitable for illnesses with shorter incubation periods, such as chemical poisonings. the vertical axis is always the actual count, or "frequency" of cases (blocks) stacked at each interval. it is often necessary to redraw the onset curve as more accurate information becomes available. if the illness is known, a rule of thumb is that the time interval used for each "block" on the x-axis should be no more than ¼ the incubation period of the disease under investigation. if the illness is not known, select an interval where the data produces a bell-shaped curve; not too flat and not too tall. construct this graph using time-of-onset data from forms c or d, employing an appropriate time scale. once all the onset times for the cases have been plotted on the histogram, determine the range as the interval between the shortest and longest incubation periods. in fig. a , the range is the day period from the th to th march. the median onset time is preferred to the mean because the latter is vulnerable to a few or even a single very small or very large value. the median on the other hand, is the midvalue of a list of all individual onset times, including duplicate entries, that are ordered in a series, from shortest to longest. if the series comprises an even number of values, the median is the mean of the two middle values. most standard reference texts on communicable diseases give onset times as median values. the mode is simply the interval having the largest number of observations. a distribution with a single "peak" is called a uni-modal distribution, while an outbreak with two peaks is called "bi-modal." subsequent modal peaks following the first may indicate either a "secondary wave" of cases or the exposure of other people at a later time. the shape of the epidemic curve helps to determine whether the initial cases originated from a single point-source exposure (such as water or food available for only part of a day), or from repeated exposures for a longer time, or even more gradual person-to-person spread. a point-source epidemic curve is characterized by a sharp rise to a peak, followed by a fall that is almost as steep ( fig. a ). an "explosive" outbreak of this type is common where a municipal water supply is the vehicle, affecting large numbers of people in a very short period of time, but without secondary cases occurring, or any evidence of onward spread within the community. propagated outbreaks are those in which the initial victims ("primary cases") manage to spread the agent to other people ("secondary cases") such as family members, patients, clients, or other contacts in crowded places through aerosols, personal contact, or contaminated water/food/utensils/surfaces, etc. propagation following a point-source exposure is demonstrated by a second increase in reported cases following the decline of the first cluster. sometimes this takes the form of a second "modal peak" separated by approximately one incubation period, but this distinction is soon lost. figure a shows no evidence of propagation; fig. b suggests that propagation may have taken place, although care must be taken to consider other explanations. in addition to ( ) true propagation, where the secondary wave can be expected to appear one incubation period after the first, secondary waves may be also explained by ( ) exposure to the same point source (e.g., food or water supply) at different, but specific times by other people; this might be a repeated offering of contaminated food or water at two or more mealtimes; ( ) a second pathogen (perhaps from the same unhygienic food or water source) which may have a different symptom profile and a different (incubation) time. slow propagation from the beginning of an outbreak with neither an obvious point-source, nor any distinctive "waves" separated by an incubation period as in fig. c , usually indicates one-at-a-time person-to-person spread through closecontact, poor personal hygiene, aerosol (e.g., influenza, or sars), or sexual transmission (e.g., hiv/aids). it can also be explained by (non-propagated) continuing exposure, for example drinking of contaminated surface water following a conflict, natural disaster or other breakdown of infrastructure. as such it is commonly associated with waterborne cholera, shigellosis, typhoid fever, or e. coli infection, and characterized by scattered cases which continue until the chain of infection is cut. slow, constant and/or intermittent exposure to persons over time to pathogenic microorganisms can also result from sewage run-off after a series of heavy rainfalls. in addition to revealing whether the outbreak was due to a single point-source, or had been spread steadily through the community by propagation in some way, another important objective in constructing the epidemic curve is to estimate the incubation period of the illness if it is not already known. with waterborne illness especially, the time of exposure may be further obscured because people usually drink water several times a day. hence, the incubation period cannot always be determined for each case, but the actual time of onset is usually available. the incubation period is the interval between exposure to food or water that is contaminated (with enough pathogens or with a sufficient concentration of toxic substances to cause illness), and the appearance of the first sign or symptom of the illness. each etiology is characterized by a typical incubation period (tables b, c, d, and g). individual onset times will vary due to immune factors, co-morbidities, the dose ingested, and other ingested materials, but the investigator can often make a rough estimate of the average incubation time by examining the aggregation of all onset times as an epidemic curve. the modal peak of a single "cluster" or distribution is the time interval in which most cases commence symptoms. in fig. a this occurs on march , and in fig. d that occurs at the double interval feb - th. where two separate modal peaks (a "bi-modal distribution") suggests secondary cases ("propagation"), then the distance between the first two modes is a good estimate of the incubation period. figure b shows about days between primary and secondary modal peaks, suggesting that the initial exposure is likely to have been days before the first mode. in fig. b this would be sometime on or near the th of the month. if the exposure point is known but the agent is not, then that estimation of the median incubation period will allow many etiologic agents to be excluded due to date of onset of symptoms: between march and april , count per day onset histogram for cases of shigellosis, march to april , illustrating slow spread through a community through either propagation (person-to-person spread via poor hygiene), or exposure by many people to a small well at different times (a non-propagated route) incubation periods that are clearly outside the range of times observed. the list of possible candidates can be further reduced by examining the symptom profile and other characteristics of the illness and suspect food or water vehicle. as time passes, the onset curve also provides an ongoing measure of the potential for propagation, and the incidence rate. all this information can be useful in deciding whether the illness in question is an infection or intoxication and thereby determining which laboratory tests should be requested (tables b, c, d, and g). note that not all water or foodborne illnesses listed in a standard reference such as the "control of communicable diseases manual" (apha ), are directly communicable person-to-person; many require a suitable substrate (food or drink) and adequate time/temperature combinations to attain sufficient numbers or the production of enough toxin to induce a pathological condition. an exposure time can sometimes be estimated from a clear, point-source, singleexposure onset distribution (fig. d ). it has no solid basis in statistics, but has sometimes been found to be useful in practice. the typical incubation periods for most foodborne and waterborne illnesses are readily available for comparison (e.g., control of communicable diseases manual, apha/cdc, ), and in this manual in tables b, c, d, and g. an incidence rate is the number of new cases of a specified disease reported during a given time period in relation to the size of the population being studied, multiplied by a constant, usually , to give percentages. thus new cases of e. coli o :h infection among the residents of a children's summer camp in july is an incidence rate of ( / ) × or ( . ) × = . % for that month. if several people have left and their state of wellness is not known, their impact should be expressed in the form of the possible range of values around the known incidence rate within the two extremes whereby they may all be well or they may all be ill. thus, where six children who were at the camp had departed around the time of the outbreak and their health is unknown, the range could be from a possible ( / ) × (or . %) if all of the six had been well, up to ( / ) × (or . %) if all had been ill. note that the "missing" six are added to the denominator only when we speculate that none were ill, whereas they are added to the numerator and denominator if we speculate that they might all have been ill. in this example, the overall incidence rate would be reported as " %, with a possible range from . to . %." depending upon the situation, it is often necessary to identify exposures which may be related to the illness, and to calculate an incidence rate for each such exposure. for example, in the summer camp illustration (above) it might be useful to enquire if gender, age, location, or some other attribute or activity increased the risk of becoming ill. this should not be interpreted automatically as implying that a given exposure would be associated with the outcome in any situation. by hypothesizing that gender was linked to the risk of illness, for example, does not imply that males are more vulnerable to the illness (the outcome) than females, but it can indicate that gender may have been related to the exposure, which in turn increased the risk. as an illustration, suppose that boys at the camp had been swimming, while the girls had gone on a nature walk. the boys may subsequently show increased incidence rate for e. coli o infection, not because they are more susceptible, but because of their activity. every proportion or percentage statement should be made with clear reference to the appropriate denominator used. incidence rates of waterborne illnesses are usually similar for both sexes at any given age group in the population, but differences in activities or dietary habits or susceptibility due to age or underlying health status can change the risk. the very young, the elderly and the immunocompromised can be at more susceptible, while in some instances, previously exposed populations may have developed a measure of immunity to an infection that may still cause more serious illness among visitors. a further complication arises where the "at-risk" population (perhaps residents at an institution, summer camp, or on a cruise ship) have generally consumed all the food and water for the extended period. careful interviewing of affected persons often uncovers one or more persons who entered the subject community shortly before becoming ill or who visited the community for a short time and became ill after leaving it. example: the south-west part of the county is served by three semi-private water systems. thirty cases of waterborne illness are being investigated in the area. when the numbers of cases are displayed for each water system, no clear grouping or clustering is evident, although the delta supply appears associated with about % more cases than the other two (table a) . however, when the analysis introduces the total population of persons who depend upon each water system (as denominators), a different scenario emerges. the incidence rates (expressed here as percentages) now allow a meaningful comparison (table b ). we can see that persons using the bravo system have roughly five times the risk of illness compared to people who are served by the other two systems. the use of the denominator is vital for most calculations. caution: numerous other factors may also explain the outbreak and these should be carefully examined. for example, the households using the bravo supply may be closer to an unhygienic corner store, drink from a cross-connected public water fountain, or their children may swim in a more polluted pond than the other communities. potential sources such as these should be eliminated before the water supply is announced as the source of the illness. sometimes a spot map may be useful in showing the location of the residence of each case, while on a larger scale, the rates of illness can be shown using city blocks, census tracts, townships, or other subdivisions. different colors or symbols to indicate cases with different time of onset periods (such as weeks) may help to support a hypothesis as to where contamination was introduced, inasmuch as the earliest cases tend to cluster around the point where contamination first occurred. the weakness of this procedure is that if the exposure had been at a restaurant, workplace, or school, plotting the relationship to the location of the home would not be useful. the investigation of waterborne or foodborne disease outbreaks invariably commences after both exposure and illness have happened. this is the classical "casecontrol" study, where a group of ill people ("cases") and a group of non-ill people ("controls") are compared in terms of their exposures. to measure the association between exposure and illness, the data are typically displayed in a × contingency table. table compares cases and controls in terms of their exposure to a suspected factor "x." the table is ready for analysis using odds ratio, as well as the chi-square or the fisher's exact tests where appropriate. one × table will be used for each possible exposure (e.g., each beverage, food, or other material). as many cases as can be identified and contacted, and as many non-ill people (controls) as can be found, should be interviewed as quickly as possible about their exposures to each suspect item. fading memories, the chance of obtaining stillavailable samples of implicated food or water, and the opportunity to obtain fecal specimens before the patient is started on the ubiquitous broad-spectrum antibiotics are all reasons for rapid response. case and control numbers do not have to be the same; the calculations compare ratios so equal numbers in each group are not needed. generally a : to : ratio of cases to controls is perfectly adequate. as interview data from cases and controls are accumulated, leading to formation of hypotheses about the source of the illness, human resources should be deployed in two additional essential tasks: ( ) tracking down and confirming the hypothesized source of the illness, and ( ) promptly issuing warnings to all affected groups about the possible risks from any source that is still accessible, with assurances that further bulletins will be issued as soon as confirmation is received. this precautionary principle is a vital component of risk management in modern public health. waiting for absolute confirmation before releasing warnings and advisories should not be an option in the twentyfirst century. the principle holds that while false alarms can be quickly forgiven, further illness should be avoided at the highest priority. failure to heed this step has contributed to needless suffering and severe damage to reputation, trust and credibility. a case-control approach is necessary because unlike the data in table b , we rarely have full information about all the attendees, and therefore the true incidence/attack rate is not available. very rarely, when all cases and controls are available for interview, we would have the true incidence rates for ill and for not-ill persons and this would allow a "retrospective cohort study" to be carried out. under such circumstances, and using table as an illustration, we could state that of persons exposed to item x, persons ( . %) had become ill compared to ill of not exposed ( . %). where incubation times are longer than a day, there is increasing likelihood that only a small proportion of the non-ill people will be available for interview, and on many occasions, not even all the ill persons can be contacted. the point here is that the investigator is usually working with sub-sets of the true cases and controls. the controls in table and possibly even the cases may have been drawn from larger groups, and therefore we cannot state the incidence rate, for example, as: "… of exposed were ill," because the " " had been artificially assembled, and may not resemble the true incidence at all. we can, however, use exposure rates, for example: "…of ill persons, ( . %) had been exposed to x," and, "…of who were not-ill, only ( . %) had been exposed to x." the overwhelming majority of waterborne or foodborne illness investigations are run as "case-control" studies (or to be more accurate, "case-comparison" studies, as very little true "controlling" is accomplished during the selection of the comparison group). a broadcasted invitation to all who might have been exposed to come forward, typically results in few non-ill persons volunteering information, because nonaffected individuals believe they have little if anything to contribute. this reduces validity even further, and more active recruitment is often necessary to convince them that their information is just as essential for the investigation as are the contributions from the less-fortunate attendees. let us examine a waterborne illness suspected as being due to the consumption of water bottled from a certain spring. you have found people who meet the case definition of illness, and another non-ill people in same neighborhood who report no symptoms at all, and who will be your controls. in table we display the data and ask the question: "is drinking this water related to the risk of illness?" whenever a × table appears, the first step is to calculate the odds ratio (or). an odds ratio tells us if there is a relationship (where or ≠ ), and the strength of the relationship (the or value itself). it also clearly indicates the direction of the relationship: was drinking or not-drinking the dangerous activity? this is easily determined by finding the dominant pair from (a × d) or (b × c). in the example above, (a × d) is greater, so cell "a" links the row "drank" with the column "ill," while cell "d" links "not drink" with "not-ill." this assumption is not as obvious as it may seem; the cause of the illness may have been whatever "other" thing was drunk by those who avoided spring water! it is important to clarify that the odds ratio yields the strength of the association, not the statistical significance. most or values (where many exposures are being assessed) will be close to . (= "no association"), while an or clearly exceeding . signifies a positive association between this exposure and illness, such that this exposure increased the risk of illness. an or < . is protective, meaning that exposure to this factor reduced the risk of illness compared to the other group. for example, an or of . means that the exposed group had only one-quarter the risk of illness compared to the non-exposed group. the non-exposed group therefore has a greater risk (by a factor of ). while this protective effect can be due to true therapeutic protection (e.g., exposure to antibiotics when you have an infection), it is frequently explained as "statistical" protection. as an example, consider an outbreak where everyone consumed only one of two possible types of bottled water. one source, a, contains a pathogen, and b does not. if the ill people were found to be five times more likely to have consumed type a (odds ratio = . ) then the not-ill would have five times the rate of consuming water b, and only one-fifth of the rate of choosing water a (or = . or %). this can also be read as the risk of illness for the non-exposed group, or as the risk of staying well by the exposed group. an easier way to interpret an or less than is to place over the or to reveal a value greater than , but clearly labeled "protective." the or is a ratio between numbers, and therefore not sensitive to the actual numbers of people in individual cells, an important consideration when the numbers of subjects are relatively small. this is illustrated by the common question: "how large does an odds ratio have to be before it is considered evidence of an association?" a popular response is "at least . ," but this must be considered with extreme caution. for instance, with very large studies, an or of . (barely more than . ) can be shown to be very highly significant statistically (p = . ), whereas in a small-n study, an or of even . may not achieve statistical significance. the odds ratio is certainly a useful measurement, and should always be used when a × table is encountered. it will quickly advise you ( ) that there is an association, ( ) the strength of that association, and ( ) the direction of the association, none of which are specifically measured by a test of statistical significance. unfortunately, it is not reliable with small cell sizes, and is unable to answer the question: "how likely is it that these numbers could happen just due to chance?" for this, we need to test the statistical significance. the best advice is to use the or (or relative risk where appropriate) together with a test of statistical significance. most online statistic calculators or laptop versions of sas, spss, epiinfo, etc. will give a selection of useful statistics (odds ratio, relative risk, several versions of chi-square, and fisher's exact test, both onetailed and two-tailed.) in keeping with all scientific enquiry, we begin by advancing the notion (the "null hypothesis") that there is no association between the exposure and the illness, and attempt to support that notion. if insufficient evidence is found to support the null hypothesis, we reject it and cautiously consider that an association may exist between the two variables. this can be described as a statistically significant association. two methods of testing are presented: the chi-square test (written χ and pronounced "ky"-square) for most × (or larger) tables, and the fisher's exact test (only for × tables) when chi-square is not valid due to the numbers in the cells being too small (the following sections give advice about this decision). the original data value in each cell we call the observed, or "o" value, and these are compared with the numbers that you would expect ("e" values) if there were no relationship at all; that is, if the variables were not related, and the data were arranged purely by chance (as stated by the null hypothesis). the chi-square test measures the difference between the o and e values. if they are close, we have to accept that there may be no real relationship; if far apart, we can reject the null hypothesis and cautiously declare that exposure and illness were probably related. numerous online statistical calculators can be used to yield ors, rrs, and chisquare values. if you prefer to do the calculation by hand, construct a × table as shown, with "observed" data, marginal totals, and the grand total. the expected "e" values are found from: to make sure the chi-square analysis is appropriate for your table, you must be sure that all "e" numbers are more than . the quickest way is to first calculate for the cell with the smallest e value; (this will be the cell with the smaller column total and the smaller row total.) in table , the smallest e value will be cell "d," and this is calculated as ( × )/ = . . as this is > , all other e values will be greater than this, so chi-sq. is valid. (note that the smallest e value did not coincide with the smallest o value). . for all four cells the sum (χ ) is: . + . + . + . = . an online statistical calculator will give you this same chi-square (χ ) value. to verify by hand whether the o vs. e difference is statistically significant, compare your chi-sq. value (for a × table only) with . . if your calculated value exceeds . , then this is unlikely to be due to chance, and thus you can begin to believe that this exposure did influence the risk of illness, and you can reject the null hypothesis. statistical results usually include a probability (p) statement. this is the probability that the null hypothesis ("no association") is correct. the . value is the minimum needed for statistical significance, where the p is less than % (p < . ). recall that the p is the probability that no real association exists between exposure and illness. by convention, if p > . (more than %) then the relationship is declared not statistically significant. where p = . or < . , then the relationship is statistically significant. the smaller the p value, (p < . , p < . , etc.) the more confidence you have that a relationship really exists. other critical values exist for assessing calculated chi-sq. values, from larger tables than × , and at more extreme levels of significance. a further chi-square calculation is shown as an appendix. where a table greater than × is found to have more than % of the cells with an e value less than , chi-square is not valid. the solution is to collapse either columns or rows to allow the e values to increase. for example in table a , two cells out of six ( %) have e values less than , but if "high dose" is merged with "medium dose" the resulting increase in observed (o) cell sizes is also reflected in greater e values, while the table becomes × (table b) . some outcome information has been lost, but the chi-square analysis can proceed. if, after trying to collapse cells and/or rows, a × table is reached still with an e value < , the fisher's test is indicated. this procedure is reserved only for × tables where one or more expected (e) values is less than , making the chi-square test not valid. our example is taken from an investigation into an outbreak of shigellosis presumed to be due to water from a well ( table ). the odds ratio has been calculated as ( × )/( × ) = . , meaning ill persons were six times as likely to have drunk well water compared to non-ill the "!" denotes a factorial, meaning that number multiplied by the next smallest number, and so on down to . (e.g.: ! = ) p persons. an attempt to use chi-square is prevented by at least one e value less than of . (cells c and d both show e values as ( × )/ = . ). the starting null hypothesis is "that no relationship exists." this is not quite the end of the calculation however. the goal is to calculate the probability of the original data occurring plus all more extreme probabilities. the original data have to be adjusted by increasing the "dominant" pair of cells by + and the others by − , while leaving the margin totals the same (table ) . because no zero has yet appeared in the matrix of cells, we continue to increase the "dominant" pair by + and obtain a zero. the next calculation is the last. (by convention, ! and ! = ) (table ) . no reference table is required. the total calculated probability ( . ) is exactly the probability that the null hypothesis ("that there was no relationship"), is correct: . %. by convention, for a result to be significant statistically, that probability (p) must be less than % (< . ), so in this instance we are not able to reject the null hypothesis and must conclude that the relationship could have occurred by chance alone more than % of the time. the odds ratio of . is explained as the number of times more likely it was for a shigellosis victim to have drunk well water than for a non-ill person. this increased risk would normally be impressive, but because of the small number of persons in the study, it has been found not to pass the test of statistical significance. a basic write up of the results might read: "a relationship exists between drinking well water and developing shigellosis. a shigellosis patient is six times more likely to have drunk water from the well compared to a non-ill person. this relationship is not statistically significant, however, and could have occurred by chance alone more than % of the time. the null hypothesis of no-relationship cannot be rejected." [ df, p > . , or: . , not statistically significant.] with the odds ratio (or) calculated for all the suspected exposures, and the chisquare test or fisher's exact test calculated for the strongest of these, all the results can be displayed in a composite table. earlier protocols for the investigation of waterborne and foodborne diseases encouraged the use of the "factor-specific attack rate table" (for example the "foodspecific attack rate table"), but where only a "convenience sample" of controls and cases are available, we are unable to derive valid incidence/attack rates. investigators are discouraged from using it as it may produce misleading results. the exposurerate table for cases and controls is preferred in all case-control studies, and compares the rates of exposure to each factor between both the ill and non-ill people. table displays six exposure factors from a hypothetical outbreak involving water contamination. exposure rates are calculated from both cases and controls. the "spring-water" data that we used for the odds ratio calculation example in table appears as the first exposure in table . the column headed "differences in exposure rates" subtracts the exposure rate among the non-ill from the exposure rate among the ill. [use: exp. rate (cases) minus exp. rate (controls), keeping the signs correct]. you are looking for a large positive difference to indicate the most likely culprit. the spring water shows the largest positive difference at + %. the odds ratio of . supports this, again the largest value, indicating that ill persons were times more likely to have drunk the spring water compared to non-ill persons in this group. hence both the large positive difference in exposure rates and the large odds ratio point to the spring water being the likely source of the illness, and it is certainly the strongest association between illness and any of the exposures shown. the chi-square value has also been added ( . ), as well as the associated p value. taken together, the evidence clearly points to this factor as the culprit. in those less-common circumstances in which all the ill and non-ill persons can be contacted for interview, the table can be rearranged to show attack rates (incidence rates) for each of the suspect factors (table ) . here, the column of "differences" shows the attack rate (exposed) minus the attack rate (non-exposed), (i e − i n ), and again a large positive difference will point to the culprit. this measure is called the attributable risk and for the spring water example we obtain + %, the largest value of all the risk factors. also, because of the availability of valid attack rates (incidence rates), the true relative risk (rr) is available, and can be substituted the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant the ("true") relative risk (also called the risk ratio) is only used when we have the true incidence data. it is calculated as the attack rate (exposed) over the attack rate (non-exposed), or i the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant for the odds ratio. the data in table shows the same data as table rearranged for easy comparison. values in the column of "differences" are not the same as for table and of course the relative risks are not the same as the odds ratios in table , but both of these results still point clearly to the suspect exposure. in both analyses, spring water is clearly the factor most strongly associated with illness. it is important to note that in both tables a high rate (exposure-or attack-) on the left side taken by itself is meaningless until it is compared with the rate from the right side of the table. this again underscores the importance of gathering complete data from the non-ill as well as the ill. an interesting phenomenon is visible in the second factor listed (soft drink). the or is listed as . , which is "protective," meaning that this factor is strongly associated with not being ill. it is the equivalent of or equal to . ( / . ), and the chi-square is seen as quite large, although not enough for statistical significance. this is sometimes seen where two factors are "in competition" with each other; if everyone had drunk one item, and the spring water was the contaminated source, then those drinking the other item would be strongly "protected" because they did not drink the spring water, and this shows clearly. all other factors have or values very close to . . most attack rate tables record some persons who did not ingest the suspect vehicle but who nevertheless became ill. plausible explanations are that (a) some people forget which beverages or foods they ingested; (b) some might have become ill from other causes; or (c) some may have exhibited symptoms with a psychosomatic rather than a physiological origin. it is also not unusual for the table to include some persons who ingested contaminated water or food but did not become ill. plausible explanations are that (a) organisms or toxins are not always evenly distributed in water or food and consequently some persons ingest small doses or perhaps none at all; (b) some persons eat or drink larger quantities than others; (c) some are more resistant to illness than others, and (d) some will not admit that they became ill, or fail to report it. whichever table is used, the combined totals for cases (ill) and controls (well) are fixed and should not change for each exposure unless there are "missing" responses from interviewees. while some procedure manuals include confidence limits around both rr and or, this may be omitted here as the use of the chi-square test or fishers exact test yield the statistical significance for both tables. illness caused by ingestion of waterborne toxicants and some pathogenic organisms can be dose-related in that the risk of developing symptoms, and their severity varies with the quantity ingested. where the suspect water is no longer available (for example, the well may have been quickly super-chlorinated to break the chain of infection before samples were taken), attack rates can be based on the amount of water usually drunk per day by each person. this is easily extended to other non-treated sources of water such as ice cubes, water-reconstituted fruit juices, and flavored crystals. a comparison of attack rates at various water intake levels may provide valuable evidence that water is, or is not, the vehicle responsible for the outbreak. for an example, see table . here, the entire group was people and we have interviewed them all, so we are justified in calculating the attack/incidence rates: in this example, the attack rate increased as the consumption of water increased, which suggests that the illness was directly related to water and the agent it contained. this is a trend established from the group as a whole, and an individual's experience may vary with factors such as (a) preferences of water ingestion, (b) intermittent contamination, (c) unequal distribution of the contaminant, or (d) varying susceptibility of individuals. these data can be compared with rates from persons who ingested no water, but only hot tea, hot coffee, soups, and/or other safe sources of liquids. if unheated water was indeed the vehicle, and the agent was a living biological agent, these persons should have attack rates showing no increase in risk of illness. (outbreaks from a toxic agent may be unaffected by chlorination, boiling, and some types of filtering.) the data can be displayed in a contingency table as follows for analysis using chi-square procedure (table ) . for this example, chi-square equals . and if calculated by computer or online, p will be shown as p = . . reference to form j confirms that for a × table ( df), the calculated chi-square ( . ) exceeds the critical value for statistical significance at the . level ( . ), allowing us to claim statistical significance at p < . . odds ratios are normally associated only with × tables, but here, the or can usefully be calculated on selective cells or groups of cells as long as you clearly explain the selection process. for instance, persons who were ill were . times more likely to have drunk three or more glasses of water per day compared to those who were well. for this calculation we collapse cells into a × table and crossmultiply: (a + c) × (f + h)/(b + d) × (e + g) = ( ) × ( )/( ) × ( ) = / = . . alternatively, because we have all people involved, we can compare the attack rates (ar) for each intake level, and observe the increasing attack rate as the intake increases: for five or more glasses/day, ar: %, for - /day, ar: %, for - / day, ar: %, and for < /day, ar: %. we might summarize as follows: "there was a relationship observed between the quantity of water consumed each day and the risk of illness. the incidence rate increased with the quantity consumed from % for < glasses/day to % for five or more glasses/day. this relationship is statistically significant. the null hypothesis of no association can be rejected." , df, p < . ] water as a vehicle can deliver pathogenic organisms in many ways beyond simply drinking a glass of water, or using a drinking fountain. investigators should be sure to ask about the preparation of ice-cubes, the mixing of fruit flavored crystal drinks, reconstituting concentrated orange juice, brushing and rinsing teeth, and washing hands, utensils, or containers. swimming or playing in muddy pools or even swimming pools have caused waterborne poliomyelitis, and naegleriasis, while swimming in saltwater inlets have allowed inadvertent infections from vibrio parahaemolyticus and v. vulnificus. unwashed plastic jugs containing poster paint residue have caused rapid illness when drink crystals are reconstituted in them, while refillable plastic containers and bottles have a long history of contamination from biological and chemical agents. in the late s, an increase of viral ear, nose, and throat infections among people who were using parkland next to a river was hypothesized to have been due to people waterskiing on the river and creating an aerosol. the river was the receiving body for effluent from a water treatment plant upstream. record all laboratory results on form i, laboratory results summary. compare epidemiological and statistical results with on-site observations, laboratory results and the information summarized on form i. the agent responsible for the outbreak can be determined by (a) isolating and identifying pathogenic microorganisms from patients, (b) identifying the same strain and/or pfge pattern or genetic sequence of pathogen in specimens from several patients, (c) finding toxic substances or substances indicative of pathological responses in specimens, or (d) demonstrating increased antibody titer in sera from patients whose clinical features are consistent with those known to be produced by the agent. when implicating the water as a likely (or presumptive) vehicle of transmission, ideally identification of a pathogen in samples of suspect water will correspond to the one found in clinical specimens from ill persons or that produces an illness that is compatible with the incubation period and clinical features of the ill who were exposed to the water. for organisms that are common in the gastrointestinal tract or that have multiple strains, compare strains isolated from ill persons with strains isolated from the suspected water. additionally, specific microbial markers (e.g., serotype, phage type, immunoblotting, plasmid analysis, antibiotic resistance patterns, restriction endonuclease analysis, nucleotide sequence analysis) or chemical markers identified by chromatography or spectrophotometry can be used for this purpose. for confirmation of water-related transmission, the same pathogen strains should be found in both the ill persons and the epidemiologically implicated water. however, due to the period of time that may have passed after the outbreak was actually reported, and to methodological issues, such as the need for concentrating pathogens in water samples, it is often unlikely that the outbreak-associated pathogen will be found in the water samples. laboratories frequently test water samples for indicator organisms, such as fecal coliforms, escherichia coli, or enterococci, rather than pathogens. the finding of these bacteria in high densities in the water may indicate contamination (from a fecal source) and implicate the water was a possible vehicle. however, the finding of increased indicators in water samples alone is insufficient evidence to confirm the water as the source of an outbreak. the probable source of contamination or the situation that allowed contamination to reach and survive in a water supply (e.g., water supply not disinfected or inadequately disinfected, inadequately filtered, or upstream to sewage or agricultural discharges; cross connection between sewerage and drinking water pipes; well improperly constructed; nearby septic tank system; or livestock in water supply) can often be identified, but the etiologic agent in the water may never be found. success in finding the etiologic agent is most likely where (a) the incubation period of the illness is short, (b) the agent is stable in water and the system is static, or (c) large amounts of the agent are being continually added to the water supply. try to recover and identify the specific agent whenever a water supply is suspected to be the vehicle of transmission, even if finding the etiologic agent is likely to be difficult and not considered practical for routine monitoring of water supplies. if water samples do not reveal a likely causal agent, clinical data as well as time, place, and person associations can cast strong suspicion on a water supply, particularly if indicator organisms are found in the water. tests other than those for pathogens, however, are frequently used to evaluate water supplies on a routine basis. organoleptic tests attempt to evaluate the total effect of all compounds present in water that can be measured by the senses of taste, smell, or sight. results cannot be expressed in terms of specific compounds present, and the measured qualities are usually a result of a mixture of compounds. these tests are often empirical and arbitrary, but changes in the physical qualities of water (such as ph, turbidity, color, odor, or taste) can indicate abnormalities of the water. outbreaks have occurred, however, when turbidity readings have met present standards and when water appeared and tasted good. chemical examination of water is useful for (a) detecting pollution (especially from industrial wastes and pesticides), (b) determining effectiveness of treatment processes, (c) evaluating the previous history of the water, (d) determining hardness, and (e) detecting the presence of specific toxins. results are usually expressed in milligrams per liter (mg/l = ppm, parts per million), or micrograms per liter (μg/l = ppb, parts per billion). historically, acute water-related outbreaks seldom involve chemical substances, so chemical tests are not requested routinely unless either (a) circumstances indicate possible chemical contamination or (b) clinical symptoms suggest chemical poisoning. flowing water in a distribution system can be monitored to determine chlorine residual. free available residual chlorine refers to that portion of the total residual chlorine remaining in chlorinated water at the end of a specific contact period that will react chemically and biologically as hypochlorous acid or hypochlorite ion. the reaction is influenced by ph and temperature. total or combined residual chlorine refers to chlorine that has reacted with ammonia or other substances and is not available for further reactions, as well as the free available chlorine. a chlorine demand exists in a chlorinated water until a free available residual is produced. a free available chlorine residual, e.g., mg/l ( ppm) or higher, maintained throughout the distribution system of a community supply is an indicator of safety from enteric bacteria but not necessarily from pseudomonads, viruses or parasites. outbreaks have occurred when chlorine residue levels have met present standards. analyses for microbial indicator organisms provide information on the microbiological quality of water and guidance as to its safety for consumption or contact. indicator organisms are easier to test for than pathogenic organisms, and some serve as a surrogate measure of fecal contamination in water. the absence of indicator organisms in the water, however, does not guarantee water safety; numerous outbreaks of water-related disease have occurred from water in which no indicator organisms were detected. evaluation of the safety of water should be based upon a combination of results of (a) an on-site study to identify sources and modes of contamination and means by which contaminants survived treatment and (b) appropriate laboratory analyses. microbiological results should be compatible with observed sources of contamination and/or treatment failures found during the investigation. although all natural waters contain bacteria, the number and kind vary greatly in different places and under different climatic and environmental conditions. the number of bacteria isolated and reported, however, often represents only a fraction of the total number present, for several reasons. colonies seen on agar plates develop from either single organisms or clusters or chains of organisms. heterotrophic bacteria represent only those that can use organic matter and grow at the selected temperature ( - °c) within - hours under aerobic/microaerophilic conditions in/on a defined medium when the standard test (spread plate, membrane filter or pour plate) is used. the hpc may also be done using different media under different incubation times/conditions. (higher counts are usually found when the longer incubation periods are used.) also, certain microorganisms are unable to grow aerobically either in or on the medium used. because of these variables, the terms total plate count (tpc), standard plate count (spc) and aerobic plate counts (apc) should not be used. hpcs serve as an index of changing sanitary conditions. in general, counts of good-quality well water are fewer than - colonies per ml. densities in surface water are higher, but quite variable, depending on water temperature, sources of pollution, amount of organic matter present, and soil that washes into the water. the sources of pathogens, toxic substances, or fecal contamination may not increase the hpc of a surface water sample as much as washings from soil. nevertheless, marked changes in the number or kind of microorganisms should be viewed with concern, at least until the reason for the change is discovered. heterotrophic plate counts greater than /ml and some specific antagonistic species may interfere with the growth or recovery of pathogenic or indicator organisms. some heterotrophic species are opportunistic pathogens that may pose a health threat to immunocompromised persons. the coliform group of bacteria comprises those from non-fecal environmental sources, and those from animal and human intestines, including escherichia coli. the environmental species of non-fecal bacteria are found in soil, on fruits, leaves, and grains, and in run-off water, especially after heavy rains. some of these species are capable of surviving in water longer than e. coli. furthermore, some coliform strains and can multiply on decaying vegetation in water, in biofilms in pipelines, or on pump packings, washers, and similar materials. therefore, finding coliforms may not be indicative of fecal contamination, although most water utilities have standards for coliforms in water. fecal coliforms are present in large densities in all human and animal feces, normally much higher than pathogens which are typically only present in infected persons and normally at lower levels. as such, high populations of fecal coliforms can indicate recent sewage pollution of water, but are not always indicative of pathogens present, particularly viruses and parasites. none of the coliform group, however persists as long as most viral or protozoan pathogens in water, and indicator bacteria described below (fecal streptococci and clostridium perfringens). typical chlorination or ozonation of water inactivates coliform bacteria. presence of the coliform group or even a high population of coliform bacteria is not proof that a treated water supply contains pathogens. however, coliforms can provide a warning that either the water treatment was inadequate or contamination occurred after treatment, and that some pathogens may be present. as mentioned above, under some conditions, pathogens may be present where there are few or no coliforms. furthermore, unlike coliforms, many parasites and viruses are resistant to normal levels of disinfectants. coliforms have little or no correlation with the presence of parasitic protozoa or pathogenic viruses. the standard test for the coliform group may be carried out by a membrane filtration technique, a multiple-tube fermentation technique (presumptive test, confirmed test, or completed test), or a presence-absence test. results of the membrane filtration technique are reported as colony forming units (cfu) per ml of water. results of the multiple-tube fermentation technique are reported as the most probable number (mpn) per ml of water. this is a statistical estimation of the total number present, but the actual number can fall within a considerable range. counts derived from these two methods are not necessarily the same, but they have the same sanitary significance. false-negative or false-positive results can also occur with the membrane filtration technique because of interfering background growth of nonfecal microorganisms. results of the presumptive test of the multiple-tube fermentation technique can be misleading, because other microorganisms frequently found in water also produce gas in laboratory media, and may thereby give false-positive results. also, especially in waters containing a large number of microorganisms, some coliforms present may produce gas slowly, leading to false-negative results. the presence of coliform bacteria is corroborated by means of the second phase of the multiple-tube fermentation technique, known as the confirmed test. positive results are usually considered confirmation of the presence of coliforms. a third phase of this test, known as a completed test, further ensures the correct identification of coliform bacteria. a simple modification of the coliform test is to analyze for the presence or absence of coliforms in a -ml drinking water sample. the "presence-absence (p/a) coliform test" allows for simple examination of a larger number of samples. when a positive sample is detected, it is advisable to measure coliform densities in repeat samples by one of the other methods to determine the magnitude of the contamination. thermotolerant coliform (fecal coliform). coliform bacteria will frequently grow at a relatively high temperature, . °c, unlike species or strains normally encountered in the environment, which usually have an optimal temperature near °c. this thermotolerant characteristic has been used in an attempt to separate coliform bacteria into those of so-called fecal and non-fecal origin. this test may provide better indication of fecal contamination than the coliform test, but it is however, unreliable. positive results are not proof that either organisms of fecal origin or pathogens are present. the number of thermotolerant coliforms is considerably lower than the number of total coliforms in contaminated water; therefore, the test is less sensitive for testing treated drinking water. furthermore, escherichia coli o :h , which has been implicated as causing water-related illness, does not grow well at . °c. escherichia coli. e. coli is common in feces of human beings, other mammals, and birds. it can also be found to grow naturally in the environment, specifically in tropical waters. comprised of the larger coliform group, its detection in water is a more definitive indicator of fecal contamination, compared to total or fecal coliforms. however, a positive test result does not identify if the fecal source is human or nonhuman. rather, the finding of e. coli in water serves as an indicator that fecal matter reached the water and provides a warning, but not proof, that pathogenic organisms may also be present. it should be noted that some strains of e. coli are pathogenic (see table b ). simple commercial p/a and quantitative tests have been developed to detect the presence of total coliforms and e. coli in hours by observing color changes and fluorescence of the media under daylight and uv light. such tests may be useful for field evaluation of microbiological water quality. another group of organisms, collectively known as fecal streptococci, is also used as an indicator of fecal contamination. enterococci (enterococcus faecalis, enterococcus faecium) are particularly used for testing recreational waters. like coliforms, enterococci are normal inhabitants of the intestinal tract of human beings and other animals. in human feces, they occur in considerably lower numbers than e. coli. some members of the group, such as e. faecalis, subsp. liquefaciens, however, have been associated with vegetation, insects, and certain types of soils. enterococci generally survive longer than coliforms in fresh water, and therefore the source of contamination may be distant in either time or place from the site where samples were obtained. their resistance is, however, less than that of clostridium perfringens, enteric viruses, and parasites. like e. coli, simple commercial p/a and quantitative tests have been developed to detect the presence of enterococci in hours by observing color changes under uv light, which may be useful for field evaluation of microbiological water quality. clostridium perfringens (sulfite reducing clostridia). c. perfringens is also of fecal origin, but it occurs in feces in much lower densities than e. coli and can also be found in soils. being a spore-former, it can survive for long durations in soil and water, and persist when all other bacteria of fecal origin have disappeared. therefore, it is a useful indicator of remote or intermittent contamination in wells that are not frequently examined by the coliform test; but, it is not, by itself, evidence of recent contamination. chlorine, in the concentration typically used in water treatment, does not inactivate all spores; and thus c. perfringens is not valuable in assessing the efficiency of chlorination for bacterial vegetative cells. its long persistence and its resistance to chlorine make this organism a potential indicator for viral and parasitic organisms that have similar resistance and disinfectant susceptibility. coliphage. coliphages, which are viruses that infect e. coli, are simpler to detect and enumerate, compared to other viruses, and are generally associated with fecal contamination. they have been considered as possible indicators of treatment effectiveness for human enteric viruses. coliphages are categorized into two groups: the somatic phages, which enter e. coli via the cell wall and the male-specific phages, which enter e. coli through the sex pili. the somatic and male-specific phages are common in sewage and the feces of human beings and other animals, but in lower densities than the common fecal indicator bacteria, fecal coliforms, e. coli, and enterococci. some strains appear to be more resistant to chemical disinfection than water-related pathogens or indicator bacteria. be aware of local standards for water distribution systems, private water systems, and recreational water. although drinking water standards, such as the total number of coliforms allowed in a water sample, vary from jurisdiction-to-jurisdiction, it is generally agreed that any fecal contamination (e.g. fecal coliforms, escherichia coli) render the water unacceptable for human consumption and may close down recreational bathing waters. there are numerous pathogens that can be transmitted by water, many of which are also able to cause respiratory symptoms, in addition to the classical gastrointestinal symptoms. for a comprehensive summary of waterborne pathogens see "american waterworks association manual of water supply practices, m waterborne pathogens, nd edition ( ) ." for several reasons, analyses for pathogens are not usually conducted during routine water testing, or are only conducted by specialized laboratories. first, tests for pathogens are pathogen-specific, expensive, and often difficult to perform because they may require specialized trained personnel. secondly, the etiologic agent of the outbreak is often unknown at the time of analysis; hence, many analyses would have to be done blindly. thirdly, pathogens are not always recovered because they are heterogeneously dispersed and diluted in the environment, and their numbers decline in water over time. as a result, they may be absent or present in low densities by the time samples are collected following an outbreak. fourthly, recovery efficiencies are often poor because microorganisms are stressed by disinfectants or the method is sensitive to interferences from the source waters environment, hence not easily recovered by routine methods. additionally, recovery efficiencies for viruses and protozoa may be poor because of the interferences of substances within the sample matrix with method reagents (concentrating l of water down to μl will also concentrate inhibitory chemicals and substances). finally, the time required for isolation and identification is often long, and the number of samples is usually too small to allow the investigator to have much statistical confidence in the results when pathogens are not found. negative results should be reported as "not detected" because they do not ensure that the water sampled was not the source of the pathogen. procedures used for many bacterial pathogens are qualitative because enrichment procedures are used. quantitative procedures (e.g., mpn) require considerable work and are less reliable than those used for coliforms because small populations may be present, and these may be unevenly distributed. despite these difficulties, pathogens that cause a syndrome similar to the one being investigated should be sought. see tables b, c, and d, for descriptions of the disease syndrome associated with the pathogens described in the following material. finding the same pathogen in specimens from patients and in water samples confirms water as a vehicle. summarize investigative data in a narrative report. describe in this report situations that led to contamination of the water and survival of etiologic agents up to the time of consumption. include all events that contributed to the outbreak to guide control and preventive measures. compare your data with the listings in table g (guidelines for confirmation of waterborne outbreaks) and table h (guidelines for confirmation of water responsible for illness), and criteria for confirmation of vehicle responsible for waterborne illness before assigning the etiologic agent and the vehicle. outbreak confirmation is based on (a) time, place, person associations, (b) recovery of etiologic agents from clinical specimens from cases and samples of water, and (c) identification of sources and modes of contamination and means by which pathogens or toxic substances survived treatment. all three of these, however, might not be found in any one investigation. complete form k (waterborne illness summary report). attach the narrative and the epidemic curve. also attach form d (case history summaries: water/laboratory data), all applicable parts of forms g, forms h, form i, and other data that will provide supplemental information to reviewers. send this report through administrative channels to the appropriate agency responsible for waterborne disease surveillance. make the final report as complete as possible, so that the agency can accurately interpret the results and develop a meaningful waterborne disease data bank. in the interest of continuing cooperation, give all participants in the investigation due credit and send each a copy of the report. also, send copies of the report through administrative channels to agencies (a) that have jurisdiction over the implicated water, (b) that initiated the alert, and (c) that participated in the investigation. those concerned with water sources, treatment and recreation, as well as with public health, should make every effort to ensure the complete investigation and reporting of waterborne diseases. without reliable, complete information, trends in waterborne disease incidence and causal factors of the disease are difficult to determine. good surveillance is essential for detecting and evaluating new waterborne disease hazards. the primary purposes of a waterborne disease investigation are to identify the cause, establish control measures, and take actions to prevent future illness. prudence may require some action before all the hypotheses regarding the water supply involved and the source of contamination are confirmed. frequently the local health authority will issue a boil water advisory if a microorganism is suspected to have contaminated the water. refer to "possible precautionary control actions" section for a discussion of these precautionary control measures. if these measures have not already been considered, consider them now. once control measures have been implemented, continue to monitor for disease to evaluate whether the measures were effective. in a waterborne event in sydney, australia (see box ) sydney water severely overestimated levels of cryptosporidium and giardia present in the water raising public alarm. boil water advisories were announced and rescinded several times. however, it is better to announce boil-water advisories than to have thousands ill, as has happened in the past, such as the cryptosporidium outbreak in milwaukee in . deficiencies in treatment must be corrected and defective parts of distribution systems must be repaired, beginning with those that either contributed to or had a high potential for contributing to the outbreak. the effectiveness of these efforts will be directly related to the thoroughness of the investigation. document the source and the manner of contamination and survival of the etiologic agent through the water treatment process. provide clear documentation of contributory factors, so that preventive measures taken will be specific to the problem. if previous sanitary surveys have revealed, or if subsequent ones reveal, that conditions which contributed to the outbreak are widespread, initiate a training and education program. these programs can be developed for water treatment plant or recreational water operators and employees, engineers, homeowners, or other appropriate groups. impress upon them the importance of proper construction and operation of facilities and proper protection, treatment, storage, and distribution of water. follow up with periodic inspections and surveys and verify by sampling, as appropriate, to determine whether faulty conditions have been corrected or allowed to be reintroduced. legal action may be necessary to ensure compliance with official standards and accepted sanitary practices. formulate solutions to problems found during outbreak investigations, and incorporate these into regulations for drinking, agricultural, industrial, domestic, and recreational waters. inform the public, through mass media and other means available to your agency, of hazardous conditions that can affect their water supply, but do so only after hypotheses are confirmed. the public must be told of any potential or actual harm that may result from ingesting or contacting contaminated water and must also be informed of measures that they can take and that official agencies are taking to correct these conditions. the water supply and recreational water facilities must be verified periodically to determine whether critical processes are being monitored and operated within limits of appropriate public health standards (see box , the walkerton outbreak). most waterborne illnesses are preventable, but prevention requires that those in the water treatment industry and in health and water-protection regulatory agencies be constantly vigilant to ensure that the hazards are understood and that questionable water treatment or delivery system construction or practices are avoided. examples investigation guidelines and investigative forms iafp manual, "procedures to investigate waterborne illness, rd ed"; copies of form c; one dozen copies each of forms e and f; two copies of form d and all parts of form g, epi-info software (cdc, atlanta). water sample bottles (bottles for chlorinated water should contain enough sodium thiosulfate to provide a concentration of mg of this compound per l of sample), plastic bags (whirl-pak ® type), ml, -l and -gal sized jars and jugs. sterile and wrapped sampling implements moore swabs (compact pads of gauze made from strips cm [ ft] by cm [ in.] tied in the center with a long, stout twine or wire-for sewer drain, stream or pipeline samples), fiberglass-epoxy bacterial filter cartridge, . μm; tongs, scoop or similar utensils for collecting ice. specimencollecting equipment (for human specimens from cases and controls) sterile containers (with lids) for stool specimens, bottles containing a bacterial preservative and transport medium, mailer tubes or styrofoam box, sterile swabs, rectal swab units, tubes of bacterial transport medium, stool preservative medium for parasites, phlebotomy supplies for blood specimens. kits for testing chemical disinfectants and ph dpd (n,n-diethyl-p-phenylenediamine) chlorine comparator with color disc for chlorine ( . ppm) and chlorine test papers; field-type ph meter or ph comparator with color disc or ph test papers; applicable ph indicator solutions and dpd reagent solution; dissolved oxygen testing unit. dye tracing study equipment fluorescein (yellow-green fluorescent) dye in powder form ( packages containing g each), in tablet form ( tablets), or in liquid form (prepared by mixing g in l of water); fluorometer; filters (primary and secondary) for use with fluorometer; sample holder for continuous sampling or individual sampling; fluorometer recorder. disinfectant and neutralizer . % w/v solution of calcium hypochlorite or . % household liquid bleach; % w/v sodium thiosulfate. virus filtration equipment for viruses and parasites b large plastic container for storing water sample prior to concentration; portable electric or gasoline powered water pump with quick disconnect brass or stainless steel plumbing adapters or hose couplings; two filter holders for -in. water filter cartridges fitted for adapters or couplings; portable water meter fitted for adapters or couplings; four lengths of fiber-reinforced garden hose fitted with adapters or couplings; one length of a strong-walled supply hose fitted with adapters or couplings; -in. prefilter ( μm nominal porosity wound polypropylene yarn filter with hollow perforated stainless steel core) cartridge filter; -in. virus absorbing filter pleated . μm porosity nylon membrane type (positively charged) for waters of ph values up to . , or pleated . μm porosity glass fiber membrane type (positively charged) for waters of ph value of . or lower (e.g., virosorb, -mds, amf/cuno meriden, pleated, . μm, glass filter); ml sterile, ph , % beef extract solution in gal wide-mouth screw capped autoclavable polypropylene container for each sample to be collected; stands to support filter holders during filtration; for parasites -in. polypropylene yarn-wound cartridge filter, . μm porosity (e.g., micro wynd ii™, amf/cuno; meriden, ct. . μm normal porosity). (continued) laptop or tablet, with software; thermocouples of varying lengths with either recording potentiometer, data logger, or digital indicator; devices to take samples below surface and sediment samples; chemical smoke kit and/ or micromanometer; occupational safety and health administration (osha) or equivalent approved respirator; sterile plastic gloves; plastic container liners for ice; waterproof marking pens; waterproof test tube rack; pencils, note pad; roll of adhesive or masking tape; labels; waterproof cardboard tags with eyelets and wire ties; flashlight; matches; test tube rack to fit tubes used; insulated chest or styrofoam container; packing material; camera with flash; spare batteries for all equipment; % ethyl alcohol; propane torch; refrigerant in plastic bags, liquid in cans, rubber or heavy plastic bags that can be filled with water and frozen; heavy-duty bags for ice, "canned ice," or cold-packs (blue ice). a assemble a kit to be kept in the agency responsible for investigating waterborne illness. it should include at least ten water sample bottles; ten -l, or gal jars or jugs; ten specimen collection containers or devices; and one each of the following supporting equipment and sterilizing equipment. date of sterilization should be marked. periodic resterilization or replacement of sterile supplies, media, or transport media is required to maintain the kit in a ready-to-use condition b similar equipment for sampling for either viruses or parasites may be available from national water, environmental, or health agencies how much to collect two rectal swabs or swabs of fresh stool from ten ill persons; samples from ten controls also can be submitted. whole stool is preferred if nonbacterial stool testing considered a fresh stool sample from ten ill persons; samples from ten controls can also be submitted. to enhanced detection, three stool specimens per patient can be collected > h apart as much stool sample as possible from ten ill persons (a minimum of ml of stool sample from each); samples also can be obtained from ten controls. a fresh urine sample ( ml) from ten ill persons; samples from ten controls also can be submitted. label each specimen in a waterproof manner and put the samples in sealed, waterproof containers (i.e., plastic bags). batch the collection and send in overnight mail to arrive at the testing laboratory on a weekday during business hours unless other arrangements have been made in advance with the testing laboratory. contact the testing laboratory before shipping, and give the testing laboratory as much advance notice as possible so that testing can begin as soon as samples arrive. when etiology is unclear and syndrome is nonspecific, all four types of specimens may be appropriate to collect c for more detailed instructions on how to collect specimens for specific parasites, please go to http://www.cdc.gov and search the website of key words d for more detailed instructions on how to collect specimens for viral testing, please go to http://www.cdc.gov and search the website of key words e the containers have been tested for the presence of the chemical of interest prior to use f unused specimen collection containers that have been brought in to the field and subjected to the same field conditions as the used containers. these containers are then tested for trace amounts of the chemical of interest table e (continued) isolation of agent from ill persons and from water and laboratory criteria for confirming etiologic agent as stated in table g . combination of on-site investigation, statistical evidence and laboratory analysis. (see entries below) presumptive vehicle on-site investigation demonstrating source and mode of contamination of water and survival of etiologic agent in water. also, desirable to have laboratory isolations from water of etiologic agent that causes syndrome similar to that observed during the investigation and other supportive epidemiologic data. if so, this might provide sufficient evidence for confirmation. or p-value for water < . when other epidemiologic data supports water hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. or odds ratio or relative risk for water greater than and the lower limit of the % confidence level greater than when other epidemiologic data supports the water vehicle hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. for calculation of data when all persons who may have been exposed to the suspect vehicle (but not all persons will have ingested every beverage/food) have been identified and interviewed (retrospective cohort) form j : chi-sq. analysis can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps: ill well totals step : create a x table as shown with observed data (o values) and marginal totals step : calculate odds ratio: (axd)/(bxc) = ( x )/( x )= . step . . chi-sq. (all four cells) = . + . + . + . = . step : compare your calculated chi-sq. value with the critical value to determine significance (table ) : begin at column p < . … your calculated chi-square value must meet or exceed the critical value to be considered statistically significant at that level. if you fail to meet or exceed the minimal value for p < . , the result is p > . , and the relationship is declared "not significant". step : summarize: exposure was related to illness. ill persons were eight times more likely to have been exposed to this factor than non-ill persons. this relationship is statistically significant. the probability of these data occurring by chance alone is less than . %. reject the null hypothesis of "no association." [odds ratio: . , % cl: . < or < . , chi-sq.: . , df, p < . ] form j : fisher's exact test can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps. ill well totals step step : calculate the probability directly... here, cell 'a' has smallest e value at . p = ! x ! x ! x ! = . ! x ! x ! x ! x ! step : the final probability (p) is the sum of all probabilities (in this case p + p + p ) or approximately . . step : summarize: exposure was related to illness. ill people were almost times more likely to have been exposed to this factor compared to non-ill people. the relationship is statistically significant. the probability of these data occurring by chance alone is less than . % (< . ). reject null hypothesis of "no association". [odds ratio: . , % cl: . < or < . , p = . ]. . when deciding which cells to increase by + , always multiply (a) × (d) and compare with (b) × (c). increase each cell of the pair with the higher product and decrease each cell of the pair with the smaller product, while keeping all marginal totals unchanged. . the final p is an "exact" p (probability) and may be reported as such (p = . ). in this example, it is also < . of course, and can be reported in this way if preferred. . the fisher's test is used when the chi-square test is invalid due to any "e" values < in a × table. in all other circumstances, chi-sq. is an excellent approximation for the fe test. . if original data include a zero in one of the cells, you will calculate only one p value. (the o.r. will be reported as "undefinable" but the direction of the effect will be very clear). . this p is calculated for a one-tailed fe test. it is adequate for this application. two-tailed fe test will require further calculation. . should a relationship not meet the critical value for significance (that is, p > . ), it is described as "not statistically significant". note that a relationship may be observed, but this result is telling you that it could have occurred by chance alone more than % of time if you were to repeat the analysis. that may still require further investigation, but from a statistical standpoint, it cannot be claimed as a statistically significant relationship. water treatment plant design, fifth edition american waterworks association. manual of water supply practices, m waterborne pathogens, second edition the flint water crisis confirms that u.s. drinking water needs improved risk management produce safety-what's going on here? national environmental health association neha-cert ep global health -division of parasitic diseases and malaria page last reviewed surveillance reports for recreational water-associated disease & outbreaks. page last reviewed on internalization and dissemination of human norovirus and animal caliciviruses in hydroponically grown romaine lettuce environmental protection agency technical guidance manual lt eswtr disinfection profiling and benchmarking environmental protection agency guidance manual for the compliance with filtration and disinfection requirements public water systems using surface water sources environmental protection agency. manual of individual and non-public water supply systems. epa no. epa no. water treatment manual: disinfection. environmental protection agency health canada risk analysis of the walkerton drinking water crisis algal bloom-associated disease outbreaks among users of freshwater lakes-us microbial resistance to disinfectants: mechanisms and significance. environ health perspect safe drinking water: lessons from recent outbreaks in affluent nations risk management for assuring safe drinking water ensuring safe drinking water: learning from frontline experience with contamination. american water works association cyanotoxin management and human health risk mitigation in recreational waters a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply private water supplies: technical manual good practice guide to the operation of drinking water supply systems for the management of microbial risk: research project two cases of keratosis follicularis squamosa (dohi) caused by swimsuit friction part two report of the walkerton inquiry: a strategy for safe drinking water public health inspector's guide to the principles and practices of environmental microbiology acute otitis externa: an update collect representative distribution system samples guidance on water supply and sanitation in extreme weather events. who regional office for europe effect of irrigation method on transmission to and persistence of escherichia coli o :h on lettuce transmission of escherichia coli o :h from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization acknowledgments the committee and association thank and cite the following persons for chloramines refer to all forms of chloramine. the ct values may be assumed to achieve greater than . % inactivation of viruses only if chlorine is added and mixed in the water before addition of ammonia. if this condition is not met, the system must demonstrate by on-site studies or other information that it is achieving at least this much inactivation of viruses key: cord- -n jvt authors: vandegrift, jillian; hooper, jennifer; da silva, allegra; bell, kati; snyder, shane; rock, channah m. title: overview of monitoring techniques for evaluating water quality at potable reuse treatment facilities date: - - journal: j am water works assoc doi: . /awwa. sha: doc_id: cord_uid: n jvt needless to say, the safety of treated water for potable reuse must be definitively ensured. numerous methods are available for assessing water quality; it's important to understand their challenges and limitations. i ncreased demand on water supplies, associated with population growth and changes in climatic patterns, has made it necessary for many utilities to consider augmenting their current drinking water sources with recycled water-i.e., potable reuse. for regions with imminent water supply shortages due to uncharacteristically long periods of drought or significant population shifts, the primary options for increasing water supply are water importation, saltwater desalination, and water reuse (snyder ) . water reuse may include a combination of potable and nonpotable uses to increase water supply resiliency. nonpotable reuse is water not intended for direct human consumption, such as irrigation or coolingtower water. potable reuse involves integrating highly treated municipal wastewater directly into drinking water systems, which may or may not include retention in an engineered storage buffer before its introduction, such as direct versus indirect potable reuse. in the united states, approximately - % of the bil gal of treated municipal wastewater effluent is recycled (usepa ). potable reuse was implemented in the united states more than five decades ago, with the first planned project at the los angeles county sanitation district's montebello forebay aquiferrecharge spreading grounds in (usepa ) . the us environmental protection agency (usepa) developed the first national policy statement on water reuse in (usepa ) , and the national research council developed the first set of water quality criteria for reuse in (nrc ) . usepa developed the guidelines for water reuse in , which went through three revisions (usepa (usepa , (usepa , (usepa , . the agency recently published a supplement titled potable reuse compendium (usepa ), appending the guidelines for water reuse to include more details for potable reuse. while there are no federal regulations specific to potable reuse in the united states, the safe drinking water act (sdwa) and the clean water act (cwa) provide the statutory requirements for water quality that apply to potable reuse scenarios. wastewater effluent must meet requirements set forth by the cwa and the national pollutant discharge elimination system. subsequently, advanced treated water must meet the requirements of the sdwa and national primary drinking water regulation (npdwr) maximum contaminant levels (mcls) and abide by nonregulatory water quality standards for aesthetics in the national secondary drinking water regulation mcls. specific regulations, policies, provisions, and/or guidance for potable reuse have been developed in states: arizona, california, florida, hawaii, idaho, massachusetts, nevada, new mexico, north carolina, oklahoma, oregon, pennsylvania, texas, virginia, and washington (usepa while several recent pilot and demonstration-scale studies have been conducted, two notable full-scale potable reuse facilities have provided water directly into the distribution system: big spring colorado river municipal water district ( . mgd) in and wichita falls ( mgd) in , both of which are in texas. there are two additional potable reuse facilities in the process of construction and permitting: village of cloudcroft, n.m., and the el paso advanced water purification facility in texas. figure shows a map of potable reuse projects in the united states. from a global perspective, potable reuse is expanding and will continue to grow as a means to meet drinking water supply needs in the decades to come (khan ) . probably the most well-known international project is the goreangab water reclamation plant in windhoek, namibia, which began in and was expanded in to produce . mgd of potable water (usepa ). this facility supplies approximately % of the city's potable water demand (nrc ) . australia was the first country to develop federal potable reuse guidelines when phase (which included augmentation of drinking water supplies) of the australian guidelines for water recycling was released in (ephc et al. ). singapore's newater facilities can provide up to % of the nation's water supply needs with water that meets or exceeds usepa's drinking water standards and the world health organization's guidelines (usepa (usepa , who ) . potable reuse requires careful monitoring of pathogens and chemical contaminants because of their potentially higher concentrations in the source water. ultimately, monitoring systems need to be accurate, technically accessible, and cost-effective to be successful (mosher et al. ). there has been great interest in further developing and adopting new monitoring techniques to incorporate indicator and surrogate three column figure max width = p (actual column width = p ) figure potable reuse projects in the united states a includes pilots, studies, and prospective projects compounds, optical sensors and biosensors, and bioassays. an indicator compound is an individual chemical or microorganism occurring at a measurable level that represents certain physiochemical and biodegradable characteristics of either a group of trace chemical constituents or microorganisms of interest (nrc , usepa , drewes et al. , who . for example, escherichia coli (e. coli) or coliphages are used as microbial indicators for evaluating disinfection log-reduction credits. a surrogate parameter is a measurable change of a bulk parameter that can assess the performance of a treatment barrier and can be consistently monitored (nrc , usepa . turbidity is often monitored for overall treatment performance and for demonstrating pathogen removal across filtration. indicators and surrogates must be selected on the basis of knowledge of suspected target contaminants. these must also be present at sufficiently high concentrations that are above their relative detection limits (crook et al. ) . drewes et al. ( ) performed an extensive evaluation of indicator and surrogate compounds for wastewater and water reclamation. they noted that a suite of indicator compounds and surrogates should be used to encompass a variety of physical-chemical properties and behaviors to help determine the removal efficacy for specific treatment barriers in instances where actual concentrations cannot be measured. in the united states, use or adoption of a monitoring tool or technique must demonstrate pathogen log-reduction credits required by state regulatory agencies. the riskbased approach used by the sdwa surface water treatment rule can apply to potable reuse scenarios to demonstrate that the appropriate level of protection of human health has been achieved (mosher et al. ) . monitoring schemes must also incorporate verification of water quality at critical control points (walker et al. , halliwell et al. , salveson et al. . figure shows examples of grab samples at critical control points through an advanced treatment scheme at a potable reuse facility. for direct potable reuse, the absence of an environmental buffer reduces failure response times, so the overall treatment process and associated monitoring program must be resilient enough to respond quickly and effectively if off-specification water is encountered (pepper & snyder . pathogens are characterized into three groups: bacteria, protozoa, and viruses. in general, bacteria can be destroyed, whereas protozoa and viruses are typically removed or inactivated. virus removal is particularly important because of the occurrence of viruses in municipal wastewater and acute health impacts (khan ) . bacteria. in general, bacteria range in size from . to μm in length, so they are large enough to be effectively removed via filtration, while they are also susceptible to chemical or ultraviolet (uv) disinfection. enteric pathogenic bacteria tend to be present in lower concentrations in wastewater than indicator species. e. coli, enterococcus spp., and fecal coliforms are often used as indicators of fecal bacteria. e. coli and enterococci are recommended by usepa as an indicator of fecal pollution in freshwater recreational waters because levels of these organisms were shown to be more accurate than fecal coliforms at predicting gastrointestinal illness (usepa (usepa , . fecal coliforms may also be selected for monitoring simply because many utilities already use fecal coliforms for routine testing. table shows typical indicator organisms for monitoring pathogenic bacteria, protozoa and helminths, and viruses (also see the photograph on page ). protozoa and helminths. most protozoan spores, cysts, oocysts, and eggs range in size from μm to more than μm and are thus substantially larger than most bacteria. protozoa and helminths can be eliminated from wastewater using either a physical removal or an inactivation process, such as ozone oxidation or uv disinfection (khan ) . clostridium perfringens has been used as an indicator for protozoa such as giardia lamblia and cryptosporidium parvum. additionally, because of c. perfringens' ability to form spores, this bacterium can be used as an indicator of pathogenic sporeforming protozoa. the methods to detect giardia and cryptosporidium are intensive, relying heavily on laboratory sample preparation and technician skill, whereas the detection method for c. perfringens is straightforward (though not usepa approved) and can distinguish between viable and nonviable spores (mosher et al. , usepa . in europe since the s, c. perfringens has been used as an indicator of fecal contamination in water (nrc ) . north carolina uses c. perfringens as an indicator in its water reuse regulations; to date it is the only state in the country that uses c. perfringens as a protozoan indicator for water reuse monitoring. florida uses cryptosporidium. viruses. ranging in size from . to . μm, viruses are far smaller than bacteria or parasites. enteric viruses replicate in the intestinal tracts of their hosts and are found in the fecal matter of those infected. monitoring for pathogenic viruses is challenging because not only are they notoriously difficult to detect, there are more than pathogenic enteric viruses and viruses (usepa ). however, viruses cannot replicate outside of their host and thus cannot multiply during wastewater or reclaimed water treatment. virus removal and inactivation is complex and species-specific, yet it can be achieved through coagulation/ flocculation/sedimentation, chemical disinfection, uv disinfection, ozone oxidation, and media or membrane filtration (myrmel et al. ) . because enteric viruses are difficult to detect, indicator viruses-most often bacteriophages (also known as phages)-are used. bacteriophages are viruses that infect bacteria; they do not infect humans. three main groups of bacteriophages are frequently used as surrogates for pathogenic waterborne viruses: somatic coliphages, male-specific or f+ ribonucleic acid (rna) phages, and bacteroides fragilis phages (lucena & jofre , who , iawprc study group . there is a lack of consensus about which group serves as the best indicator of enteric viruses. advantages and disadvantages of each category are presented in table . pathogen detection methods. the best methods for monitoring pathogens are rapid, highly sensitive, selective, and can distinguish between viable and nonviable organisms. no single method achieves all of these goals, so a suite of methods is recommended (mosher et al. ). indicator organisms must be present in sufficient concentrations to assess removal and must either strongly correlate to or be conservative with respect to the pathogen of interest. samples are typically concentrated to improve sensitivity using membrane filters, magnetic beads, or microfluidic devices that discriminate particles on the basis of charge or size. molecular biological methods are highly sensitive-i.e., able to detect single copies of a genome. one concern with molecular methods is that they do not distinguish viable from nonviable cells or infectivity of viruses. immunological methods tend to be more rapid than molecular biology methods because they don't require as much sample pretreatment, but they are less sensitive (connelly & baeumner ) . biological molecule assays bioenergy molecules such as adenosine triphosphate (atp) or nicotinamide adenine dinucleotide phosphate (nadp + ) are present in all living organisms. atp and nadp + are not present in viruses. hydrolase enzymes, which represent a large class of microbiological household enzymes, may also be used for quantifying biomass. immunological assays these assays are highly selective but not highly sensitive. examples include the enzyme linked immunosorbent assay and serum neutralization tests. polymerase chain reaction (pcr), quantitative pcr (qpcr) pcr allows for the identification of the deoxyribonucleic acid (dna) or ribonucleic acid (rna) of pathogens or indicators present in a water sample. qpcr allows for the dna or rna to be quantified and is relatively rapid (less than hours). nucleic acid sequence-based amplification (nasba) nasba uses isothermic conditions for dna or rna amplification (rather than the temperature cycles used in pcr and qpcr), and therefore may be somewhat faster than pcr-based methods. droplet digital pcr samples are partitioned into thousands to millions of nanoliter or picolitre volumes inside small chambers on a chip (i.e., chamber digital pcr) or within a water-in-oil droplet (i.e., droplet digital pcr) before pcr amplification. the frequency of positive partitions is used with poisson statistics to estimate the number of target copies in the original sample. this technology starts with a single-stranded pcr product to which a primer is added to initiate a dna-sequencing reaction. turbidity higher-turbidity water may have a higher likelihood of having pathogens present, though correlations are not straightforward or ensured. light scattering suspended particles can be detected because of their ability to scatter light in water. multi-angle lightscattering technology uses lasers and light-scattering detectors to determine the shape and size of particles. microscopic identification an example microscopic identification method is usepa method for detecting cryptosporidium parvum and giardia lamblia. a sample is filtered, and then the oocysts and cysts are separated from the material captured using magnetic beads conjugated to anti-c. parvum and anti-g. lamblia antibodies, then eluted, stained, and counted with differential interference contrast microscopy. biosentry is another tool for microscopic identification and can detect between and spores/ml. raman spectroscopy involves ultraviolet through visible to near infrared light excitation. surfaceenhanced raman spectroscopy (sers) can identify specific microorganisms. a optical tweezers can isolate bacteria, allowing discrimination between different strains of bacteria. b fourier transform infrared (ftir) spectroscopy involves the excitation of molecules by infrared light and the unique signature spectra is measured. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry detects the mass to charge ratio of particles to provide a "fingerprint" of specific microorganisms. c flow cytometry (fc) fc can be combined with the use of nucleic acid probes or fluorescent antibodies to rapidly identify and quantify specific microorganisms. researchers have presented methods to identify pathogenic e. coli o :h , c. parvum, and nonpathogenic e. coli in water. d-f sensitivity has improved through the amplification of viral signals as a result of the use of a novel nucleic acid dye, sybr-gold. these recent advancements make near-real-time measurements for total bacteria and viruses in water a practical possibility. ware et al. processes and their ongoing performance for removing pathogens and other chemicals (crook et al. ) . though each unit treatment process has its own bulk surrogate parameters that lend information on treatment performance, some are applicable across multiple methods. such surrogates include conductivity, boron, calcium, magnesium, uv absorbance, total organic carbon (toc) or dissolved organic carbon, specific uv absorbance, fluorescence excitation/emission matrix spectroscopy, total nitrogen, ammonia, nitrate, and concentration times time (crook et al. , drewes et al. . table shows a list of conventional parameters used to assess performance. solid-state instruments, including those for conductivity, toc, ph, turbidity, and temperature, continue to be the most reliable methods for measuring changes in drinking water quality in real time (storey et al. ) . for potable reuse scenarios that include an environmental buffer, a wastewater tracer test can assess dilution and contaminant degradation in receiving water bodies. wastewater tracers must be recalcitrant and not easily removed during treatment. for example, primidone and carbamazepine are not easily biodegraded and exhibit poor removal (< %) during soil-aquifer treatment (sat) (drewes et al. ) . these compounds can be used to track wastewater contributions to groundwater during sat. artificial sweeteners, such as sucralose and acesulfame-k, are also relatively nondegradable in nature yet abundantly present at levels above their detection limits, and they serve as conservative tracers for the presence of wastewater in surface water and groundwater (anderson et al. ) . sucralose is useful for evaluating the removal of water-soluble, uncharged chemicals with moderate molecular weights (crook et al. ) . trace chemical constituents. trace chemical constituents encompass an array of compounds, including industrial, manufacturing, and consumer products, as well as personal care products and pharmaceuticals; example constituents are described in table . their concentrations in raw, secondary, and tertiary treated wastewater are wide-ranging (drewes et al. ) . a survey of drinking water treatments in the united states demonstrated maximum finished-water constituents of emerging concern (cecs) at concentrations below ng/l, except for the flame retardant tris ( -chloro- -propyl) phosphate (commonly known as tcpp), which had a maximum concentration of ng/l . in california, the water quality control for recycled water, also known as the recycled water policy, was adopted by the state water board in and amended in to include cec monitoring recommended by its science advisory panel (anderson et al. ) . the final list of recommended compounds included cecs (drewes et al. ) . four cec indicator compounds were recommended for potable reuse monitoring by the national water research institute panelnamely, cotinine, meprobamate, carbamazepine, and estrone, because of the following characteristics (crook et al. ): • cotinine: low molecular weight that is likely charged to some degree at ph values employed in water treatment • meprobamate: low molecular weight with a basic structure similar to alkanes and occurs abundantly and in sufficient concentrations • carbamazepine: low molecular weight, unique structure, recalcitrant, abundant in wastewater and occurs at adequate concentrations • estrone: natural steroidal hormone that is present at higher levels than β-estradiol or ethinyl estradiol chemical constituent detection methods. as shown in table , different analytical methods are needed to quantify trace chemical constituents because of their diverse physicalchemical properties. despite considerable progress in detection methods, there are no standard methods for quantification. when interpreting (drewes et al. ) . bioanalytical tools. through drinking water, human exposure to chemicals does not happen singly but rather occurs as a complex mixture of dissolved chemicals and particulate matter. often chemical toxicity is nonadditive and may be synergistic or antagonistic. scientific studies have demonstrated that combinations of chemicals that would not elicit an effect on their own can induce significant toxicity when combined (margiotta-casaluci et al. ) . conversely, antagonistic effects between suites of chemical constituents may occur. because of the complex nature of constituents in wastewater, in addition to potential synergistic effects of mixtures of compounds, biological assays that can rapidly and comprehensively screen water for a suite of toxicological end points may be useful. however, biological assays tend to be subjective and thus should be used appropriately. a comprehensive survey of bioassays indicative of a wide range of responses has been published (escher et al. ) . these include assays that were sensitive to induction of specific modes of toxicity, such as mutagenicity and genotoxicity, xenobiotic toxicity, reactive toxicity, cytotoxicity, and endocrine disruption, among other modes of action. the conclusions of the study demonstrated that while there are limitations to bioassay techniques, they are a valid tool for water quality assessment that complement chemical analyses. a recent study incorporating bioassays covering biological endpoints indicated that a suite of bioassays can be useful in characterizing multiple toxicological pathways relevant to human health and can guide decision-making with regard to the treatment processes (jia et al. ) . the science advisory panel, which convened in to evaluate risks from cecs in water treated for reuse, recommended that estrogen receptor alpha and the aryl hydrocarbon receptor bioassays be used to respectively assess estrogenic and dioxin-like biological activities in recycled water (drewes et al. ). however, these technologies are relatively immature and require further development in order to identify, create, and standardize bioanalytical tools. advanced-treated recycled water can be beneficially reused for a variety of purposes, including augmenting drinking water supplies. multibarrier approaches, including advanced treatment, have been demonstrated to achieve highquality water, even when the source water is significantly degraded (nrc ). however, appropriate monitoring schemes are needed to ensure public health protection. a challenge for potable reuse monitoring is how to effectively characterize pathogens, chemical constituents, and emerging contaminants in advanced treated water in an appropriate time frame. water quality monitoring must incorporate conventional monitoring schemes that apply to wastewater and drinking water treatment; it must also incorporate high-frequency indicator and surrogate compounds to assess pathogen and chemical removal efficacy. complex monitoring programs tracking trace chemical constituents aren't required, but indicator compounds such as sucralose or cotinine can be useful to characterize removal of more recalcitrant compounds. commonly used bulk indicators, such as turbidity and conductivity, remain useful to assess removal of pathogenic organisms, but they have limitations. additional characterization of pathogen removal using indicator organisms (e.g., total coliform bacteria, clostridium perfringens, viral indicators, bacteriophages), incorporating a combination of physical detection, cell culture, and/or molecular biological assays, gas chromatography (gc) and mass spectrometry (ms) gc/ms is one of the most widely used combined techniques because of its sensitivity and selectively. ms measures an analyte dependent on its mass to charge ratio. the compound must be charged before it enters the machine. gc/ms employs two methods to charge or ionize analyte, electron ionization (ei) and chemical ionization (ci). ei results in molecular fragmentation, which gives each compound its own "fingerprint" and allows for individual detection; however, sensitivity is lost. ci involves ionization of a gas and results in less fragmentation than ei. liquid chromatography (lc) and tandem ms lc/ms differs from gc/ms in that the separation of analytes occurs in the liquid phase. lc/ms uses three ionization techniques: electrospray ionization (esi), atmospheric pressure chemical ionization, and atmospheric pressure photoionization. esi is the most common technique. isotope dilution lc and tandem ms lc/ms/ms involves solid-phase extraction and lc/ms/ms using esi in positive and negative modes. urobilin is a metabolic byproduct of heme metabolism that is excreted through human and animal feces in water. urobilin has been shown to be a beneficial biomarker for detection of fecal contamination. fluorescence detection of urobilin is achieved when urobilinogen-zinc, a chelation complex, is excited by blue light and exudes a green fluorescence. may provide useful information to enhance characterization of water quality and subsequent advanced water treatment. monitoring strategies for chemicals of emerging concern (cecs) in recycled water: recommendations of a science advisory panel. california state water resources control board comparative survival of indicator viruses and enteric viruses in seawater and sediment bio-sensors for the detection of waterborne pathogens examining the criteria for direct potable reuse, recommendations of an nwri independent advisory panel (wrrf - ) development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of escherichia coli :h in aqueous samples and comparison of the assay with a standard microbiological method monitoring strategies for constituents of emerging concern (cecs) in recycled water: recommendations of a science advisory panel development of indicators and surrogates for chemical contaminant removal during wastewater treatment and reclamation the application of a recently isolated strain of bacteroides (gb- ) to identify human sources of faecal pollution in a temperate river catchment benchmarking organic micropollutants in wastewater, recycled water and drinking water with national health and medical research council, & natural resource management ministerial council bacteriophage as pollution indicators utilization of hazard analysis and critical control points approach for evaluating integrity of treatment barriers for reuse (wrrf - ) bacteriophages as model organisms in water treatment f-specific rna bacteriophages are adequate model organisms for enteric viruses in freshwater f-specific rna bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin bacteriophages and indicator bacteria in human and animal faeces distinguishing human from animal fecal contamination in water by typing male specific rna coliphages genotyping male-specific rna coliphages by hybridization with oligonucleotide probes bacteriophages as model viruses in water quality control in vitro bioassays to evaluate complex chemical mixtures in recycled water is the replication of somatic coliphages in water environments significant phage methods drinking water through recycling: the benefits and costs of supplying direct to the distribution system simple and rapid f+ coliphage culture, latex agglutination, and typing assay to detect and source track fecal contamination potential use of bacteriophages as indicators of water quality and wastewater treatment processes mode of action of human pharmaceuticals in fish: the effect of the -alpha-reductase inhibitor, dutasteride, on reproduction as a case study enteroviruses and bacteriophages in bathing waters potable reuse research compilation: synthesis of findings (wrf reuse - ). water environment & reuse foundation enteric viruses in inlet and outlet samples from sewage treatment plants water reuse: potential for expanding the nation's water supply through reuse of municipal wastewater indicators for waterborne pathogens. the national academies press quality criteria for water reuse. the national academies press pathogens in water: value and limits of correlation with microbial indicators monitoring for reliability and process control of potable reuse applications (wrrf - ) achieving reliability in potable reuse: the four rs detection of viruses: atomic force microscopy and surface enhanced raman spectroscopy assessment of techniques to evaluate water quality from direct and indirect potable facilities. project . the water research foundation assessment of techniques to evaluate and demonstrate the safety of water from direct potable reuse treatment facilities risk reduction for direct potable reuse (wrrf - ) microbial source tracking: current methodology and future directions emerging chemical contaminants: looking for greater harmony toxicological relevance of edcs and pharmaceuticals in drinking water (wrf ) advances in on-line drinking water quality monitoring and early warning systems. water research swrcb (state water resources control board), . investigation on the feasibility of developing uniform water recycling criteria for direct potable reuse. swrcb bacteriophages active against bacteroides fragilis in sewage-polluted waters human origin of bacteroides fragilis bacteriophages present in the environment potable reuse compendium guidelines for water reuse. epa/ /r- / guidelines for water reuse guidelines for water reuse guidelines for water reuse epa policy statement on water reuse and water reuse background statement critical control point assessment to quantify robustness and reliability of multiple treatment barriers of a dpr scheme evaluation of an alternative ims dissociation procedure for use with method : detection of cryptosporidium in water applications of whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry in systematic microbiology potable reuse: guidance for producing safe drinking-water water, sanitation and hygiene links to health: facts and figures. who water quality: guidelines, standards, and health detection of escherichia coli o : h , salmonella typhimurium, and legionella pneumophila in water using a flow-through chemiluminescence microarray readout system spectroscopic differentiation and quantification of micro-organisms in apple juice • planning for direct potable reuse: operational aspects of an integrated drinking water system these resources have been supplied by journal awwa staff. for information on these and other awwa resources key: cord- -uklnrgt authors: black, e. kathleen; finch, gordon r. title: detection and occurrence of waterborne bacterial and viral pathogens date: - - journal: water environ res doi: . /j. - . .tb .x sha: doc_id: cord_uid: uklnrgt nan the th edition of standard methods for the examination of water and wastewater was published during . changes in the microbiological examination section include modifications in the proposed detection of staphylococci in recreational waters, the total coliform multiple-tube fermentation technique and enumeration, virus concentration methodology, as well as a proposed chromogenic substrate test for coliforms. environmental microbiology was a monograph published in featuring a broad range of topics related to environmental microbiology, including detection of several waterborne bacterial, protozoan, and viral pathogens (singh and mcfeters) . the occurrence of waterborne bacterial pathogens is summarized in table . water types included in this table are drinking water (including bottled water), surface water, groundwater, and wastewater. the most frequently reported bacteria were escherichia coli.. followed by the coliform group. these indicator bacteria are included in this review because of their use as indicators of pathogens. attempts were made to correlate the presence of coliform bacteria with pathogens, such as salmonella. vibrio parahaemolyticus, and aeromonas hydrophila (martinez-manzanares et al.; o'shea and field; stelzer and jacob; and townsend) . the correlation between salmonella and coliform june bacteria or enterococci was unclear in a survey of two pools in the australian wet-dry tropics (townsend) , in an estuary of a river in spain (martinez-manzanares et al.) , and in storm water runoff (o'shea and field). v. parahaemolyticus and a. hydrophila in shellfish were correlated with total coliforms (martinez-manzanares et al. ) , and aeromonads increased relative to coliforms in treated drinking water (stelzer et at.) . studies on the persistence of pathogens and indicators in water were reported. the survival of e. coli (gauthier et al.; korhonen and martikainen, ; prabu and mahadevan; roberts; and terzieva and mcfeters, ) , salmonella species (townsend) , campylobacter species (korhonen and martikainen, and terzieva and mcfeters) , legionella pneumophila (cargill et al., and paszko-kolva et al., ) , and h elicobacter pylori (west et al.) was examined in surface water, groundwater, seawater, and under varying saline conditions. surveys of the incidence of vibrio vulnificans indicated that this organism shows seasonal variance in numbers and in habitat (o'neill et al.. tamplin and capers; and vanoy et al. ) , increasing in numbers only when the temperature of the water is greater than °- °c. during the cold weather months, v. vulnificus was detected only in sediments, whereas at higher water temperatures, such as found during the summer months, the organism was detected in oysters, sediment, and seawater (vanoy et at.) . a survey of pools in the australian wet-dry tropics revealed that the levels of salmonella. coliforms, and enterococci varied through the year, being lowest during the dry season (townsend) . the concentration of salmonellas varied by as much as two log cycles,and the concentration of coliforms and enterococci varied by as much as three log cycles. the high reported levels of salmonella were attributed to the high carrier rate in reptiles and marsupials and the high water temperatures. the norwalk agent and hepatitis a were implicated in several outbreaks of waterborne illness in north america during - (health and welfare canada, and herwaldt et al., ) . the occurrence of viruses in water are summarized in table . surveys of river water and recreational water showed that virus levels varied throughout the year (hughes et al., and tani et al.) . poliovirus presence in river water was found to be associated with the application of the polio vaccine, however, the presence of echoviruses, reoviruses, coxsackieviruses, and adenoviruses did not vary throughout the year (tani et al.) . in coastal and inland seawater, coxsackieviruses were most frequently isolated. the other viruses that were identified were polioviruses (hughes et al.) , possibly derived from immunized children. the authors noted, however, that identification and isolation of viruses in their study were limited by the cell culture used and the number of plaque forming units seeded. the infectivity of viruses from lawns irrigated with wastewater was examined using an animal model (deming et al.) . fewer piglets exposed for hours to lawns irrigated with x % cell-culture infectious dose (ccid so ) virus particles became positive than piglets inoculated with ccid so virus particles. references (leung et al.; martinez-manzanares et al.; moyer et al.; stelzer et al.) (korhonen and martikanen, ; stelzer and jacob; terzieva and mcfeters, (terzieva and mcfeters, ) a survey of municipal wastewater reported that the survival of human immunodeficiency viruses in wastewater was less than that of polioviruses (casson et at.) . the occurrence of g. lamblia and two other protozoa, cryptosporidium parvum and acanthamoeba. is summarized in table . the survival of c. parvum oocysts was reported under various environmental conditions and it was found to be a robust organism in all of the water types examined (robertson et al.v. acanthamoeba is distributed widely in the freshwater environment. studies have shown that the detection of l. pneumophila in water is more probable if acanthamoeba species are present also (sanden et at.) . representative literature from the past year related to the detection of waterborne pathogens are summarized in table . several articles investigated differences in recovery of waterborne pathogen detection methods, particularly with respect to quantity and speed of analysis (barrell; covert et al.; foster et al.; schets and havelaar, ; warburton et al.; and whipple et al.) . viable but nonculturable organisms present a problem when detecting organisms in water (mccarty et al.; mckay; and singh and mcfeters) . preenrichment and selective enrichment of samples, as well as the newer technologies of gene probes and immunoassays, improve the detection of injured and stressed organisms. methods for quantifying pathogens continue to improve, resulting in improved detection of stressed and injured organisms and reducing the time committed to identification (bifulco and schaefer; covert et al.; jacob and stelzer; oliver et al.; and warburton et al.) . developments in gene probe and immunoassay technologies are making these detection methods more accessible to routine water analysis laboratories (atlas et al.; kerr et al.; sano et al.; thorns et al.; and yamamoto, k. et al.i. gene probes are rapid, sensitive, and specific, however, the probes do not always detect viable cells, and in some cases the technology requires special equipment. an extensive overview of gene probe technology was published during (atlas et al.) . immunoassays can detect toxins produced by organisms or the organisms themselves, however, viability determination of the detected cells is not reliable. (brown et al.) a new technology that shows promise is the combined use of conductance and immunology (parmar et al.v . beads coated with the antigen for a specific pathogen are exposed to the organism. after a short incubation, the beads are separated, washed, and resuspended in broth. the change in conductance of the broth is measured, and the resulting curve is specific for the organism. salmonella typhimurium can be detected from a preenrichment broth within hours using this method (parmar et al.) . the sensitivity of the immunobead assay is yet to be determined, specialized equipment is necessary, and competition table - etection of waterborne pathogens. between the salmonella species studied and some species of citrobacter was reported. characteristic substances produced by mycobacterium species have been detected by gas chromatography-mass spectrometry (alugupalli et al.) . although this method is extremely sensitive and considerably faster than current recovery methods, the cost of the detection equipment is beyond the capabilities of most laboratories. entis and boleszczuk, ; foster et al.; kerr et al.; martinez-manzanares et al.; parmar et al.; todd et al., ; townsend; tsen et al., ) shigella f exneri x (bej et al., ) staphylococcus sp. x x x (jaulhac et al.; martinez-manzanares et al.; park et al.; ruzickova, ; shinagawa et al., ; stelz and (hastie et al.; isaacrenton et al.; mahbubani et al., ; salmonella detection in marine waters using a short standard method application of gas-chromatography massspectrometry for rapid detection of mycobacterium xenopi in drinking water universal preenrichment broth for the simultaneous detection of salmonella and listeria in foods. . food prot a comparison between tryptone bile agar and membrane lauryl sulphate broth for the enumeration of presumptive escherichia coli in water polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples iii ( ) serial subcultivation of giardia lamblia in keister modified tyi-s- medium containing ultroser-g recovery of enteroviruses from water and sediments of lake maracaibo, venezuela detection of campylobacter upsaliensis in diarrheic dogs and cats, using a selective medium with cefoperazone viability of cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes detection ofsalmonellas by dna hybridization with a fluorescent alkaline phosphatase substrate effects of culture conditions and biofilm formation on the iodine susceptibility of legionella pneumophila cyanobacteria secondary metabolites-the cyanotoxins hiv survivability in wastewater update: cholera-western hemisphere, comparing defined-substrate coliform tests for the detection of escherichiacoliin water an animal model to assess the potential for viral disease transmission from lawns irrigated with wastewater instability of fecal coliform populations in waters and bottom sediments at recreational beaches in arizona rapid detection of salmonella in foods using ef- agar in conjunction with the hydrophobic grid membrane filter rotavirus detection by dot blot hybridization assay using a non-radioactive synthetic oligodeoxynucleotide probe a combined pcr and selective enrichment method for rapid detection of listeria monocytogenes modified colorimetric dna hybridization method and conventional culture method for detection of salmonella in foods-comparison of methods sensitivity of escherichia coli cells to seawater closely depends on their growth stage a study of the incidence of different fluorescent pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water zeekoevlei-water chemistry and phytoplankton periodicity concentrating giardia cysts in water by tangential row filtration compared with centrifugation gastroenteritis outbreak at an industrial camp-british columbia waterborne-disease outbreaks rapid determination of members of the family enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen enteroviruses in recreational waters of northern ireland comparison of an in vitro method and an in vivo method of giardia excystation comparison of two media for the isolation of thermophilic campylobacters from waste waters of different quality synthetic dna probes for detection of genes for diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of salmonella survival of escherichia coliand campylobacter jejuniin untreated and filtered lake water biochemical fingerprinting of water coliform bacteria, a new method for measuring phenotypic diversity and for comparing different bacterial populations use of an oligonucleotide probe to detect vibrioparahaemolyticus in artificially contaminated oysters detection of the light organ symbiont, vibrio fischeri. in hawaiian seawater by using lux gene probes bacterial pathogens and indicators in catfish and pond environments a rapid method for detection of yersinia enterocolitica serotype - in pig feces using monoclonal antibodies row cytometric detection of prochlorophytes and cyanobacteria in the gulf of policastro a membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water. . appl. bacteriol i) detection of giardia cysts by using polymerase chain reaction and distinguishing live from dead cysts differentiation of giardia duodenalis from other giardia spp. by using polymerase chain reaction and gene probes relationship between indicators of fecal pollution in shellfish-growing water and the occurrence of human pathogenic microorganisms in shellfish detection of virulence factors in culturable escherichia coli isolates from water samples by dna probes and recovery of toxin-bearing strains in minimal ortho-nitrophenolbeta-d-galactopyranoside- -methylumbelliferyl-beta-d-glucuronide media evaluating a commercially available de-water environment research test for recovery of chlorine-treated escherichia coli viable but non-culturable forms of potentially pathogenic bacteria in water-review direct detection by polymerase chain reaction (pcr) of escherichia coli in water and soft cheese and identification of enterotoxigenic strains application of ribotyping for differentiating aeromonads isolated from clinical and environmental sources seasonal incidence of vibrio vulnificus in the great bay estuary of new hampshire and maine detection and disinfection of pathogens in storm-generated flows detection oflisteria monocytogenesusing polyclonal antibody use of colistin-polymyxin b-cellobiose agar for isolation of vibrio vulnificus from the nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (tecra) for detection of detection and differentiation of bovine group-a rotavirus serotypes using polymerase chain reactiongenerated probes to the vp gene use of a hydrophobic grid-membrane filter dna probe method to detect listeria monocytogenes in artifically-contaminated foods survival of escherichia coli -h in drinking water associated with a waterborne disease outbreak of hemorrhagic colitis coliform bacteria from aquatic sources in fiji survival of cryptosporidium parvum oocysts under various environmental pressures a rapid polymerase chain reaction (pcr)-based assay for the identification of listeria monocytogenes in food samples application of phage typing to the identification of sources of groundwater contamination detection of bovine adenovirus nucleic acid sequences in nasal specimens by biotinylated dna probes rapid method for detecting thermostable nuclease in staphylococci incubation of water samples containing amoebas improves detection of legionellae by the culture method immuno-pcr-very sensitive antigen detection by means of specificantibody-dna conjugates comparison of indole production and beta-glucuronidase activity for the detection of escherichia coliin a membrane filtration method detection of staphylococcal enterotoxin-a by sandwich enzyme-linked immunosorbent assay with monoclonal antibodies evaluation of an enzyme immunoassay kit for detecting cryptosporidium in faeces and environmental samples detection methods for waterborne pathogens detection of rotavirus in stools of diarrhoeic children in belo-horizonte, mg occurrence and frequency of staphylococci in bottled mineral water the occurrence of campylobacterin a mountain river a study of the prevalence of aeromonads in a drinking water supply persistence of vibriovulnificus in tissues of gulf coast oysters, crassostrea virginica, exposed to seawater disinfected with uv light enteric virus levels in river water survival and injury of escherichia coli. campylobacterjejuni, and yersinia enterocolitica in stream water development of monoclonal antibody elisa for simultaneous detection of bovine coronavirus, rotavirus seregroup-a, and escherichia coli k antigen in feces of calves evaluation of salmonella antisera for an optimum enzyme-linked antibody detection ofsalmonella using hydrophobic grid membrane filters solid-phase polymerase chain reaction-applications for direct detection of enteric pathogens in waters the relationships between salmonellas and faecal indicator concentrations in pools in the australian wet dry use of the polymerase chain reaction for specific detection of type-a, type-d and type-e enterotoxigenic staphylococcus aureus in foods salmonella detection in meat and fish by membrane hybridization with chromogenic/phosphatase/biotin dna probe ecology of vibrio vulnificus in galveston bay oysters, suspended particulate matter, sediment and seawater-detection by monoclonal antibody immunoassay most probable number procedures a one-minute oxidase test to detect vibriostrains isolated from cultures on thiosulphate-citrate-bile salts-sucrose (tcbs) medium s rrna-based probes and polymerase measurement of pollutants chain reaction method to detect listeria monocytogenes cells added to foods comparison of methods foroptimum detection of stressed and low levels of listeria monocytogenes. food microbial effect of physical environment on survival of helicobacter pylori comparison of a commercial dnaprobe testand cultivation procedures fordetection ofmycobacterium paratuberculosis in bovine feces contamination of shellfish by stool-shed viruses-methods of detection detection of enterotoxin and verocytotoxin genes in escherichia coli from diarrhoeal disease in animals using the polymerase chainreaction factors stimulating propagation of legionellae in cooling tower water enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) of vibrio parahae molyticus. can. j. microbiol., , . reginald goo monitoring effluent monitoring data collected under the national pollutant discharge elimination system (npdes) program were used to estimate priority pollutant loads to the san francisco estuary (davis et al. ) chromium, copper, nickel, and zinc from four municipal wastewater treatment facilities accounted for approximately % of the total loads for these elements to the estuary. silver concentrations were measured in coastal surface waters in the southern california bight (sai'iudo-wilhelmy and flegal). based on mass balance calculations, anthropogenic silver inputs from the point lorna discharge off san diego, calif., account for essentially all of the silver in coastal waters along the united states-mexico border during summer conditions. grimalt et al. reported a study of a sediment sampling network encompassing six coastline perpendicular transects, each situated in front ofa river mouth. variance analysis has shown this type of sampling scheme can recognize the main coastal pollution discharge sites, but is not useful for tracing their area of influence.aichinger et al. used electrolytic respirometry to measure the biodegradability of six phthalate esters and four polynuclear aromatic hydrocarbons. dissolution appeared to govern the degradation rate when these compounds were present in amounts exceeding their solubility. alexander and fu found microbial mineralization of styrene rapid in wastewater and certain types of soil. they suggested that styrene will be rapidly destroyed by biodegradation in most aerobic environments and the rate may be slow in environments of low ph. chaudhuri et al. studied the effect oftemperature on the incubation time in biochemical oxygen demand (bod) tests. they concluded that bod s. o is equivalent to bod • and should be used in tropical countries.fendlinger et al. published an analytical method for the determination of alkyl sulfate surfactants in natural waters. their monitoring results indicated that alkyl sulfate surfactants removal is similar to bod removal and was at least % at two trickling filter wastewater treatment plants sampled. field et al. published a method for the determination of sulfonated aliphatic and aromatic surfactants in wastewater sludge. concentrations of secondary alkanesulfonate and linear alkylbenzene sulfonate surfactants in wastewater sludges ranged from . to . and from . to . g/kg, respectively.comprehensive air emission tests were conducted on two multiple-hearth incinerators (hentz et al.) . lead and cadmium control efficiencies were significantly higher than those assumed in proposed regulations. despite low sludge concentrations and high control efficiencies, arsenic and cadmium emissions are expected to exceed limits proposed in recent regulations. huang et al. reported dibromopolychlorodibenzo-p-dioxins and dibromopolychlorodibenzofurans characterization in municipal waste incinerator fly ash. concentrations of these compounds ranged from low to high parts per trillion depending on the location where the samples were collected.brodbelt proposed an interface for on-line process monitoring of gas and liquid samples using a mass spectrometer that was applied to freon, benzene, and pentachloropyridine. reinelt and grimvall reported that different loading estimation methods can yield significantly different results, even if sampling during peak flows occurs. theoretical calculations showed that sampling strategies with evenly spaced sampling intervals may systematically over or underestimate the true loading. characteristics of the collected data, relative influence of point sources, and desired detail of loading estimates influence the choice of the estimation method. martin et al. compared surface-grab and cross-sectionally integrated sampling methods for determining stream water quality. surface grab samples underrepresented concentrations of suspended sediments and some sediment-associated constituents.foreman et al. encountered analytical interference from mercuric chloride-preserved water samples. this interference occurred in the continuous liquid-liquid extracts that were analyzed by gas chromatography-mass spectrometry. poor recoveries of spiked samples were observed when using a closed-loop stripping method. clement described a classroom experiment that illustrates sampling difficulties and analytical errors that can occur in trace environmental determinations. a practical guide for environmental sampling and analysis was presented by keith. sperling et al. (a) published an on-line flow injection preconcentrator coupled to an electrothermal atomic absorption spectrometer for the determination of chromium(vi) and total chromium. sodium diethyldithiocarbamate was used as the key: cord- -ruizhgwh authors: elliot, elisa l; colwell, rita r title: indicator organisms for estuarine and marine waters date: - - journal: fems microbiol lett doi: . /j. - . .tb .x sha: doc_id: cord_uid: ruizhgwh abstract the use of indicator organisms for estuarine and coastal waters has been reviewed. the natural flora of the environment must be considered in selecting an indicator organism, but, more importantly, recent work which shows a viable but non‐recoverable stage of pathogens entering the marine environment demonstrates that the conventional detection of indicator microorganisms is misleading, if not inaccurate. results suggest that the newly developed epifluorescent/immunofluorescent direct detection of pathogens in the environment may be the most reliable method for determining public health hazards in marine and estuarine waters. the use of indicator organisms for estuarine and coastal waters has been reviewed. the natural flora of the environment must be considered in selecting an indicator organism, but, more importantly, recent work which shows a viable but non-recoverable stage of pathogens entering the marine environment demonstrates that the conventional detection of indicator microorganisms is misleading, if not inaccurate. results suggest that the newly developed epifluorescent/immunofluorescent direct detection of pathogens in the environment may be the most reliable method for determining public health hazards in marine and estuarine waters. the microbiology of the world oceans and coastal waters has been under study since the late nineteenth century. among the earliest investigations concerning the number of bacteria and their * to whom correspondence should be addressed. distribution in seawater was that of russell [ ] [ ] [ ] who conducted a survey of the distribution of bacteria in the gulf of naples. the early workers established general distributional patterns for bacteria in the sea, using agar plate and dilution methods for the enumeration of bacteria. bacteria were found in small numbers, uniformly distributed in water several miles from shore and at all depths. early workers also found that the water of harbors and bays may contain large numbers of bacteria, but that the number of bacteria becomes very small at comparatively short distances from shore [ , ] . the work of fischer is, of course, classic, since he investigated sea water over a wide area of the atlantic ocean and found bacteria throughout, the largest numbers being found near land or in the presence of floating seaweed [ , ] . seasonal studies were done by lloyd [ ] in the clyde sea, where the waters are relatively shallow. the surface waters were richest in bacteria, the number decreasing with depth but usually increasing slightly at the bottom. a variety of studies was done in the early )'ears of marine microbiology, summarized by benecke [ ] and zobell [ ] . the observation that direct microscopic examination of seawater showed bacteria to be much more abundant in seawater than cultural methods would - / /$ . © federation of european microbiological societies suggest, was made as early as by cholodney [ ] . marine bottom deposits are known to contain much larger numbers of bacteria than the water column. russell [ ] showed that bacteria were distributed in sediments of the bay of naples at depths ranging from - m below the surface of the water. zobell and morita [ ] , in a study carried out about years ago, showed that bacteria are indeed present in sediment cores taken in the phillippines and marianas trenches. some years earlier, reuszer [ ] had demonstrated that bacteria are much more numerous in bottom deposits than in overlying water, and that the number of bacteria decreases with depth into the sediment. dale [ ] showed a strong relationship between bacterial numbers and sediment properties, i.e., the number of bacteria is highly correlated with grain size and other granulometric properties. sampling methods for microbiological analyses of water include the j-z sampler and the niskin sampler [ ] . deep ocean environmental samplers have also been developed in recent years [ , ] . sediment samples are unfortunately still obtained by core or grab [ ] . animals are collected by means of traps and trawls. the methods for the enumeration of microorganisms, estimating their biomass, and measuring their activities in the ocean, were the basis of a recent symposium [ ] , and include the classic methods of plate count and filtration. more recently developed methods include atp measurement, a biochemical technique for estimating total microbial biomass as a function of the luciferinluciferase reaction, which is quantitatively dependent on the amount of atp in the sample [ ] , and bacterial cell wall component analyses [ ] . several different approaches to microscopic bacterial counts are now available, all employing fluorochromes and epifluorescent microscopy. some of these methods enumerate total (i.e., living and non-living) bacteria [ ] [ ] [ ] , whereas others purport to count only living bacteria [ ] [ ] [ ] . recently, tabor and neihof [ ] compared of the direct viable count methods with amino acid uptake, and found that amino acid uptake consistently measured a larger number of cells. the epifluorescent-immunofluorescent method offers exciting new opportunities for .determining directly the presence of pathogens in water, even when these organisms are viable but non-recoverable [ ] [ ] . the results of the work of pioneers in marine microbiology at the turn of the century have been firmly established. more recently, sieburth [ ] surveyed seawater from the pacific, caribbean, and atlantic oceans, culturing bacteria in agar roll tubes indicated within . °c of the temperature of the water from which they were obtained. the isolates were keyed to genus, and a characteristic bacterial flora was reported in the ~m-thick surface film overlying 'low-count' waters off the west coast of central america in an area known for upwelling; in all the samples, pseudomonads were dominant. however, very recently, the same areas have been re-examined and the dominant organisms, especially at the ocean dumping sites, have been identified as vibrio spp. [ ] . murchelano and brown [ ] found that heterotrophic bacteria in the long island sound water column showed bacterial concentrations ranging from - per ml, with variations in seasonal concentration mirroring that of the phytoplankton. gram-negative rods constituted % of the isolates. genera were identified, but . % were a chromobacter, flaoobacteriurn, and pseudomonas, with pseudomonas dominant in the summer and flaoobacterium in the spring. again, more recent work suggests a far more prominent role for vibrio [ ] . there is a wide oceanic distribution of bacterial genera associated with geomicrobial cycling in nature. for example, nitrogen-fixing, nitrite-and nitrate-forming, nitrate-reducing, cellulose-decomposing, agar-decomposing, and chitin-decomposing bacteria have been isolated from seawater and sediment [ ] . denitrifying bacteria in shallowwater marine sediment were documented by patriquin and knowles [ ] . nitrogen-fixers have been isolated, including nitrogen-fixing vibrios ( [ ] ; m.l. guerinot and r.r. colwell, in press). thiobacillus-like bacteria, i.e., reduced sulfuroxidizing bacteria (thiosulfate or elemental sulfur), have been isolated from seawater and sediment in the atlantic ocean, black sea, and other areas [ ] [ ] [ ] . yeasts are readily isolated from coastal waters [ ] , especially in association with seaweeds [ ] . actinomycetes, mycelial bacteria, can also be isolated from bays, coastal and, offshore waters [ . however, more recent work [ ] suggests that the actinomycetes are perhaps more commonly of terrestrial origin, especially at ocean dumping sites. a very interesting group of bacteria is the 'filterable form' or 'mini-bacteria'. the occurrence of filterable bacteria in sea water was reported by oppenheimer [ ] , who found up to viable cells per ml of sea water filtrate after passage through a membrane with a pore diameter of approx. . /lm. brisou [ ] , using filtration procedures, showed that l-forms of bacteria can be isolated from marine molluscs. anderson and hefferman [ ] used a process of double filtration of sea water, filtering first through a . #m membrane and then through a . #m pore diameter filter. a group of microorganisms, not usually encountered by the conventional techniques of pour-plates or one-stage filtration, was obtained. many could not be identified, but the largest single group belonged to the genus spirillum. other isolates were placed in the genera leucothrix, flavobacteriura, cytophaga, and" vibrio. a group of microorganisms also often overlooked is the oligotrophic bacteria. these include organisms which require prolonged incubation periods or low nutrient conditions. such organisms have been studied in chesapeake bay and were found to include hyphomicrobium, nocardia, and sheathed bacteria [ ] . recent work of martin and macleod [ ] suggests that the oligotrophic bacteria might better be defined as organisms with narrowly defined patterns of nutrient utilization. for centuries man has used the sea as both a dumping ground, with wastes dumped into rivers and streams or directly into the ocean, and as a source of fish for food. when populations in the settlements along coastal regions of the world were small and sparse, and fishermen harvested offshore, the risk of contamination of fish by domestic and/or industrial wastes was low. nevertheless, even in the earliest recorded times, disease was widespread and life expectancy short. with the world population increase, and concomitantly more densely inhabited cities, domestic and industrial waste problems have magnified. the spread of disease via fecally contaminated water and foods in many countries is guarded against by hygienic treatment of sewage wastes. despite such efforts, outbreaks of disease still occur. as a result, standards have been proposed and employed to maintain water quality for specific use, i.e., drinking, agriculture, mariculture and recreation. standards are developed to ensure that water supplies are either free of disease-causing organisms, including pathogenic bacteria, viruses, and other microorganisms, or carry concentrations of pathogens at a level that does not pose an unacceptable risk to health. the list of potentially pathogenic organisms found in water is long, and even now, remains incomplete, since new pathogens are constantly being described, as for example, the legionella spp. the task of assaying fresh and salt waters for all such organisms on a regular basis would be monumental. for this reason, those concerned with water potability, as well as food safety, including microbiologists, sanitary engineers, and medical personnel, have devised schemes using indicator organisms as a warning of the unsafe condition of waters and foods, the protocol specifying the use to which the water is to be put. most indicator organisms were selected for fresh water systems, since river, lake, and ground waters are the most common sources of community water supplies. however, sewage wastes are dumped all too frequently into estuaries and oceans, as well as into rivers. although the oceans cover three-quarters of the earth's surface, the dilution of contaminated influent waters is rarely sufficient or rapid enough to rid estuarine, coastal and deep marine waters of microbial health hazards and the effect of pollutants carried in wastes. thus, indicator organisms appropriate for measuring the quality of estuarine and marine waters are needed. questions to be answered, with regard to estuarine and marine water standards include: (i) for which activities are water quality standards needed; (ii) are corresponding fresh water indicators adequate and appropriate indicators of health hazards or increased risk of disease arising from the presence of pathogenic organisms of fecal origin; and (iii) are such indicators appropriate and satisfactory, even for fresh water? of greatest importance is defining what the indicators will be used to indicate, i.e., fecal pollution or risk of disease. in addition to public health problems associated with infectious microorganisms transmitted by water, it has become evident that there are also risks of disease caused by organisms which occur naturally in the aquatic environment. gastroenteritis or wound infections are caused by vibrio parahaemolyticus and related vibrio spp., as well as by pseudomonas and aeronomas spp., none of which are necessarily associated with fecal pollution. it is increasingly evident that indicators of sanitary quality are not appropriate markers for the presence of pathogens that are autochthonous to the aquatic or marine environment. therefore, yet another indicator or set of indicators is required. historically, an appreciation of the spread of water-borne pathogens, e.g., salmonella spp. and vibrio cholerae, was developed in the late s. a dramatic example is the cholera epidemic in london. in the late s, klebsiella pneumoniae and klebsiella rhinoscleromatis were isolated from the feces of patients by von fritsch. escherich [ ] isolated bacillus coil, now classified as escherichia coil, and a erobacter aerogenes ( enterobacter aerogenes) from feces. these bacteria and other coliforms were used in the past as indicators of water-borne pathogens, that is, the presence of fecal contamination being correlated with the occurrence of pathogens, for which direct detection methods were not available. thus, 'indicator organisms' are markers of health hazards and/or water (or food) quality. mossel [ ] , when discussing markers in foods, differentiates indicators from index organisms, the latter suggesting the risk of occurrence of pathogens, indicators being more general and suggesting inadequate bacteriological quality. cabelli [ , ] also differentiates fecal indicators from water quality indicators. fecal indicators would be inadequate as markers of pollution by organisms from fecal wastes if these organisms could multiply in the environment. a water quality indicator would be necessary to indicate these organisms, in addition to those organisms which do not have a fecal source, but are health hazards and are present in the environment [ ] . indicators must fulfil basic requirements, including: (i) specificity to the source of pathogens or fecal contamination; (ii) sensitivity of detection; and (iii) resistance to disinfection and to the environment that either equals or slightly exceeds that of the pathogen [ , - ]. since an indicator of fecal pollution is usually considered to be an index of the risk of the presence of fecal pathogens, the indicator must be present when the pathogen is present, and its source should be feces. an indicator must be sufficiently abundant in feces that the probability of detection is high, and it should be present in higher concentration than the pathogens. also, an indicator should be as resistant, or more resistant, than the most resistant fecal pathogens, and it should not multiply in the aquatic environment, whether freshwater or marine. in addition to these basic requirements, simple techniques must be available for detection and quantification of the indicator. the ideal indicator, therefore, is the pathogen itself [ ]. in fact, salmonella spp. has been suggested as an index of pollution in fresh water [ ] . the major arguments against using pathogens directly as indicators have been that they are difficult to isolate and identify, which is in most cases no longer a problem, and that the absence of one pathogen does not rule out the presence of another, nor does it have predictive value for the concentration of the pathogen [ ] . indeed, testing for the presence of every possible pathogen would be very expensive in time, effort, and resources. indicators are needed for application in the marine environment to help protect the quality of shellfish beds and primary contact recreational waters, and to prevent the spread of enteric disease. sewage treatment and subsequent dilution obtained upon entry into rivers or by direct disposal into coastal waters via marine outfalls and sludge dumping is monitored by bacteriological methods. unfortunately, marine and estuarine waters present special problems, since the behavior of pathogens and indicators differs from that in fresh water. indicators used in freshwater environments are not always appropriate for salt water. estuaries are less saline than the ocean, and salinity, as one parameter, can range from % at the mouth of a river to ~ where the estuary meets the ocean. the waters .may be stratified into a cold saline bottom layer and a less saline top layer in the typical salt-water wedge type of estuary [ ] . most ocean waters have a salinity of - %~. the water temperature of estuaries is higher and more variable than that of the ocean. tidal flushing is controlled by influent fresh water and ocean water, as well as by the physical shape of the estuary itself. biologically, estuaries are more productive and densely populated than oceans, with the populations including microorganisms, plants, and animals. freshwater organisms and other material entering estuaries and oceans are subject to competition with, and predation by, the autochthonous flora and fauna, in addition to effects of changes in the chemical constituents and physical structure of the surrounding waters. clearly, any proposed indicator must be examined in the water in which it will be monitored and/or used as a regulatory aid. many groups of microorganisms have been proposed as indicators of water quality and fecal pollution. indicators discussed separately, and in detail in the symposium proceedings edited by hoadley and dutka [ ] , include: coliforms, fecal coliforms, clostridium perfringens, pseudomonas aeruginosa, vibrio spp., bifidobacteria, candida albicans, salmonella spp., fecal streptococci, and klebsiella spp. bacterial and biological indicators were also the subject of a symposium, the presentations of which were published by james and evison in [ ] . the coliform group, i.e., total coliforms, are aerobic and facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas formation within h at °c [ ] . total coliforms are the most universally used indicator group, but include bacteria, in addition to escherichia coil, that are not specifically associated with fecal pollution, i.e., klebsiella spp., citrobacter spp., and enterobacter spp. [ ] . fecal coliforms are differentiated from other coliforms by their ability to produce gas from lactose at . °c within h [ ] . several techniques are available for enumeration of total and fecal coliforms, including tube enrichment and membrane filter methods, which are continually being compared and modified. although a positive fecal coliform test has been equated with the presence of e. coli and the presence of fecal pollution, fecal coliform-positive klebsiella pneumoniae [ ] [ ] [ ] [ ] and enterobacter aerogenes [ , ] have also been isolated from the environment. hendricks [ ] pneumoniae, alone, as indicative of fecal pollution, and the concurrent isolation of fecal coliform positive k. pneumoniae and e. coil as indicative of recent fecal pollution. they concluded that the ability of bacteria to grow in ec broth at . °c was not sufficient proof of a fecal source, and that whether or not k. pneumoniae was correlated with fecal pollution, it was indicative of a potential health hazard. enterococci have been proposed as indicators of fecal pollution for coastal bathing waters [ ] . enterococci are gram-positive cocci which grow at temperatures between - °c, and survive exposure to °c for at least min, grow at ph . and also grow in . % sodium chloride [ , ] . the enterococci are part oft he genus streptococcus, and include certain group q streptococci (e.g., [ , ] . the enterococci group excludes streptococcus boois and streptococcus equinus, which are not usually found in human feces [ ] . recently, schleifer and kilpper-balz [ ] proposed that s. faecalis and s. faecium be transferred to the genus enterococcus nom. rev. as enterococcus faecalis comb. nov. and enterococcus faecium comb. nov. fecal streptococci comprise all these organisms, as well as streptococcus mitis, and streptococcus safiv-arius, and are, therefore, less indicative of human fecal pollution. in fact, moore and holdeman [ ] found that s. salivarius ranked th on a list of human fecal bacteria, making it the most frequently encountered fecal streptococcus in human feces, and placing just behind e. coli which ranked th (the most abundant species was bacteroides vulgatus ). fecal streptococci are characterized by their ability to grow in the presence of azide and ethyl violet at °c, in azide dextrose broth and ethyl violet azide broth, or to produce red or pink colonies on kf streptococcus agar, lack the cataiase enzyme, and grow in bile broth at °c [ . the enterococci have a fecal source, survive sewage treatment better than coliforms, and are present in concentrations of - times less than e. coli in treated sewage [ ] . the survival of enterococci in sea water is longer than that of e. coli and total coliforms, but growth does not usually occur in sea water, regardless of the total salt concentration [ ]. enterococci have not been considered to be ubiquitous in the natural environment or to multiply in water [ ], even though biotypes of s. faecalis have been isolated from insects and plant surfaces. the above cited qualities, in the view of some investigators, make enterococci useful indicators of fecal pollution. the use of fecal coliform-fecal streptococcus ratios was proposed by geldreich and kenner [ ] to differentiate between human and other animal fecal pollution. since fecal streptococci survive longer than fecal coliforms in fresh water, the changing ratio of fecal coliform-fecal streptococcus makes the use of this ratio of limited and questionable significance [ , ] . the most widely used indicators for primary contact waters and shellfish waters are total and fecal coliform estimates. the concentration of e. coil is also used in shellfish guidelines. although coliforms are used as indicators, their effectiveness is now being questioned for both fresh and salt water applications. one major problem with fecal coliforms is their low survival rate in sea water, compared to other indicators and pathogens. dilution alone does not account for the rapid decrease in coliform counts near sewage outfalls in marine waters [ ] . various mechanisms have been proposed for the 'die-off, including ultraviolet (uv) radiation [ ] , predation [ ] , competition [ ] , osmotic stress [ ] , antibiosis [ ] , and heavy metal toxicity [ , ] . solar radiation is considered by some investigators to be a significant factor responsible for the bactericidal action of seawater. in field studies, mortality rates of coliform bacteria exposed to seawater are much greater in daylight than in the dark [ , , ] . gameson and gould [ ] report that half of the lethal effect of light is due to wavelengths below nm, while the remainder results from light wavelengths of - nm. however, fujioka et al. [ ] stress the effect of visible light as more significant than that of uv light, with respect to the bactericidal action of light on both fecal coliforms and fecal streptococci in seawater. high turbidity, which blocks the penetration of sunlight, slows the rate of bacterial mortality [ ] . reduction in coliform counts has been shown to be correlated with intensity of sunlight and has been concluded to be related to diurnal and seasonal variations. these variations should be taken into consideration in designing ocean outfalls and sampling protocols for coastal waters [ . the mechanism of bacterial 'die-off caused by exposure to light may be related to the presence of sensitizers in cells, which absorb light and cause cell damage [ ] . another theory is that light energy reacts with oxides to form superoxides, which may damage cells [ ] . kapuscinski and mitchell [ ] demonstrated that, in e. coli, sunlight damages the catalase enzyme system, which is necessary for the degradation of peroxide. long uv and visible wavelengths affect membrane permeability and damage active transport [ ] . inhibition of amino acid uptake, traced to the effects of uv and visible light, has also been demonstrated for estuarine bacteria [ ] . interestingly, the effect of sunlight is a function of the salinity of water [ ] . fecal coliforms and fecal streptococci are considerably more resistant to sunlight when diluted in fresh streams than in marine waters [ . chojinowski et al. [ ] reported that the mortality rates of coliforms in fresh water exposed to light are approximately half those obtained in experiments carried out using estuarine water, and one third of those when ~ sea water was employed. osmotic stress alone adds to the 'die-off" of coliforms in sea water. sublethal stress has been recorded for e. coli after exposure in test media to sea water of various salinities and incubation for up to days in the dark [ ] . the time taken to reach ~ mortality for sewage coliforms in river water ( ~ salinity) was twice that for coliforms in sea water (> % salinity) incubated in the dark [ ] . studies of non-illuminated, highly saline water taken from great salt lake (ut), which has an ionic composition similar to that of seawater, showed a higher death rate for e. coii in water from the more concentrated north ann of the lake ( g/ chlorides) than in water from the southern portion ( g/l chlorides) [ ] . an inverse relationship was also found between salinity of estuarine water and survival of an e. coli strain isolated from the rhode river estuary in maryland, u.s.a. [ ] . temperature is directly related to coliform 'dieoff" rates in both fresh and sea water [ , ] . a °c increase in temperature doubles the mortality rate of e. coil [ ] and other coliform bacteria [ ] . in a multiple linear regression analysis of the effects of temperature, dissolved oxygen and salinity on e. coli survival in diffusion chambers in the rhode river estuary, temperature was associated with over ~ of the variation in numbers of viable e. coil [ ] . in highly saline water collected from great salt lake, low temperature had a sparing effect on mortality rates [ ] . cold temperatures allowed a lower death rate of e. coli in water collected from the more saline arm of the lake. however, hussong et al. [ ] suggest that numbers of false-positive total coliforms, i.e., presumptive total coliforms which are not confirmed at the completed step, are increased in sediment when the water temperature is < °c. parasitism by bacteriophage was suggested by mitchell [ ] as a factor responsible for the destruction of fecal microorganisms entering aquatic environments. interestingly, roper and marshall [ ] demonstrated that sorption of bacteria, and their specific phage, to saline sediment interfered with viral infections, thereby allowing for persistence of bacteria in sediment. destruction by bacteriophage is also thought to occur in sewage treatment plants, although predation may be more important in the removal of enteric pathogenic bacteria from the final clarified effluent [ ] . predation has been suggested as another mechanism contributing to the bactericidal effect of sea water on non-marine bacteria [ ] . ciliated protozoa were found to be responsible primarily in reducing the survival time of e. coli in activated sludge [ ] . in estuarine waters, protozoan predators, rather than lyric bacteria, were associated with the decrease in numbers of e. coil [ , ] . as an extension of this hypothesis, mccambridge and mcmeekin [ ] reported that sunlight and microbial predators acted synergistically to reduce the numbers of enteric bacteria. fresh and stored sea water yield higher mortalities than artificial seawater. thus, it appears that there are substances in seawater, in addition to the inorganic salts, which increase 'die-off" of e. coli [ ] . these include antibiotics and other antagonistic chemicals produced by marine bacteria and plankton. catalase-sensitive lipopolysaccharides, produced by some strains of pseudomonas and chromobacterium, are active against terrestrial bacteria [ ] . sunlight-induced injury to the catalase enzyme [ ] would render terrestrial bacteria more susceptible to these antibiotics. marine phytoplankton also secrete antagonistic substances during most of the year [ ] , which may affect the survival of bacterial pollutants. acrylic acid, produced from the breakdown of dimethyl-js-propiothetin of some marine algae, has an antibacterial effect both in sea water and in the intestinal tract of penguins [ ] [ ] [ ] [ ] . oyster extracts and sea water containing acrylic acid produced by phaeodactylum tricornutum were shown to have an antibacterial effect against e. coil [ ] . thus, the presence of acrylic acid in oysters would have the potentially dangerous effect of masking a possible health hazard posed by the presence of human pathogens. reduction in counts of bacteria in seawater can also be caused by sedimentation, in addition to 'die-off". in laboratory experiments, using natural seawater, the concentration of e. coil was shown to decrease because of starvation. interestingly, the addition of nutrients resulted in floc formation, followed by adsorption of the cells onto suspended particles, with concomitant precipitation [ ] . ogawa suggested that precipitation was a factor in the 'disappearance' of coliforms in coastal waters, and that, in sediment, these bacteria survive longer because of the higher concentrations of nutrients present in sediment. however, xu et al. [ ] reported that e. coli survived for at least weeks in salt water microcosms, but were not culturable (see below). sedimentation is considered to be the most important factor associated with reduction in numbers of bacteria in the water column in the deep ocean [ ] . the presence of indicator bacteria in sediments has been reported by investigators [ ] [ ] [ ] [ ] . bottom sediments were found to contain larger numbers of e. coli [ ] and enterobacter aerogenes [ ] than the overlying seawater, because of a higher concentration of organic matter in sediment. in laboratory studies using autoclaved estuarine sediments and seawater, e. coli has been shown to increase in number [ ] , whereas when non-autoclaved sediments and water were employed, persistence of e. coli was noted, with limited growth of the organism. lalibert~ and grimes [ ] made similar observations on the in situ growth and persistence of e. coli inoculated into dialysis bag-contained sediment. gerba and mcleod [ ] suggest that the type of nutrient and microflora present in sediment may differ from those present in sea water, allowing for greater competition with e. coil in sediment. the particle size of sediment was found to be inversely proportional to the concentration of nutrient, and to survival of enterobacter aerogenes [ ] . since the survival of fecal coliforms in sediment samples was prolonged at °c, the temperature of sediment in situ is also an important factor in their persistence [ ] . since the concentration of fecal indicators [ , , , ] , microbial pathogens including salmonella spp. [ , ] , and enteric viruses [ , ] are higher in sediment than in the overlying waters, samples of sediment, rather than water, should be taken for examination of the presence of indicator bacteria in the case of primary contact water and shellfishing areas. although de flora et al. [ ] found greater numbers of viruses and bacteria in surface water than in bottom water or sediment samples collected from moderately or grossly polluted coastal sites, they also suggested that sediment was a transient reservoir for viruses and that viruses survived longer in some sediments than in sea water. wave action, tides, currents, storms, run-off, and human activities, including dredging and pleasure-boating, can disturb sediments, stirring up particulates to which organisms which persist in these sediments are adherent. resuspension of sediment allows the sediment microorganisms to enter the water column which may previously have contained negligible numbers of indicator bacteria and pathogenic organisms. it has, in fact, been documented that dredging resulted in the release of sediment-bound fecal and total coliforms and fecal streptococci in the mississippi river [ ] [ ] [ ] . although the preceding section has focused on (i) die-off of e. coli and other indicator bacteria in seawater, and (ii) their persistence in sediment because of various factors associated with the particulate nature of the latter, it must be emphasized that recent evidence disputes claims that coliforms are rapidly killed by sea water. coliforms appear to be injured, stressed, or debilitated to such an extent that special techniques may be required to recover them [ ] [ ] [ ] . methods for recovery of stressed coliforms have been proffered for fresh water [ ] and for frozen or heated foods [ ] . for stressed coliforms in marine waters, the methods presently available which allow 'repair' include filtration through membrane filters which are subsequently placed on two-layered plates [ ] , modification of the most probable number (mpn) method requiring transfer of all presumptive tubes showing growth whether or not gas is produced [ ] , use of special membranes that 'nestle' the bacteria [ ] , and pour-plating followed by overlaying with a selective medium [ ] . the pour-plate method also has been used for enumeration of fecal coliforms and enterococci in seafood samples. dawe and penrose suspended dialysis tubes filled with bacterial suspensions or untreated samples of water in sea water ( - °c), and found that, although the rate of 'injury' for coliforms was high, the rate of survival was also high, based on atp content per cell [ ] . it was suggested that sea water, rather than being bactericidal, acts to protect 'injured' bacteria from death. recently, work in our laboratory has shown that e. coil cells in saltwaten microcosms remain viable, yet 'non-culturable' on laboratory media [ ] . viability of cells was determined using a direct viable microscopic technique [ ] . other microcosm experiments employing v. cholerae in salt water [ ] and salmonella enteritidis in fresh water showed the same result, i.e., all the cells remained viable, but were non-culturable on routine laboratory media, as had been observed for e. coli [ ] . this result was documented for at least days after inoculation. the apparent 'dormancy' of these enteric bacteria, possibly arising from depletion of available nutrients, compromises the use of e. coli and related coliforms as indicators of fecal pollution in fresh and salt waters, and renders highly questionable the phenomenon of 'dieoff' or genuine mortality of these bacteria. finding an appropriate indicator for the presence of enteric viruses, i.e., poliovirus, coxsackievirus a, coxsackievirus b, and echovirus [ , ] , in sea water, is a vexing problem, especially for those responsible for regulating the use of sewage-contamined sites, including fresh, estuarine, and marine water and sediment, and shellfish harvested from these waters. fecal coliforms and total coliforms have been used as indicators of contamination by domestic sewage, and therefore of the risk associated with exposure to bacterial and viral pathogens. however, in fresh water and waste water effluents [ ] [ ] [ ] [ ] [ ] [ ] [ ] , marine waters [ , , [ ] [ ] [ ] , and in shellfish meats [ , , ] , the total coliform and fecal coliform counts cannot be used to indicate the presence of enteroviruses. the lack of association of these indicators with enteric viruses is due to the fact that the latter survive longer in the 'recoverable' stage than fecal coliforms in fresh water [ , . ] and marine water [ ] , resulting in continuously changing ratios of viruses to the fecal indicators. however, goyal et al. [ ] observed a positive correlation between the concentration of virus in sediment and total coliform concentration. although de flora et al. [ ] found significantly larger numbers of viruses and bacteria in surface waters than in bottom water or sediment from polluted coastal waters, they also concluded that sediment was a transient reservoir for viruses and that survival of viruses was longer in some sediments than in sea water. in open ocean water, viruses persist longer when associated with, or attached to, solids, than when not associated with solids [ ] . viruses also survive longer in sediment than in overlying water [ ] , resulting in greater concentrations of enteric viruses recorded as being present in sediment than in water samples [ ] . since adsorbed viruses remain infectious [ ] , viruses in sediment and resuspended sediment are potential health hazards. most viruses also survive chlorination better than bacteria. grabow et al. [ ] reported that hepatitis a virus was more sensitive to chlorination than mycobacterium fortuitum, coliphage v , and poliovirus type , but more resistant than e. coil, streptococcus faecalis, coliphage ms , reovirus type , and rotavirus sa . total and fecal coliforms are also destroyed by chlorine more quickly than viruses [ ] . cold temperatures and factors associated with gross pollution appear to prolong virus survival in rivers [ ] . since fecal streptococci and enterococci survive longer than fecal cofiforms, in both fresh water and sea water, fecal streptococci and enterococci have been suggested as better indicators of enterovirus contamination in fiver water [ , ] , relatively less polluted waters [ , ] , and coastal recreation water [ ] . however, the viability in virus titers recorded for sediment and sea water samples is less than that of any of the bacterial indicators examined [ ] . isolation of enteric viruses when the number of indicator bacteria was extremely low has been reported for sediment samples [ ] , marine water [ ] [ ] [ ] , ] , and ground water [ ] . it is important to point out the disturbing finding of gerba et al. [ ] that enteroviruses can be present about % of the time in approved recreational and shellfish harvesting waters, approval being based on total or fecal coliform standards. although the number of total coliforms was reported to be correlated significantly with the number of viruses present in water samples, the amount of variation associated with the indicator was such that it could not be considered a good predictor. a positive correlation between coliforms and enteric viruses was found for ocean waters, but only for samples collected close to a sewage outfall. at further distances from the outfall, viruses could be isolated, even in the absence of fecal indicator bacteria [ ] . vaughn et al. [ ] found little difference in the viral quality of open and closed shellfish waters. furthermore, echovirus, the vaccine strain of poliovirus, and other, unidentified viruses, could be isolated from clams collected from both areas [ ] . contamination of shellfish with enteric viruses has been well documented, and the subject has been reviewed extensively by several investigators [ ] [ ] [ ] . enteric viruses have been isolated from shellfish collected from approved shellfishing waters. for example, fugate et al. [ ] reported the isolation of echovirus type , and poliovirus types and , from oysters collected in approved waters off the louisiana and texas coast. the coliform mpn of water and coliform mpn, e. coli mpn, and aerobic plate count of the oysters, thus, were not indicative of the presence of viruses [ ] . no correlation was found between fecal coliforms in water and the number of viruses isolated from oysters collected from approved and prohibited oyster harvesting sites in coastal mississippi [ , ] . rather than using fecal coliforms as indicators, it was proposed that a virus, such as poliovirus type , could be helpful, and that concentrations of a viral or bacterial indicator in the sediment might also be valuable as a predictor of health risk from viral contamination. similarly, goyal et al. [ ] found no significant correlation between virus concentration in oysters harvested from approved shellfish sites and the bacteriological quality of water and shellfish. the relationship between risk of viral disease and presence of viruses in water is not well known. the fact that a small number of viruses constitutes an infective dose has prompted the recommendation of less than one viral infectious unit per gallons ( . ) of water as acceptable [ ] [ ] . methods for the detection of viruses in water are still being improved, and recoveries are low. therefore, the detection of one virion indicates the presence of many more viruses which go undetected. sobsey and glass [ ] have developed filtration methods which have an average recovery efficiency for poliovirus from tap water of %, ranging between - %. in a comparison of positively charged microporous filters with conventional standard methods, i.e., negatively charged filters, for the recovery of enteric viruses from tap water, conventional methods averaged less than % recovery efficiency, whereas the new methods averaged % and . %, for -and -stage protocols [ ] . sobsey et al. [ ] have also devised methods for recovery of viruses from shellfish, with virus recovery efficiencies ave'raging %. but, as has been noted, epidemioiogical studies are needed for primary contact and shellfish harvesting waters [ , , , ] . to date, the evidence available suggests that the presence of viruses in shellfish can be linked to viral gastroenteritis and hepatitis outbreaks. enteric viral disease outbreaks, traced to swimming in polluted waters or eating contamined shellfish, have made it clear that coliform standards are not sufficient for monitoring the health hazards posed by the presence of viruses in sewage or the presence of viral pathogens in freshwater, marine waters, or in shellfish. viruses which may be transmitted by water, and are currently of interest, include enteroviruses, adenoviruses, reoviruses, hepatitis a virus, rotaviruses, coronaviruses, and the norwalkvirus [ , ] . total and fecal coliforms, e. coil, enterococci, fecal streptococci and s. faecalis do not survive as long as the viruses employed in studies accomplished to date, and therefore cannot be relied upon to be present and recoverable when enteric viruses are also present. coliforms can be a useful indicator for very recent contamination, but clearly not for contamination after a prolonged period of time after occurrence. fecal streptococci and enterococci, which survive longer in seawater than coliforms, but have not been studied as thoroughly as the coliforms, have been proposed as indicators for the presence of enteric viruses [ , ] , but, as stated above, cannot be relied upon to indicate the presence of viruses. viral indicators, suggested for waste-water, include sabin vaccine poliovirus [ , [ ] [ ] [ ] , cyanophage [ , ] , and coliphage [ , ] . the difficulties associated with the use of a virus as an indicator are many, the most important of which are: no single human virus is always present in every sewage-impacted area; viruses are often present only in relatively small numbers; and better methods for virus concentration are badly needed [ ] . total virus count was recommended as a method preferable to enumeration of poliovirus for those countries with high enterovirus infection rates [ ] . katzeneison and kedmi [ ] found that poliovirus was absent in about % of waste-water samples tested, although the total virus concentrations were high. they also recommended that a system be developed to isolate and identify total viruses in water. smedberg and cannon [ ] proposed the use of lpp-cyanophages, i.e., viruses of blue-green algae of the general lyngbya, phoridium and plectonema [ ] as indicators of the presence of human enteric viruses and reliability of disinfection for treated wastewater. cyanophages can be found throughout the year in wastewater. furthermore, they are more resistant to chlorine than coliforms, and can be correlated with the number of fecal coliforms present in wastewater [ , ] . coliphages have also been proposed as indicators [ , ] . correlations between the number of fecal bacterial pathogens and indicator bacteria and their respective bacteriophages in fresh and marine water have been reported [ ] . the concentration of coliphages was reported to be at least as high as the number of enteroviruses in waste-water and other waters [ ] . in estuarine waters, the survival time of coliphages is comparable to that of enteric viruses [ ] . coliphages are more resistant to chlorine than enteroviruses and survive longer in sand columns than poliovirus type [ ] . a rapid test for coliphages, as a water quality indicator for bacteria, was developed for fresh water [ ] . using this test, a linear relationship for the number of fecal or total coliforms with coliphages has been reported [ ] . the value of coliphages has also been investigated, with respect to indication of enteric viruses in shellfish and shellfish-harvesting waters [ ] . as a result of their studies, vaughn and metcalf [ ] recommended the use of coliphages as an indicator for raw sewage and treated effluents, but not for estuarine sheufishing waters, because coliphages were found to increase in number by replication in estuarine waters. furthermore, they were accumulated by oysters to a greater extent than enteric viruses, and showed shifts in dominant type over time [ ] . thus, the presence of coliphages was concluded not to be related to the presence of enteric viruses in estuarine water [ ] . in freshly shucked oysters contaminated with poliovirus and stored at °c, the virus persisted longer than days [ ] . in fact, viruses in contaminated oysters can survive processing, refrigeration, and freezing [ ] . in addition to bivalves, other marine invertebrates, including crabs, must be suspect agents in the transmission to man of enteric viruses present in polluted waters. even crabs caught in unpolluted waters may be contaminated with viruses accumulated when the crabs were exposed to polluted waters, either by swimming and/or feeding on contaminated shellfish in polluted waters [ ] . in laboratory studies, digirolamo et al. [ ] found that crabs concentrated high titers of poliovirus from contaminated mussels and filtered sea water that had been artificially seeded. in similar experiments, polioviruses were isolated from all parts of crabs, being present in highest concentrations in the hemolymph and digestive tract of the animal [ ] . poliovirus survived for up to days in crabs held at °c. recovery of poliovirus type , simian rotavirus sa , and echovirus type was reduced by . % in crabs after they had been boiled for rain [ ] . as the human populations located along urban coastal areas have increased, so too has the amount of material discarded into the ocean. it is now well known that larger concentrations of bacteria are found near the mouths of rivers and bays in highly populated areas and other coastal regions than in the open ocean. to protect bathers from waterborne enteric diseases acquired by swimming in coastal waters, some indication of the extent and amount of fecal pollution is needed. epidemiological studies are valuable for determining whether or not swimming in sewage-polluted waters poses a health risk, and, also, what organism or organisms are best suited to predict such health hazards. in addition to assessing the risk of enteric viral and bacterial diseases such as infectious hepatitis, typhoid fever, shigellosis, and cholera, the risk of contracting diseases affecting the eyes, ears, nose, throat and skin, as well as wound infections, is a matter of serious concern. organisms which cause the latter group of diseases are not necessarily sewage-associated, and may not be signalled by the traditional drinking water indicator bacteria [ ] . an important aspect of epidemiological studies is the determination of the infectious dose for a particular pathogen, and its virulence. in a review of water-borne pathogens, geldreich [ ] focused on enteric pathogens, including salmonella, shigella and leptospira (freshwater) species, enteropathogenic e. coli, francisella tularensis, vibrio cholerae, mycobacterium tuberculosis, hepatitis a virus, poliovirus, other human enteric viruses and parasitic protozoa and worms. not all of these organisms are necessarily of concern for the marine environment. in addition to the organisms listed by geldreich [ ] , moore [ ] cites as being communicable, otitis media, sinusitis, and gastroenteritis of unknown etiology, but dismisses certain organisms as irrelevant for coastal bathing waters, including staphylococcus aureus, clostridia, mycobacterium spp. and candida spp., based on their biology. however, staphylococci, especially staphylococcus hominis and staphylococcus epidermidis, and including s. aureus, have recently been isolated from coastal, offshore, and deep ocean sites [ , ] . also in conflict with moore [ ] are the frequent isolations of clostridium botulinum and clostridium perfringens from marine sediment [ ] [ ] [ ] . according to moore [ ] , typhoid fever is of concern at the present time only in areas of high enteric morbidity. wound and enteric infections caused by bacteria of the vibrionaceae and skin rashes caused by trematodes, acquired from swimming in estuarine and ocean waters, were also included in a review of recreational water use published by cabelli [ ] . most of the epidemiological studies of bathing water quality were done in the s in the u.s.a. and u.k. notable of these are the reports of stevenson [ ] in the united states and moore [ ] in britain. indicator organisms used to compare to the incidence of disease in both the american and british studies were total coliforms. however, the authors of these reports reached different conclusions. stevenson [ ] reviewed results of surveys of swimming pools and beaches on the ohio river, lake michigan, and long island sound, and concluded that a positive correlation existed for fresh waters between high total coliform concentrations and illness in swimmers, i.e., more swimmers than non-swimmers became ill, with more than one-half of the illnesses being ear, eye, nose, and throat diseases, one-fifth gastrointestinal, and the remainder skin or other types of infections. on the other hand, moore [ ] , who reported on an epidemiological study of bathing waters carried out on the coasts of england and wales, found no evidence to support the conclusion that sewage-contaminated water was a significant cause of illness. it should be pointed out, however, that isolation of salmonella spp. from seawater was interpreted as being evidence of the wide dissemination of this organism in the population at large, rather than a risk of infection for bathers. in a later publication, moore [ ] concluded that the environmental hazards of swimming are difficult to assess and require statistical proof of association, causation, and specificity of disease with cause of the disease. two other factors important in epidemiological studies were cited, these being the incubation time of the pathogen, and dose response of morbidity related to frequency of swimming [ ] . the most recent report of results of an epidemiological study of coastal bathing waters was of a study conducted over a -year span in the s at beaches in new york city, lake pontchartrain, la and boston, ma [ ] . a direct linear relationship was observed between swimming-associated gastrointestinal disease and water quality. other types of disease were not reported. bacteria enumerated in the waters included enterococci, total and fecal coliforms, e. coli, klebsiella sp., enterobacter sp., citrobacter sp., clostridium perfringens, pseudomonas aeruginosa, aeromonas hydrophila, and vibrio parahaemolyticus. the concentration of enterococci was strongly correlated with gastrointestinal symptoms, while fecal and total coliforms were found to be poorly correlated. as reported in earlier studies in the u.s.a. by stevenson [ ] , swimmers showed a higher rate of symptoms than non-swimmers [ ] , even in marginally polluted marine waters, the latter being indicated by the presence of enterococci. although total coliforms have been used as indicators of primary contact water quality, this approach has been discontinued in many areas in favor of the more fecal-specific fecal coliforms [ ] . enterococci have been suggested as better than either the fecal coliforms or e. coli as an indicator of fecal pollution for bathing waters since the correlation of enterococci with gastrointestinal symptoms was highest [ , ] . a wide array of indicator organisms has been proposed for brackish, estuarine, and coastal waters. it is clear that no single indicator, index or reference organism exists for titering public health safety of these waters. with the development of immuno-epifluorescent microscopy methods for direct detection of specific human pathogens, and considering the 'viable but non-eulturable' stage which allochthonous human pathogens undergo when released to natural bodies of water, it is concluded that the direct viable 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coliform contamination of gulf coast oysters detection of virus spread by the water route viruses in water poliovirus concentration from tap water with electropositive adsorbent filters concentration of poliovirus from tap water using positively charged microporous filters improved methods for detecting enteric viruses in oysters viral pollution of seawater at barcelona viral gastroenteritis fecal streptococcus as an indicator in disinfected water and waste water polioviruses and other enteroviruses in urban sewage from laval (canada): presence of nonvaccinal strains of poliovirus polioviruses as indicators of virological quality of water a survey of enteric viruses in domestic sewage cyanophage analysis as a biological pollution indicator-bacterial and viral serological typing and chlorination resistance of waste water cyanophages current concepts of indicator bacteria some thoughts concerning water pollution indicators unsuitability of pofioviruses as indicators of virological quality of water etude des bactr'riophages typhiques. viri dans les eaux sur le choix des souches talons pour la d~tection du bacille typhique dans les eaux par la recherche des bact*riophages sprcifiques human enteric viruses and bacteriophages as indicators of sewage pollution coliphages as indicators of enteric viruses in shellfish and shellfish raising estuarine waters rapid determination of the presence of enteric bacteria in water evaluation of coliphage detection as a rapid indicator of water quality persistence of polioviruses in shellstock and shucked oysters stored at refrigeration temperature the effects of freezing on the survival of salmonella an escherichia coil in pacific oysters concentration of rotavirus and enterovirus from blue crabs (callinectes sapidus) preliminary observations on the uptake of poliovirus by west coast shore crabs uptake and survival of enteric viruses in the blue crab, callinectes sapidus the coliform count as a measure of water quality water-borne pathogens the present status of diseases connected with marine pollution a note on the isolation and numeration of gram-positive cocci from marine and estuarine waters numerical taxonomy of staphylococci isolated from the marine environment clostridium perfringens in the environment ciostridium perfringens as a water pollution indicator distribution of clostridium botulinum ) studies of bathing water quality and health sewage contamination of coastal bathing waters in england and wales: a bacteriological and epidemiological study a marine recreational water quality criterion consistent with indicator concepts and risk analysis key: cord- -yq wsugy authors: anim, desmond ofosu; ofori-asenso, richard title: water scarcity and covid- in sub-saharan africa date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: yq wsugy nan lv et al recently predicted that many countries could face similar covid- situation as experienced in hubei in china [ ] . several factors may impact their prediction. current evidence suggest that the covid- virus is transmitted via respiratory droplets or contact [ ] . contact transmission happens when contaminated hands touch the mouth, nose, or eyes. consequently, hand hygiene (regular hand washing) is extremely recommended to control the spread of covid- virus [ ] . in this paper, we highlight the issues that characterize water services amid the covid- pandemic in sub-saharan africa (ssa) and discuss avenues for improving water management during this pandemic and future infectious disease outbreaks. in response to the promotion of hand hygiene by the world health organization (who) and national public health agencies as a means to curbing the spread of covid- , water service providers (wsp) in most developed countries have outlined drastic measures with the goal of ensuring continuous provision of essential water and sewerage services to all during this pandemic. for example, in the us and australia, wsp have suspended water shutoff with service to be temporarily restored to thousands of households disconnected [ , ] . access to water is a key determinant for infectious disease control and prevention; thus, limited access creates a challenge for transmission control [ ] . nevertheless, across many ssa countries where inequalities in access to safe water is pervasive [ ] , there is a need to be worried in light of covid- pandemic. nearly million people in ssa live in water stressed environment (table ) [ ] . this present a major challenge towards controlling the spread of the covid- virus. indeed, poorly developed water and sanitation systems was reported to be a key determinant of the rapid spread of the ebola outbreak, as well as an underlying factor in the high number of deaths [ ] . so how do the recommended precautionary measures relative to covid- fit within the everyday practices in ssa countries characterised by overwhelming water scarcity? as of may ( : gmt), only lesotho remains a covid- -free country in ssa (supplementary figure s ). in response to the increasing threat from covid- , most ssa cities have instituted a lockdown (partial in most places). however, residents are concerned about a potential increased spread of covid- due to water rationing. in ghana (www.youtube.com/watch?v=kl v a toyg) and kenya (www.bbc.com/news/world- ) for example, many households struggle to comply with the advice to 'frequently wash hands under running water' because of the water rationing. it is worth noting that social distancing is almost impossible as residents are likely to queue to access or buy water. notably, one of the key public health preventive messages for covid- is: 'washing hands with soap and water for seconds, repeatedly throughout the day, is critical to prevent transmission of the virus' [ ] . in many ssa settings, this is an unimaginable luxury due to the inequalities that characterise the provision of water services as well as the limited opportunity to wash their hands regularly at home. consequently, in this period, it is important to reflect on the water and sanitation services situation in the ssa region. in particular, the ongoing covid- pandemic provides an opportunity to remind water authorities and the respective governments of the with the water demand pressures from rapid population growth and urban expansion in ssa [ ], there is the urgent need to increase the efficiency of use by implementing strategies for improving the conservation of available water. green or nature-based solutions can help to improve water storage and supply, thus increasing water availability. this is particularly needed today considering expectations that water shortage will worsen in ssa due to climate change and risk of droughts causing the decline of water levels of dam and freshwater supply sources [ , ] . water scarcity and security issues will be exacerbated by recent trends of climate variability and consequent rise in droughts. thus, climate resilient water resource management will require an integrated strategy to ensure resilience for water-related policy making to address both short-and long-term impacts of climate change by balancing robustness with flexibility. with future uncertainties and the likelihood of other potential infectious disease outbreaks, there is the need for robust adaptation options that have the primary objective of supporting sustainable water resources use. ensuring affordable access to safe water, sanitation and hygiene (wash) services is important to address the current covid- and future pandemics. particularly, improved access to wash facilities could help to minimize transmission, and reduce healthcare and other societal costs. for example, the ghana government is currently spending money to use water tanks to provide water for poor communities severely hit by the pandemic. residents in these communities are unable to socially distance or conform to lockdowns for reasons such as the need to get out to access water and toilets. in response, long-term efforts should focus on addressing wash access issues among the poor communities especially in urban areas. most people living in informal settlements as is the case for many ssa cities rely on communal water stands and toilets. high cost coupled with limited access could stop generous use of water for hand washing. leaving homes to use communal services and queuing for access also makes social distancing difficult to implement. the poor access to water in ssa presents a major barrier to effective containment of the covi- outbreak. it is essential that this unfolding moment trigger collaborative efforts between all stakeholders in rethinking and acting for improved water services during this pandemic and future infectious disease outbreaks. notes: population living in water scarcity (i.e. with less than m per capita per year) and stress (i.e. with less than , m per capita per year) in selected ssa countries. water scarcity reflects the lack of sufficient available water resources to meet demand of usage within a region whereas water stress refers to the inability to meet human and ecological demand for water. source: world data lab (https://worldwater.io/about.php; accessed on th april ) global covid- fatality analysis reveals hubei-like countries potentially with severe outbreaks features, evaluation and treatment coronavirus (covid- ) us cities and states suspend water shutoffs to tackle coronavirus pandemic here's how australian water utilities are responding to covid- progress on household drinking water, sanitation and hygiene - . special focus on inequalities water scarcity and related environmental problems in parts of sub-saharan africa: the role of the transboundary environmental impact assessment convention the emergence of ebola as a global health security threat: from 'lessons learned' to coordinated multilateral containment efforts covid- hand wash timer water crisis in africa: myth or reality? international journal of water resources development the projected timing of climate departure from recent variability key: cord- -gw mgg m authors: junter, guy-alain; lebrun, laurent title: cellulose-based virus-retentive filters: a review date: - - journal: rev environ sci biotechnol doi: . /s - - - sha: doc_id: cord_uid: gw mgg m viral filtration is a critical step in the purification of biologics and in the monitoring of microbiological water quality. viral filters are also essential protection elements against airborne viral particles. the present review first focuses on cellulose-based filter media currently used for size-exclusion and/or adsorptive filtration of viruses from biopharmaceutical and environmental water samples. data from spiking studies quantifying the viral filtration performance of cellulosic filters are detailed, i.e., first, the virus reduction capacity of regenerated cellulose hollow fiber filters in the manufacturing process of blood products and, second, the efficiency of virus recovery/concentration from water samples by the viradel (virus adsorption–elution) method using charge modified, electropositive cellulosic filters or conventional electronegative cellulose ester microfilters. viral analysis of field water samples by the viradel technique is also surveyed. this review then describes cellulose-based filter media used in individual protection equipment against airborne viral pathogens, presenting innovative filtration media with virucidal properties. some pros and cons of cellulosic viral filters and perspectives for cellulose-based materials in viral filtration are underlined in the review. abstract viral filtration is a critical step in the purification of biologics and in the monitoring of microbiological water quality. viral filters are also essential protection elements against airborne viral particles. the present review first focuses on cellulosebased filter media currently used for size-exclusion and/or adsorptive filtration of viruses from biopharmaceutical and environmental water samples. data from spiking studies quantifying the viral filtration performance of cellulosic filters are detailed, i.e., first, the virus reduction capacity of regenerated cellulose hollow fiber filters in the manufacturing process of blood products and, second, the efficiency of virus recovery/concentration from water samples by the viradel (virus adsorption-elution) method using charge modified, electropositive cellulosic filters or conventional electronegative cellulose ester microfilters. viral analysis of field water samples by the viradel technique is also surveyed. this review then describes cellulose-based filter media used in individual protection equipment against airborne viral pathogens, presenting innovative filtration media with virucidal properties. some pros and cons of cellulosic viral filters and perspectives for cellulose-based materials in viral filtration are underlined in the review. virus capture/purification/concentration is critical in a number of biopharmaceutical and clinical applications. validation of virus clearance is essential in the manufacture of therapeutic proteins, in particular blood products (bryant and klein ; klamroth et al. ; de mendoza et al. ; radosevich and burnouf ; shukla et al. ). on the other hand, large-scale, efficient purification schemes of viruses/ virus-like particles are required for the production of prophylactic vaccines and gene therapy vectors (rodrigues et al. ; segura et al. segura et al. , vicente et al. a, b) . virus inactivation technologies are commonly used to fulfil viral safety. they include physical (e.g., heat application, ultraviolet-and gamma irradiation) and chemical methods (e.g., solvent/detergent treatments) or their combination (e.g., exposure to photosensitizer plus uv light) (bryant and klein ; klamroth et al. ; klein and bryant ; pelletier et al. ; prowse ; radosevich and burnouf ; solheim ) . common methods for virus capture include filtration (charcosset ; grein et al. ; liu et al. ; van reis and zydney ) and chromatography in column or membrane configurations (charcosset ; gottschalk ; liu et al. ; orr et al. ; van reis and zydney ; segura et al. ). viral filtration is usually the final purification step in the downstream processing of biopharmaceutical products, e.g., monoclonal antibodies ( fig. ) , following one or more chromatography ''polishing'' steps that contribute to the overall virus removal efficiency of the process before concentration of the purified product. filtration technologies are also extensively used for the capture and concentration of waterborne viral pathogens (gibson ) from drinking, environmental, recreational or waste water samples (cashdollar and wymer ; ikner et al. ). in addition, particulate air filters are used in personal respiratory protective equipment (i.e., face masks and respirators) cohen and birkner ; rengasamy et al. ) to ensure shortrange protection of wearers against airborne pathogens-which include a number of viruses (tang et al. )-and in air purifiers/cleaners to limit longrange aerosol transmission of infection in healthcare settings (hyttinen et al. ; tang et al. ) . cellulosic membrane microfilters have been used routinely for ages in laboratories to perform the socalled ''sterile filtration'' (cold sterilization), i.e., the absolute removal of bacteria, yeasts and molds but not viruses (walsh and denyer ) from heat-sensitive liquid media. however, many virus filtration devices currently implemented are made from cellulose and its derivatives. some eighty years ago, gradocol, graded collodion (cellulose nitrate) membrane filters (elford ; bauer and hughes ) have been extensively used in ultrafiltration to estimate the size of several medically important viruses such as foot-and-mouth disease (galloway and elford ) , vaccinia (elford and andrewes ) , herpes (elford et al. ) , poliomyelitis (elford et al. ) and influenza (elford et al. ) (see also ferry ( ) for an exhaustive review). later on, commercial mixed cellulose ester filters with very low pore size, namely vf (virus fine), vm (virus medium) and vc (virus coarse) grade filters produced by millipore (billerica, ma, usa-a subsidiary of merck kgaa, darmstadt, germany), have also been applied to virus ultrafiltration for grouping by size assessment (hsiung ). as part of a series of papers surveying the antiviral applications of polysaccharide-based materials, the present review focuses on viral filtration using cellulosic filter media-most of which are commercially available products that have been extensively tested over the past twenty years for the removal/concentration/purification of viral particles from liquid samples, i.e., biopharmaceutical (namely blood products) and environmental (raw or treated) water samples. the application of cellulose-based filters in individual respiratory protective devices protecting the wearer against airborne viral pathogens is also detailed. polysaccharide-based chromatographic adsorbents for viral clearance or recovery/purification of viruses or virus-like particles will be presented elsewhere. in the processing of biological products, virus removal from the product stream while providing maximum product recovery is a critical task. it is more particularly difficult to eliminate small viral particles such as parvoviruses, which may contaminate blood products and mammalian cell cultures used in the production of recombinant proteins (charcosset ) . viral filters developed to answer this challenge are typically membrane (screen) filters ensuring a size-based rejection of viral particles via a sieving mechanism. given the size of viruses, ranging roughly from (parvoviridae) to nm (poxviridae) (segura et al. ) , viral filtration stands between microfiltration and ultrafiltration among pressure-driven filtration processes (fig. ) , though it is frequently but incorrectly (van reis and zydney ) classified as nanofiltration (burnouf et al. ). . regenerated cellulose hollow fiber (hf) membrane filters common virus filtration membranes are made from poly(ethersulfone) (pes), poly(vinylidene fluoride) (pvdf), and regenerated cellulose (burnouf and radosevich ; carter and lutz ; liu et al. ; van reis and zydney ) . among the latter, the commercial planova tm filters (asahi kasei medical, tokyo, japan) have been widely used to clear viruses from biologically produced pharmaceuticals, in particular blood products. planova filters are composed of cuprammonium regenerated cellulose hf (bemberg tm cupro fibers), prepared from cellulose cuprammonium spinning solution via microphase separation under precise spinning conditions (tsurumi et al. b, c) . the wall of each hf has a threedimensional web structure of pores consisting of large, bulky void pores interconnected by fine capillaries (tsurumi et al. a, b, c) (fig. ). during filtration, as the feed solution containing the product of interest is circulated through the hf bore, viruses accumulate in the large, bulky void pores of the fiber network while the product solution passes through the capillary pores. since the hf wall is several tens of micrometers thick (fig. ) , viruses are captured gradually inside the porous structure that can be considered multi-layered ( - layers) (hongo-hirasaki et al. ) . hence, planova filters behave like ''membrane depth filters'' (walsh and denyer ) operating on the basis of size exclusion. they offer a choice of nominal mean pore sizes, namely nm ( n), nm ( n), nm ( n) and nm ( n)-the large pore n model being essentially used as a prefilter to remove impurities or aggregates prior to final virus filtration-and can be operated in normal (dead-end/flow through) or tangential flow (cross flow) filtration mode (phillips et al. ). planova filters have emerged to answer a publichealth problem of worldwide magnitude, i.e., blood contamination by hiv (hamamoto et al. ; manabe et al. ) and hepatitis viruses (yuasa et al. ; sekiguchi et al. sekiguchi et al. , . table gives examples of viral clearance studies assessing the capacity of planova filters to remove viruses from blood products. in these viral clearance assays, product streams resulting from successive, scaleddown purification steps representative of the manufacturing process of the product (kundu and reindel ) , were artificially contaminated (''spiked'') with model viruses of different sizes (table ) (at much higher concentrations than what might be commonly found in the product intermediate) before being submitted to filtration. the virus removal efficiency of the filtration step was expressed as lrv (log reduction value, i.e., log ratio of the viral load in the spiked product feed stream to that recovered in the product filtrate), which implies that residual virus infectivity can never be reduced to zero but may be greatly reduced mathematically (ich ) . minimum lrv values were most frequently given. they were estimated when no viruses could be detected in filtered samples taking into account the detection limit of the assay (viral titer estimated as infectious unit per sample volume). from the measured lrvs, the filtration operation is usually classified as effective (lrv [ ), moderately effective ( \ lrv \ ) or makino et al. ( ) rev environ sci biotechnol ( ) ineffective (lrv \ ) (phillips et al. ). since filtration complements other viral clearance methods in the downstream processing of biotherapeutics (brorson ; klamroth et al. ; shukla et al. ) , several studies quoted in table also report lrvs for common virus reduction steps such as inactivation by pasteurization (gröner ; gröner et al. ; terpstra et al. ) or solvent/detergent treatment (dichtelmüller et al. ; terpstra et al. ) , and chromatographic capture (gröner ; gröner et al. ) . virus removal during the plasma fractionation process (bryant and klein ) leading from the crude plasma pool to the product (immunoglobulin here) stream has also been quantified (dichtelmüller et al. ; koenderman et al. ; terpstra et al. ) . overall virus reduction factors obtained by addition of successive lrvs are given in these works, as illustrated by table . in the virus spiking studies quoted in table , the actual viral filtration step was preceded by prefiltration to eliminate viral and/or protein aggregates that might be present in the spiked product solution. the presence of aggregated viruses may lead to overestimation of size exclusion effectiveness while contributing with protein aggregates to the fouling of viral filters, which reduces the membrane hydraulic permeability and may affect virus retention (phillips et al. ). pre-filtration was performed on the spikevirus stock suspension (caballero et al. ; gröner et al. ; roberts et al. ) on the product intermediate before virus inoculation (gröner et al. ; koenderman et al. ) , and/or on the virus furuya et al. ( ) and terpstra et al. ( ) cpv canine parvovirus b v gröner ( and terpstra et al. ( terpstra et al. ( , spiked material (chtourou et al. ; dichtelmüller et al. ; koenderman et al. ; terpstra et al. terpstra et al. , . for pre-filtration of the virus preparation, micro filters with a pore size adapted to the spike virus size, i.e., slightly lower than the virus size, were used to remove cell aggregates but not single viral particles-usual pore sizes ranging between . and . lm. micro filters with . - . lm pore size (koenderman et al. ; terpstra et al. ; , but also n (dichtelmüller et al. ) and n (chtourou et al. ) . planova viral filters were used to pre-filtrate the spiked product solution. in the latter work, the association in series of n and n filters led to efficient removal of small viruses such as ppv (chtourou et al. ). this was not the case when n was used as the main viral filter after pre-filtration with n (dichtelmüller et al. ) , which confirms previous date showing that the nm pore size was too large to retain the smallest viral particles (furuya et al. ; hongo-hirasaki et al. ). both n (caballero et al. ; furuya et al. ; gröner ) and n (caballero et al. ; roberts et al. ; terpstra et al. ; planova filters and their combination (gröner et al. ; koenderman et al. ) showed effective virus removal over a wide range of viral particle sizes (table ) . as a general rule, the removal efficiency of the filters increased with the ratio of pore size to virus size, as illustrated by gröner ( ) and terpstra et al. ( ) . this was also demonstrated by roberts et al. ( ) by filtrating poliovirus type (a picorvanirus) through planova filters manufactured with different pore sizes, ranging between (lrv = . ) and nm (lrv = . ). the two-filter combinations were globally more efficient than single filters at removing small viruses from spiked protein intermediates, though two n filters connected in parallel for tangential filtration (koenderman et al. ) yielded lrvs quite similar to those obtained using n alone (terpstra et al. ). care should be taken when comparing vertically the data collected in table , however. in addition to the sizes of viral particles and filter pores, the results of such virus-spiking studies are dependent on a number of process parameters such as the filtration operating conditions (filtration mode, transmembrane pressure, volume per filter area, tempera-ture…), the characteristics of the product feed stream (ph, conductivity, nature and concentration of the product…), or else the purity level and concentration of the virus spike. moreover, several studies include pre-filtration (dichtelmüller et al. ) or viral inactivation (koenderman et al. ; terpstra et al. ) in the calculation of lrvs. this variability in test conditions for filtration-based virus removal makes difficult any comparison between lrvs collected from multiple sources as in table , even though some results (caballero et al. ; hongo-hirasaki et al. ; roberts et al. ) contradict its influence on filtration performance, underlining the robustness of the filtration process. also, some variations in virus removal capacity may occur between commercial filter modules owing to their manufacture from different batches of hf, as illustrated by roberts et al. ( ) for n filters. like membrane microfiltration, depth filtration is widely used as a clarification step in biopharmaceutical purification to ensure removal of cell debris, large aggregates and contaminants from the product stream prior to purification processes such as column/membrane chromatography and viral filtration/inactivation steps (liu et al. ; van reis and zydney ; vicente et al. vicente et al. a, b) . commercially available depth filters currently employed in bioprocessing are composed of cellulose fibers with a porous inorganic filter aid and a resin binder, generally cationic. for instance, the zeta plus tm filter media ( m purification inc.-formerly cuno, meriden, ct, usa) is composed of a cellulose fiber depth matrix containing silica-based filter-aid material and positively charged by chemical bounding of a cationic charge modifier (ostreicher ) . varying retention ratings are available ( . - . lm). the triple layered a hc filter media from emd millipore include two depth filtration layers (cellulose ? diatomaceous earth) of different grades and a . lm-pore-size microporous membrane pre-filter made of mixed cellulose esters (van reis and zydney ). hence, these cellulosic depth filters rely on both size exclusion and electrostatic adsorptive binding to effect separation (liu et al. ) . in viral spiking studies (barnette et al. ; dichtelmüller et al. ; zhou et al. ) , they were found to retain viral particles whose surface is negatively charged over a wide range of ph as their isoelectric points (pi) range most frequently between . and . , with a mean value of . ± . (michen and graule ) . viruses are recognized as a major cause of waterrelated disease (bosch et al. ; fong and lipp ; gibson ; hamza et al. ) . enteric viruses, more particularly, which are associated primarily with gastrointestinal illness (bosch et al. ; fong and lipp ; gibson ; hamza et al. ) , are implied in most waterborne viral outbreaks (gibson ; hamza et al. ). all types of water, including waters used for drinking (surface or ground supplies), recreational waters (fresh, marine, and swimming pool), agricultural waters for irrigation (rivers and groundwater) and waste waters (sewage or industrial effluents), have been shown to be potential vehicles for virus transmission (bosch et al. ) . contamination is most frequently of fecal origin (bosch et al. ; wong et al. ) . although viral levels may be high at the contamination source, e.g., concentrations ranging between and virus particles per gram of feces are referred to in the literature (michen and graule ) , viral concentrations as low as - viral particles per liter may be found in environmental water (julian and schwab ) . since enteric viruses display high infectivity (fong and lipp ; julian and schwab ) , such titers may constitute a health risk and should be detected for reliable surveillance of viral pathogens in water. as a consequence, efficient concentration methods are needed to capture viruses in large-volume water samples and release the retained viral material in concentrated form. most common methods are based on filtration processes (cashdollar and wymer ; ikner et al. ). in particular, virus adsorptionelution (viradel) filter methods have been widely implemented since the s (cashdollar and wymer ) . briefly, in a viradel method, viruses from aqueous samples are reversibly adsorbed to microporous filters and then eluted from the filters in a small liquid volume (apha, awwa and wef ). adsorbent filters carry electrical charges and virus retention occurs via electrostatic interaction rather than by size exclusion. in other words, viradel filters act as depth filters rather than sieves. contrary to viral filters that retain viruses by a sieving mechanism to achieve complete viral clearance, their pore size lies in the microporous range, which allows the high flow filtration necessary for virus capture in large water samples. positively or negatively charged filters can be used in a viradel procedure, among which those made of cellulosic materials have been dominant over the last few decades. since their pi is below the ph of natural water (i.e., around neutrality) ( huang et al. ( ) table continued filter type a water nature no. [ tested earlier for concentration of viruses in water samples of different origins (table ). more recent studies made use of zeta plus tm mds microfilters, however (table ). in these works, pure (e.g., distilled), tap, environmental or sewage water samples were seeded with varying enteric viruses that are common waterborne pathogens, i.e., members of the families picornaviridae (polioviruses, coxsakieviruses, teschoviruses), adenoviridae (adenoviruses), caliciviridae (noroviruses, caliciviruses) and reoviridae (rotaviruses) (fong and lipp ) , or bacteriophages (enterobacteria phages) that are considered as alternative indicators of fecal contamination and as index organisms for the presence of enteric viruses in waters (goodridge and steiner ). naturally contaminated waters containing sufficient levels of indigenous viruses, more particularly bacteriophages, were also used in addition to spiking studies sensu stricto (table ) . following sample filtration, viral particles adsorbed to the filter were eluted in concentrated form using a variety of eluting solutions, the most common eluent consisting of a slightly alkaline (ph . - . ) protein solution (i.e., beef extract), frequently buffered with glycine-naoh or another amino acid solution (sometimes supplemented with salt to aid disruption of electrostatic interactions between viruses and filters (shields and farrah ) , a chaotropic agent (e.g., urea) or a surfactant (e.g., tween ) to affect virus-filter hydrophobic interactions . eluted viruses were quantified-using plaque titer assays, % tissue culture infectious dose (tcid ), quantitative realtime pcr (qpcr) and reverse-transcription pcr (rt-qpcr) (hamza et al. )-to assess the recovery efficiency of the filtration step by comparing to input titers. this efficiency depends on a number of process parameters including the filter type and filtration conditions (e.g., filtration rate and pressure), the elution buffer composition and eluting conditions, the nature, input titer and titration method of the tested virus-in addition to the water matrix characteristics that may affect virus quantification and filter performance (borchardt et al. ) . hence, tables and display a wide range of recovery yields. in practical tests aimed at the detection of naturallyoccurring viruses in field, large-volume water samples, the filtration (adsorption-elution) step is usually a primary concentration step which is followed by a secondary one to reach the virus detection threshold, e.g., organic flocculation or peg precipitation (lewis and metcalf ) . the recovery yield of such two-step concentration processes was evaluated in several studies using seeded or naturally contaminated water samples (tables , ) . as a general rule, the viral loss due to secondary concentration was balanced by a huge increase in concentration factor allowing virus detection in water concentrates from large-volume samples, as reported by chang et al. ( ) and raphael et al. ( ) for indigenous enteroviruses in wastewater and rotaviruses in sewage-polluted surface water, respectively. after determining the optimal elution conditions for mds disk-adsorbed noroviruses following filtration of spiked distilled water samples, lee et al. ( ) (table ) applied the optimized procedure to the detection of noroviruses in environmental water. they combined adsorptive filtration using mds cartridge with organic flocculation to enrich viruses from largevolume surface ( l) and ground ( l) water samples. this integrated two-step process led to high volume reduction factors, i.e., , and , for surface and ground water samples, respectively ( - for filtration, for organic flocculation), but its recovery efficiency was not evaluated by spiking studies. in the same way, beside spiking woo et al. ( ) . a ratio of the virus survival factor in the treated filter to that in the untreated filter, where the virus survival factor in a filter is the number of viruses recovered by elution from the filter divided by the number of viruses removed by the filtration process. b pf pp filter from commercial surgical mask (dupont tm n), ccf coarse pore cellulose filter paper (whatman tm grade , lm pore size), fcf fine pore cellulose filter paper (whatman tm grade , . lm pore size) studies like those detailed in table , mds filter cartridges have been widely applied to concentrate enteric viruses in water samples of different origins (table ) , ranging from large-volume samples of weakly contaminated water intended for drinking or drinking water production (borchardt et al. (borchardt et al. , lee et al. ; sedmak et al. ; verheyen et al. ) to wastewater samples with high viral content-to assess the virus removal efficiency of wastewater treatment plants (kuo et al. ; simmons et al. ) . in these tests of field water samples for enteric viruses, mds filtration was mainly combined with second-stage concentration by organic flocculation as detailed by fout et al. ( ) in the usepa (united states environmental protection agency) manual of methods for virology. the elution of filter-adsorbed viral particles was mostly performed with an alkaline beef extract solution supplemented or not with glycine (buffers eb and eb in table ). the work by verheyen et al. ( ) is an exception to these common procedures. to concentrate viruses in small-volume samples from drinking water sources, these authors used two filter cartridges in series with no additional concentration step and an alkaline powdered milk solution as elution buffer. they found that % ( / ) and % ( / ) of surface and ground (well) water sources, respectively, were contaminated with adenoviruses, with very few samples positive to rotavirus ( / surface water samples, / well water sources). despite the differences in virus concentration methods, these results compare with those obtained by xagoraraki et al. ( ) and cheong et al. ( ) for adenovirus detection in surface ( / samples, %) and ground water ( / samples, %), respectively. they also agree with data reported by borchardt et al. ( ) and cheong et al. ( ) showing the low contamination level of ground water by rotavirus. it is worth noting here, however, that the number of positive samples reported in these viral analyses of water (and the others quoted in table ) is dependent on the sensitivity of the detection techniques used to assess the presence of viruses, i.e., nucleic acid-based amplification methods such as conventional pcr, reverse-transcription pcr (rt-pcr), nested pcr, real-time pcr/rt-pcr or integrated cell culture pcr (icc/pcr) to determine virus infectivity (fong and lipp ; hamza et al. , mattison and bidawid ; watzinger et al. ) with different detection thresholds. it emerges from this glance at literature that cellulose-based electropositive filters are still commonly used for viral monitoring of water. these filters are expensive, however, and face competition with cheaper products, in particular nanoalumina fiber filters and glass wool filters (cashdollar and wymer ; wong et al. ) . the former, nanoceram tm filters manufactured by argonide corporation (sanford, fla., usa), are composed of nanosized ( nm diameter), alumina-based {mainly boehmite, c-alo(oh)} fibrilles dispersed in a microglass fiber matrix, resulting in an electropositive filter media with - lm average pore size kaledin , ) . the latter consist of commercial sodocalcic glass wool coated with mineral oil (type bourre qn/tech loose wool, isover saint-gobain, courbevois, france), hand packed into columns or filter holders in the laboratory (vilaginès et al. ) . these glass wool filters harbor electropositive sites while presenting hydrophobic surface characteristics. efficient enrichment of (seeded or/ and) indigenous viruses from various water samples using nanoceram (gibbons et al. ; ikner et al. ; pang et al. ) , and glass wool (deboosere et al. ; lambertini et al. ; wyn-jones et al. ) filters has been reported in recent years (see also cashdollar and wymer ; wong et al. ) . compared with mds filters (table ) , nanoceram filters showed similar , slightly lower (mcminn ) or higher (karim et al. ) virus concentration efficiency. commercially available under different pore sizes ( . - . lm), mixed cellulose ester membrane filters are negatively charged over a wide range of ph values, their overall negative charge increasing with ph (kessick and wagner ) . microporous filters in the . - . lm pore size range have been used for ages in laboratory and industry for size-based filtration of bacterial particles and cell debris (surface filters). these and larger pore size filters have been shown to retain enteroviruses, however, despite the much smaller size of viral particles compared to the nominal mean diameter of filter pores (cliver ). the presence of salts enhanced virus adsorption to cellulose ester membranes, this effect increasing with the cation valence (i.e., al ? [ mg ? [ na ? ) (wallis et al. ) . acidification of the viral suspension also improved virus adsorption efficiency, even in the absence of exogenously added salts (sobsey et al. ). these early results were later confirmed by lukasik et al. ( ) , who investigated the influence of mono-, di-, and trivalent salts (nacl, mgcl , and alcl ) on the adsorption of poliovirus and enterobacteria phages to the mf-millipore tm membrane filter type ha ( . lm pore size) and the electropositive m tm zeta plus tm mds microfilter at neutral or acidic ph. at ph , salts promoted virus adsorption to ha filter while affecting adsorption to mds filter. at ph . , more than % of the viruses tested adsorbed to ha filter with or without salt added to the viral suspension-the salts interfering again with viral adsorption to mds filter. furthermore, the addition of urea or tween to the salt solution affected virus adsorption to both filters at ph . and ha filter at ph . in agreement with previous studies by the same group (haramoto et al. (haramoto et al. , lukasik et al. ( ) explained these results by the antichaotropic effect of salts that increased hydrophobic interactions between filters and viruses and was impaired by the chaotropic agent or the detergent. at neutral ph, charge screeening by salt addition reduced electrostatic attractive and repulsive forces between viruses and mds or ha filter, respectively. cation-mediated cross-complexation between negative groups on virus and filter surfaces (kessick and wagner ) and, more particularly, strengthened hydrophobic virusfilter interactions contributed to improving the adsorption efficiency of ha filter. at acidic ph, viruses displayed a positive surface charge and their electrostatic interactions with ha filter switched from repulsive to attractive-and inversely for electropositive mds filter. the presence of salt probably affected these interactions but was balanced by the promoting effect of salt on hydrophobic interactions. hence, most tests reported so far for virus concentration from water samples using cellulose ester filters (essentially ha filters) have been performed after addition of multivalent cations (mainly mg ? ) to the water samples with or without ph adjustment to an acidic level. according to katayama et al. ( ) , the virus-loaded filters were rinsed with an acidic solution to eliminate remaining cations before elution with naoh or other alkaline buffers. haramoto et al. ( haramoto et al. ( , haramoto et al. ( , a proposed a variation in the method that consisted in pre-conditioning the filter with al ? ions. aluminum chloride was passed through the filter, making an electropositive ion coating, which avoided cation addition to the water sample. the recovery efficiency of both protocols has been evaluated by seeding water samples with enteric viruses and bacteriophages and quantifying eluted viruses (table ) . similarly to spiking studies (tables , ) and viral analyses of field water samples (table ) with electropositive filters, the ha-based filtration/elution process was frequently associated with a secondary concentration step to increase virus concentration factors. here, centrifugal ultrafiltration (cu) using commercial millipore (centriprep Ò , centricon Ò or amicon Ò ) concentrators was the elective concentration method. these cu units contain a low adsorptive regenerated cellulose membrane (ultracel Ò ) whose nominal molecular-weight cutoff ranges between and kda-a mwco value of kda being most frequently selected for secondary concentration of eluted viruses. they are routinely used in laboratories to purify and concentrate biomacromolecules such as peptides, proteins, and nucleic acids from small-volume biological samples (e.g., - ml for the centriprep filter unit). tested or not for virus recovery by spiking experiments, the combined concentration procedures have been extended to the detection of enteric viruses in field water samples including tap (haramoto et al. ), sea (katayama et al. ) , river (surface) (fong et al. ; hamza et al. ; haramoto et al. ) and waste (fong et al. ; katayama et al. ) water (table ) . samples with different volumes were collected according to the water source, i.e., sample volumes increased as the expected contamination level decreased. for instance, katayama et al. ( ) applied ha filtration, followed by acid rinse of the filter, viral elution with naoh and secondary concentration of the eluate by cu, to concentrate naturally occurring viruses (noroviruses, enteroviruses and hav-hepatitis a virus) in seawater samples. based on the volume reduction factor ( -l sample/ ml final concentrate, i.e., ) and the recovery yield of the two-step process (table ) , a virus concentration factor of was reached. only hav virus was not detected in any sample tested. later on, katayama et al. ( ) followed the same protocol to concentrate enteric viruses in the raw influent of a wastewater treatment plant. samples of -ml volume were collected and submitted to the two-step concentration process, yielding a volume reduction factor of c. . the four tested kinds of enteric viruses were detected in all wastewater samples but one lacking norovirus gi (nov gi). using peg precipitation as the secondary concentration step, hamza et al. ( ) tested river water samples for contamination by enteric viruses and bacteriophages. a volume reduction factor of ( -l sample/ ml final concentrate) and virus concentration factors ranging between (norovirus) and (adenovirus) (see table for recovery yields) were obtained. all analyzed samples were found positive for enteric viruses. human adenovirus and norovirus were detected in . and % of the samples, respectively. haramoto et al. ( ) illustrated the ability of al ? -coated filters to concentrate viruses from large-volume freshwater samples without salt addition by detecting noroviruses in tap water from tokyo university. tap water samples ( -l average volume) underwent two successive filtration/elution steps using -mm and -mm diameter ha filters prior to concentration by cu (final volume: . ml), which ensured a volume reduction factor higher than , . however, the virus recovery efficiency of the -step concentration process was not evaluated. ten of the tested samples were found positive for noroviruses. these results compare to those reported by lee et al. ( ) ( table ) using mds cartridge with organic flocculation to enrich noroviruses from large surface and ground water samples. despite these promising data, the two viradel methods based on electronegative filters (i.e., addition of mgcl to water samples or filter coating with al ? ions before filtration) have been essentially implemented for viral analysis of waters containing high amounts of indigenous viruses, requiring limited sample volumes (table )-electropositive filter cartridges being more adapted to virus concentration from large volumes of weakly contaminated water (table ). beside the above-mentioned work by haramoto et al. ( ) , only two studies among those detailed in table describe virus detection in environmental water samples with low viral content, namely drinking water sources after chlorination or without treatment (rigotto et al. ) and groundwater from artesian wells (chironna et al. ) . both studies used small-volume samples and cu as secondary concentration step, yielding a modest volume reduction factor of . very few from artesian well water samples were found positive to the tested enteric viruses: none for hav and enterovirus, for rotavirus and for norovirus (chironna et al. ) . a large proportion ( %) of samples from drinkingwater supplies was positive to adenovirus, however (rigotto et al. ) . these data are compatible with viral analyses of groundwater and water intended for drinking performed using mds filtration as the first virus concentration step (table ). it should be noted, however, that the same remark applies to data collected in table as to those in table concerning the numbers of virus-positive samples, i.e., their dependence on the virus detection method. this can be illustrated by the two following examples. using icc-pcr (measuring infectious viruses), katayama et al. ( ) detected enteroviruses in of seawater samples from bathing beach, but no sample was found positive by direct rt-pcr. de paula et al. ( ) found / river water samples positive for hav by quantitative real-time rt-pcr, but only / by nested rt-pcr. as attested by the large body of literature data quoted above, virus adsorption-elution methods using electropositive or electronegative filters are routinely applied to the primary concentration of waterborne viruses, each type of filter possessing its own advantages and drawbacks. ultrafiltration is considered another filtration-based option to concentrate viruses from water samples (cashdollar and wymer ; ikner et al. ). it has been indicated earlier that cu with microconcentrators based on cellulose ultrafiltration discs is commonly used as a secondary concentration step following the viradel process. hollow fiber ultrafiltration (hfuf), however, is considered a potential technique for primary concentration of viruses from large-volume water samples, yielding better virus recoveries than the viradel method performed with either electropositive or electronegative filters (cashdollar and wymer ) . commercial hfuf devices operated in cross-flow mode have been applied to the simultaneous concentration of biological particles, including viruses, spiked in tap (polaczyk et al. ) or reclaimed (liu et al. ) water samples, to virus recovery from seeded tap (rhodes et al. ) and estuarine (hernandez-morga et al. ) water samples, and also, more rarely, to virus recovery from field water samples (grassi et al. ; hernandez-morga et al. ). most of these devices are dialyzers that contain synthetic hf made from polysulfone. following the early work by belfort et al. ( ) showing that polysulfone hf membranes were superior to cellulose acetate ones for virus concentration, virus concentration experiments using hfuf dialyzers equipped with hf manufactured from cellulose are scarce. an example is given by liu et al. ( ) . they showed that the exeltra plus cellulose triacetate hf dialyzer (baxter healthcare corp., deerfield, il, usa) and the optiflux Ò f nr polysulfone dialyzer (fresenius medical care, walthamm, ma, usa) provided similar recovery efficiencies for ms and ux bacteriophages, escherichia coli, clostridium perfringens spores, and cryptosporidium parvum oocysts from spiked -l and -l samples of reclaimed water. an array of viral infections can be transmitted by the airborne route, in particular via aerosols (droplet nuclei) tang et al. ; tellier ; verreault et al. ). even though virus inactivation rates in the atmosphere are generally higher than those of bacterial and fungal contaminants, virus-containing aerosols can spread worldwide (després et al. ) . the threat of viral outbreaks and pandemics such as those caused by sars coronavirus (yu et al. ) and highly pathogenic strains of influenza a virus (tellier ) , allied with the fear of bioterrorism using viruses (e.g., smallpox and hemorrhagic fever viruses) (barras and greub ) as biological weapons, has encouraged the search for efficient protection equipment against aerosolized virus-containing particles. individual respiratory protective devices consist in respirators, i.e., air-filtering face masks designed to protect the wearer against inhalation of a hazardous atmosphere (here airborne infectious aerosols)-opposing to surgical masks designed to protect the environment from contaminants generated by the wearer's exhaled breath (i.e., prevention of surgical infections) . currently, most ''filtering facepiece'' (ffp) respirators have a multilayer composite structure with a central filtering layer displaying electret properties (gralton and mclaws ) . these electret filters (thakur et al. ) are produced by imparting an electrostatic charge to a nonwoven fibrous mat composed of synthetic polymer fibers such as poly(propylene) (pp) (mainly), poly(butylene terephthalate), poly(tetrafluoroethylene) and poly(carbonate) fibers. electrostatic charging of the filter media is commonly obtained by corona discharge, triboelectrification and electrostatic spinning (tsai et al. ) . electret filters collect particles through the combined action of mechanical and electrostatic forces (podgórski ; wang (cen ) should retain respectively at least and % of influent particles (nacl particles are used as nonbiological surrogate particles) (rengasamy et al. ). first worn by surgical teams a hundred years ago to prevent bacterial contamination of patient's open wounds, early surgical masks were constructed from layers of cellulose materials, more particularly cotton cellulose (gauze) (haller and colwell ) and derivatives (arnold ) (see also belkin ) . much more recently, face masks made from cotton fabrics have been tested as alternative respiratory protective equipment against pandemic outbreaks such as influenza (dato et al. ; davies et al. ; rengasamy et al. ) . like respirators, current commercially available surgical face masks include several layers of non-woven fabrics with, frequently, a cellulose inner layer in contact with the wearer's face to improve wearer's comfort. however, the filtering layer, usually made of meltblown fibers (ghosh ) , is devoid of electret properties. since particulate filtration is only mechanical, i.e., less efficient than that of respirators, most surgical masks are not certified for use as respiratory protective devices, standard tests to evaluate their filtration efficacy being less stringent (oberg and brosseau ) . while a number of studies have confirmed that surgical masks logically offer lower protection than respirators against aerosol particles, the effectiveness of both types of face masks at preventing viral respiratory infection, in particular influenza, is still a matter of controversy (bin-reza et al. ; cowling et al. ; gralton and mclaws ) . the improper facial fit of respirators coia et al. ; weiss et al. ) may affect their protection efficiency against infectious aerosols. it allows particulate flows outside the filtration area of the mask, resulting in face-seal leaks, i.e., the leakage of infectious particles around the edges of the mask (grinshpun et al. ; lei et al. ) . mask seal leakage can be minimized by applying existing guidelines for correct donning and fit checking of respirators . moreover, human face and head form models have been proposed as a tool for designing respirators with improved protection efficiency, simulating interactions between faces and facemasks and describing their fit (golshahi et al. ; lei et al. ) . the accumulation of viral particles at the surface and within the filtration media of respirators, where they can remain viable and infectious for extended periods of time (coulliette et al. ; sakaguchi et al. ) , is another significant problem with which respirator wearers are confronted in the prevention of viral transmission and spread. incorrect mask handling by the wearer may lead to accidental self-inoculation, cross-contaminations affecting both other healthcare workers and patients, and contamination of fomites (casanova et al. ) . furthermore, the risk of virus reaerosolization from respirators during extended use, if limited, cannot be ruled out (fisher et al. ) . recommendations for respirator doffing do exist but have proven insufficient to prevent virus transfer from respirator to healthcare employees' hands and clothing (casanova et al. ) . adding antiviral properties to the filtration process may contribute to limit the risk of viral transmission by improper handling of used respirators. a number of virucidal facemasks have been developed and patented over the past years (tiliket et al. ) , some of which are commercially available. in the spectrashield tm plus respirator masks designed by nexera medical inc. (fort lauderdale, fla.) (haas ), for instance, two layers of filter media are sandwiched between (inner and outer) antimicrobial layers where a silver-copper zeolite (carrier) antimicrobial agent (agion Ò antimicrobial, sciessent llc, wakefield, mass.) is embedded around the core of synthetic fibers (fosshield Ò , foss manufacturing, hampton, nh). the biofriend tm biomask tm ffp respirator, manufactured by filligent ltd. (hong kong) and distributed by medline industries, inc. (mundelein, ill.) , is another four-layered device in which the melt-blown pp filtration layer is inserted between an inner layer of spunbond pp and two antiviral layers, namely a cellulose/polyester layer containing copper and zinc ions and an outer layer of spunbond pp treated with a low ph (citric acidacidified) hydrophilic plastic coating (davison ; stewart et al. ) . the outer layer absorbs infectious aerosol droplets and viruses are denaturated by exposure to citric acid. in the second layer, inactivation of viruses with damaged structures is completed by the virucidal effect of divalent metal cations. contributing to the overall antiviral efficiency of the structure, the cellulosic component of this layer is a sulphated or sulfonated rayon fabric (stewart et al. (stewart et al. , to which a variety of viral human pathogens bind via the cationic sites of viral envelopes/capsids. beside the commercial biomask tm respirator, various composite structures designed for filtrationinactivation of airborne microbial contaminants, where cellulosic materials play an active antimicrobial role, have been patented in recent years bernard ; nakamura and nakamura ; tsutsumi ; zhang et al. ; zhong ) . several of them are based on bacterial cellulose layers/coatings provided with an antimicrobial component, i.e., silver (zhong ) or zeolite-supported silver (nakamura and nakamura ) nanoparticles, and chitosan (zhang et al. ) . the respiratory protective mask described by zhong ( ) comprises a three-layer bacterial cellulose membrane whose middle layer contains silver nanoparticles. the antimicrobial facemask invented by nakamura and nakamura ( ) includes two base cloth elements both of which are made of a woven textile (gauze), a nonwoven cellulose fabric (e.g., rayon), or a porous sheet (e.g., a . - . mm sliced sheet of urethane sponge). the first base cloth is filled with bacterial cellulose nanofibers that retain silver zeolite and a humectant (e.g., trehalose or , -butylene glycol) in their network structure. additional antimicrobial properties are provided to the mask by impregnating the second base cloth with a carboxylic acid (e.g., citric acid). zhang et al. ( ) also used bacterial cellulose in a composite antimicrobial material suitable for respiratory protective masks. a nonwoven polymer fabric rev environ sci biotechnol ( ) : - was coated with a film of bacterial cellulose mixed with poly(vinyl alcohol)-to improve the film-forming properties, mechanical strength and air permeability of the coating-and chitosan, a non-sulfated polysaccharide produced commercially by deacetylation of chitin that displays strong antibacterial potency (kong et al. ; rabea et al. ) and, to a lesser extent, antiviral activity (rabea et al. ; wang et al. ). in the antimicrobial air filtration device proposed by bernard ( ) , cellulose derivatives-more particularly, cellulose acetate phthalate (cap), a common pharmaceutical excipient for enteric coating of tablets and capsules with antiviral activity (pirrone et al. )-were used as ''biocidal prophylactic compounds'' to provide the classical filter media (e.g., a nonwoven pp web, electrostatically charged or not) with antimicrobial properties. cellulose derivatives could be incorporated as fibers or particles (micronized fibers) into the air filtration device either in a separate layer positioned before or after the filter media, or deposited onto the outer surface of the filter media relative to the air flow direction. to illustrate this invention, handsheets prepared from cap fibers intermixed or not with cap particles were successfully tested for virucidal efficacy by exposure to an aerosol challenge of enterobacteria phage Øx . by exploiting the antimicrobial properties of dialdehyde polysaccharides such as starch and cellulose dialdehydes, baney et al. ( ) proposed a new method for producing low-cost virucidal filters suitable for respiratory protective masks. the treatment of a standard cellulose filter paper (whatman tm grade filter paper) with sodium periodate improved its antiviral potency due to oxidation of some cellulose to dialdehyde cellulose inside the filter. challenged with aerosols of ms enterobacteria phage at high relative humidity ( % at °c), the treated filter showed higher filtration efficiency than a control, untreated one, i.e., better removal of viable viral particles (plaque forming units) joined to a lower resistance to air flow (pressure drop)-due to increased pore size distribution (woo et al. ) . higher inactivation of ms virus by the treated filter was also highlighted, confirming an improvement of its disinfection capability (woo et al. ). however, the filtration and inactivation of airborne viruses were less effective at lower moisture content of the filter, i.e., at air humidity levels commonly encountered in hospital settings, in particular operating rooms (balaras et al. ). furthermore, the inhalation resistance of the treated filter remained too high for application to respiratory protective masks. the same pros and cons apply to cellulose paper filters treated by immersion into aqueous suspensions of dialdehyde starch (das) (woo et al. ) . while this treatment did not affect the removal efficiency of viable ms viruses from aerosols at high relative humidity, virus survival on the treated filters was reduced (fig. ) and the pressure drop decreased as das concentration increased. the drop in air flow resistance remained insufficient for practical application to respirators. das treatment of a pp filter from a commercial ffp mask did not modify the filtration parameters (virus removal efficiency and pressure drop) of the filter but significantly improved its biocidal efficacy (fig. )-an opening of this treatment towards the development of virucidal facemasks. another example of cellulose filter material treated with cellulose derivatives to yield antiviral properties is given by tsurumi (tsutsumi ). this inventor devised a low-cost virucidal mask filter made of a standard cellulose nonwoven fabric impregnated with water extracts of sulfonated or aminated styrenegraft cellulose. to reinforce the water retention properties of the filter media, a superabsorbent resin such as poly(acrylic acid) was added to the graft cellulose extract before impregnation of the fabric or incorporated into a separate layer covering the impregnated fabric. low (hydrogen ions from sulfonic acid groups) or high (hydroxyl ions generated by aminated groups) ph conditions inside the hydrated material were claimed to make it virucidal against influenza a virus and caliciviruses. the patent gives no illustration of such antiviral properties, however. studies published by tiliket et al. ( ) and catel-ferreira et al. ( ) present another type of cellulosebased material for airborne virus filtration in which a low-cost nonwoven cellulose material, i.e., commercial kimwipes Ò (kw) wipes (kimberly-clark worldwide, inc., dallas, tex.), was chemically modified by coating with a synthetic polymer, i.e., poly(ethylenimine) (pei) (tiliket et al. ) or grafting of an antiviral agent, i.e., catechin (catel-ferreira et al. ) . the filtration efficiency of the modified filter media was first tested on aerosolized t d viruses (enterobacteria phage t , doermann's strain t d). then the treated filter was inserted inside a commercial medical mask in place of its cellulosic layer (kolmi m mask, kolmi-hopen, saint-barthélémy-d'anjou, france), and the reconstructed mask was challenged with td aerosols to evaluate its virus removal efficiency. both treatments significantly improved the virus capture factor (ratio of upstream to downstream pfu contents) of kw cellulose wipes and of reconstructed commercial masks compared to original masks and to masks reconstructed with untreated wipes. in these studies, the breathability of reconstructed masks was not quantified, nor was the virus survivability on the modified filter media. however, additional filtration experiments using aerosols of influenza a virus (low pathogenic h n strain) showed that challenged viral particles accumulated in pei functionalized kw layers with no loss in number and infectivity (tiliket et al. ) . thus these modified masks showed no virucidal effect, the peimodified layers behaving like an electret media owing to the polycationic character of pei. on the other hand, since catechin and catechin-grafted wipes showed antiviral activity against t d viruses in liquid media (catel-ferreira et al. ) , it may be assumed that part of viruses accumulated in excess in the treated wipes were inactivated by the polyphenol agent, i.e., the reconstructed masks were actually virucidal. despite these few examples, the use of cellulosic media in filtration devices aimed at the capture of airborne viral particles remains now very limited compared to filtration of virus-contaminated liquid samples. while patented, several cellulose-based, virucidal filter media designed for insertion in air filtering face masks have not been commercially developed yet. besides the wearing of personal respiratory protective equipment, another aspect of the fight against airborne infections lies in the decontamination of indoor air in healthcare facilities, and, more generally, of air processed through heating, ventilating and air conditioning (hvac) systems in the built environment. high efficiency particulate air (hepa) filters are the primary technology used for particulate removal in these collective protection devices-allied with uv irradiation, ozonation or air ionization to yield indoor air decontamination (bolashikov and melikov ; jacob et al. )-and may be complemented with a photytocatalyst such as tio to improve the inactivation of accumulated microorganisms pigeot-remy et al. ). since hepa filters are essentially made of glass fibers, they are beyond the scope of this review and their virus-retentive properties will not be detailed further. most frequently associated with other virus reduction (virus clearance of biopharmaceuticals) or concentration (viral analysis of water) steps, size-exclusion and/ or adsorptive (charge mediated) filtration is an essential tool to fight against viral contaminants in aqueous media. owing to inherent properties of cellulose, including mechanical strength and hydrophilicity (that opposes protein adsorption and biofouling), allied with widespread availability and biocompatibility, cellulose-based materials are still widely present in viral filters. developed commercially over the past thirty years, several cellulosic filter media have been and continue to be used for both routine analyses in laboratories and academic research studies aimed at improving the filtration performance, generating a wealth of data quantifying the efficiency of virus removal or recovery/concentration from biologic or water samples, respectively. as concerns biopharmaceutical compounds, these data arise from validation studies of the filtration step performed by spiking intermediate biologic solutions (mostly blood proteins) from multistage purification processes with known titers of viral particles. regenerated cellulose hf planova filters n -n , operated in the dead-end mode as a single filter or two units in series, were shown to provide lrv values ranging between and , on the average. increasingly tested since their launching in , these cellulosic filters compete with filter media made of synthetic polymers such as pvdf (e.g., viresolve Ò nfp-normal flow parvovirus-from emd millipore; ultipor Ò vf grade dv /dv from pall corporation, port washington, ny) or pes (e.g., millipore viresolve Ò pro; virosart Ò hs/hc/cpv from sartorius stedim biotech, aubagne, france). the hydrophilic nature of cellulose fibers is a definite advantage of planova filters over others for virus removal from protein solutions, minimizing flux decline due to proteindominated filter plugging (cake formation) at constant pressure (normal-flow) filtration (rathore et al. ). however, they are usually operated at lower flow rate than filters made of synthetic polymers whose operating pressure is higher. hydrophilic modified pvdf membrane filters designed for virus removal from high-concentration protein solutions at high flow rate are being developed by several manufacturers, including asahi-kasei (i.e., planova tm bioex filters, launched in ). many studies have also been reported to assess the virus concentration efficiency of the viradel method using spiked water samples, prior to the detection of waterborne viral contaminants in field water samples. in this method, cellulosic filters, whether positively or negatively charged, are used for adsorptive capture of viruses before virus recovery by elution with a lowvolume eluent. electropositive filters are more particularly suitable for virus capture in low-contaminated, high-volume water samples, e.g., samples of groundwater or source water for drinking water production ( table ) . though routinely used, these filters are expensive and face competition with nanoalumina fiber filters (e.g., the patented ahlstrom disruptor Ò electroadsorptive filter) (levi ) and glass wool filters, which are cheaper. on the other hand, efficient virus adsorption by electronegative filters-the standard, cost-effective mixed cellulose ester membrane microfilters-requires impractical sample pre-treatment: their use is limited to viral analysis of highlycontaminated waters, for which small-volume samples are sufficient (table ) . cellulose nanomaterials may represent a promising perspective for cellulosic materials in their application to viral filtration of liquid samples, yielding filtration membranes with higher mechanical strength, water permeability, surface hydrophilicicy and resistance to biofouling (carpenter et al. ) . ma et al. ( ) have presented a composite membrane consisting of an electrospun poly(acrylonitrile) nanofibrous scaffold deposited on a non-woven poly(ethylene terephthalate) support. this two-layered membrane was coated by a layer of cellulose nanofibers and tested for retention of ms- phages. at acidic ph, phage particles were adsorbed by the negatively charged cellulose nanofiber coating, yielding a lrv value [ . . later on, the same nanofibrous composite membrane was doped with cellulose nanofibers functionalized by grafting with poly(vinylamine) . ms- phages were adsorbed at neutral ph onto positively charged amino-modified nanofibers, with a lrv value of . metreveli et al. ( ) and asper et al. ( ) have presented a size-exclusionbased filter paper made of pure cellulose nanofibers for removal of swine influenza a and murine leukemia viruses, respectively. these non-woven materials could be tailored to ensure efficient virus retention (gustafsson and mihranyan a). self assembled into nanosheets to yield ''mille feuille'' structures, they showed efficient removal of parvoviruses (gustafsson et al. b) : maybe a new filtration media for viral clearance? a number of air filtration devices designed for individual protection against airborne viruses also include cellulosic materials. while published data on the filtration performance of cellulosic media for removal of viruses from air are scarce, industrial research is more apparent, in particular in asian countries (china, japan) where airborne pollution is a matter of great concern-the main objective being to add virucidal properties to the filtration media so as to inactivate accumulated viral particles. therefore, some 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and processing water emerging viral infections-a potential threat for blood supply in the st century hepatitis a virus in environmental water samples from the amazon basin development and validation of a concentration method for the detection of influenza a viruses from large volumes of surface water primary biological aerosol particles in the atmosphere: a review effective virus inactivation and removal by steps of biotest pharmaceuticals igiv production process a new series of graded collodion membranes suitable for general bacteriological use, especially in filterable virus studies filtration of vaccinia virus through gradocol membranes the filtration of herpes virus through graded collodion membranes the size of the virus of poliomyelitis as determined by ultrafiltration analysis the sizes of the viruses of human and swine influenza, as determined by ultra-filtration respiratory protective devices-particle filters-requirements, testing, marking effect of chaotropic and antichaotropic agents on elution of poliovirus adsorbed on membrane filters ultrafilter membranes and ultrafiltration reaerosolization of ms bacteriophage from an n filtering facepiece respirator by simulated coughing enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools quantitative detection of human adenoviruses in wastewater and combined sewer overflows influencing a michigan river concentration and processing of waterborne viruses by positive charge mds cartridge filters and organic flocculation detection of rotavirus a in sewage samples using multiplex qpcr and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration implementation of a -nm pore-size filter in the plasmaderived factor viii manufacturing process filtration of the virus of footand-mouth disease through a new series of graded collodion membranes composite nonwovens in medical applications evaluation 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large volumes of water development of a virus concentration method and its application to detection of enterovirus and norwalk virus from coastal seawater one-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in japan organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water electrophoretic mobilities of virus adsorbing filter materials detection and genetic analysis of human sapoviruses in river water in japan relative abundance and treatment reduction of viruses during wastewater treatment processes-identification of potential viral indicators pathogen inactivation and removal methods for plasma-derived clotting factor concentrates pathogen-reduction methods: advantages and limits virus safety of plasma products using nm instead of nm filtration as virus removing step antimicrobial properties of chitosan and mode of action: a state of the art review evaluation of viral clearance in purification processes assessment of human adenovirus removal in a fullscale membrane bioreactor treating municipal wastewater concentration of enteroviruses, adenoviruses, and noroviruses from drinking water by use of glass wool filters evaluation of electropositive filtration for recovering norovirus in water incidence and molecular characterization of hepatitis a viruses in korean surface water between headform and n filtering facepiece respirator interaction: contact pressure simulation and validation simulation and evaluation of respirator faceseal leaks using computational fluid dynamics and infrared imaging fluid purification media and systems and methods using same polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis a virus and human rotavirus, from oyster, water, and sediment samples concentration of viruses from environmental waters using nanoalumina fiber filters recovery and purification process development for monoclonal antibody production hollow-fiber ultrafiltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water rapid concentration of bacteriophages from large volumes of freshwater: evaluation of positively charged, microporous filters influence of salts on virus adsorption to microporous filters ultra-fine polysaccharide nanofibrous membranes for water purification concentration of live retrovirus with a regenerated cellulose hollow fiber mechanism of human immunodeficiency virus (hiv) removal by regenerated cellulose hollow fiber (bmm) analytical methods for food and environmental viruses optimization of adenovirus and recovery from tap water using small disk filters a size-exclusion nanocellulose filter paper for virus removal molecular detection and characterization of gastroenteritis viruses occurring naturally in the stream waters of manaus, central amazonia, brazil isoelectric points of viruses antibacterial mask, antibacterial filter for the 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from -l tap water samples component pathogen inactivation: a critical review chitosan as antimicrobial agent: applications and mode of action intravenous immunoglobulin g: trends in production methods, quality control and quality assurance second-step concentration of viruses in drinking and surface waters using polyethylene glycol hydroextraction rotavirus concentration from raw water using positively charged filters mechanistic modeling of viral filtration respiratory protection against bioaerosols: literature review and research needs comparison of nanoparticle filtration performance of niosh-approved and ce-marked particulate filtering facepiece respirators simple respiratory protection-evaluation of the filtration performance of cloth masks and common fabric materials against - nm size particles evaluation of hollow-fiber ultrafiltration primary concentration of pathogens and secondary concentration of viruses from water assessment of adenovirus, hepatitis a virus and rotavirus presence in environmental samples in florianopolis, south brazil virus removal from factor ix by filtration: validation of the integrity test and effect of manufacturing process conditions purification of retroviral vectors for clinical application: biological implications and technological challenges the impact of combined sewage overflows on the viral contamination of receiving waters comparison of microporous filters for concentration of viruses from wastewater maintenance of influenza virus infectivity on the surfaces of personal protective equipment and clothing used in healthcare settings nine-year study of the occurrence of culturable viruses in source water for two drinking water treatment plants and the influent and effluent of a wastewater treatment plant in downstream processing of oncoretroviral and lentiviral gene therapy vectors overview of current scalable methods for purification of viral vectors possibility of hepatitis b virus (hbv) removal from human plasma using regenerated cellulose hollow fiber (bmm) an attempt to prepare hepatitis b virus (hbv)-free plasma by ultrafiltration using microporous regenerated cellulose hollow fiber influence of salts on electrostatic interactions between poliovirus and membrane filters downstream processing of monoclonal antibodies-application of platform approaches removal of human enteric viruses by a full-scale membrane bioreactor during municipal wastewater processing poliovirus concentration from tap water with electropositive adsorbent filters concentration of enteroviruses from large volumes of water pathogen reduction of blood components composition for use in decreasing the transmission of human pathogens devices and methods for decreasing human pathogen transmission factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises review of aerosol transmission of influenza a virus nanosize electropositive fibrous adsorbent a high-performance electropositive filter viral safety of nanogam Ò , a new nm-filtered liquid immunoglobulin product viral safety of c -inhibitor nf electret air filters a new material for airborne virus filtration effective detection of human noroviruses in hawaiian waters using enhanced rt-pcr methods different electrostatic methods for making electret filters mechanism of removing monodisperse gold particles from a suspension using cuprammonium regenerated cellulose hollow fiber (bmm hollow fiber) structure of cuprammonium regenerated cellulose hollow fiber (bmm hollow fiber) for virus removal structure and filtration performances of improved cuprammonium regenerated cellulose hollow fiber (improved bmm hollow fiber) for virus removal mask and graft polymer extract. japanese patent no detection of adenoviruses and rotaviruses in drinking water sources used in rural areas of methods for sampling of airborne viruses large-scale production and purification of vlpbased vaccines rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: current advances evaluation of an adsorptionelution method for detection of astrovirus and norovirus in environmental waters glass wool for virus concentration at ambient water ph level virus concentration on aluminum and calcium salts enterovirus concentration on cellulose membranes ayliffe's principles and practice of disinfection, preservation and sterilization, th edn electrostatic forces in fibrous filters-a review the antiviral activities and mechanisms of marine polysaccharides: an overview nanofibrous microfiltration membranes capable of removing bacteria, viruses and heavy metal ions detection and monitoring of virus infections by real-time pcr disrupting the transmission of influenza a: face masks and ultraviolet light as control measures application of enteric viruses for fecal pollution source tracking in environmental waters evaluation of the performance of dialdehyde cellulose filters against airborne and waterborne bacteria and viruses use of dialdehyde starch treated filters for protection against airborne viruses a simple and novel method for recovering adenovirus in small volumes of source water surveillance of adenoviruses and noroviruses in european recreational waters occurrence of human adenoviruses at two recreational beaches of the great lakes evidence of airborne transmission of the severe acute respiratory syndrome virus the particle size of hepatitis c virus estimated by filtration through microporous regenerated cellulose fibre antivirus bacterial cellulose protective material and its preparation method. chinese patent no air filter type bacterial cellulose mask and production method thereof. chinese patent no viral clearance using disposable systems in monoclonal antibody commercial downstream processing key: cord- - xu hj n authors: ahmed, w.; gyawali, p.; toze, s. title: evaluation of glass wool filters and hollow-fiber ultrafiltration concentration methods for qpcr detection of human adenoviruses and polyomaviruses in river water date: - - journal: water air soil pollut doi: . /s - - - sha: doc_id: cord_uid: xu hj n pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. they are difficult to concentrate from environmental waters due to low numbers and small sizes. rapid enumeration of viruses by quantitative polymerase chain reaction (qpcr) has the potential to improve water quality analysis and risk assessment. however, capturing and recovering these viruses from environmental water remain formidable barriers to routine use. here, we compared the recovery efficiencies of human adenoviruses (hadvs) and human polyomaviruses (hpyvs) from -l river water samples seeded with raw human wastewater ( and ml) using hollow-fiber ultrafiltration (hfuf) and glass wool filter (gwf) methods. the mean recovery efficiencies of hadvs in river water samples through hfuf were and % for and ml of seeded human wastewater, respectively. in contrast, the estimated mean recovery efficiencies of hadvs in river water samples through gwf were . and % for and ml seeded raw human wastewater, respectively. similar trends were also observed for hpyvs. recovery efficiencies of hfuf method were significantly higher (p < . ) than gwf for both hadvs and hpyvs. our results clearly suggest that hfuf would be a preferred method for concentrating hadvs and hpyvs from river water followed by subsequent detection and quantification with pcr/qpcr assays. pathogens have been found in environmental water sources as a result of fecal pollution from wastewater treatment plants (wwtps), storm water drains, defective septic systems, and run-off from livestock and wildlife (abdelzaher et al. ; sidhu et al. ; ahmed et al. ) . identification of the source(s) of fecal pollution provides the first step in initiating remediation efforts and minimizing human health risks. this can be achieved by applying rapid microbial source tracking (mst) tools to identify and quantify host-specific genes or markers targeting bacteria, protozoa, and viruses (harwood et al. ) . development of numerous mst markers has been reported in the literature (harwood et al. ). among the enteric viral markers, human adenoviruses (hadvs) and human polyomaviruses (hpyvs) have been most widely used to detect human wastewater pollution in environmental waters (fong et al. ; hundesa et al. ; mcquaig et al. ). hadvs are responsible for a wide array of diseases such as gastroenteritis, respiratory infections, eye infections, acute hemorrhagic cystitis, and meningoencephalitis (videla et al. ; echavarría ). on the other hand, hpyvs are unique to humans and generally produce asymptomatic viruria, especially in immunocompromised people (polo et al. ) . hpyvs are frequently excreted in urine from healthy individuals. due to the high abundances of hadvs and hpyvs in human feces and urine, they have received significant attention as mst markers (fong et al. ; hundesa et al. ; mcquaig et al. ; ahmed et al. ) . generally, polymerase chain reaction (pcr) and quantitative pcr (qpcr)-based assays are used to detect and quantify these viral markers in environmental samples (mcquaig et al. ; staley et al. ; rusiñol et al. ) . enteric viruses need to be concentrated from environmental water samples prior to pcr/qpcr analysis. the most commonly used concentration methods are hollow-fiber ultrafiltration (hfuf) (rodriguez-diaz et al. ), ultracentrifugation (nordgren et al. ), adsorption-elution-based protocol with glass wool filter (gwf) (lambertini et al. ) , and positively and negatively charged membranes (katayama et al. ; bennett et al. ) . the ability to recover maximum numbers of viruses from various water matrices, however, can be highly variable depending on the concentration methods used (haramoto et al. ; dubois et al. ) . enteric viruses are relatively difficult to concentrate from environmental waters due to their low occurrence and small size (maier et al. ) . therefore, recovery of viruses from environmental waters requires filtration on the scale up to l of sample depending on the magnitude of fecal pollution. among the most commonly used concentration methods, hfuf has been used widely to recover viruses from environmental waters with recovery rates ranging from to % (morales- morales et al. ; hill et al. ; hill et al. ; polaczyk et al. ) . sodocalcic gwf also offers a promising alternative as an adsorptive material for virus concentration. gwf has been used to concentrate viruses from human wastewater (gantzer et al. ) and environmental waters (hot et al. ; ehlers et al. ) . albinana-gimenez et al. ( a) compared gwf and ultrafiltration cartridge to recover known quantities of hadv and john cunningham polyomavirus (jcpyv) in source water and drinking water using quantitative pcr. both methods produced similar recovery efficiencies for hadv (gwf . %, ultrafiltration . %) but ultrafiltration had higher efficiencies ( %) for jcpyv compared to gwf ( . %). based on the results, the authors concluded that the gwf method produced acceptable and reproducible recovery efficiencies, whereas the ultrafiltration method yielded variable recovery efficiencies. it has to be noted that albinana-gimenez et al. ( b) seeded cultured human adenoviruses type (hadv ) and jcpyv obtained from plaque assays. in a real-world scenario, fecal pollution of environmental waters would occur via human wastewater/septic overflows. little has been documented on the recovery efficiencies of hfuf and gwf methods for concentrating hadvs and hpyvs markers from environmental water samples seeded with raw human wastewater. the aim of this study was to compare the performance of hfuf and gwf concentration methods to recover hadvs and hpyvs from river water samples. qpcr assays were used to measure the concentrations of these viruses in deoxyribonucleic acid (dna) samples extracted from river water seeded with human wastewater. a two-liter human wastewater sample was collected from the primary influent of a wastewater treatment plant (wwtp). the wwtp has a flow capacity of megaliter day - and treats human wastewater from approximately , people. the treatment process consists of primary treatment, secondary treatment (activated sludge), and disinfection with chlorine and ultraviolet (uv) prior to being discharged into the brisbane river. a -l river water sample (clear color) was collected from the upstream in the brisbane river in carboys at a depth . to m. this site receives overflow of water from the wivenhoe reservoir after precipitation. the suspected sources of fecal pollution in this site include wildlife. human wastewater and river water samples were stored at °c for no more than h before processing. for each separate trial (n = ), and -ml volumes of human wastewater samples were seeded into two batches ( . and . l) of river water samples in triplicate (final volume of l). the ph and turbidity of the river water sample were . ± . and . ± . nephelometric turbidity unit (ntu), respectively. the background concentrations of hadvs and hpyvs in human wastewater and river water samples were also determined using qpcr assays (see below for details methodology). in brief, -ml raw human wastewater samples (n = ) were amended with hcl followed by passing through the . -μm ha negatively charged -mm membranes (hawp ; merck millipore ltd, sydney, australia). two-milliliter dna was extracted from the membranes using a powermax® soil dna isolation kit (mo bio laboratories, inc., carlsbad california, usa). river water (n = ) samples were concentrated using the hfuf method described below in details. hadvs and hpyvs were concentrated using a tangential flow hfuf method (hill et al. ) . general procedures are shown in fig. . the method involves concentrating water samples using a hemoflow hf s dialysis filter (fresenius medical care, bad homberg, germany). briefly, each -l water sample was pumped with a peristaltic pump in a closed loop with high-performance, platinum-cured l/s silicone tubing (masterflex; cole-parmer instrument co., chicago, illinois, usa). at the end of the concentration process, pressurized air was passed through the filter cartridge from the top to recover approximately to -ml concentrated sample in the retentate container. to improve recovery, after each sample was processed through the hfuf, ml of a surfactant solution ( . % tween , [sigma-aldrich, st. louis, missouri] . % napp, and . antifoam a [sigma-aldrich, st. louis, missouri, usa]) was recirculated through the filter until the system started to draw air. this elution solution was collected and added to the retentate to achieve a final volume of approximately - ml and stored at °c. a new filter cartridge was used for each sample. the ph of the concentrated sample was adjusted to . using . n hcl. the sample was then passed through . -μm ha negatively charged -mm membranes (hawp ; merck millipore ltd, sydney, australia) (mcquaig et al. ) attached to a glass membrane holder (merck millipore ltd., sydney, australia). dna was extracted from the membrane using a powermax® soil dna isolation kit (mo bio laboratories, inc., carlsbad california, usa) with slight modification . extracted dna was eluted through the spin filter membranes by adding ml solution c and stored at − °c until processed. the gwf method involves preparing glass wool as described elsewhere (millen et al. ) . general procedures are shown in fig. . washed glass wool was packed into cam lever couplings (banjo, crawfordsville, indiana, usa) using a metal plunger. the cam lever coupling packed glass wool was flushed with ml of sterile phosphate-buffered saline (ph = . ) using a catheter-tipped syringe. a water sample with a ph > . was adjusted to ph . - . by adding . n hcl. each human wastewaterseeded water sample ( l) was passed through the glass wool using a peristaltic pump. viruses were eluated in the opposite direction to the original flow with × ml % beef extract in . m glycine buffer with a ph of . . the first eluent was allowed to soak the glass wool for min before adding the second eluent, which was immediately pushed through the filter by air to obtain approximately to -ml concentrated sample. the ph of eluate was adjusted to . - . using . n hcl. concentrated sample ( to -ml) was further re concentrated using a jumbosep™ (molecular weight cut-off = kda) centrifugal device (pall corporation, east hills, ny, usa) to obtain a final volume of - ml. dna ( ml) was extracted directly from the - -ml concentrated sample using a powermax® soil dna isolation kit (mo bio laboratories, inc., carlsbad california, usa) with slight modification as described earlier. to assess for cross-contamination, one negative process control ( l of unseeded tap water) was processed for each method in parallel to river water samples seeded with human wastewater. a sketa real-time pcr assay was performed to determine the level of pcr inhibition in dna samples extracted from river water seeded with human wastewater (haugland et al. ; ahmed et al. ) . river water dna samples were seeded with a known amount ( pg) of oncorhynchus keta dna (sigma chemical co., st. louis, mo). dnase-and rnase-free water samples were also seeded with pg o. keta dna. the threshold cycle (c t ) values for seeded o. keta dna ( pg) were determined in a pcr run for both river water dna samples and dnase-and rnase-free water. the o. keta c t values obtained for dnase-and rnase-free water were compared to the c t values obtained for river water dna samples to obtain information on the pcr inhibition level. a c t delay was considered as having pcr inhibitors. the hadvs-and hpyvs-positive controls (dna) were isolated from adenovirus strain (atcc vr- ) and raw human wastewater, respectively. the pcr-amplified products were purified using a qiaquick pcr purification kit (qiagen, valencia, california, usa) and cloned into a pgem-t easy vector system ii (promega, madison, wisconsin, usa). recombinant plasmids with corresponding inserts were purified using a plasmid mini kit (qiagen, valencia, california, usa). standards for qpcr assays of hadvs and hpyvs were prepared from the plasmid dna, ranging from × to × (for hadvs) and × to × (for hpyvs) copies. the amplification efficiency (e) was determined by analysing the standards and was estimated from the slope of the curve as e = − /slope . qpcr assays were performed using previously published assays using the bio-rad cfx thermal cycler (bio-rad laboratories, richmond, california, usa). the primer and probe sequences, concentrations, qpcr reaction volumes, and cycling parameters are shown in table . sketa real-time pcr amplifications were performed in -μl reaction mixtures containing μl ( pg) of o. keta dna using iq supermixes (bio-rad laboratories, richmond, california, usa). hadvs qpcr amplifications were performed in -μl reaction mixtures containing -μl dna samples using ssofast evagreen supermix (bio-rad laboratories, richmond, california, usa), and hpyvs amplifications were performed in -μl reaction mixtures containing -μl dna samples using taqman universal pcr master mix (applied biosystems, foster city, california, usa). all qpcr reactions were performed in triplicate. to minimize qpcr contamination, dna extraction and qpcr set-up were performed in separate laboratories. a method blank was included for each batch of tap and river water samples. a reagent blank was also included during dna extraction. for each qpcr experiment, corresponding positive (standards) and negative controls (dnase-and rnase-free water) were included. the qpcr lower limit of quantification (lloq) was determined from the c t values obtained for each standard. to determine qpcr lloq, tenfold serial dilutions of standards ( × to copies for hadvs and × to copies for hpyvs) were tested in triplicates. the minimum concentration of copies from the standard series detected in / qpcr reactions was considered qpcr lloq. the recovery efficiency of virus concentration method was calculated as follows: recovery efficiency % ¼ concentration of copies recovered concentration copies seeded f or each virus  a paired t test for equal means was conducted to determine the difference between hadvs and hpyvs concentrations obtained through hfuf and gwf methods. the mean concentrations of hadvs and hpyvs in raw human wastewater sample were . × ± . × copies and . × ± . × copies ml - , respectively. none of the viruses were detected in l of river water sample. the standards had a linear range of quantification from × to × (for hadvs) and × to × (for hpyvs) copies μl - of dna extract. the slope of the standards ranged from − . to − . (for hadvs) and − . to − . (for hpyvs). the amplification efficiencies ranged from to % (for hadvs) and to % (for hpyvs), and the correlation coefficient (r ) ranged from . to . (for both hadvs and hpyvs). lower limit of quantification of qpcr assays was determined using the standards. the qpcr lower limits of quantification were and copies for hadvs and hpyvs, respectively. the mean c t value and standard deviation for the o. keta-seeded dnase-and rnase-free water were . ± . . c t values for o. keta-seeded river water samples were comparable to dnase-and rnase-free water for both hfuf (c t = . ± . ) and gwf (c t = . ± . ) methods, suggesting the samples were potentially pcr inhibitors free. the mean recovered hadv copy numbers in l river water seeded with ml ( . × ± . × copies) and ml ( . × ± . × copies) human wastewater obtained through hfuf were much higher than those river water samples obtained through gwf (fig. ) . similar results were also obtained for hpyvs. the mean recovered hpyv copy numbers in l river water seeded with ml ( . × ± . × copies) and ml ( . × ± . × copies) human wastewater obtained through hfuf were also much higher than those obtained through gwf method. hfuf method recovered significantly higher concentration of hadvs (p = . ; p = . ) and hpyvs (p = . ; p = . ) compared to gwf method for river water samples seeded with and ml of human wastewater, respectively. the mean recovery efficiencies of hadvs and hpyvs in river water samples processed using hfuf and gwf are shown in table . our data suggest that hfuf outperformed gwf in terms of recovering efficiencies of both hadvs and hpyvs. hfuf method involved concentrating viruses from l river water to obtain a manageable volume ( - ml) of sample. consequently, the ph of the concentrated sample was adjusted to . (below the isoelectric point of the viruses). the concentrated samples were further passed through negatively charged ha membranes which adsorb the positively charged viruses present in the sample. hfuf has been used for concentrating hpyvs, human bacteroides hf , and methanobrevibacter smithii from l of environmental water samples seeded with or . ml raw human wastewater (leskinen et al. ) . leskinen et al. ( ) extracted dna directly from the -mm membranes using a powersoil® dna isolation kit. one limitation of using smaller diameter membranes is that they may get clogged depending on the turbidity of the water. as a result, multiple membranes may be required which may increase the sample processing time and may reduce recovery efficiency. in addition, dna extraction using a powersoil® dna isolation kit does not utilize all supernatant which may also influence the recovery efficiency in the downstream application. in view of these, we processed river water samples through . -μm, -mm diameter negatively charged membranes. the larger diameter membranes provide larger net area ( . times more than -mm membranes) which allow to process relatively large volume of concentrated water samples ( - ml). for dna extraction, we used powermax® soil dna isolation kit which can easily accommodate -mm diameter membrane. unlike powersoil® dna isolation kit, powermax® soil kit utilizes all supernatant and because of that better recovery is expected . a limitation of such approach can be the presence of potential pcr inhibitors on the membranes. however, the powermax® soil kit is equipped with inhibitor removal technology which potentially removes % humic substances and other inhibitors as specified in the manual. this was supported by the sketa real-time pcr assay which indicated the absence of pcr inhibitors in river water samples seeded with human wastewater obtained through both methods. it should be noted that -ml dna sample was obtained using the powermax® soil kit which is - times higher than powersoil® dna isolation kit or other commercially available dna extraction kits such as qiagen stool kit. this may reduce sensitivity of the qpcr assay. however, to increase qpcr sensitivity, -ml dna can be reconcentrated further to a suitable volume (if required). between the two methods, hfuf had higher recovery than gwf for river water samples. these results were consistent for both viruses. the mean recovery efficiencies of % (hadvs) and % (hpyvs) of hfuf obtained in this study can be considered quite efficient for simultaneous detection or quantification of these two viral markers in environmental waters. our findings are in accordance with research by morales-morales et al. ; hill et al. ; and polaczyk et al. who also reported that hfuf can be effective for higher recovery ( - %) of viruses from various water matrices. in addition, hfuf is rapid and it does not require the preparation of extensive chemicals. the method also simultaneously retains bacteria, protozoa, and viruses in a single step which is an added advantage when analysis of multiple pathogens is required (kfir et al. ; oshima ; morales-morales et al. ) . gwf method used in this study was originally developed for detecting viruses and later it was used to detect agricultural zoonotic pathogens in large volume of drinking water, surface water, groundwater, and agricultural runoff (vilagines et al. ; millen et al. ) . this approach has been successfully used to provide information of the recovery efficiencies of bacteria and viruses in l of environmental water samples amended with variable amount of agricultural soil (abd-elmaksoud et al. ) . the recovery efficiencies of bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus type and type using gwf have been reported to range from to % (at turbidity . ntu), to % (turbidity ntu), and to % (turbidity ntu), respectively. however, the standard deviations for each water type and virus varied significantly. the mean recovery efficiencies of hadvs and hpyvs in this study for gwf ranged from . to . % for river water samples. our findings are in accordance with albinana-gimenez et al. b who also reported . % hadvs and . % hpyvs recovery through gwf method. francy et al. also reported . % recovery of hadvs from lake water samples. lower recovery efficiencies of other viruses such as h n influenza and feline calicivirus f through the gwf method have been reported (gassilloud et al. ; deboosere et al. ). caution should be exercised when comparing published studies on recovery efficiency of viral concentration methods as several factors such as adsorption of viruses to glass wool filters, glass wool filter type, seeding materials (raw human wastewater vs. intact plaques), environmental sample type (ground water vs. river water), sample volume ( l vs. l), and sensitivity of qpcr assays can influence recovery efficiencies albinana-gimenez et al. a; li et al. ) . the low recovery efficiencies of hadvs and hpyvs could be due to the fact that viruses adsorbed on the glass wool filters were not effectively eluated. this assumption needs to be tested by directly extracting dna from a segment of glass wool. the amount of glass wool packed into cam lever couplings may have affected the recovery efficiencies. for example, vilagines et al. used -g glass wool which resulted in % poliovirus recovery compared to menut et al. ( ) who used -g glass wool, which resulted in . % recovery. glass wool filters are currently packed by hand and not commercially available in a column format. this makes qa/qc difficult as packing may vary from person to person, resulting in large variability in results (cashdollar and wymer ) . in addition, viruses in the elution buffer also underwent an additional concentration step in a jumbosep before dna extraction. reconcentration of viruses is commonly used approach because the levels of viruses in environmental waters could be low. reconcentration methods have some disadvantages, such as these methods do not produce consistent recovery efficiency for different viruses (lewis and metcalf ) . in a previous study, centriprep filter concentrators provided high and stable recovery yields ( %) of polioviruses (haramoto et al. ) . another study reported the % recovery of adenovirus through centricon filters (wu et al. ) . to determine the recovery efficiency, we seeded raw human wastewater compared to the other studies that seeded cultured viruses obtained from plaque assays (hill et al. ; albinana-gimenez et al. b; millen et al. ; francy et al. ; abd-elmaksoud et al. ) . in real-world scenario, fecal pollution of environmental waters occurs via sewer/septic overflows and surface run off containing fecal matters from various animals. therefore, it is deemed necessary to obtain recovery efficiency of hadvs and hpyvs by seeding raw human wastewater that potentially contains naked (genetic materials from defective virions) and intact viral genomes than other studies which seeded intact viruses. from the mst context, there is no need to elute bintact^viruses since the objective is to determine whether the sample contains the signature of human fecal pollution. our data suggest that hfuf method provides better recovery for hadvs and hpyvs compared to gwf method used in this study. therefore, we recommended that hfuf method should be used to concentrate water samples for mst field studies. the advantage of hfuf method is that these filters are readily available for use in dialysis treatment of patients, and sample-processing time is relatively shorter than the gwf method. although, low turbid river water samples were tested in this study, the results may be applicable to stormwater, drinking water, and recreational water. simultaneous concentration of bovine viruses and agricultural zoonotic bacterial from water using sodocalcic glass wool presence of pathogens and indicator microbes at a non-point source subtropical recreational marine beach evaluation of bovine feces-associated microbial source tracking markers and their correlations with fecal indicators and zoonotic pathogens in a comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters distributions of fecal markers in wastewater from varying climatic zones for human fecal pollution tracking in australian surface waters 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utilising the same concentration techniques for the detection of protozoan parasites and viruses in water concentration of enteroviruses, adenoviruses, and noroviruses from drinking water by use of glass wool filters hollow-fiber ultrafiltration and pcr detection of human-associated genetic markers from various types of surface waters in florida removal of viruses in sewage treatment: assessment of feasibility concentration of viruses from environmental waters using nanoalumina fiber filters environmental microbiology quantification of human polyomaviruses jc virus and bk virus by taqman quantitative pcr and comparison to other water quality indicators in water and fecal samples association of fecal indicator bacteria with human viruses and microbial source tracking markers at coastal beaches impacted by nonpoint source pollution poliovirus recovery from tap water after concentration over glass powder and glass wool glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens optimization of a reusable hollow fiber ultrafiltration for simultaneous concentration of enteric bacteria, protozoa, and viruses from water prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant efficient and predictable recovery of viruses and cryptosporidium parvum oocysts from water by ultrafiltration systems ultrafiltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from -l tap water prevalence and patterns of polyomavirus urinary excretion in immunocompetent adults and children detection and characterization of waterborne gastroenteritis viruses in urban sewage and sewage-polluted river waters in caracas application of human and animal viral microbial source tracking tools in fresh and marine waters from five different geographical areas prevalence of human pathogens and indicators in stormwater runoff in performance of two quantitative pcr methods for microbial source tracking of human sewage and implications for microbial risk assessment in recreational waters acute lower respiratory infections due to respiratory syncytial virus and adenovirus among hospitalized children from argentina glass wool for virus concentration at ambient water ph level a simple and novel method for recovering adenovirus in small volumes of source water acknowledgments this research was funded by southern water, tasmania. we thank dr. jatinder sidhu for providing technical expertise key: cord- -u rc f authors: ishaq, sadia; sadiq, rehan; farooq, shaukat; chhipi-shrestha, gyan; hewage, kasun title: investigating the public health risks of low impact development at residential, neighbourhood, and municipal levels date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: u rc f abstract low impact developments (lids) employ a series of vegetative techniques to retain rainfall close to the origin. although lids offer sustainable runoff management, these infrastructures can be considered a risk to public health due to the presence of pathogens in the runoff and human exposure to contaminated water held in and transported by lids. the objective of this study is to examine the disease burden from exposure to lids at the residential, neighbourhood, and municipal levels. the authors conducted a meta-analysis of literature on three water features: ( ) harvested rainwater obtained from lids, ( ) surface water, and ( ) floodwater. a set of studies were systematically selected to collect values of risks of infection and expressed as the disease burden, i.e. disability adjusted life years (dalys). the results showed that the percentage of gi illness exceeding the health guidelines were high for harvested rainwater, i.e. % of annual disease burden exceeded the who guidelines ( . dalys/ persons), and % exceeded the us epa guidelines ( . dalys/ bathers). among the six exposures for harvested rainwater, exposure to spray irrigation, exceeded us epa guidelines whereas; five exposures, i.e. flushing, hosing, daily shower, spray irrigation, and children playing, surpassed the who guidelines. considering lid treatment, the values of annual disease burden from all the selected barriers were below us epa guidelines however, these values exceeded the who guidelines for three barriers i.e. water plaza, grass swale, and open storage ponds. these findings provide a broader perspective of the disease burden associated with lids and emphasise to consider the type of exposures and required treatment barriers for developing lid infrastructures in urban areas. j o u r n a l p r e -p r o o f requires consideration and management of human exposure to runoff water and reducing public health risks. the presence of pathogenic microorganisms in runoff water has been well documented (albrechtsen h. j., ; sakamoto et al., ; selvakumar and borst, ; sidhu et al., ; van heijnsbergen et al., ) . these pathogens entering the water system belong to various sources such as animal waste, (un)treated sewage, and solid waste mixing with rainwater when it flows over the urban surfaces (arnone and walling, ; hofstra et al., ) . exposure to water-borne pathogens during domestic use of water and recreational activities can cause mild health problems (e.g. gastrointestinal, respiratory, skin, ear, and eye infection) or more severe conditions (e.g. hepatitis and meningitis) (white et al., ) . the occurrence of health problems incur economic burden in terms of lost productivity, visits to healthcare, hospitalization, sequelae, and mortality. for example, in the us, recreational water-borne illness is estimated to cost a substantial economic burden of approximately usd . billion/year (deflorio-barker et al., ) . since lids are meant to store and transport runoff water, the presence of pathogenic microbes can influence the risk of infection and investigations are needed to compare human exposure versus occurrence of disease and identify critical factors. a substantial amount of research shows the ability of water-borne pathogens to survive on leafy greens and enter the human body during consumption (dicaprio et al., ; esseili et al., ; ethelberg et al., ; hirneisen and kniel, ; warriner et al., ) . for example, zoonotic pathogens can persist and internalize leaves of lettuce or spinach foliage due to protection from solar irradiation and desiccation (markland, ) . similarly, leguminous tree and bryophytes are associated with a large number of facultative human pathogens, including staphylococcus, salmonella, and j o u r n a l p r e -p r o o f residential and neighbourhood levels. surface water includes urban tributaries, rivers, and lakes, which receive runoff and are mainly used for recreational purposes (lim et al., ) . floods include rainfallgenerated surface runoff, and/or from the flooded storm sewers, which contain a variety of microbial pathogens and pose risks to public health. the process of systematic review started with the identification of scientific literature on qmra modeling of runoff water flowing through lids, surface water, and floods. for this purpose, a systematic literature search was performed using four electronic databases including science direct, pubmed, engineering village, and google scholar. the combinations of keywords used in this search included the following: "runoff"/"rainwater"/"surface water"/"stormwater"/"floods" and "low impact development"/"green infrastructure"/"water sensitive urban design" and "quantitative microbial risk assessment"/"public health risk"/" disease burden". to identify additional records, initially identified articles were searched for forward/reverse citation. all original articles referenced in reviews were retrieved including qmra of water sensitive urban design (wsud) and green infrastructure. the initial identification of articles was followed by the removal of duplicate records. the screening of articles was performed to determine the eligibility of the articles as per inclusion-exclusion criteria (table s; supplementary document -s ). the titles and abstracts of identified articles were reviewed for the selection of relevant records and, full-text pdfs were obtained for those articles, formatted with adobe acrobat x pro® for text recognition. the selected articles were imported into mendeley library to further proceed with the data extraction and analysis. the key output of this systematic review was a collection of peer-reviewed literature on qmra modeling of runoff water flowing through j o u r n a l p r e -p r o o f from each of the selected studies, the data on risk of infection estimated via qmra modeling was extracted by a single reviewer. in the context of this research, the details of qmra steps are as follows. the health hazards are the water-borne pathogens i.e. virus, bacteria, and protozoa, present in urban runoff; lid infrastructures e.g. rain barrels, grass swales, biofilters; surface water; and floodwater (step ). during exposure assessment (step ), all processes of human-water interaction are quantitatively described through the estimation of concentration of pathogens, volume of water ingested/inhaled, and annual exposure frequency (ahmed et al., ) . once an episode of humanwater interaction occurs at any of the selected water features, water-borne pathogens transmit into human body via mouth, nose, and/or skin and may cause an infection. in the case of harvested rainwater at residential level, human-water interaction may occur when rainwater is reused during irrigation and non-potable activities, such as toilet flushing, showering, etc. regarding surface water, human-water interaction occurs via primary and/or secondary contact activities. primary contact activities indicate that the whole body is submerged in water (e.g. swimming, bathing, diving, water skiing, etc.) and secondary contact activities indicate that only limbs of an individual is exposed to water (e.g. boating, rowing, fishing, sailing, etc.) (dorevitch et al., ) . in dose-response assessment (step ), mathematical models specific to the type of pathogen are used to calculate the probability of infection (chhipi-shrestha et al., ) . this research covered the complete suite of water-borne pathogens responsible for gi infection and the relevant equations for estimating the probability of infection are beta-poisson (eq. ) and exponential (eq. ) dose-response models. lastly, risk characterisation (step ) involves the integration of exposure assessment and dose-response assessment for a specific pathogen and exposure route. typically, the risk of infection is measured for a single-event (p inf ), followed by the estimation of annual risk (p inf(a) ) over a period of one year by using an annual exposure frequency for the exposure activity under consideration (eq. campylobacter, and salmonella (haas et al., ) . exponential dose-response model used for adenovirus (vergara et al., ) , norovirus (schmidt, ) , and cryptosporidium and giardia (robertson et al., ) . where p inf represents probability of infection to an individual exposed to a single pathogen dose "d"; d is the pathogen dose; p inf(a) refers to annual probability of infection from "n" exposures/ year due to a single pathogen dose "d"; "α" and "r" are parameters referring to pathogen infectivity constant which characterise dose-response relationships; n is the median infective dose, i.e., the dose required to infect % of the exposed population. following data extraction, geometric mean was calculated to estimate central tendency if the data on risk of infection were available as a set of more than one value. in addition to the risk of infection values, the record of review included the details of location of the study, water feature, sampling site, pathogen prevalence, detection method, recovery efficiency, pathogen concentration, exposure assessment, and dose-response model. based on the estimation of annual risk of infection, the annual risk of illness (p ill ) and disease burden (daly) were calculated as follows (eq. and ): j o u r n a l p r e -p r o o f where p ill|inf refers to risk of disease given infection, i.e., morbidity. for the purpose of standardisation, the data were expressed as the burden of disease i.e. daly (eq. and ). daly is the estimation of the number of healthy years lost due to disease or early death of an individual; a disease with higher values of dalys indicates unhealthy impacts whereas lower values of dalys are beneficial for population health (murray and lopez, ) . in this research, the risk is expressed in units of dalys/ persons/year. this unit provides an estimate of the risk of illness or disease cases for a population of individuals in a year and offers a more accurate comparison of the disease cases and health targets. often, the incidence of infection has been considered as the assessment endpoint of risk but, it may lead to over or underestimation of health risk. to overcome this discrepancy, dalys measurement was used to represent the health risks from exposure to lids or related infrastructures. the conversion of risk of infection into dalys requires three conversion factors, i.e. annual exposure frequency, infection to illness ratio, and dalys loss per disease case (table s ; supplementary document-s ). the values of these conversion factors were obtained from the literature as given in supplementary document-s (table s , s and s , respectively). for the purpose of analysis, the health risks from exposure to surface water during primary (e.g. swimming, bathing) and secondary (e.g. rowing, sailing) recreations were assumed comparable to lids present in neighbourhoods and municipalities in terms of water quality, exposure type, and pathogen. the data were analyzed for the disease burden exceeding the health targets, most critical pathogens, water features, exposure activity, and age groups. two widely recognised health risk guidelines were used to examine the risk assessment outcomes, which includes ) the acceptable disability-adjusted life the current research presents an attempt of examining the public health risks associated with lids at three levels i.e. residential, neighbourhood, and municipal. at the residential level, the use of cisterns and rain tanks is a common practice to overcome water scarcity in many parts of the world such as australia, pakistan, and india. similarly, in urban communities, the development of grass swales, permeable pavement, and retention ponds are considered a sustainable solution to manage rainfall and runoff close to the site of origin thus reducing pollutant loading and stormwater quantity in the drainage system (fletcher et al., ) . the water management benefits of lids are well recognised, but the assessments of public health risks associated with these infrastructures are scarce. water-borne pathogens are an increasing public health concern and substantial research is available on quality management of drinking water and wastewater treatment systems. this study builds upon the previous work to determine whether or not the health risks from exposure to lids during recreational and nonpotable activities meet public health guidelines of the who and us epa. a summary of the results from a systematic review are given in tables and for ) harvested rainwater, ) surface water, and ) floodwater). the information on geographical location of the articles included in this review is shown in table . the data on risk of infection and burden of disease were collected from articles ( j o u r n a l p r e -p r o o f data were most abundant for cryptosporidium (n= ), followed by giardia (n= ), and norovirus (n= ). regarding floodwater, the data were obtained from five articles and, qmra analysis was mostly performed for cryptosporidium and campylobacter (n= for each of these pathogens), followed by giardia (n= ). table here>> << please insert table here>> the comparison of cumulative probability of exceedance and the annual burden of disease from exposure to the selected water features is shown in fig. . in general, the percentage of diarrheal illness exceeding the two guidelines was highest for floodwater exposures, followed by recreational exposure in surface water, and non-potable exposures to harvested rainwater. in the case of harvested rainwater, almost % of annualised disease burden exceeded the who ( . dalys/ persons), and approximately % exceeded the us epa guidelines ( . dalys/ bathers). the results of surface water revealed that the percentage of exceedance for recreational activities in surface water was considerably high, surpassing the who guidelines up to % of the time and us epa guidelines up to . % of the time. considering floodwater, the probability of exceeding the two health guidelines was significantly high, and approximately % of the dataset exceeded the who guidelines and . % exceeded the us epa guidelines. these findings support the fact that microbial contamination is more pronounced in floodwater and so is the likelihood of gi illness during wet weather, thus the burden of gi illness or diarrheal problems needs to be considered while developing lids in urban settings. previous studies have confirmed that microbial contamination of floodwater far exceeded the acceptable levels as given in guidelines pertaining to microbial quality of recreational water (xiao et al., ) whereas, the harvested rainwater was reported to exhibit a lower level of microbial contamination that lies below the health guidelines (ahmed et al., ) . floodwater impacted by j o u r n a l p r e -p r o o f animal waste and human sewage exhibited the highest likelihood of gi illness during withdrawal and the clean-up of contaminated water (fewtrell et al., ) . similarly, surface water bodies were reported to receive a high concentration of water-borne pathogens from (un)treated sewage, defecation from animals, sewer overflows which could lead to the widespread occurrence of gi illness during recreational activities (ahmed et al., ; gannon and busse, ; lechevallier et al., ; noble et al., ; rajal et al., ) . in contrast, harvested rainwater was recorded to be less polluted as the sources of microbial pollution included bird/insects droppings, leaves in the gutter, and condition of the roof, piping, and storage (brodie et al., ; evans et al., ; huston et al., ; lye, ). the analysis of data of floodwater and surface water presented a worst-case scenario of the disease burden of gi illness, and harvested rainwater depicted the situation of least contamination of runoff water leading to less burden of gi illness. it is important to note that these results do not provide an exact measure of the burden of gi illness from lids. however, broadly, one can infer that the development of lids may negatively affect public health in urban neighborhoods. this study covered the complete suite of pathogens that were examined in the selected literature and can cause gi illness and legionellosis. figure summarises the total annual burden disease of water-borne pathogens from exposure to the selected water features. from exposure to harvested rainwater, the total annual burden of disease, the sum of annual disease burden from each pathogen, was . dalys/ persons. the pathogen with the highest burden was adenovirus, with an estimated value of . dalys/ persons/year, followed by campylobacter, and rotavirus. these three top-ranking pathogens accounted for % of the disease burden when harvested rainwater was used for non-potable activities (fig. a) . j o u r n a l p r e -p r o o f considering surface water, the total annual burden of water-borne pathogens from exposure to this water feature was . dalys/ persons. the pathogen with the highest burden was giardia, with an estimated annual value of . dalys/ persons, followed by adenovirus, and cryptosporidium. these three top-ranking pathogens accounted for . % of gi illnesses when the population was exposed to surface water through primary or secondary contact recreational activities (fig. b ). regarding floodwater, the total annual disease burden from water-borne pathogens was alarmingly high, i.e., . dalys/ persons. the pathogen with the highest burden was virus, having an estimated value of . dalys/ persons, followed by cryptosporidium, and campylobacter. these three top-ranking pathogens accounted for . % of the disease burden of water-borne diseases when the population was exposed to urban floods during clean-up and withdrawal of contaminated water (fig. c) . overall, it is found that the total annual burden of disease for water-borne pathogens vary greatly across the three water features. another important finding was that the total annual disease burden was highest for the viral pathogens, followed by protozoal and bacterial pathogens (fig. ) . these << please insert fig. here>> the comparison of disease burden at individual and population level is given in fig. . for the pathogens examined within the selected water features, rotavirus stood out for having a high impact on individual health, whereas, two bacterial pathogens, campylobacter and salmonella, were identified to have a high impact on population health. overall, viruses make a large contribution to the disease burden at the individual and population level in the selected water features. for prevention-orientated management of water-borne diseases from these water features, intervention strategies must focus to manage risks from viruses, cryptosporidium, and giardia. for harvested rainwater at the residential level, the results of annual disease burden in relation to exposure activity and pathogens are presented in figure . at this level, the analysis of six activities showed that the disease burden from all the exposures were below us epa guidelines ( considering lid treatment train in the neighborhood, the value of annual disease burden was below the us epa guidelines for the selected treatment barriers but, it exceeded the who guideline for three lid barriers i.e. water plaza, grass swale, and open storage ponds (fig. ) (an analysis of annual disease burden in relation to lid treatment train, water-borne pathogens, and exposure activity is given in fig. s; supplementary document s ). these treatment lid barriers provide desirable habitat to birds and animals, which leads to contamination of water and infects the local population (ahmed et al., ; petterson et al., ) . on the other hand, the rainwater from biofilters met the who guidelines for use in toilets, showering, and crop irrigation (fig. ) . toilet flushing using non-potable water is a common practice in many parts of the world. generally, it involves aerosolisation of human waste and vomitus and limited human-water interaction makes the use of harvested rainwater safe in toilet flushing. similarly, the disease burden of cold shower was far below the acceptable level of who guidelines. but, the social acceptance of using harvested rainwater for showering is argued due to close contact of water with the human body. since rainwater flowing in neighbourhoods may contain a high concentration of zoonotic fecal and human fecal pathogens, appropriate treatment of water depending on the level of contamination is recommended before using for showering or other activities that involve close human-water interaction. fig. here>> j o u r n a l p r e -p r o o f i.e. playing in water, jump, and fall ( fig. ) (an analysis of surface water annual disease burden in relation to water-borne pathogens and exposure activity is given in fig. s; supplementary document s ). these high values of disease burden can be attributed to the level of contamination, volume of ingested water during these contacts, exposure duration, and exposure frequency. considering floodwater, the disease burden from one exposure activity, residents' withdrawal of floodwater, surpassed us epa guidelines. the median values of disease burden from water splash were below who guideline whereas, these values for three exposures were above this guideline and included floods clean-ups, splash and play, and playing in floodwater ( fig. ) (an analysis of flood-based annual disease burden from flood water in relation to water-borne pathogens and exposure activity is given in fig. s; supplementary document s ). these findings were similar to the previously observed trends in surface water and can be associated with the high microbial concentration of floodwater and longer exposure duration of these activities. overall, the burden of gi illness in relation to age groups and three water features has shown unevenness. the inconsistency of results for the surface water and floodwater can be attributed to the lack of data as number of data points for children was less than adults in these two water features. considering adult population, the likelihood of developing sequelae or death is higher at old ages (dumic et al., ) . also, the disease burden of immune-compromised population cannot be neglected. further research on the role of lids in developing gi infection among these groups would be beneficial for appropriate stormwater management. fig. here>> this research aimed to analyze the burden of respiratory illness from legionella. there was a lack of information and data on the risk of legionellosis from exposure to selected water features. only a few data points were available from previous studies on harvested rainwater, and this discrepancy limited scientific understanding of legionellosis from exposure to lids and related infrastructures. from the available literature, the risk of legionellosis was reported from three exposure activities i.e. daily showering, hose irrigation, and children play in water plaza ( fig. and ). these three activities are related to the generation of aerosols and inhalation of contaminated droplets that may cause respiratory infection. overall, the burden of disease from legionella seems to be low but the unavailability of dataset impedes an in-depth understanding of the burden of legionellosis. more research is necessary to draw any conclusion about the burden of legionellosis for the population exposed to lids in urban neighbourhoods. journal pre-proof j o u r n a l p r e -p r o o f the use of lids is becoming increasingly popular in urban stormwater management. considering the water management benefits of lids, in many developed countries, stormwater management guidelines urge the development of lids for quantity and quality management of urban runoff. for example, the stormwater planning guidelines of british columbia, canada, state that total runoff volume should be % of rainfall and % must be returned to the natural hydrological cycle through percolation, evapotranspiration, and reuse (finnie et al., ) . this means permeable cover should be % of the total watershed area. the availability of large amount of green spaces in urban settings combined with increased water retention would possibly offer the most suitable habitat to water-borne pathogens, thus increasing the risk of gi infection and subsequent disease burden. in rotterdam, the netherlands, sales-ortells & medema ( ) reported the campylobacter disease burden of . dalys pppy (i.e. . pppy) for kids playing in water plaza (an infrastructure that combines the storage of runoff with water recreational activities). in another study, nguyen et al., ( ) reported the campylobacter disease burden of . dalys pppy for children playing in floodwater. based on these estimates, the authors assumed that during the rainy season, the disease burden of gi illness from exposure to a lid treatment train at neighbour level (i.e. green play areas, grass swales, interactive fountains, retention ponds) for all pathogens could be approximately % of the disease burden from floods. considering this assumption, figure shows the annual disease burden of gi illness for children from exposure to neighbor level lids treatment train and expected probability of exceedance for gi illness against who and us epa guidelines. the plot revealed that the percentage of exceedance was considerably high, i.e. . %, for who guidelines whereas, us epa guidelines was met all the time. therefore, it can be deduced that the development of lids in urban communities is associated with the likelihood of gi infections and people exposed to these infrastructures may suffer water-borne gi illness. this research is the first attempt to estimate the disease burden of gi illness and legionellosis from exposure to lids at the residential, neighbourhood, and municipal levels. the authors adopted a meta-analysis methodology and estimated the public health risks of these infrastructures. the review of literature showed a dearth of qmra modeling for lids at neighbourhood and municipal levels, which was overcome by including surface water and floods from rains in the review process. however, understanding the risk of infection associated with lids at these two levels is necessary to obtain insights into the disease burden experienced by the exposed population. this study establishes the conceptual foundation of the effects of lids on public health and presents a wider perspective of the disease burden from exposure to lids. the results revealed that the probability of gi illness exceeding the who and us epa guidelines was highest for floodwater, followed by recreation in surface water, and non-potable exposures to harvested rainwater. among the various microbes, the most critical pathogens of concern were adenovirus, cryptosporidium, campylobacter, and giardia. considering exposure activity, the most critical exposures of concern were hosing and spray irrigation from harvested rainwater at residential and neighbourhood levels. considering lid treatment train in the neighbourhood, the estimated disease burden met the us epa guideline for the selected treatment barriers, but it exceeded the who guideline for three lid treatment alternatives, i.e. water plaza, grass swale, and open storage ponds. within the different population groups, it was found that children could ingest a larger amount of contaminated water and suffer more severe illness from exposure to harvested rainwater. moreover, the likelihood of developing sequelae or death is also an important concern, particularly in elderly and immune-compromised individuals. when designing j o u r n a l p r e -p r o o f future lid in the urban neighborhood, it is recommended to consider the type of exposure activity and lid treatment train. in future work, this research can be extended to assess the safety of these infrastructures in urban areas using modeling tools. in the long-term, an assessment of the disease burden from exposure to lids under the conditions of climate change is important to identify best practices that help to reduce negative health impacts. respectively. the horizontal lines identify the exceedance rate for these two guidelines. legend'a' describes exposure activities and legend'b' describes lid treatments. evidence of septic system failure determined by a bacterial biochemical fingerprinting method health risk from the use of roof-harvested rainwater in southeast queensland, australia, as potable or nonpotable water, determined using quantitative microbial risk assessment † microbiological investigations of rainwater and graywater collected for toilet flushing waterborne pathogens in urban watersheds urban aerosols harbor diverse and dynamic bacterial populations microbial quality of reclaimed water for urban reuses: probabilistic risk-based investigation and recommendations quantitative assessment of infection risk from exposure to waterborne pathogens in urban floodwater estimate of incidence and cost of recreational waterborne illness on united states surface waters internalization and dissemination of human norovirus and animal caliciviruses in hydroponically grown romaine lettuce health risks of limited-contact water recreation gastrointestinal tract disorders in older age comparative study of enteric viruses, coliphages and indicator bacteria for evaluating water quality in a tropical high-altitude system binding of human gii. norovirus virus-like particles to carbohydrates of outbreaks of gastroenteritis linked to lettuce wind, rain and bacteria: the effect of weather on the microbial composition of roof-harvested rainwater the microbiology of urban uk floodwaters and a quantitative microbial risk assessment of flooding and gastrointestinal illness stormwater planning: a guidebook for british columbia suds, lid, bmps, wsud and more -the evolution and application of terminology surrounding urban drainage health risk implications from simultaneous exposure to multiple environmental contaminants disease burden of selected gastrointestinal pathogens in australia progress and data gaps in quantitative microbial risk assessment quantitative microbial risk assessment an outbreak of legionnaires disease associated with a decorative water wall fountain in a hospital canadian guidelines for domestic reclaimed water for use in toilet and urinal flushing human pathogens on plants norovirus surrogate survival on spinach during preharvest growth quantifying the impact of climate change on enteric waterborne pathogen concentrations in surface water this review comes from a themed issue on aquatic and marine systems edited by characterisation of atmospheric deposition as a source of contaminants in urban rainwater tanks state of provincial regulations and guidelines to promote low impact development (lid) alternatives across canada: content analysis and comparative assessment microbial water quality in the upper olifants river catchment: implications for health effect of rainfall on giardia and cryptosporidium assessment of public health risk associated with viral contamination in harvested urban stormwater for domestic applications rooftop runoff as a source of contamination: a review a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply investigating behaviors of zoonotic and human pathogens on plants molecular detection and characterization of gastroenteritis viruses occurring naturally in the stream waters of manaus the global burden of disease. a comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in and projected to microbial risk assessment of tidal−induced urban flooding in can tho city (mekong delta, vietnam) enteroviruses detected by reverse transcriptase polymerase chain reaction from the coastal waters of santa monica bay, california: low correlation to bacterial indicator levels multitiered approach using quantitative pcr to track sources of fecal pollution affecting santa monica bay new solutions for sustainable stormwater management in canada the bryophyte genus sphagnum is a reservoir for powerful and extraordinary antagonists and potentially facultative human pathogens drainage and stormwater management strategies for low-income urban communities. incidence of cryptosporidiosis at the community level in ontario integrated storm-water management for watershed sustainability detection of enteric viruses and bacterial indicators in german environmental waters molecular quantitative analysis of human viruses in california stormwater the potential for acquiring cryptosporidiosis or giardiosis from consumption of mung bean sprouts in norway: a preliminary step-wise risk assessment health impact assessment of new urban water concepts microbial health risks associated with exposure to stormwater in a water plaza norovirus dose-response: are currently available data informative enough to assessing pathogen risk to swimmers at non-sewage impacted recreational beaches variation of microorganism concentrations in urban stormwater runoff with land use and seasons rehabilitation of aging water infrastructure systems: key challenges and issues prevalence of human pathogens and indicators in stormwater viruses in recreational water-borne disease outbreaks: a review survival of infectious poliovirus- in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and poliovirus- genome viable legionella pneumophila bacteria in natural soil and rainwater puddles risk assessment of noroviruses and human adenoviruses in recreational surface waters energy and environmental sustainability solutions for megacities-e s view project risk assessment of noroviruses and human adenoviruses in recreational surface waters diverse endophytic bacteria isolated from a leguminous tree conzattia multiflora grown in mexico internalization of human pathogens within growing salad vegetables blue space: the importance of water for preference, affect, and restorativeness ratings of natural and built scenes occurrence and potential health risk of cryptosporidium and giardia in the three gorges reservoir survival of salmonella enterica serovar newport in manure and manure-amended soils evaluating the effects of low impact development practices on urban flooding under different rainfall intensities the authors would like to thank natural sciences and engineering research council of canada for supporting this research. thanks are extended to ms. anber rana for assistance with the charts. the authors would also like to thank the editors and anonymous reviewers for their helpful and constructive comments that greatly contributed to improving the quality of this content analysis. key: cord- -n t by r authors: maurya, anurag; singh, manoj kumar; kumar, sushil title: biofiltration technique for removal of waterborne pathogens date: - - journal: waterborne pathogens doi: . /b - - - - . - sha: doc_id: cord_uid: n t by r pathogenic agents such as protozoa, bacteria, virus, and helminths disseminate among human community via ingestion of contaminated water and pose a serious concern to public health. biological filtration method of disinfection is the key step in centralized multistep and decentralized point-of-use water treatment system. a biofilm of indigenous microorganisms fixed on solid surface, called biofilter, is an eco-friendly and economic technique for removal of pollutants and pathogens. stone, gravel, sand, soil, compost, mesoporous mineral stone, wood chips, etc., exemplify solid bed medium which provide enhanced surface area for microbial adherence and water retention. slow sand filter (ssf) characterized by slow flow rate ( . – . m( )/h) and a biological layer over filter bed called schmutzdecke is fairly efficient in removal of coliform microorganisms such as giardia, cryptosporidium, salmonella, escherichia coli, total coliform and fecal coliform, fecal streptococci, bacteriophage, and ms virus from wastewater. rapid sand filter characterized by coarser sand and higher flow rate than ssf removes indicator microorganisms efficiently. stormwater biofilter is popular and effective method for removal of pathogen and involves biological activity and physical retention of pathogen on filter media. microbial diversity, inoculum size, ph and moisture of medium, temperature, and nutrient content are key parameters governing optimum performance of biofilter system. therefore, biofiltration can be an efficient bioremediation technology for pathogen removal. water is an important natural resource utilized for domestic, industrial, recreational, and agricultural purpose by human society. utility of water is negatively affected by contamination of various pollutants. pollutants are physical, chemical, and biological in nature and they deteriorate respective qualities of water after contamination. physical properties of water includes electrical conductivity (ec), total dissolved solids, and suspended solids. chemical properties are given by composition of various minerals, carbon content, dissolved oxygen, nitrogen and phosphorus. biological property refers to presence of various types of microbes and pathogens specially viruses, bacteria, algae, protozoan, nematodes, insects, and their propagules. based on the source of pollution, wastewater is broadly classified as stormwater runoff, agricultural runoff, industrial wastewater, and domestic wastewater. stormwater is a kind of raw water formed by natural contamination of pollutants in rain catchment areas like agricultural field, pond, and forest, etc. other examples of raw water are groundwater abstracted through borehole, rivers, natural and man-made lakes, and reservoirs (scholz, ) . the raw water can be supplied for potable use after simple filtration steps and disinfection. after domestic or industrial usage, water is discharged in sewage system. this water, burdened with pollutants and pathogenic microbes, is called as wastewater. domestic wastewater is categorized as greywater and blackwater. former is generated from kitchen, laundry, and washrooms, while latter includes human excreta, i.e., feces and urine discharged from toilets. well-engineered wastewater treatment plant and zero-energy constructed wetlands are employed to treat domestic wastewater so water can be discharged back to waterbodies (lewandowski and boltz, ) . waterborne disease refers to outbreaks or cases of disease of which pathogenic agents spread through ingestion of water. human excreta contains wide variety of pathogenic agents including protozoa, bacteria, and viruses which can contaminate water source. ingestion of water polluted with pathogen causes infection. infection can be categorized as symptomatic (with clinically observable syndrome) and asymptomatic (without clinical manifestation). disease is a symptomatic infection in which cause agent is known. asymptomatic infection is also a serious concern for public health. many bacteria and viruses that cause asymptomatic infections may spread directly in person via food or water and may result in an epidemic condition (table . ) (riley et al., ; bridle, ; leclerc et al., ) . diverse kinds of pathogens are impractical to monitor for assessment of a wastewater treatment system. therefore, suitable indicator organism which may be pathogenic or nonpathogenic is selected for assessment of pathogen contamination level. one widely used indicator organism is coliform bacteria, quantified either as total coliforms or fecal coliforms. fecal coliform indicates the contamination of fresh fecal matter. possible indicators for protozoa used in the various studies include aerobic and anaerobic spores and particle size distribution. similarly, helminths' ovum is implicated as indicator for monitoring helminths abundance in wastewater. t coliphage virus is used as indicator for viruses as it is similar to adenoviruses, reoviruses, rotaviruses, and coronaviruses. most common indicator organism for pathogen removal is coliform. the coliforms designate the genera escherichia, citrobacter, klebsiella, and enterobacter belonging to family enterobacteriaceae. ec (escherichia coli) is considered as a more specific indicator organism because several of coliforms are abundant in unpolluted soils and water and their presence cannot specify fecal pollution. e. coli is considered the most sensitive, reliable, and specific indicator of fecal pollution because it is nearly exclusive to fecal microflora and constitutes more than % of the coliform flora of the human gut. because of huge number of pathogenic microorganisms, their removal is expressed in terms of log removal value (lrv). lrv can be defined as follows: log removal ¼ Àlog inflow concentration outflow concentration thus, -log removal designates % reduction in microorganisms and log removal is a % reduction and so on. in some reports, turbidity is also taken as a crude parameter for pathogen removal because suspended microorganisms behave as colloidal particles. however turbidity includes concentration of suspended solids along with microbes, cysts, and ova. biofiltration has been proved an essential constituent of treatment process for air, wastewater, and raw water. major sources of potable water in megacities are treated surface water. raw water is not suitable for human consumption because of unwanted micropollutants, pathogenic microorganisms, organic matter, and other growth-supporting nutrients present in it. storage raw water may lead to biofouling, adverse taste and odor development, and pathogen growth. in multistep treatment process of centralized wastewater treatment plant, biofiltration constitutes one of the steps primarily for removal pollutant and in part for removal of pathogen. biofilter is applied as sole process to make raw water potable in remote areas. trickling filter or granular activated carbon (gac) or sand filter is a common form of biofilter in wastewater treatment plant; gac and sand filter are applied for treatment of raw water; rain garden and soil filter are examples of low-impact development filters for management of stormwater; horizontal rock filters are applied to mitigate polluted stream (hammes et al., ; lewandowski and boltz, ) . a biofilter can be defined as any type of filter media with attached microbial community on surface in the form of biofilm performing at least one essential functions of treatment process. however, some studies referred filter media as biofilter without confirming role of indigenous microorganisms or biofilm in removal of pollutants and pathogens. biofilter was introduced in wastewater treatment plant as trickling filter where filter media was slag or rock. nowadays, various kinds of filter media such as rock stones, gravel, sand, gac, or synthetic plastic media, etc., have been successfully applied for treatment of domestic and industrial wastewater. necessity of applying biofilter for treatment of drinking water arose after the discovery of microbial regrowth in supply pipelines. though microbes in drinking water pipelines were not found pathogenic but their presence makes water biologically not stable and unfit for drinking purpose. cause of microbial regrowth in drinking water supply was due to pertinent micropollutants such as biodegradable organic matter (bom), nh þ , fe þ , mn þ , no À , dissolved h , reduced form of sulfur by-products and disinfection by-products, etc. these pollutants act as nutrient for microbial growth. primary role of biofilter is to remove bom, secondary is to reduce pathogenic load. removal of pollutants on biofilter impairs microbial growth. organisms present in biofilm of filter absorb and utilize most organics to fulfill their nutrition and energy requirements (lewandowski and boltz, ) . ancient egyptians dug along the nile to get filtered water for drinking purpose. river sand acted as filter. john gibb (scotland, ) and james simpson (england, ) prepared first industrial sand filter for drinking water treatment. the system could effectively reduce turbidity and contaminant of cholera in drinking water; however, microbial nature of filter was not known. current knowledge of indigenous microorganism in drinking water treatment arose years back by several studies done on presence and impact of benign microbial population on filters. first trickling filter was installed in in madison, wisconsin, for large water treatment system. understanding the microbial community of filter and their role in water treatment is a continuing process, assisted with advent of advance tools for the analysis of microbial diversity and increased understanding of microbial growth in oligotrophic condition and the consideration of potable water as a complex ecosystem (bureau of safe drinking water, ; daigger and boltz, ; lewandowski and boltz, of safe drinking water). slow sand filtration is a low-cost and simple-to-operate technique for removal of chemical contaminants and pathogens. essentially an ssf is composed of following vertically arranged layers of components. topmost layer is the supernatant water which is subjected for filtration. water column provides enough hydrostatic pressure for its percolation through filtration system. second is a thick layer of actual filtration medium that is of fine sand (effective size . e . mm). it is a low-cost durable medium for filtration. because of its smaller particle size ( . e . mm) fine sand provides large surface area for filtration as well as for the formation of biofilm, however its small voids size decreases flow rate ( . e . m/h) through ssf. topmost portion of sand is enriched with microbial growth because of better availability of oxygen as compared to lower portion. most of the decontamination occurs in this active biological layer, also called as schmutzdecke. microbes form of biofilm on inert sand particles and aid the biofiltration process. bottom to sand is a layer of gravel which provides free passage to treated water to exit the bed. gravel supports sand bed and prevents exit hose pipe from clogging. usually the four layers make up m thick column of the biofilter. slow filtration rate of ssf allows longer retention time for supernatant water and water percolating through bed which allows ample filtration and biological activity. biological activity at the top layer results in schmutzdecke, which extends cm above the bed as a slimy matrix. schmutzdecke consists of mineral precipitates and colonized microorganisms comprising bacteria, fungi, protozoa, and even some larger eukaryotes. intermittently scrapping of schmutzdecke is essential because its overgrowth blocks percolation process. a gap period of ripening of schmutzdecke follows the scrapping to achieve a fully active ssf. a well-developed schmutzdecke is necessary for pathogen removal. schmutzdecke is primary contributor to the filtration process while secondary contributors are microbes attached to sand particle in deeper part of ssf (verma et al., ) . ssf efficiently removes various waterborne pathogens including viruses, bacteria, and protozoan cysts of giardia and cryptosporidium enteroparasites. removal of pathogenic bacteria had been up to %e . %. however, efficiency is site-specific depending on operating parameters such as temperature, filtration rate, particle size of medium, and bed depth. log removal for viral population ranges from to . removal efficiency enhances with bed depth but reduces with temperature and rate of filtration. removal of enteric bacteriophages ms- and ped-i was comparatively lesser as they absorb poorly to sand surface. parasites such as giardia and cryptosporidium were removed efficiently ( . %) in ssf (bauer et al., ; lewandowski and boltz, ) . sand bed retains and inactivates microbes from seeping raw water. both physical and biological mechanisms are involved in retention and inactivation of microbial retention on sand bed. physical phenomenon and interactions are involved in straining and adsorption. size of bacteria ( . e mm) and viruses ( . e . mm) is much smaller than pore size of media, hence straining is ineffective on top. organic matrix of schmutzdecke and exopolysaccharides (eps) secreted indigenous bacteria makes adsorption of pathogen effective. deeper in the bed, other mechanisms of transport (inertia, impaction, sedimentation, interception, hydrodynamic action, and diffusion) become effective. pathogens carried to the particle are retained on filter media (hammes et al., ) . biological mechanisms contribute significantly to the removal of pathogens because of slow flow rate. this mechanism is dominant in upper layer of ssf. slow percolation facilitates enough time for interaction of pathogens with biofilm present on sand particle. predation is the main activity in mature biofilm responsible for removal and inactivation. for example, bacteria removal in ssf has been attributed to grazing by protozoa and worms. grazing of adsorbed bacteria in biofilm frees the site for further retention of incoming bacteria. e. coli population in the treated water was found to be negatively correlated with the diversity and size flagellate and ciliate populations in the filter which signifies the importance of protozoa in bacteria removal. meiofaunal species ( . e mm in size) also predates on individual bacterial or algal cells, suspended particles, or other species. adsorption on sticky biofilm assists the removal of biological particles in deeper region. moreover, natural death, inactivation, and metabolic breakdown (i.e., reduction of organic carbon) also predominate in lower part (guchi, ) . natural form of bacterial inhabitant in ssf is oligotroph. they thrive on wide variety of organic substrates present in low concentration. dense growth of oligotrophic bacteria grown in biofilm is sometimes referred to as zooglea. zooglea is created and stabilized by sticky secretions from bacteria that are extracellular polymeric substances (eps), polysaccharides, and proteins. this layer improves adsorption capacity of filter media. viral particles were demonstrated to be better removal in presence of zooglea. secreted polymers are proposed to flocculate organisms and destabilize clay and bacteria to facilitate attachment (guchi, ) . some natural inhabitant bacteria called "autochthonous" bacteria outcompete pathogenic bacteria in oligotrophic condition. moreover, they may secrete toxic chemicals against pathogenic microbes. the phenomenon is called as "bioantagonism." for example, survival of cryptosporidium declined in the presence of autochthonous microorganism. antagonistic mechanism is also hypothesized to be responsible for oocyst decay in ssf by autochthonous bacteria (guchi, ) . indigenous microorganisms reported in the sand bed are aerobic bacteria, flagellates, ciliates, rotifers, flatworms (microturbellaria), gastrotriches, nematode (round worms), annelids, and arthropods (harpacticids), as well as algae, protozoa, and higher order eukaryotes. however, microbial flora is dominated by gram-negative pigmented bacteria such as pseudomonas and aeromonas. each layer of the sand bed has its own inactivation potential depending on the vertical distribution of biomass. for example, prokaryotes and eukaryotes were active throughout the filter bed in inactivating enteric microorganisms (e. coli); however, inactivation potential was highest near the surface of filter bed. series operation of ssf enhances pathogen removal efficiency. also, air saturation of water aids the pathogen removal. use of lava rock, silica sand, and gac instead of sand enhanced pathogen removal in greywater. continuous mode of operation was better for removal of escherichia coli and ms than intermittent mode of operation. few studies on ssf are reported to remove virus also. it was able to remove -log to -log of echovirus- and e . -log of bacteriophage, . -log/h of ms , and . -log/h of prd- . less than h of operation time was found ideal time for removal of infectious viruses. ssf was able to do . -log removal for phages, fecal bacteria, and enteric adenoviruses also. pathogen removal efficiency of ssf reported in various studies is mentioned in table . (bauer et al., ) . all investigated filter configurations achieved substantial mean removal for e. coli ( . log), enterococci ( . log), clostridium perfringens spores ( . log), coliphages ( . log), and aerobic bacterial load (heterotrophic plate counts, hpc) ( . log) in secondary effluent (seeger et al., ) . hpc is a broad indicator of bacterial load in water. sometimes it is applied to assess general efficiency of water treatment systems. rapid sand filter (rsf) evolved at end of th century in the united states of america. it became popular in s because it required lesser necessary facilities with respect to ssf. unlike slow sand filters, rsf involves only physical process because of absence of biological layer (biofilm) on filter media. coarse-grained sand and gravels efficiently remove suspended solid by straining and adsorption. rsf must be aided with pretreatment (sedimentation and flocculation) and posttreatment (disinfection) steps to remove pathogens and prevent fouling. it requires lesser area for construction as compared to ssf for treatment of unit volume of water. rsf is constructed in a rectangular tank usually made up of concrete. three to five layers of graded gravel are installed at the bottom of tank over a network of drainage pipes placed on the floor. filter media that is coarse sand with a diameter ranging from . to . mm is filled over gravel layer. as coarse sand provides larger void as compared with fine sand of ssf, rsf achieves a higher rate of filtration. gravel layer prevents sand from being drained out during filtration. also, it facilitates even distribution of water through filtration media during backwash. top of the rsf is either open for supernatant water (gravity filter) or closed (pressure filter) (o'connor and o'connor, ) . rsf is not as good as ssf for pathogen removal because pore size of medium is larger and it lacks biofilm. however, rsf removes suspended solid along with biological particles. prominent biological particles retained by rsf include algal microcolonies ( e mm), protozoan cysts ( e mm), bacterial cells ( . e mm), and virus particles ( . e . mm). rose ( ) reported removal of giardia and cryptosporidium. the deposition of microorganisms and other particles in filters depends on transportation efficiency and retention in surface pore of filter media. theoretical model for collection of microorganism on anthracite and sand media suggested lowest removal of individual bacterial cells in comparison to free suspended viruses, protozoa, or microbial aggregates and other particulates. in fact, removal of nanoscale particles such as viruses is governed by diffusion while protozoans are removed by cumulative effect of sedimentation and interception. removal mechanism for suspended bacterial cells involves diffusion, differential sedimentation, and interception. effective grain size is an important factor of collection of viruses and bacteria on media surface, whereas removal of protozoa and microbial aggregates is chiefly influenced by hydraulic loading rates. therefore, the model suggests that smaller grain size media is major factor for removal of freely suspended viruses and other nanosized particles, and lower hydraulic loading rates would be improving removal efficacy for protozoan pathogens. other factors that were not included in the model such as net surface charge on the filter media and microbial surfaces; media properties (type, size, and depth); hydraulic loading rates; upstream chemical use (oxidants and/or coagulants); water quality variables; flow control; and backwashing and postbackwashing practices may also significantly influence pathogen removal efficiency of filter media. additional factors such as ph, ionic strength, temperature of effluent; concentration, molecular size, and charge density of dissolved organics; and particle characteristics influence removal efficiency. for example, high ionic strength reduces the electric double layer around microorganisms and filter media, thereby increasing attachment efficiency between the two. backwashing of filter media in rsf may release pathogen from rsf granules. pathogen removal in water treatment system was observed in many experimental studies. removal of giardia cysts and cryptosporidium oocysts was shown to be affected by extent of filter maturation and application of coagulant chemicals. treatment of coagulated primary effluent through rsf demonstrated approximately log unit decrease in fecal coliform, pathogenic bacteria (salmonella and pseudomonas aeruginosa) and enteroviruses, %e % of protozoan (giardia and entamoeba histolytica) cysts, and %e % of helminth ova (adelman et al., ; hoslett et al., ; jiménez et al., ). stormwater biofilter refers to bioretention system, bioinfiltration swale, and rain garden. it is trending as part of urban landscape because of its multifaceted implications such as filtration, groundwater recharge, evapotranspiration, mitigation of urban heat island, and esthetic value. the system is prepared by filling soil, sand, and gravel media in low-lying area of landscape and vegetated to slow down urban runoff for better percolation while reducing the contamination load in downstream receiving zone. usually bioretention systems are consist of five vertically arrenged zones. ponding zone or detention area constitutes the topmost layer where received water is kept before filtration. an overflow drain is installed to avoid flooding. ponding zone is followed by vegetation-rich biological zone performing pollutant uptake from water. biological zone rests over filter media (sand, sandy loam, or loamy sand) which facilitates high infiltration rate and pollutant removal capacity. zone of filter media is followed by a lower transition stratum of coarse sand and the lowest drainage layer of fine gravel. bottom of bioretention system can be pervious or impervious. besides aforesaid five layers, a biofilter system may also be configured with a submerged zone (sz) covering the basal portion. sz is also called internal water storage zone as it remains saturated with water between storms. sz can be prepared by connecting a pipe to the drainage exit and rising the pipe opening to the transition level so that water leaves the exit only when sz is fully saturated. level of transition can be moved up or down by maintaining the drainage exit at respective level. sz creates anaerobic microenvironment where denitrifying bacteria act to reduce nitrate content in water. however, this zone also affects removal of pathogenic bacteria (rippy, ) . a comparative metaanalysis was done to evaluate pathogen removal efficiency of stormwater biofilters with or without sz. studies performed on biofilters including designs with szs and designs without szs were compiled to compare log removal of e. coli (ec), fecal coliforms (fc), and enterococci (ent) in laboratory mesocosm and in field conditions. stormwater monitoring usually focuses these three groups of bacteria as indicator of pathogen load (table . ). fecal indicator bacteria (fib) includes klebsiella and escherichia genera. ec is a subgroup of fc that comprises both nonpathogenic and pathogenic members and ent include species of grampositive diplococcoid bacteria, some of which are human pathogens. fib is taken as proxy for pathogen load in stormwater. pathogens exhibit high infectivity even at their low abundance in water. low abundance poses difficulty in detection; hence proxy like fib is essential. fib concentration in stormwater remains high, sometimes several order higher than standards for potable and recreational purpose of water. fib contamination in stormwater occurs by human fecal (sewage), nonfecal, and animal sources. it poses serious challenge for public health and therefore to stormwater management system like biofilter. average fib removal efficiency of biofilter with sz was higher (approximately eightfold) than the one without sz (peng et al., ) . physicochemical nature of filter media strongly affects filtration of microorganisms. traditional filtration media, represented by a mixture of fine and coarse sand, compost, and an overlying layer of mulch, shows poor microbe removal ability under field conditions despite sufficient hydraulic conductivity, good removal efficiencies for many pollutants, and acceptable support for vegetative growth. many reasons might be responsible for poor efficiency such as operating conditions, maintenance issue including clogging and short circuiting, overloading of stormwater, and microbial regrowth. laboratory-scale sand biofilters removed indicator bacteria in range of . log e . log . log removal capacity for protozoan indicator (c. perfringens) and viral indicators (f-rna coliphages) was . and . , respectively, as reported in another study. removal efficiency can be enhanced by altering physical and chemical properties of filter media. smaller grain size and inclusion of secondary geomedia, e.g., activated carbon, zeolite, improve filtration rates while chemical modification of media enhances transport, attachment, and die-off process (martin et al., ) . microbial removal of gac-amended biofilters shows . log net leaching to more than log ec removal. high surface area and exceptional sorption capacity make gac a better medium for contaminant removal. gac-amended biofilters facilitate microbial growth and potentiate leaching in the course of intermittent flow, thereby encouraging formation of biofilm in pore spaces on the collector grains. moreover, biofilm adds further heterogeneity to the surface which may influence infiltration by altering hydrodynamics of the flow through the porous media. extracellular polymeric substances secreted on biofilm alter roughness, hydrophobicity, and electrokinetic properties of the collector surface. another geomedia studied for amendment is zeolite. zeolites are porous aluminosilicate minerals commonly used as adsorbent because of its exceptional sorption and ion-exchange characteristics. it is an effective adsorbent for chemical compounds owing to high specific surface area of porous surface. nevertheless, zeolite is useful for microbial removal. zeolitemodified stormwater biofilter studies explored the effect of zeolite particle size, surface modification, and the presence of vegetation on e. coli removal. another common amendment for sand media is biochar. similar to gac, biochar is prepared by pyrolysis of organic material under oxygen-limited condition. unlike gac, preactivation of biochar is not necessary for its use. physicochemical properties such as hydrophobicity and adsorption characteristics of biochar depend on variable of preparation method, e.g., feedstock, pyrolysis duration, and temperature, etc. biochar modified sand show reduction of the remobilization of e. coli under intermittent flow conditions. smaller particle sizes are better for e. coli removal. physically weathered (by wetedry or freezeethaw cycles) biochar-amended biofilter exhibited improved e. coli removal capacity. despite of abovementioned three amendments, expanded shale and red cedar wood chips (rc) have also been applied with sand media. they exhibited . e . and . e . log removal efficiencies respectively for e. coli depending upon e. coli concentrations (from to cfu/ ml) in the influent water (schifman et al., (schifman et al., , torkelson et al., ) . surface property of sand filter influences transport and filtration mechanism of pollutant and microbe removal. surface coating with metal, metal oxides, metal hydroxides, and chemicals with antimicrobial properties are reported to increase microbial removal during sand filtration in drinking water systems. metal and metal oxide coating imparts positive charge and coating of hydroxide or polymeric modification reduces negative charge to filter media surface. both kinds of coatings facilitate microbial attachment to filter media wooing to net negative charge on cell-membrane. iron oxideecoated sand media significantly removed e. coli and enterococci from urban stormwater as compared to uncoated counterpart (mohanty et al., ; zhang et al., ) . amendment of sand with surface-modified gac with metals (cu, zn) or metal oxides (cuo, tio ) and metal hydroxides (cu(oh) , zn(oh) ) shows improved removal of indicator bacteria. addition of surface-modified zeolite with metal, metal oxide, or metal hydroxide enhanced e. coli removal. some nanometallic or polycationic coatings utilized for coating of collector surface exhibit antimicrobial properties. such coating enhances die-off of microbes transported to filter. ec and rc media when coated with antimicrobial agents that is -(trimethoxysilyl)propyldimethyloctadecyl ammonium chloride or silver nanoparticle enhanced e. coli log removal in amended biofilters. biofilm plays significant role in removal of microbes following similar mechanism as described for ssf. macrofauna: earthworms, potworms, springtails, mites, fly larvae, beetles, millipedes, centipedes, isopods, ants, spiders, and snails constitute common taxa of stormwater biofilter. earthworms make most considerable biomass contributing % of total invertebrate abundance together with potworms, springtails, and mites. macrofauna is associated with fragmentation and decomposition organic matter, plant nutrient uptake and growth, and infiltration in biofilter. indirect role of macrofauna in microbial removal is suggested to play by impacting biofilter residence time and/or plant root architecture. for example, earthworm's burrowing activity increases soil infiltration activity e -fold. earthworm's activity is also proposed to increase plant growth and expansion of plant roots and has significant effects on pathogen when used for composting of biosolids or sludge. the activity was found associated with increased microbial diversity, better transport of pathogen, and removal of fecal coliforms, salmonella, enteric viruses, and parasitic worm eggs. micro/mesofauna: protozoans ( e mm) and nematodes ( mme mm) are grazing organisms of the biofilter composing micro-and mesofauna. protozoa are specialized grazers, that is, they capture and graze on targeted population of specific suspended or attached microbe. conversely, nematodes are generalized grazers ingesting all kinds of suspended microbes including protozoa within a limited size range. primarily, protozoa influences structure of microbial community while nematode alters community size. protozoae e. coli interactions experiment for stormwater biofilter performed in glass column demonstrated higher bacterial removal by protozoa-supplemented sediment as compared to sterile sediment or sediment with microbial community deficient of protozoa or biologically immature sediment. plantation of grasses, sages, and small shrubs, etc., is done in bioretention system for esthetic purpose. plant roots procure nutrition from organic and inorganic nutrients from the system and simultaneously influence soil processes such as nutrient cycling, nutrient availability, soil structure, moisture content, and stability. besides, vegetation affects soil microbiome, biofilm growth, porosity, and hydraulic retention time by creating preferential flow paths, which in turn influences pathogen removal. rhizosphere exudates influence diversity and abundance of soil infauna (macro-, meso-, and microfauna). infauna directly impacts pathogen removal process. biofilter planted with shrubs (melaleuca incana, leptospermum continentale) and grasses (paspalum conjugatum and buchloe dactyloides) showed improved e. coli removal. the removal was enhanced due to reduced infiltration rates in vegetated biofilter system. plants selected for biofilter must have morphological and physiological plasticity toward changing microclimatic condition (peng et al., ) . microbial-earthworm ecofilters (mees) was developed for the first time in at the university of chile by professor jose thoa. it is a low-cost wastewater treatment system suitable for developing countries, especially rural area. it is a natural engineered system referring to a passive engineered wastewater treatment system in which traditional vermicomposting system is inoculated with potentials of earthworms. earthworms and microorganisms function symbiotically to remove pollutant and pathogen from wastewater. different kinds of pollutant removal processes, that is, sedimentation, filtration, adsorption, precipitation, volatilization, and uptake by earthworm and microbe, occur in mee. primarily, microorganisms are responsible for biochemical degradation of pollutants, while earthworms regulate microbial population and activity. the latter degrade and homogenize ingested material by muscular actions of their foregut and add mucus to it. thus, the activity of earthworm conditioning of the filter media results in improved microbiological activity. besides, earthworms regulate microbial biomass directly and/or indirectly via three main mechanisms: comminution, burrowing, and casting; grazing; and dispersal (samal et al., ) . of the various filter media (river bed material, wood coal, glass balls, and mud balls) evaluated for performance of mees, river bed material showed the best activity for removal of pollutants and pathogens. pathogen removal efficiency of mee was studied at a pilot scale for treatment of domestic wastewater (arora et al., a,b) . a -month study revealed that mees could proficiently remove bod, cod along with total coliform, fecal coliforms, fecal streptococci, and other pathogens. in addition, study also suggested that antibacterial activity of the isolated microorganisms might be responsible for the removal of pathogens. another pilot-scale study found that mee is dominated byproteobacteria. however, major turnover of microbial biomass was chiefly affected by earthwormassociated bacterial groups including g-proteobacteria, bdellovibrio, lysobacter, and myxococcales. sinha et al. ( ) also reported pathogen removal in a small-scale plant for treatment of toxic wastewaters from the petroleum industry. earthworm species and filter media types are key factors for mees performance because they are the chief vital components of mees which directly or indirectly affect removal processes of contaminants over time. other important factors are worm load, chemical factors, hydraulic loading rate, and seasonal temperature and operating parameters, such as hydraulic loading rate, nutrient load, packing bed height, and design of setup which can alter removal process. in addition, biological activity of worm and microbe is sensitive to temperatures, ph, ammonia, and sodium. filter media makes external environment and affects structure and function of the earthworm's body wall. earthworm respires through its body wall and hence filter media is a regulatory factor for metabolic and physical activity of earthworm. for example, quartz sand media caused less injury to earthworm's body wall as compared to ceramsite media. river bed material was found best as compared with other natural filter media (wood coal, glass balls, mud balls, slagecoal cinder ceramsite, and quartz sand) for pathogen removal through mee (xing et al., ; kumar et al., ) . this filter media showed maximum removal of pathogen such as total coliform, fecal coliform, fecal streptococci, and e. coli. arora et al. ( a,b) also reported river bed material as the best media for removal of the aforementioned pathogens. vermicompost and manure aids to buffering capacity (ph rang ¼ . e . ) of filter media and brings ph in tolerance range for earthworm's survival. ammonium with ammonium sulfate had no influence on mortality at g/kg, although ammonium chloride exhibits comparatively low toxicity (lc for ammonium of . g/kg) to the worm. other chemical factor is salt stress of nacl. earthworms showed ability to detoxify moderate concentrations of nacl if exposed for a long time. seasonal variation in ambient temperature was demonstrated to significantly affect removal of pathogen and indicator organisms, earthworm population, and bacterial and actinomycetes number. the optimum temperature range for earthworm (eisenia fetida) is e c in which the organisms show best activity, growth, and reproduction. however, slightly higher temperature positively influences pathogen removal efficacy of mees (jiang et al., ; sinha et al., ) . biofilter is promising and an economic aid or alternative to physical and chemical disinfection of wastewater. based on the solid media, source of wastewater and flow rate biofilter can be slow sand filter, rsf, mee, etc. indigenous, oligotrophic microbes of biofilm pose competition to pathogen, release antimicrobial agents, feed on pathogen, and assist in transportation and filtration processes. variation of biofilter with additional macrofauna like earthworm and/or plants further improves removal efficiency of indicator organisms. solid sand filter, rsf, mee, and stormwater biofilter were able to efficiently remove indicator organisms such as giardia, cryptosporidium, salmonella, e. coli, total coliform, and fecal coliform, etc. application of synthetic and natural biodegradable filter media, analysis of indigenous flora and fauna of biofilter, and optimization of operating condition are the areas of current research in this field. stacked filters: novel approach to rapid sand filtration antibacterial and enzymatic activity of microbial community during wastewater treatment by pilot scale vermifiltration system a comparative study for pathogen removal using different filter media during vermifiltration removal of bacterial fecal indicators, coliphages and enteric adenoviruses from waters with high fecal pollution by slow sand filtration wastewater treatment plant operator training, module : trickling filters. pennsylvania department of environmental protection trickling filter and trickling filter-suspended growth process design and operation: a state-of-the-art review review on slow sand filtration in removing microbial contamination and particles from drinking water biotreatment of drinking water surface water filtration using granular media and membranes: a review the use of microbial-earthworm ecofilters for wastewater treatment with special attention to influencing factors in performance: a review wastewater treatment for pathogen removal and nutrient conservation: suitable systems for use in developing countries evaluation of vermifiltration process using natural ingredients for effective wastewater treatment microbial agents associated with waterborne diseases biofilms in water and wastewater treatment scaling bacterial filtration rates in different sized porous media engineering solutions to improve the removal of fecal indicator bacteria by bioinfiltration systems during intermittent flow of stormwater rapid sand filtration indicator and pathogen removal by low impact development best management practices. water (switz.) , e biological approaches for addressing the grand challenge of providing access to clean drinking water meeting the criteria: linking biofilter design to fecal indicator bacteria removal occurrence and significance of cryptosporidium in water treatment of wastewater by vermifiltration integrated with macrophyte filter: a review bacteria removal from stormwater runoff using tree filters: a comparison of a conventional and an innovative system new antimicrobially amended media for improved nonpoint source bacterial pollution treatment wetland systems to control urban runoff, wetland systems to control urban runoff removal of pathogen indicators from secondary effluent using slow sand filtration: optimization approaches earthworms: nature's chemical managers and detoxifying agents in the environment: an innovative study on treatment of toxic wastewaters from the petroleum industry by vermifiltration technology investigation of quaternary ammonium silane-coated sand filter for the removal of bacteria and viruses from drinking water slow sand filtration for water and wastewater treatmentea review a comparative study of synchronous treatment of sewage and sludge by two vermifiltrations using an epigeic earthworm eisenia fetida the capture and destruction of escherichia coli from simulated urban runoff using conventional bioretention media and iron oxidecoated sand key: cord- - qawjquv authors: lara, r.j.; islam, m.s.; yamasaki, s.; neogi, s.b.; nair, g.b. title: aquatic ecosystems, human health, and ecohydrology date: - - journal: treatise on estuarine and coastal science doi: . /b - - - - . - sha: doc_id: cord_uid: qawjquv this chapter treats two main topics: the relationship between human health, aquatic ecosystems, and water use; and the necessity of interdisciplinary approaches for the development of water management policies and disease control. main waterborne diseases, mostly affecting developing countries and relevant in terms of water management and changes in land use, such as malaria, schistosomiasis, or cholera, are discussed stressing links to the global water crisis. also, the role of artificial and natural wetlands in influenza epidemics is treated. the effects of increasing water use and scarcity on human health are discussed considering historical and contemporary incidence of diarrheal diseases in european and south asian megacities, relationships between dams and on waterborne diseases in asia and africa, and intensive agri- and aquaculture resulting in man-made ecotones, fragmented aquatic ecosystems, and pathogen mutations. it is emphasized that the comprehension of the multiple interactions among changes in environmental settings, land use, and human health requires a new synthesis of ecohydrology, biomedical sciences, and water management for surveillance and control of waterborne diseases in basin-based, transboundary health systems. surveillance systems should monitor changes in water management, ecotones, and hydrological cycles and shifts in, for example, the outbreak timing of strongly seasonal diseases. these indicators would provide criteria for the development of innovative water management policies, combining methods of vector control and the safe creation of water reservoirs, irrigation systems, and wetland habitats. this chapter is not intended to provide an extensive review of water-borne diseases, since there are already excellent examples of these in the literature. rather, the intention is to call atten tion to particular aspects of the principal water-borne diseases and related water management issues. the emphasis is often on controversial aspects which it is hoped will stimulate an open, undogmatic, and fruitful discussion on possibilities and cur rent and future challenges for ecohydrology in relation to human health. ecological integrity is central to health (epstein, ) . pollution, disturbed environments, habitat loss, and climate change promote disease emergence in a number of ways. threats to human health arising from man's interaction with aquatic ecosystems can originate from multiple factors, which can be broadly grouped into the following major categories: • natural biological cycles in which humans can act as hosts of pathogenic microorganisms (protozoans, bacteria, etc.); • consequences of the management of aquatic resources (e.g., wetlands drainage or creation, aquaculture, and dam construction); • effects of water pollution (chemical, microbiological, radio active, and thermal) on man and on the physiology of individual organisms; and • the impact of global changes affecting climate and hydrolo gical cycles (e.g., habitat degradation, warming, increased rainfall, and storms). clearly, these are interlinked and specific case studies are likely to present evidence of more than one causal factor. thus, an effort has been made to integrate these different topics into sections covering the most relevant factors related to diseases that can be considered indicators of determined hydrological processes. globally, approximately % of deaths due to infectious dis eases are water related. this amounts to . million deaths per year, % of which are caused by diarrhea, a disease killing around million people every year, mostly children in devel oping countries (wwd, ) . thus, it is crucial to develop an integrated water and health management system, as well as the tools required to identify and predict interconnected trends in the evolution of aquatic ecosystems and diseases. although the importance of integrating natural sciences with socioeconomic research is stressed and recommended in every forum dealing with sustainable use of natural resources, human health, one of the most valuable public goods, is seldom included as a significant issue in coastal or basin management programs. a frequent goal of cooperation between socioeconomic and natural sciences research is the evaluation of the sustainability of resource use and the vulnerability of the coastal zone. the seven step assessment framework of the international panel on climate change (ipcc, ) is the customary tool used for this purpose (figure ) . similarly, the health map program from the world health organization (who) provides criteria and a software platform for linking epidemiological data with layers of geographic information (who and unicef, ) . however, these two types of research programs mostly lack any substantial interaction. ecohydrology can provide the conceptual framework for establishing such links. this involves a scale (the river basin and the coastal zone), an integrative process (flooding dynamics), and an integrative tool such as digital elevation models (dem) of the study area (figure ) . high-resolution topography is essential for data integra tion in low-lying areas. for this purpose, a closer interaction of socioeconomic studies, climate, hydrology, ecology, micro biology, biogeochemistry, molecular biology, and medicine is required, as exemplified in the case of cholera (discussed in section . . . . ). further, it is necessary to generate a common language to facilitate and promote inter-and transdisciplinary communication. several concepts related to disease dynamics, such as outbreak, epidemics, and pan demics, are commonly used in a qualitative way. yet, the creation of interdisciplinary databanks or the compilation of information from diverse sources (e.g., hydrological events and outbreak intensity) including historical ones would ben efit from objective category definitions. in the following sections, some examples are dealt with, which will appear in this chapter and in other related literature. it is considered that an epidemic occurs when new cases in a given human population, during a given period, sub stantially exceed what is expected, based on recent experience (the number of new cases in the population during a speci fied period of time is called the 'incidence rate'). an epidemic may be restricted to one locality (an outbreak), be more general (the usual 'epidemic'), or even global (pandemic). common diseases that occur at a constant but relatively low rate in the population are said to be 'endemic'. an example of an endemic disease is malaria in some parts of africa, where a large portion of the population is expected to contract malaria at some point in their lifetime. these somewhat subjective definitions require more precisionand a wider diffusion among the populationwhen, for aquatic ecosystems, human health, and ecohydrology figure integration of socioeconomic, ecological, and medical research within the ecohydrology framework. example, public funds have to be allocated for prevention, alleviation, or fight against determined diseases or for insur ance policies. an increasing number of direct or indirect cases of water-borne diseases are due to global warming, increasing frequency and intensity of storms, flood control, and environmental management (dam construction, irriga tion, use of fertilizers, etc.), and this has greatly widened the spectrum of stakeholders with an interest in the use of water and its impact on health. this underlines the need for agree ment on and diffusion of definitions and criteria between users and managers to facilitate the quantification of impacts of disease, ecological damage, and poor sanitation, as well as for cost-benefit analysis of proposed prevention or remedia tion measures. the global burden of disease analysis (who, ) pro vides a comprehensive and comparable appraisal of mortality and loss of health due to diseases and injuries, and risk factors for all regions of the world. the overall burden of disease is assessed using the disability-adjusted life year (daly), a timebased measure that combines years of life lost due to premature mortality and years of life lost due to time lived in states of less than full health. worldwide, about million dalys per year are due to water-borne diseases (pruess et al., ) . a relevant specific example is the definition and evaluation of the impact of disease related to drinking water quality. this concerns the presence of chemicals or pathogenic microorganisms that are transmitted when contaminated drinking water is directly con sumed. if contaminated drinking water is used in the preparation of food, it can be the source of food-borne disease through consumption of the same microorganisms, often in the form of gastrointestinal illnesses. according to the who (who, a) , diarrheal disease accounts for an estimated . % of the total daly global burden of disease and is respon sible for the deaths of . million people every year. it is estimated that % of that burden is attributable to unsafe water supply, sanitation, and hygiene. at the national level, surveillance systems are the primary source of data concerning the scope and effects of water-borne diseases on persons. these data can be organized to provide information for political and administrative units such as counties or federal states, and/or geomorphological units, that is, basins. in the united states, since , the centers of disease control and prevention (cdc), environmental protection agency, and the council of state and territorial epidemiologists have maintained a collaborative surveillance system which monitors the occurrence and causes of water borne-disease outbreaks (wbdos) (calderon et al., ). the surveillance system includes data for outbreaks associated with drinking water and recreational water. state, territorial, and local public health departments are primarily responsible for detecting and investigating wbdos and voluntarily reporting them to cdc on a standard form. the unit of analysis for the wbdo surveillance system is an outbreak, not an individual case of a water-borne disease. two criteria must be met for an event to be defined as a wbdo. first, more than two persons must have experienced a similar illness after either ingestion of drinking water or exposure to water encountered in recreational or occupational settings. this cri terion is waived for single cases of laboratory-confirmed primary amebic meningoencephalitis and for single cases of chemical poisoning if water-quality data indicate contamina tion by the chemical. second, epidemiologic evidence must implicate water as the probable source of the illness. the integration of national information on water-borne disease incidence into collaborative regional, transboundary basin databank networks of open access is crucial to allow a fair and efficient implementation of water management regula tions related to, for example, dam operation and international irrigation agreements. this is particularly relevant in tropical countries where large river basins (e.g., amazon, nile, mekong, indus, ganges, and yarlong-brahmaputra) represent the main source of income, water, and food for millions of people. the rivers originating in the tibetan plateau ( figure ) are particu larly relevant as they flow through several, densely populated tropical and subtropical countries with quite different degrees of development. in general, water-borne disease can be caused by protozoa, viruses, or bacteria, many of which are intestinal parasites. in the following tables, some relevant examples of various disease types are grouped and summarized, including those which mostly affect developing countries, in many cases in tropical and subtropical regions. in the following sections, diseases have been selected that are relevant in terms of numerical incidence, water management, and changes in land use, parti cularly under the ecohydrological approach. these are malaria, schistosomiasis, lymphatic filariasis, onchocerciasis, cholera, spotted fever, and those illnesses induced by toxins produced during cyanobacteria blooms due to increased nutrient loads. we will provide a general overview of these diseases, stressing the links to the global water crisis, possible contributions of ecohydrology, and challenges to its classical approach. parasitic infections, particularly malaria and schistosomiasis, represent some of the most universal health problems. the who estimates that million people are infected and another million people are at risk of infection (who, ) . in fact, only malaria accounts for more cases of disease than schis tosomiasis, and both are closely related to water management (who, ) and will be extensively treated in this chapter. water scarcity and uncoordinated water management go hand in hand with poverty and disease. russel ( ) explains the links clearly: bilharziasis and malaria are both debilitating diseases and debility is not conducive to good farming. throughout history, malaria and bilharziasis have interfered with the use of arid land and indeed, calcified eggs of bilharzia worms were found in the kidneys of two mummies of the th dynasty - - b.c. there is a crucial chain reaction that in some instances has contributed to the virtual abandonment of irrigation systems: the more debility, the less canal maintenance, therefore the more anopheles mosquitoes and the more snails transmitting more malaria and more bilharziasis, thus leading to more debility, and so on…. (russel, : ) . table provides a summary of relevant water-borne diseases produced by protozoan parasites. in this section, we will con centrate on malaria, currently still the world's most important parasitic disease. today, malaria is one of the world's deadliest diseases and occurs mostly in tropical and subtropical countries, being transmitted from one person to another through the bite of female anopheles mosquitoes. the who estimates that there are - million cases of malaria, with over million deaths each year (who, a) . t he main burden of malaria (more than %) is in africa, south of the sahara. two-thirds of the remaining burden affects six countries: brazil, colombia, india, solomon islands, sri lanka, and vietnam. the ecology of the disease is closely associated with the availability of water, as the larval stage of mosquitoes develops in different kinds of water bodies. the mosquito species vary considerably in their water-ecological require ments (sunlit or shaded, with or without aquatic vegetation, stagnant or slowly streaming, and fresh or brackish; discussed in section . . . . . ), and this, to a great extent, determines the ecology of the disease. in many places, the nat ural habitat sustains intense malaria transmission; in others, the development of water resources (irrigation, dams, and urban water supply) has exacerbated the intensity of transmission and caused the disease to spread. in yet others, for example, the central asian republics of the commonwealth of independent states, malaria has returned as a result of a breakdown in water management and maintenance problems of local irrigation systems (who, ) . climate change (global warming) appears to be moving the altitudinal limits of malaria to higher elevations, for example, in the east african highlands and madagascar. further, in some of the tropical regions of the developing world, the incidence of malaria has increased in recent years as the mosquito and the malaria parasite it transmits have evolved more resistance to sewage, nontreated drinking water, flies in water supply (hand-to-mouth) untreated water, poor disinfection, pipe leaks, groundwater pollution, sharing of water source by humans and wildlife. beavers and muskrats create ponds that act as reservoirs for giardia (oral-fecal, hand-to-mouth) encephalitozoon intestinalis has been detected in groundwater chills, period fever attacks; debility, spleen and liver enlargement; anemia, jaundice; clogging of brain vessels can lead to death flu-like symptoms, watery diarrhea, loss of appetite, substantial loss of weight, bloating, increased gas, and nausea abdominal pain, fatigue, weight loss, diarrhea, bloating, and fever diarrhea, bloating abdominal discomfort, and flatulence diarrhea and wasting in immunocompromised individuals alternatives to dichlorodiphenyltrichloroethane (ddt) and to the medications used to prevent or treat the disease. in some regions (e.g., south africa), ddt is again increasingly used to control mosquitoes (thurow, ) . in general, land reclama tion for agriculture, deforestation, and changes in land use are probably the principal causes of the climatic and habitat changes responsible for these developments. yet, it should be borne in mind that it was the drainage of wetlands for agricul tural purposes in the nineteenth century which contributed most significantly to the eradication of malaria in europe (reiter, ) . these are crucial aspects to be carefully evaluated when wetland creation and management policy are being considered as an ecohydrological tool, for example, for sequestration of nutri ents in estuaries and for preventing toxic algal blooms. these issues will be discussed extensively in the following section (also discussed in section . . . ). it is often erroneously thought that larvae of malariatransmitting mosquitoes can only develop in freshwater. it is also frequently forgotten that malaria is not restricted to the tropics, and that only in did the who declare that europe was free of malaria. about years ago, malaria was a leading cause of death in many marshland commu nities along the coast of southern england. there, extensive salt marshes provided high-quality grazing for sheep and cattle, but were also a favored habitat for anopheles atroparvus, a highly effective malaria vector, which prefers to breed in brackish water along river estuaries and in the presence of abundant algae. until the nineteenth century, malaria was a major mortality factor in the netherlands. however, by the end of that century transmission had dropped precipitously in the more prosperous countries of north europe. a major factor contributing to this decline was that the mosquito habitat had been eliminated by improved drainage and extensive land reclamation. major epidemics still occurred in russia and poland in the s, with high death rates reaching regions near the arctic circle (wolanski et al., and references therein) . today, malaria is again common in many parts of central america, northern south america, tropical and subtropical asia, some mediterranean countries, and many of the republics of the former ussr. this spread of the disease has been attrib uted to, among other factors, forest clearance, irrigation, ecological change, population increase, deterioration of public health services, resistance of mosquitoes to insecticides, and resistance of the malarial parasite to antimalarial drugs (reiter, ) . thus, policies on wetland creation or restoration must take account of not only the benefits of the reestablishment of lost ecological services, but also the potential consequences of increased areas of slow-flowing or stagnant waters on disease vector proliferation, particularly under a scenario of increasing temperatures. the related ecohydrological concepts of system robustness and flushing dynamics can make a major contribu tion to the integrated analysis and handling of this issue, seeking equilibrium between the necessary water residence time, for example, efficient nutrient sequestration, and the minimization of mosquito reproduction. the choice cannot be formulated as 'mosquitoes in the basin' or 'toxic algal blooms in the estuary'. there are techniques such as 'runneling' that have been used with varying success to control mosquito proliferation in cre ated or natural wetlands. as mosquitoes can be vectors for several other diseases besides malaria, this will be treated in a separate section (discussed below in section . . . . . ). table provides a summary of relevant water-borne diseases produced by worm infections. in this section, we will concen trate on schistosomiasis, lymphatic filariasis, and river table main water-borne diseases produced by worms blindness because of their strong links to water management measures, such as irrigation and dam operation schemes. schistosomiasis is considered the second most important para sitic infection after malaria in terms of public health and economic impact. it is a chronic debilitating disease that is estimated to affect between and million people in countries. as many as million live in endemic areas. also known as bilharzia, bilharziosis, or snail fever, it is caused by several species of fluke (trematode) of the genus schistosoma. (gryseels et al., ; figure ) . infection with any of the five species of schistosome worms is rarely fatal. although it has a low mortality rate, schistosomiasis is often a chronic illness that can damage internal organs and, in children, impair growth and cognitive development. the urinary form of schis tosomiasis is associated with increased risks for bladder cancer in adults (hodder et al., ) . human infections are most common in asia, africa, south america, or the middle east, especially in areas where the water contains numerous fresh water snails, which are intermediate hosts, that is, may carry the parasite. however, trematodes can be found anywhere human waste is used as fertilizer. the disease affects many people in developing countries, particularly children who may acquire the disease by swim ming or playing in infected water, and field workers in arid or semiarid regions where agriculture depends heavily on irrigation. first infection with schistosomiasis usually occurs during the early school years and is a frequent cause of absen teeism. it is not uncommon for % of a school's student population to be infected in some highly endemic areas in africa. in addition to its effect on children, schistosomiasis has a major impact on the agricultural workforce and on national economic productivity. in egypt, where % of the people are infected, economic losses due to lost work are estimated to exceed $ million a year (anonymous, a) . development, both planned and unplanned, has resulted in a number of changes in the epidemiology of the disease that threaten to increase its spread and the number of people infected, reduce economic productivity, and compromise development gains. water development schemes, including dam building and irrigation systems, with slow water flows and aquatic vegetation, have created new breeding sites for snails. intensive agriculture has encouraged people to migrate to urban and peri-urban areas that are ill-prepared to meet their needs for sanitation and water. in these areas, snail-infested streams and canals are often the most convenient water sources. new agricultural systems that emphasize irrigation, double cropping, and other intensive cultivation practices have increased farmers' exposure to infection. the emergence or reemergence of schistosomiasis as a result of large-scale hydroprojects has been reported from the egyptian aswan high dam (khalil, ; strickland, ) , the sudanese gezira-managil dam (amin, ; omer, (teklehaimanot and fletcher, ) . in china, the danling dam in the province of sichuan and the huangshi dam in the province of hunan have all had adverse effects by increasing local schistosome transmis sion (zhang and guo, ) . however, not all dam regions suffer from schistosomiasis. for example, when the ertan dam in the province of sichuan became operational, local schistoso miasis control centers collaborated with dam management offices and government ministries to actively monitor and pre vent the spreading of schistosome worms. as a result of these efforts, potential schistosome transmissions in the ertan region were successfully averted (gu et al., ) . schistosomiasis infection in humans, the definitive hosts, is caused mainly by three species of flatworm, namely schistosoma haematobium, s. japonicum, and s. mansoni. infection occurs when free-swimming larvae penetrate human skin. the larvae develop in freshwater snails, which serve as intermediate hosts for the parasite. humans are infected when they enter larvaeinfested water for domestic, occupational, and recreational purposes and the larvae of the parasite penetrate the unbroken skin. the life cycle is continued when people infected with schistosome worms deposit urine or fecally borne eggs into the water. the disease particularly affects children who may acquire the disease by swimming or playing in infected water (hodder et al., ) . the life cycle of s. mansoni provides a simplified example for all species of schistosomes and helps understand the potential contribution of water management to snail proliferation. after the eggs of the human-dwelling parasite are emitted in the feces, into the water, the ripe miracidium hatches out of the egg. the miracidium searches for a suitable freshwater snail to act as an intermediate host and penetrates it. following this, the parasite develops, via a so-called mother-sporocyst and daughter-sporocyst generation, into the cercaria. the purpose of the growth in the snail is the numerical multiplication of the parasite. a single miracidium can produce several thousand cercaria, each one of which is capable of infecting humans. the cercaria propel themselves in water with the aid of their bifurcated tail and actively seek out their final host. when they recognize human skin, they penetrate it within a very short time. following a migration through the body within the bloodstream, they develop into sexually mature adults. the larvae enter through the skin, migrate via the blood vessels, and mature in the lungs. from there they travel to the veins of the upper or lower intestine or bladder and, if they find a partner of the opposite sex, they reproduce. there are specific host/parasite combinations that lead to typical forms of bilhar ziasis in endemic areas in different regions of the world, frequently associated with a particular economic activity. there are also region-specific control measures with positive and negative consequences or side effects (mannesmann and fuchs, ) as discussed in section . . . . . . in these regions, the snail bulinus truncatus, b. globosus, and b. forskali can be hosts of s. haematobium, which produces uro genital bilharziasis (lang, ) . the two clearest examples of large-scale irrigation systems spreading schistosomiasis are found in africa, especially in the nile valley. in upper egypt south of cairo, it has been known for a long time that the shift from basin irrigation by the floodwaters of the nile to perennial irrigation results in a dramatic increase in schistosomiasis. basin irrigation allows the land to dry out seasonally, killing almost all snails. under perennial irrigation, the land is wet all year round. soon after the construction of aswan low dam, four locations that changed irrigation methods in the mid- s experienced a surge in s. haematobium infections (khalil and abdel azim, ; khalil, ) . infection rates increased from - % in to - % in . urinary schistosomiasis had a pre valence at % in areas of perennial irrigation in upper egypt and only % in areas with basin irrigation (scott, ) . in the s, prevalence ranged from % to % in three districts with perennial irrigation, while in two districts with basin irriga tion, it was % and % (wright, ) . in the s, after the construction of the aswan high dam, the nile delta became a major breeding habitat for the snail hosts of both urinary and intestinal schistosomiasis. irrigation canals and drains ( figure ) harbored stable populations of these snails throughout the year. this resulted from the elimina tion in these canals of the so-called 'winter closure'. before the construction of the dam, the closure was enforced for about days, during which the canals were closed and dried up, and the silt deposited on their beds during the nile flood was dredged out together with the snails and aquatic weeds (malek, ) . the other large-scale nile valley irrigation system impli cated in schistosomiasis transmission is the gezira irrigation scheme in the sudan. the major increase in prevalence of schistosomiasis in gezira came after , when the cropping rotation changed to include 'winter' wheat. farmers kept the canals filled with water from march to may, when they were previously dry (fenwick, ) . another factor was the creation of the adjoining managil extension irrigation system which left tenants without adequate water supplies or sanitation facilities. also, there was an influx of migrant laborers in the original gezira scheme, who lived near irrigation canals, under very poor sanitary conditions, leading to the propagation of further water-borne diseases (tameim et al., ) . in north africa and the middle east, research has demon strated the association of even small-scale irrigation plans, decentralized at the village level, with increases in schistoso miasis transmission. malek ( ) reports that in sudanese villages along the nile, north of khartoum, prevalence of urin ary schistosomiasis in children was - %, compared to an average of less than % in other areas. wright ( ) reports that in the rural area around baghdad, iraq, prevalence of schistosomiasis increased from % to % following the installation of lift pumps. . . . . . asia in east and southeast asia, intensification and expansion of irrigated rice production systems over the past decades have increased the habitats for snail and schistosoma. the host/para site combinations tricula aperta/s. mekongi and oncomelania hupensi/s. japonicum produce intestinal bilharziasis predomi nantly in rural communities living close to irrigation ditches. the association of asian schistosomiasis with rice-growing areas has been reported by different authors. however, in many cases, rice fields themselves did not seem to be breeding habitats; rather snails found in the rice fields appeared to have spread from nearby irrigation canals. cattle and water buffalo can also be important reservoir hosts. various methods have been applied to substantially reduce schistosomiasis in rice agriculture, which will be discussed later in section . . . . . in this part of the world, it is mainly the combination of s. mansoni with biomphalaria glabrata snails that causes bilhar ziasis. brazil, with million people living in the endemic areas and million infected, is the most affected country in the americas (chitsulo et al., ) . however, it is not obvious to what extent hydrological factors, including large dams or irrigation systems, contribute to spreading schistosomiasis in brazil. the african slave trade was probably responsible for the introduction of schistosomiasis in brazil soon after the country was discovered by european explorers, and internal migration was responsible for spreading it from seashore to interior. nowadays, schistosomiasis transmission occurs over a vast endemic region, from maranhão to espírito santo, and minas gerais, and there are further areas with a high risk of endemic expansion (araujo, ; paraense, ) . there are also iso lated foci in the federal district and in the states of pará, goiás, rio de janeiro, são paulo, paraná, and rio grande do sul. some examples from the literature point to patterns of labor and household migration as significant factors in the incidence of disease in brazil, probably more than irrigation schemes. cases of disease importation from endemic areas have been registered over almost the entire country, mainly in the states that are considered migration destination areas, such as rondônia (coura and amaral, ) . a study involving irrigation projects in the semiarid region of five northeastern states of brazil (coutinho et al., ) found that schistoso miasis transmission was not a major problem in the areas studied. socioeconomic-sanitary analysis identified the pre sence of migrant farm workers coming from endemic areas of schistosomiasis and living in poor sanitary conditions in the irrigation areas as a main factor of epidemiological importance. however, continued epidemiological surveillance is essential in all irrigated areas of northeastern brazil if schistosomiasis control is to be maintained, as well as improvement in the water supply and sanitation measures for migrant workers (coutinho et al., ) . the potential association between irrigation levels and the occurrence and spread of s. mansoni infection was investigated (martins and barreto, ) in the state of bahia, where two forms of irrigation have been developed. the first is capital intensive and mechanized, requiring little manual labor. the second is labor intensive and characterized by limited mechan ization. according to the study, the municipalities with the largest irrigated areas are not the ones with the highest s. mansoni infection rates. in most of these counties, irrigation is capital-and technology intensive. according to these find ings, unlike africa, irrigation in the state of bahia has had little impact on the spatial profile of the schistosomiasis endemism. regarding the impact of schistosomiasis on labor-intensive irrigation schemes, some research points to the importance of the household in disease transmission, as a result of the cluster ing of domestic activities associated with water collection, storage, and usage. such activities can result in the sharing of water-contact sites and water-contact behavior, which expose all members of the household to an increased risk of infection. in previous studies in brazil (bethony et al., ) , it was deter mined that shared residence accounted for % of the variance in schistosoma fecal egg excretion rates. further, shared residence accounted for % of the variation in total water contacts per week. it also accounted for a large proportion of the variation in individual water-contact behavior: for example, agricultural con tacts ( %), washing limbs ( %), or bathing ( %). these results implicate the household as an important composite measure of the complex relationships between socioeconomic, environmental, and behavioral factors that influence water-con tact behavior and, therefore, the transmission of schistosomiasis. these results also support the idea of focusing on safe water supply and household density in the implementation of schis tosomiasis prevention and control measures. the role of aquatic animals in maintaining the schistosome life cycle in brazil requires further clarification. the influence of s. mansoni on a population of the water rat, nectomys squamipes, was studied at sumidouro, rio de janeiro, and brazil (d' andrea et al., ) . the population dynamics of parasites was studied. water contamination (i.e., the source of miraci dia), abundance of the intermediate host, and rodent migration were found to be related to schistosome prevalence. the n. squamipes population was not obviously influenced by the infection, as shown by the high number of reproductive infected females, high longevity of infected individuals, and the absence of a relationship between recruitment or survivorship rates and the intensity of schistosome infection. the data indicate that n. squamipes can increase transmission of s. mansoni in endemic areas and carry it to noninfected areas. furthermore, this rodent can be used as an indicator of trans mission foci. in the caribbean, b. glabrata can be found in shallow ponds with abundant vegetation or with fallen banana leaves. it has been also found in drains around banana plantations on the west indian island of st. lucia (sturrock, ) . this is a mosquito-borne parasitic worm infection, a debilitating parasitic disease, which affects million people in the tropical and subtropical areas of southeast asia, south america, africa, and the islands of the pacific. while filariasis is rarely fatal, it is the second leading cause of permanent and long-term disability in the world. a person with the disease tends to have more bacterial infections in the skin and lymph system. this causes hardening and thickening of the skin, which in its most dramatic form is expressed in the symptoms of elephantiasis, the accumu lation of lymph, usually in legs. it is not a killer disease, but causes severe debilitation and social stigma. the who has named filariasis one of only six 'potentially eradicable' infectious diseases and has embarked upon a -year campaign to eradi cate the disease. in addition to consistent long-term treatment by oral medicines, eradication efforts focus on controlling the pro liferation of mosquitoes in aquatic environments, which will help to reduce the transmission of lymphatic filariasis, as well as that of malaria, which is prevalent in many of the same communities in africa (cdc, ) . onchocerciasis or river blindness is the world's second leading infectious cause of blindness. it is found in countries in africa as well as in guatemala, southern mexico, some areas of venezuela, small areas in brazil, colombia, and ecuador, and in the arabian peninsula. a total of million people are affected worldwide. the disease is caused by onchocerca volvulus, a parasitic worm that breeds in water and that can live for up to years in the human body. controlling insect breeding sites in rivers is one of the pillars of prevention. the disease is trans mitted person to person by bite of a blackfly (simulium), which breeds solely in fast-flowing waters. symptoms of the disease in a person usually begin to show - years after infection. each adult female worm, which can be more than half a meter in length, produces millions of microscopic young worms (micro filaria). the microfilaria migrate through the skin and, upon death, cause intense itching and depigmentation of the skin (leopard skin), lymphadenitis, resulting in hanging groins and elephantiasis of the genitals, serious visual impairment, and blindness when they reach the eye. the disease blinds between % and % of its victims and seriously undermines the economic productivity of communities in endemic areas (who, b; figure ). figure onchocerciasis also affects the development of exposed children. their school performance is affected by the unrelenting itching associated with this disease. many youth are deprived of their childhood as they are often forced to guide and look after elderly relatives blinded by the disease. photo credit: bill vanderdecker in: http://www.pqmd.org/cms/node/ unlike malaria and schistosomiasis, transmission of river blindness is usually found along fast rivers or streams with white-water rapids and cascades. the species of blackflies, which transmit this blinding parasite, require well-aerated, high-velocity flow to deposit their eggs, usually on rocks or overhanging vegetation. the larval stages are filter feeders and need large flows passing their habitat to obtain sufficient food and oxygen for development. in many parts of africa, people living near rivers migrate out of the fertile river valleys because of the painful bites of the flies and the eventual blindness resulting from this parasite (kim and merritt, ) . river blindness has historically plagued the fertile valleys of west africa, but it was the arrival of europeans that unleashed the full force of the disease upon the region's inhabitants. traditional taboos had kept people from settling along river banks or visiting streams in broad daylight, when blackflies are most active. white colonists, however, insisted on recreating the riverside towns they remembered from home. by removing longstanding cultural prohibitions, they made onchocerciasis more prevalent than it had been before. by the s, several hundred thousand people had been blinded by the disease. also tragically for the region, the most fertile farmlandan area roughly the size of michiganwas abandoned due to the risk of contracting the disease (wong, ) . currently, river blindness has been eliminated as a major public health problem in west african countries. despite success in west africa, million people remain at risk of contracting river blindness in the countries of central, eastern, and southern africa (anonymous, ) . due to the tight connec tions between the dynamics of this disease and changes in flow velocity, we will return to it in section . . . in the discus sion of the effect of dams on human health. table provides a summary of relevant water-borne diseases produced by bacteria. we will concentrate on cholera and other vibrio illnesses for the reasons explained below. the case of cholera has been considered paradigmatic of the links between global climate change and infectious diseases. it offers an excellent example of how information about environ mental factors permits better understanding of disease virulence, transmission, and epidemiology. therefore, and due to the links to wetlands and other coastal ecosystems, aquaculture, and water management in megacities, this disease will be discussed in detail in this and the following sections, in relation to the dynamics of its causative agent in aquatic ecosystems. cholera is still an important cause of morbidity and mor tality in many countries in asia, africa, and latin america due to lack of safe water supply and poor hygienic practices (colwell, ) . cholera is endemic in the ganges and brahmaputra deltas. it was originally endemic to the indian subcontinent but spread worldwide along the trade routes, and mostly affects developing countries, particularly in coastal zones. in the last ∼ years, there have been seven pandemics and there is evidence of accelerated change of vibrio and disease dynamics as consequence of environmental changes and increased global connectivity (faruque et al., ) . the cur rent pandemic, which started in , is the most extensive in geographic spread and duration. cholera epidemics were reported from over countries during , the largest scale ever recorded in human history (who, ) . vibrio cholerae, a gram-negative comma-shaped gammapro teobacterium, is the causative agent of cholera ( figure ). vibrios are aquatic bacteria of marine and estuarine origin but can survive and be pathogenic in freshwater ecosystems. apart from v. cholerae, some other vibrios (v. parahaemolyticus, v. vulnificus, etc.) are also responsible for incidences of diarrhea, gastroenteritis, necrotizing fasciitis, and septicemia, which afflict human populations (table ) all over the world (chakraborty et al., ) . besides, many vibrios can also cause diseases in fish, shrimp, corals, and other aquatic organisms (thompson et al., ) . there are convergent approaches to the investigation of coral and human diseases. these include the research into the figure the causative agent of cholera, vibrio cholerae, is a faculta tively anaerobic bacterium, . - . µm to . - . µm, and a natural inhabitant of temperate/tropical estuaries, salt marshes, mangroves, coastal waters, and reefs. photo credit: moredun animal health, ltd/ science photo library/photo researchers, inc. same bacteria genus (vibrio), responsible for coral bleaching and for cholera, the use of remote sensing of ocean-surface temperature, climate research, and an emerging common cur rent of epidemiological thinking. traditionally, the association between water temperature and coral bleaching has been stressed, and only recently it has been discovered that this is probably triggered by a vibrio bacterium, and that it could be transmitted by a coral-feeding worm, acting as 'vector'. it is the first time a vector has been found for a coral disease (rosenberg and falkovitz, ) . table main water-borne illnesses produced by bacteria at the same time, the study of cholera disease has begun to encompass marine and estuarine research. the association of v. cholerae with plankton was established only recently, allow ing analysis of patterns of cholera epidemics, especially in those regions where it is endemic. the sporadic and erratic nature of cholera epidemics can now be related to climate-ocean coupling events, such as el niño (colwell, ) . since zoo plankton has been shown to harbor the bacterium and zooplankton blooms follow phytoplankton blooms, remote sensing can be employed to determine the relationship between cases of cholera and ocean chlorophyll concentration, as well as sea-surface temperature, ocean height, nutrient con centrations, salinity, and turbidity. during each pandemic, cholera has struck coastal regions before spreading inland. the principal agents responsible for cholera epidemics are the o and o serogroups of v. cholerae, of more than serogroups so far identified. within the o serogroup, the classical biotype caused the pre vious six pandemics, while the el tor biotype is associated with the present seventh pandemic. a coupling of vibrio dynamics to that of aquatic, brackish ecosystems is strongly suggested by the marked cholera seasonality in coastal villages of the bay of bengal (colwell, ) . the role of coastal areas in maintain ing endemicity is clearly a significant feature of cholera ecology. however, it was mainly laboratory research that provided evi dence for the existence of aquatic environmental reservoirs where v. cholerae survives for long periods of time and from which a toxigenic form may emerge to support epidemic con ditions (miller et al., (miller et al., , (miller et al., , barua and greenough, ) . the o serogroup emerged in the coastal zone in through a natural genetic alteration of the o el tor biotype. a survey in bangladesh established that this strain was mainly found in southern coastal marshes (faruque et al., ) . however, systematic, interdisciplinary field studies on the relationship between habitat characteristics and vibrio diversity and virulence are scarce. little has been done to widely monitor v. cholerae and study its ecology from a basin perspec tive in different related coastal environments such as mangroves, marshes, and estuaries, despite clear evidence that this could be one significant step forward for early warning and understanding human vulnerability to the disease (collins, ) . as early as , robert koch suggested that the bay of bengal, especially the sundarban mangrove forest, was the main source of cholera, noting that the combination of a brackish, organic-matter-rich environment, with a high den sity of human population represented the ideal conditions for the proliferation of v. cholerae (koch, ) . however, since then, there have been no extensive surveys of this transbound ary ecosystem (bangladesh and india), which represents the largest unitary mangrove and marsh system worldwide (akhtaruzzam, ) . difficult access, cyclones, floods, and the difficulty of obtaining funding for long-term research have precluded the systematic spatiotemporal monitoring of these habitats. most of the few available works are based on one-site samplings and do not provide information on seasonal trends. plankton is a significant marine reservoir of v. (huq and colwell, ) . in most cases, phytoplankton and zooplank ton are spatially and temporally associated (kiorboe and nielsen, ) and their abundance can be estimated by remote sensing, which has been related to cholera incidence in the bay of bengal region by lobitz et al. ( ) . living zooplankton can be a reservoir for vibrios, which attach them selves to the zooplankton's chitinaceous exoskeleton (kaneko and colwell, ) . nevertheless, watkins and cabelli ( ) found that growth and survival of v. parahaemolyticus was more stimulated by addition of pulverized chitin, than by living zooplankton, which however had a greater effect than the addition of sewage or other nutrients. in addition to salinity, nutrients, and plankton, suspended particle load and sediment resuspension also influence the vibrio amount in the estuary. recent investigations (lara, unpublished results) on seston size fractionation in sunderbans waters showed that the largest amounts of chitin and cultivable vibrio spp. were generally present in the fractions correspond ing to micro-, nanoplankton, microdetritus, and silt/clay particles, and not in zooplankton. it is still an open question whether the vibrio serotypes o and o , as well as the viable but not cultivable, and nonviable forms are preferen tially associated with determined seston size classes or wetland ecosystem compartments. better understanding of vibrio diversity in aquatic environ ments such as estuaries, marshes, and mangroves can lead to new insights into their genetic expression and co-regulation in the environment where they interact with each other. however, most vibrio studies treat species of pathogenic importance (e.g., v. cholerae, v. parahaemolyticus, and v. vulnificus) and not the diversity of the genus itself. a further key issue is the compre hension of the seasonality, life cycle, and dormant phases of bacterial population in nature. the environmental persistence of v. cholerae may be facilitated by entering a dormant state in which it remains viable but becomes nonculturable (vbnc) in conventional laboratory media (colwell et al., ) . the v. cholerae cells attached to plankton enter into the vbnc state as survival strategy (colwell and huq, ) . therefore, the detection of the nonculturable state is crucial to understanding v. cholerae ecology. however, the mechanisms that cause the organism to associate with plankton or other particles, to form biofilms, or to enter dormant or free-swimming phases are not yet completely understood. in the karnaphuli estuary, bangladesh, recent studies (lara, unpublished) showed that suspended particulate matter (spm) other than zooplankton contained significant amounts of chitin, especially in the size class < µm. the quantitative contribution of respectively nano-or bacterioplankton, microdetritus of biological origin, or resuspended sediment particles to the chitin pool in that size class is still unknown. particle load together with salinity significantly influenced estuarine vibrio distribution. we compared the microbial landscape dur ing a pre-monsoon situation and after a strong cyclone: the amount of cultivable vibrio, and its relative contribution to total aerobic bacteria, increased dramatically after the cyclone. amounts of spm also increased and there were higher salinities along the estuary. sediment resuspension and salt intrusion can thus strongly influence the abundance and distribution of estuarine vibrio population. the above findings call attention on essential questions relating estuarine dynamics and human health: are vibrios in sediment part of a benthic community with its own character istics or do they basically consist of a fraction of a pelagic population reaching the sediment after sedimentation of the particles to which they are attached? nair et al. ( ) also addressed the role of sediments as a possible vibrio reservoir in freshwater environments in calcutta. in florida, there was a predominance of non-o v. cholerae infections at the time the organisms flourished in the sediment (williams and larock, ) , which was detected down to -cm depth. higher sedi ment vibrio concentrations were associated with organic matter flocs occurring after the seasonal phytoplankton productivity maximum and during the zooplankton decline. this suggested that the flourishing of vibrio in the sediments was related to the presence of organic matter input from plankton detritus. thus, although plankton itself is an important aquatic vibrio reser voir, its relevance for fueling benthic vibrio seasonal cycles has probably been overlooked. the relevance of seasonally driven sediment resuspension in relation to annual cholera cycles in endemic regions deserves more attention. the incorporation of particle-bound vibrios and porewater nutrients into the water column could favor a sharp vibrio increase, even at otherwise unfavorable salinities (singleton et al., ) . future studies should investigate the links between the spatiotemporal estuarine variability and the ecology, diversity, and spreading mechanisms of vibrios including v. cholerae. a key question is how and to what extent these microorganisms persist in the transition from a brackish to a freshwater envir onment, involving strong gradients in salinity, ph, inorganic nutrients, dissolved and particulate organic matter, turbidity, plankton, and wetland vegetation. further, an ecohydrological research approach should focus on how the distribution of the above factors influences the relative abundance of v. cholerae compared to the total vibrio population and other bacterial groups in aquatic environments of west bengal, including water and sediment compartments. there have been efforts to predict cholera outbreaks through models relating disease incidence and environmental variables such as seawater temperature, chlorophyll content, height of the ocean surface, and rainfall, as well as by remote sensing (e.g., lobitz et al., ) . recently, an empirical model relating multiyear data of the number of cholera cases, rainfall, and chlorophyll was able to successfully reproduce outbreaks of cholera in kolkata and matlab over the time span of the data set (magny et al., ). yet, these authors stated that a finer temporal resolution (submonthly) in environmental data col lection was needed to improve mechanistic models and account for short-term variability, especially for the kolkata region. there is also evidence that cholera cases increased following a rise in ocean-surface temperature. however, a direct correla tion exists only for the spring peak, while during the rest of the year there are lags and even an inverse correlation between the two variables (colwell, ) . simple (lag) correlations between the seasonality of cholera and that of climate variables such as monsoon rainfall merely confirm that cholera is seaso nal (bouma and pascual, ) . these authors hypothesized that there would be two different aquatic habitats: the marineestuary type and the inland water bodies, with potentially different driving factors. although much has been speculated about possible ocean/ land interactions based on empirical correlations, the causal links between ocean parameters and cholera incidence in ripar ian inland villages are tenuous, as discussed in the following. zooplankton in freshwater has been considered a main trans mission means of v. cholerae to humans . although tidal transport of vibrio-carrying marine zooplankton toward the inland is likely and has been proposed by these authors as a possible infection source, until now there have been no studies demonstrating that marine copepods survive long enough to represent an infection source in freshwater, which is what people drink finally, and not seawater. simultaneous variation of two parameters does not imply causality and, although the existing mechanistic models can be useful as predictive tools, they have not significantly contributed to explaining the reasons for cholera endemicity or the existence of bimodal and unimodal occurrence patterns in the indian subcontinent. for example, sea-surface tempera ture in the bay of bengal shows a bimodal cycle similar to the seasonal pattern of cholera in dhaka, bangladesh (colwell, ; figure ) , and is therefore often used, directly or through covarying parameters, with success for such models. however, this seems to mask the fact that strong unimodal patterns are observed elsewhere in india (e.g., north bengal and vellore; jesudason et al., ; bouma and pascual, ) without any obvious relationship to the marine environment or even to temperature and rainfall. several studies found that the effect of rainfall on cholera incidence does not show any univocal pattern (glass et al., ; bouma and pascual, ; ruiz-moreno et al., ) . even in dhaka, which has a strong bimodal seasonality, there is no clear relationship between rainfall and cholera. the number of cases peaks before the monsoon (high rainfall period) and at its end, with a strong decrease in the middle of the monsoon. recently, hashizume et al. ( ) showed that the weekly number of cholera cases in the period - in dhaka did not show any direct relationship to rainfall, and suggested that river levelbelow flooding levelalso plays a role. river discharge is controlled not only by rainfall but also by snow melting in the himalayas and can control the survival of the bacterium through determining salinity and ph levels (bouma and pascual, ) . hashizume et al. ( ) stated that, because of the observed and potential effects of chemical changes in surface waters due to rainfall on vibrio survival and toxicity, it is necessary to quantify the level of v. cholerae in aquatic environments at the same time as measuring rainfall and river levels ( table ) . this section deals with examples of diseases derived from or influenced by high nutrient loads, particularly those derived from toxic algal blooms, as well as with the general effect of global changes in nutrient cycles on parasitic infectious diseases according to toxin type and exposure: skin irritation, stomach cramps, vomiting, nausea, diarrhea, fever, sore throat, headache, muscle and joint pain, liver damage, and kidney disease in the body nitrate is reduced to nitrite, which reacts with hemoglobin forming methemoglobin, and reducing blood ability to carry oxygen; affects mostly infants and old persons (pids). although arsenicosis affects millions of people, parti cularly in bangladesh, the problem arises from using water from wells reaching layers where groundwater naturally con tains high as levels and there is, to date, no obvious way that ecohydrological methods could help solve this problem. for this reason, we will not discuss this disease further in this chapter. eutrophication in water reservoirs or semi-enclosed water bodies leads to the formation of intensive phytoplankton blooms. during recent years, both the incidence and intensity of such blooms appear to be increasing, when examined at the global scale. this increasing severity of algal blooms may be a consequence of increasing levels of nutrient enrichment as a result of sewage disposal, increased agricultural runoff, and changes in hydrological regimes potentially related to climate change (unep, ) . blooms caused by toxic cyanobacteria (harmful algal blooms) lead to outbreaks of disease and dete riorated recreational and aesthetic values, causing both economic losses and illness and death of both humans and animals. in evolutionary terms, cyanobacteria are one of the oldest organisms on earth, dating back to more than ma (schopf, ) . they are oxygenic photosynthetic prokaryotes possessing the ability to synthesize chlorophyll a as their photosynthetic pigment. the nitrogen-to-phosphorus ratio (n:p) has been frequently used as a key indicator in predicting algal biomass and compositions, and its seasonal succession in lentic systems (tilman, ; kilham, ) . smith ( ) pointed out that bloom-forming cyanobacteria had a tendency to dominate in a lake when the n:p ratio was less than . the significance of the n:p ratio as a critical factor, however, is still controversial, due to the variability of the other chemical char acteristics and phytoplankton composition within a geographic region. for example, cyanobacteria blooms can be induced by increases in the phosphorus concentration instead of by a decrease in the n:p ratio (trimbee and prepas, ; sheffer et al., ) . cyanobacteria ( figure ) produce a variety of toxic com pounds known as cyanotoxins. the impacts of cyanotoxins on human health have been of increasing concern, as the impacts of cyanobacterial blooms on water supplies as well as nearshore marine ecosystems have become better understood. outbreaks of poisoning from cyanobacterial blooms can be catastrophic, such as the death of dialysis patients following exposure to inadequately treated water from the tabocas reservoir in brazil (pouria et al., ) or the hospitalization of children and adults who drank cyanobacterially contaminated water impounded by the solomon dam in australia (hawkins et al., ) . cyanobacterial toxins have also been implicated in mortality in wild fisheries and terres trial mammals, and can accumulate in the ecosystem . toxins are classified by how they affect the human body (who, c): hepatotoxins (which affect the liver) are produced by some strains of the cyanobacteria microcystis, anabaena, oscillatoria, nodularia, nostoc, cylindrospermopsis, and umezakia. neurotoxins (which affect the nervous system) are produced by some strains of aphanizomenon and oscilatoria. cyanobacteria from the species cylindroapermopsis raciborski may also produce toxic alkaloids, causing gastrointestinal symptoms or kidney disease in humans. not all cyanobacteria of these species form toxins and it is likely that there are other as-yet unrecognized toxins. further, cyanobacteria produce a wide range of secondary metabolites, among which new cya notoxins continue to be found. besides the recognized effects of the toxins mentioned above, a recent study opened up a discussion about possible new causes of known neurodegenerative illnesses. cyanobacterial strains were found to produce the neurotoxic nonprotein amino acid, β-n-methylamino-l-alanine (bmaa) (cox et al., (cox et al., , . the bmaa is considered a possible causative agent of human motor neuron disease, amyotrophic lateral sclerosis parkinsonism/dementia complex (als/pdc), and has been found to accumulate in brain tissues of patients with progressive neurodegenerative illness (murch et al., ) . this was first detected in humans as a consequence of dietary habits and biomagnification in terrestrial ecosystems in guam (cox et al., ) . however, sometime later, bmaa was detected in aquatic ecosystems, such as in seawater in the area of a trichodesmium bloom and in freshwater and brackish waterbodies (metcalf et al., ) . cox et al. ( ) isolated bmaa in several species of free-living cyanobacteria. possible implications of these findings (cox et al., ; ince and codd, ) are that bmaa of cyanobacterial origin might occur in diverse natural and controlled environments where massive cyanobacterial populations occur. because these environments can include waterbodies used for drinking and recreational use, bmaa in drinking water may contribute to chronic intoxication. this may involve incorporation of the neurotoxin into an endogenous protein reservoir with slow release (murch et al., ) and a possible lag between expo sure and effect of years or decades. since protein-associated bmaa can accumulate within food chains, it is possible that biomagnification of bmaa occurs in marine ecosystems in a way similar to that in terrestrial ecosystems (banack et al., ) . thus, production of bmaa by marine cyanobacteria may represent another route of human exposure to this neuro toxin. cox stated that the bmaa produced by the algae may act as a slow toxin. for these reasons, it would be advisable to monitor bmaa concentrations in drinking waters contami nated by cyanobacterial blooms and in fish and animals that may be ingesting the microbes, even at low environmental densities. this can be particularly relevant in semiarid or arid regions that are highly dependent for their drinking water supply on reservoirs, in which cyanobacteria can regularly occur on a seasonal basis, normally in summer. fertilizer use, widespread cultivation of leguminous crops, and fossil fuel burning have produced strong changes in the global n cycle (galloway et al., ) . more than half of all n fertili zers ever used in earth's history has been applied in the past two decades (howarth et al., ) . moreover, increased n loading in the environment often occurs together with that of phosphorus. in freshwater ecosystems in particular, a com bined increase in both n and p is highly likely to drive eutrophication and associated significant ecological change. mckenzie and townsend ( ) reviewed direct and indirect evidence that changing global nutrient cycles are influencing the dynamics and incidence of pids ( figure ). some of the disease agents discussed earlier in this chapter could react particularly sensitively to increased n and p loads. although generalizations are difficult, the evidence presented by these authors, together with past reviews by lafferty ( ) and townsend et al. ( ) , suggests a trend in which most asso ciations between increased nutrients and disease are positive. a parasite or pathogen may respond to nutrients directly, that is, without requiring an intermediate or vector host; or an inter mediate or vector host may respond to nutrients, mediating the overall disease response. there can be an increase in vector host population density as a result of increased primary productivity, or a decrease in population due to some disturbance related to nutrient addition, in both cases resulting in changes in transmis sion success of the parasite or pathogen (anderson and may, ; dobson, ; arneberg et al., ) . besides the above-mentioned increase in cyanobacterial blooms, increased nutrients could particularly affect ecological processes associated with, for example, malaria, schistomiasis, cholera, and filariasis, leading to a higher occurrence of these diseases. for example, vector-borne pathogens that cause malaria and schistosomiasis involve intermediate hosts (mos quitoes and snails) whose growth and reproduction depend on algae or weed abundance in their respective environments. where n additions cause greater plant growth and/or changes in plant species composition, the density of these intermediate hosts is also likely to be affected, with cascading consequences for the risk of disease. lafferty ( ) reviewed parasitic responses to a suite of environmental changes and found that, in most cases, eutrophi cation caused an increase in parasite abundance. johnson and carpenter ( ) suggested that planorbid snails in nutrient-rich environments grow faster. larger individuals are able to produce significantly more trematode cercariae at a faster rate. the avail able evidence suggests that, in general, nutrient additions should favor trematode development and increase disease risks. in the case of cholera, the bacteria display a strong associa tion with marine plankton, and, therefore, factors that cause increases in plankton primary productivity can also increase the prevalence of v. cholerae. thus, nitrogen-based eutrophication of coastal regions (nrc, ) has been linked to increased cholera risks (epstein, ; colwell and huq, ) , prob ably as an indirect response to plankton dynamics. changes in the n cycle are not globally uniform (galloway et al., ) . the largest changes have occurred in industria lized countries but the focus of global change is shifting to tropical and subtropical countries, where greatest relative changes in the n cycle over the next years are expected (galloway et al., ; dentener, ) . for example, the booming soybean agriculture in brazil in the last decade is leading to rapid increases in regional n deposition and nutrient loading to aquatic ecosystems (martinelli et al., ) . similar increases are occurring throughout tropical and subtropical portions of asia and central america. these same regions harbor the greatest diversity of human pids (guernier et al., ) , including those that currently cause the majority of pidrelated human deaths (who, b) . thus, the next decades will probably bring an increase in the potential for nutrients, especially n, to affect parasitic and infectious diseases in these regions. the changes in temperature and rainfall regime in temperate regions and the changes in land use resultant from them or from market shifts can exacerbate that potential. diseases produced by viral infections can emerge, reemerge, and be transmitted by several different, but often interlinked, mechanisms involving tight interactions between man and domestic and wild animals sharing artificial or natural aquatic systems. table summarizes some viral diseases of worldwide relevance. in the following section, we will focus on avian influenza due its multiple and complex connections with the dynamics and use of wetlands by man and animals. . . . . influenza: human and animal links between artificial and natural wetlands nutrient reduction in wastewaters or surface waters is a prior ity for reducing risks to human health. nevertheless, from the point of aqua-and agriculture, the nutrients contained in wastewater are a valuable resource, in particular in arid and semiarid regions. wastewater form has been used for aquaculture, for example, in duck ponds, in several countries, mainly in asia, for centuries to produce human food. organizations such as who, food and agriculture organization (fao), etc., have developed guidelines for the safe use of wastewater, excreta, and gray water in agriculture and aquaculture in order to provide a basis for the develop ment and implementation of health risk assessment and management approaches, including standards and regula tions, to address hazards associated with human waste-fed aquaculture (edwards, ) . however, wastes and fecally polluted surface water are often used without any pretreat ment or assessment of the presence of pathogens. various hazards are associated with waste-fed aquaculture: excretarelated pathogens (bacteria, helminths, protozoans, and viruses), skin irritants, vectors that transmit pathogens, and toxic chemicals. wastewater systems were also developed independently in india from the s, in china from the s, and in vietnam from the s onward, but they were designed primarily for aquaculture, not to treat wastewater. few engineered waste water-fed aquaculture systems have been developed recently. systems primarily engineered to treat wastewater that incorpo rated aquaculture were developed in germany from the end of the nineteenth century, but only the munich system remains, and only for tertiary wastewater treatment and currently as a bird sanctuary (edwards, ) . in this, the following aspects in the dynamics of bird popu lations are of ecohydrological and biomedical relevance. during migratory movements, birds carry pathogens that can be transmitted between species at breeding, wintering, and stopover places where numerous birds of various species are concentrated, such as wetlands. a study by jourdain et al. ( ) focused on bird migration routes to the camargue in relation to risk of pathogen dispersion into western mediterranean wetlands. they considered two pathogens clo sely associated with wild birds: avian influenza (ai) virus and west nile virus (wnv). the ai viruses have a water-borne transmission, and ducks are their main natural reservoirs (easterday et al., ; alexander, ) ; wnv has a vectorborne transmission, and passerines are believed to play a major role in the amplification cycle (hurlbut, ; malkinson and banet, ) . despite different transmission cycles and ecology, both viruses are known to be carried by reservoir birds during migration and have been associated with emer ging disease transmission risk for humans and domestic animals (rappole et al., ; reed et al., ; olsen et al., ) . environmental conditions, avifauna abundance, and diversity, as well as the interactions among birds from different species and departure sites in stopover wetlands, may be of key importance in terms of virus communication (hudson et al., ) . for water-transmitted pathogens such as ai viruses, the risk of transmission may be associated with the number of ducks congregated on the same water body, particularly in autumn and winter. this crowding of wintering species, in addition to the permanent presence of a transient population of birds using wetlands to stop off during migration, could provide the conditions for the circulation and rapid dissemi nation of ai viruses. for vector-transmitted pathogens such as wnv, transmission possibilities depend both on reservoir bird density and on the dispersion capability and activity periods of the arthropod vectors. the risk for interspecific transmission of disease is particularly problematic when wild and domestic species are involved. ducks are the aquatic birds most likely to come in contact with free-range poultry, especially because the presence of congeners can induce migrating wild ducks to make a stopover (jourdain et al., ) . the study by jourdain et al. ( ) showed that western mediterranean wetlands are a hub for birds from several differ ent origins in central asia, siberia, northern and eastern europe, western africa, and the mediterranean basin. as exam ple for the potential of wetlands for introduction or reemersion of these viruses, they state that wnv dispersion by birds migrat ing from sub-saharan africa might explain why an outbreak occurred in in the camargue, even though the virus had not been observed there since the s. besides migration, breeding ducks in the aquaculture ponds can increase virus circulation between ducks, water, sediment, fish, and man. in a study (markwell and shortridge, ) of the occurrence and persistence of influ enza viruses (hong kong type) within domestic duck communities, the virus was isolated throughout the year from feces or pond water or both, indicating a cycle of water-borne transmission. infection was asymptomatic and virus persistence in the duck community appeared to be dependent upon the continual introduction of ducklings sus ceptible to infection onto virus-contaminated water, since the feces of ducks - days old were generally virus-free despite the ducks' exposure to the virus in pond water. the normal practice of raising ducks of different ages on the same farm, where the water supplies are shared (figure ) , appears to be instrumental in maintaining a large reservoir of influenza viruses in the duck communities. domestic ducks may act as a silent reservoir for the h n ai virus. the concern is greatest in rural areas of affected countries, where traditional free-range ducks, chickens, and wildlife frequently share the same water source. domestic ducks can harbor the virus for long periods and without showing any sign of illness. an altered role for domestic ducks is further supported by evidence that the h n virus circulating in parts of asia has increased its virulence in chick ens and mice (a laboratory model for mammals), and has expanded its host range to include larger mammals (e.g., cats and tigers), not previously considered susceptible to infection (fao, ) . the assessment of respective roles of routes and timing of wild waterbird migration and poultry imports is of utmost importance in order to objectively identify the origin and pos sible evolution of an outbreak in a determined country. for example, the avian flu outbreak in nigeria in may have been caused by the supply of infected live poultry including day-old chicks from different sources, including east asia and turkey, and not by wild waterbirds. this is supported by sam plings of wild waterbirds in african wetlands, in which no evidence of the h n virus was found, indicting that wild birds probably played a relatively minor role in the spread of ai in that region. northward migration of wild birds from africa to europe in the northern spring of did not cause any major outbreaks. nor do wild birds seem to play a role in indonesia, where h n has been present for some years and several cases of human infections have been recorded. however, although not many major outbreaks took place in europe in , there is evidence to suggest that wild birds did play a significant role in spreading the disease on the european continent (aiweb, ). useand scarcityon human health: some examples from aquaculture, megacities, dams, and intensive agriculture aquaculture in or close to wetlands is increasing worldwide. besides being responsible for massive wetland destruction, aqua culture itself faces serious problems, arising from several diseases that can affect shrimp ponds. among the groups of microorgan isms that cause serious losses in shrimp culture, the best known are bacteria because of the devastating economic effects they have on the affected farms. as mentioned before, vibrio organ isms attach themselves preferentially to chitin surfaces, such as in zooplankton and shrimp exoskeletons. bacterial diseases, mainly due to vibrio, have been frequently reported in penaeid shrimp culture systems. at least vibrio species are implicated in disease outbreaks in shrimp, including vibrio harveyi, v. alginolyticus, v. vulnificus, v. parahaemolyticus, and v. cholerae (non-o ) (venkateswara, ; figure ). vibrios can produce different chitinases to degrade various chitin types (svitil et al., ) in marine, estuarine, and figure example of vibrio disease affecting shrimp. in such severe cases, extensively melanized black blisters can be seen on the carapace/ abdomen of the infected animals. copyright national institute of oceanography, dona paula, goa, india, . pond and irrigation canal next to live poultry markets. in http://www.eastwestcenter.org/index.php?id= &print= freshwater environments. further, meibom et al. ( ) found that v. cholerae can acquire new genetic material by natural transformation during growth on chitin. thus, natural compe tence occurring in chitin-attached bacterial communities can act as a powerful driver of v. cholerae evolution, which could be accelerated by environmental events such as high nutrient input, giving rise to copepod blooms. it is still unclear whether growth on a determined type of chitin substrateand the production of the corresponding specific chitinasespromotes the capture of external genetic material by vibrios. this empha sizes the need for biogeochemical characterization of different aquatic microhabitats, such as different types of chitin-contain ing particulate matter, besides living zooplankton, in environmental studies of vibrio diversity and virulence. as organic-matter-rich aquatic environments contain multiple microbial strains and species and high concentrations of bac teriophage and free dna, horizontal gene transfer (hgt) provides the most likely explanation for why vibrionaceae have developed high levels of genomic diversity (meibom et al., ) . interestingly, the cholera toxin gene (ctx) is a phage-mediated mobile genetic element that is transferrable to genetically closely related bacterial strains. in the aquatic environment, vibriophages can regulate seasonal disease out breaks, for example, seasonal cholera epidemics in dhaka were inversely correlated with the prevalence of environmental cho lera phages (faruque et al., ) . the v. cholerae (non-o ) is frequently isolated from sewage, estuarine waters, and seafood in cholera-endemic and noncholera-endemic countries. it has been associated with sporadic episodes of diarrhea worldwide, but has not caused pandemics. interestingly, in , there was the first report of an epidemic of diarrhea caused by v. cholerae non-o that produces heatstable toxin, affecting khmers in a camp in thailand. in con trast to the v. cholerae o isolated from the same camp, in % of the cases, the non-o were resistant to three or more anti biotics (bagchi et al., ) . this calls attention to several important aspects in relation to threats to human health. the fact that the toxin is heat stable could imply a higher diarrhea risk for humans consuming cultured seafood as cooking would not completely eliminate toxin activity. its higher resistance to antibiotics compared with v. cholerae o potentially increases the disease hazards associated with these non-o strains. further, as mentioned previously, the abundance of chitinac eous substrate can favor mutations. thus, just as v. cholerae o evolved from a nonpathogenic to a pandemic-causing form (faruque et al., ) , it cannot be excluded that the high individual density of aquaculturein the same way as megacitiescould be favoring the emergence of new and highly pathogenic vibrio types. in many of the world's cities, water management and sanitation are in crisis and will dramatically worsen with the continuing growth of cities and slums. sewage pollution is the largest and most common type of pollution and one of the most common causes of illnesses. illnesses caused by sewage pollution are estimated to affect the health of more than million people at any one time. contaminated water, inadequate sanitation, and poor hygiene cause over % of all disease in developing countries; diarrhea is the world's second most serious killer of children, but paradoxically in % of cases it could be easily prevented or treated. pollution of water sources by sewage contributes to billion cases of diarrhea in the world each year, killing some . million children under the age of . poor sanitation currently affects . billion people, % of the world's population, who lack access to even the most minimal toilet facilities. the number of people without sanita tion will double to almost billion in , as the world becomes more urbanized (anonymous, ) . some people are moving to cities from the country side every day. at least million people in africa, asia, and latin america now live in squatter settlements without any sanitation whatsoever. the pollution of rivers and groundwater by sewage spreads disease and causes environmental degrada tion. in latin america, as a whole, only % of sewage receives any treatment. in asia, the level of sewage in rivers is times higher than the united nations (un) guidelines. levels of suspended solids in asia's rivers almost quadrupled since the late s. every minute . million liters of raw sewage are dumped into the ganges river (vidal, ) . most megacities are in asia. dhaka's population rose from in the early s to ∼ million today, including the metropolitan area, and will probably reach million in . in the same time, kolkata's population grew from to million; including the metropolitan region, its population is million and is predicted to reach million in . pakistan experienced one of the highest growth rates of popu lation worldwide: it quadrupled in only years to over million in (prb, ) . according to the world bank, karachi is one of the fastest-growing megacities of the world and is expected to rank seventh by the year (kamal, ) . the country faces a serious situation in terms of water availability, depletion, and pollution of its water bodies and irrigation systems as well as a severe degradation of its coastal ecosystems. this complex and multifaceted setting will be clo sely analyzed in the following sections. it has been reported that at least of the towns of the city are supplied with water unfit for human consumption, in most cases infected with escherichia coli. only around % of the total sewerage generated by karachi at present is treated. the e. coli, found in human feces, and other bacteria found in drinking water could cause life-threatening diseases, including diarrhea and cholera. the bulk of the drinking water concerned is taken from the indus river. bacteria easily enter the drinking water as the pipelines are rusted and leaking (irin, ) . lowintensity seismic activity, though normally not felt by people, probably further damages worn-out pipelines. additionally, due to the water shortages, the pipelines remain empty for a considerable amount of time daily, during which time they develop negative pressure and absorb moisture and sewage that has leaked from the nearby, similarly worn-out sewers. additionally, after rains, rainwater mixes with sewage and gar bage can enter the pipelines through the leaks to contaminate the drinking water supply, making people vulnerable to numer ous health hazards (hasan, ) . karachi is situated in a desert. however, in a study by sheikh et al. ( ) the analysis of microbiological data for the period - showed the permanent presence of cholerawith seasonal periodicityin karachi. cholera cases peak each year between may and august in both epidemic and nonepidemic years. both v. cholera o and o serogroups were involved in the outbreaks in and ; o disappeared in . the role of rain in disease seasonality and incidence is not clear. rainfall is scarce and sporadic, usually only between june and august. in , the city could have been flooded, with wide spread overflowing sewers, whereas, in , there was virtually no rain at all. cholera appears each year before the rains, and epidemic years in the - data set appear to have occurred independently of rainfall. like most enteric diseases in an endemic setting, cholera in karachi is a disease of young children. the mean age of patients with acute cholera in karachi closely resembles that in the rest of the indian subcontinent where social conditions are com parable. despite this association with poverty, % of the patients in the study were admitted to expensive private rooms in good hospitals. this shows that, in a city with a sanitary infrastructure like that of karachi, personal wealth affords no protection (sheikh et al.. ) . visitors and tourists are also at risk. in the same context, despite the common perception that bottled water was safe and pure, microbiologi cal tests showed that about % of samples of water supplied in many bottled brands tested all over the city was unfit for human consumption (hasan, ) . according to the same above-mentioned study, in , the new strain of v. cholerae, serogroup o , established itself in karachi and during and karachi experienced over lapping, but distinct epidemics of both strains. the serogroup o never wholly replaced serogroup o , and by it had disappeared. at that time, it was still unclear whether the dis appearance would be permanent, or whether o would reemerge in subsequent epidemic years. karachi is in a semi desert area, and the strain may not be able to maintain itself outside the human host (sheikh et al., ) . this strain has also shown diminished ability to maintain its epidemic potential in bangladesh, and it has been suggested that one reason for this may be that it is less able to persist long term in the aquatic environment (faruque et al., ) . however, years later, in july and june , cholera outbreaks were detected by a diarrhea surveillance system in a fisher village near karachi (siddiqui et al., ) . the first outbreak was caused by v. cholerae o and the second one by serotype o . it would be erroneous to conclude that, because of the relatively small number of persons affected, these cases are not relevant in terms of public health. on the contrary, they should be considered extremely valuable indicators of environmental change, especially of aquatic systems, which provide an early warning of possible future trends for policymakers and sanitar ians. water source was a risk factor only in the first outbreak: a reservoir in the village containing brackish water was only used for washing utensils and clothes and for bathing. only illness caused by v. cholerae o was associated with the use of reservoir water, while o cases were not. washing the clothes of infected persons may have introduced the pathogen into the reservoir. this implies that v. cholerae o was probably able to survive for a time outside the body and in water long enough to infect other people. this may be partly because the water was salty and that v. cholerae is a salt-loving bacterium. in summary, fecal pollution, increased nutrients, turbidity, and sodium content create favorable conditions for the propagation of v. cholerae in pakistan's coastal zone, megacities, and irrigation systems. when the increasing pollu tantchemical and microbiologicalload reaches the coastal region, it encounters a disturbed wetland ecosystem (discussed in section . . . . ), where vibrios could potentially multi ply and mutate to new pathogenic types. a close regular surveillance of vibrios at basin level, including in the coastal region, rivers, and channels, and in humans is essential in this region, which has the potential to become an epidemic center. further, restoration of riparian forests and wetlands habitat should be a priority to avoid further habitat loss, and potential host shifts, although salinization would probably preclude the reinstallation of the same species existent before dam building and population explosion. the hoogly river is the most important source for the water supply of kolkata. through an agreement with bangladesh, only a determined amount of water from the ganges can be diverted into the hooghly river during the dry season. although this does not increase the amount of pollution in the river, it does increase its concentration. during the mon soon, rubbish and feces are washed out from the city into the ground and into the river (karthe, ) . around % of the water supply is lost through leakages in the obsolete distribu tion network, reservoirs, and public water tap connections. however, the disposal of sewage is an even greater threat to human health. reduced capacity of the inadequate sewer net work, aggravated by obstruction caused by mud or garbage, as well as flooding during the monsoon produces pollution of surface and groundwater with enteric bacteria. as in karachi, interruptions or shortages in the water supply produce a negative pressure in the pipelines, which then absorb polluted water that had leaked from sewers and the surface, especially during the monsoon season. before and after the monsoon, water quality increases, that is, bacterial load decreases. following several cholera epidemics, the chlor ination of unfiltered water supply began in . this reduced cholera incidence, but supply of potable water to many parts of the city is still insufficient. people in shanties frequently get untreated water from hydrants, or from the river (hensgens, ) . cholera seasonality in kolkata is further discussed in section . . . . . the situation in london years ago resembled current conditions in many megacities in developing countries. by , half of the population of britain was living in townsthe first society in human history to do so. over the previous years, britain's population had risen at an unpre cedented rate. large towns were desperately unhealthy, with death from sickness at a level not seen since the black death (daunton, ) . london had a large scavenger class living off the refuse of the citya group so numerous that it could have formed the fifth largest city in england. new epidemics affected the citiescholera and typhoid were carried by polluted water, typhus was spread by lice, and 'summer diarrhea' was caused by swarms of flies feeding on horse manure and human waste. london suffered from recurring epidemics of cholera and in - more than londoners were killed by the disease (johnson, ) . the frequent occurrence of cholera in london gave impetus to legislation, enabling the metropolitan board to begin work on sewers and street improvements. by most of london was connected to a sewer network brilliantly devised by joseph bazalgette (bbc, ) . the flow of foul water from old sewers and underground rivers was intercepted and diverted along new, low-level sewers, built behind embankments on the river front and taken to new treatment works. by , both the albert and the victoria embankments had been opened. the victoria embankment protected bazalgette's low-level sewer from the hydraulic pressure from the thames estuary. the chelsea embankment was completed in . the public health act of required local authorities to implement building regulations, or bylaws, which insisted that each house should be self-contained, with its own sanitation and water. this change in the design of housing complemented the public investment in sewers and water supply. cholera never reappeared in london after that. london was the largest city on the planet in , but now it is on the small side, in comparison to, for example, mexico city, são paulo, or mumbai. massive shantytowns have exploded at the margins of today's megacities. in such places, the water-borne diseasesincluding cholerathat plagued victorian london are still widespread, thanks to insufficient public health and sanitation resources. worldwide, up to a billion people live in shantytowns and according to some projections this will increase to a quarter of the world's popula tion by (johnson, ) . despite enormous progress in the molecular biology of v. cholera, still little is known about basic forces, such as spatial biogeochemical gradients, seasonal rainfall variations, or cyclones, driving its abundance, diversity, and virulence in the basins of rivers and estuaries of the indian subcontinent. the most recent detailed studies on seasonal variations of estuarine salinity and related urban cholera incidence are from the s (chatterjee and gupta, ) . these compare the river systems kolkata-hoogly (a main branch of the ganges) and london-thames (data from the nineteenth century), and are discussed in miller et al. ( ) . an important aspect of the s data is that minute but clearly delimited salinity oscillations (e.g., . - . and . - ppt) in the hoogly river tightly correlate with cholera incidence in kolkata. several authors reported significantly higher salinity ranges for the growth and persistence of v. cholerae in the environ ment, for example, . - ppt (miller et al., ) or - ppt (singleton et al., ; louis et al., ; randa et al., ) . salinities < ppt were considered suboptimal (miller et al., ) . however, kolkata's data clearly indicate cholera out breaks at much lower salinities. such oscillations could also be a proxy of other processes occurring at basin level, which were responsible for triggering the cholera outbreaks. vibrio survival at low salinities can be facilitated by adsorption onto algae, zooplankton, or by high nutrient concentrations. unfortunately, this information mostly originated from stag nant water bodies or short-term investigations (e.g., islam et al., , and references therein) , but no studies are available about the seasonality of hydrology, biogeochemistry, and vibrio dynamics in flowing waters of the large rivers in this region through which vibrios most likely spread from the coastal zone toward inland habitats. since the s, seven large dams for irrigation purposes have been constructed in india. the farakka barrage, complete in , diverts the ganges river water into the hooghly river during the dry season to flush out the accumulating silt in the port of calcutta. it cuts off bangladesh's water supply, elevating salinity, and has affected fisheries, caused desertification, and hindered navigation, and poses a threat to water quality and public health (wolf, ) . there is evidence of changes in cholera seasonality due to hydrologic disturbances. before the s, the peak cholera season in dhaka was november-february; now it is september-november. in kolkata, season ality has changed twice since the mid- s (niced, . these shifts may be related to changes in salinity, particle load, and associated estuarine biogeochemistry due to, for example, the construction of the farakka barrage on the river ganges (mirza, ) or increased melting of himalayan glaciers (unep, ) . more that years ago, miller et al. ( ) postulated that dam construction in india could influence vibrio dynamics by salt intrusion. these aspects deserve further investigation; apart from the s data, there are no other published data series systematically relating these parameters in the rivers of the region with cholera incidence. this informa tion is essential in order to evaluate the transboundary effects of dam construction and water management. dam construc tion in india has reduced riverine discharge in bangladesh, inducing desertification in its northern sector and facilitating salt intrusion into its estuaries, particularly in the southwestern region (wolf, ; adel, ) . this resulted in changes in land use from rice cultivation to shrimp farming in the southern bay of bengal (gebauer, ) (discussed in section . . . ). salinization of inland water bodies can facilitate the spreading of the halophilic vibrio organisms and affect drinking water availability. on the other hand, global warming increases glacier melting and associated riverine runoff. both factors, in a frame of increasing intensity and frequency of cyclones and flooding events, create an extremely complex situation in the coastal zone that could result in shifts in seasonal cholera patterns. despite the different overall climatic setting of pakistan as com pared to india and bangladesh, increasing water needs in pakistan are leading to a similar situation in the coastal zone, including increased cholera incidence. the common factors are the halophilic character of vibrio cholerae and other vibrios and the salinization of estuaries and inland waters. water needs for irrigation of desertic and semidesertic areas, as well as for drinking water supplies, mainly for karachi and islamabad, have led to the construction of several dams along the indus river. within the sindh province, there are three major barrages on the indus-guddu, sukkur, and kotri. severe reduction of water flow below the kotri barrage started affecting environments in the area from s onward, with the following consequences in the river basin: ( ) drying up and death of riparian forests, figure the indus delta faces major degradation threats, whose major cause is the reduction in the flow of freshwater from the indus river. as the delta dries up and the mangrove forests decline, the sea is slowly sweeping in. which occurred soon after ; ( ) reduction of the area under fruit and vegetable crops; ( ) destruction of natural pastures causing a reduction in animal populations; and ( ) desertifica tion leading to a shifting of human settlements. in the coastal region, intrusion of seawater in the river bed to a distance of km upstream from the shore, with percolation of saline water from the riverine areas into groundwater of adjoining irrigated areas, has turned shallow water lenses brackish ( figure ) . another reason for accelerated salinization in the indus river is the saline water discharges from the salinity control and reclamation program in the north-west frontier province, punjab, and india (hasan, ) . along the coast, the increase in salinity of seawater along the whole coastline of sindh has resulted in damage to man groves, colonization by other halophilic species, and the abandonment of ∼ ha of land reserved for shrimp farming by the government of sindh. sea shrimp can survive within salinity range of - of water, but in the space of years , salinity rose beyond the tolerance limit. water salinity in sea creeks and estuaries increased from ∼ to over ppt, making estuaries inhabitable for some shrimp and other species of commercial interest. in conditions of low salinity, shrimp farming could have been established all along km of seacoast. sea fish and prawn catch has declined considerably and severe erosion due to reduced sediment load occurs along the coast. in the coastal region, the livelihood of fishing communities and the fishing industry as a whole depends on ecosystem integrity. however, this has already been devastated by reclamation of former marine areas, and of mangrove marshes and mudflats. it desperately needs to be protected, for no city that destroys the ecology of the region where it is situated can be sustainable. the south asian tsunami gave ample proof of this and so did the flooding of karachi, much of which is the result of reclama tion from mangrove marshes, creeks, and natural drainage channels for elite real estate (hasan, ) . in addition to the deeply disturbed aquatic ecosystems described above, only % of cities with a population of over have wastewater-treatment facilities. of the wastewater generated daily, % is used in agriculture and % is disposed of into rivers or the arabian sea (iwmi, ) . directly or indirectly, % of the people of sindh, in rural or urban regions, drink water from the indus. salt content at kotri reaches . ppt in winter months, and supplies to karachi range from . to . ppt (panhwar, ) . the combina tion of a riverine environment with increasing salinity, growing populations, lack of sanitation, input of untreated sewage, drinking water contaminated with enteric bacteria, and rela tively high sodium content is very similar to that in london during the cholera epidemics in the nineteenth century (see section . . . . ). recently, a cholera outbreak in mirpur khas, a district of sindh province, has been reported (anonymous, ) . scientists at the university of health sciences found out that around % of the patients were affected by cholera instead, as was previously thought, by gastroenteritis. the authorities have been requested to pay particular attention to this outbreak of cholera, which could spread to the other areas of the province. mirpur khas has a population of ∼ , has successful agriculture, and is con nected to the indus via irrigation canals. the irrigation system of pakistan has been considered "one of the largest contagious systems of the world" by gachal et al. ( ) . the reservoir formed by the three gorges dam (tgd) is the largest in the world at over km in length. studies about the impact of parasite dynamics have been restricted to medically important species such as schistosoma japonica (zheng et al., ) . these studies have focused on the distribution of the intermediate host oncomelania hupensis within the tgd area and associated downstream water bodies. before construction of the dam, neither o. hupensis nor s. japonica occurred in the reservoir region. however, it is widely predicted that the dam will lead to the introduction of s. japonica into the tgd region, while downstream both positive and negative effects on schistosomiasis transmission will occur (zheng et al., ) . nevertheless, a survey of the recently filled reservoir (jobin, ) concluded that because of the very steep shoreline along most of its length and subsequent narrow photic zone, which provides only a limited area for the growth of plants, there was little chance of o. hupensis, principally a marshland snail, becoming established. however, these unfavorable topo graphic conditions do not preclude other mollusk species colonizing the shoreline (morley, ) . for example, former gravel quarries converted into lakes have a steep-sided profile with a narrow discontinuous zone of plant life but support a diverse molluskan-trematode community (adam and lewis, ; morley et al., ) . river blindness is an important parasitic disease around tropi cal dams in africa, along the red sea in arabia, in central america, and in parts of south america. the rapids of the upper nile river used to be a classic focus of this blinding parasite, spread by the bite of a species of blackfly, which breeds in white-water habitats and on dam spillways. in uganda (jobin, ) , the history of river blindness can be traced to the owen falls dam in the upper nile river. this dam flooded out owen falls and also ripon falls near the outlet of lake victoria. the history of the impact from the owen falls dam on onchocerciasis over the last years shows the importance of optimal water current velocity regulation for the avoidance of reduction of this and other water-borne dis eases such as schistosomiasis. prior to construction of the owen falls dam (figure ), river blindness was endemic among the buganda people, downstream along the nile. in , the prevalence of the parasite was %. to protect the workers, weekly applications of ddt were made at the outlet of lake victoria, treating the entire flow during the construction phase, and eliminating the blackflies for at least km downstream. after dam completion in and discontinuation of ddt applications, the black flies did not return to their former habitats downstream of the dam. by , the prevalence of the parasite had decreased to . % among the populations along the river. the fact that blackfly populations did not return to former levels indicates that there must have been a change in the basic habitat condi tions in the river. the dam had two major hydraulic effects on the river: one effect of turbine operation is a reduction of velocities downstream. the preferred range of water velocity for breeding of the east african species of blackfly involved in river blindness is between . and m s − . at present, the mean velocities downstream of owen falls dam are between . and . m s − , slightly less than the required velocities. prior to dam construction, the mean velocity was roughly twice the present, and thus highly suitable for blackfly breeding. yet, the major ecohydrologic effect of the dam reservoir was the complete submergence of owen falls and ripon falls upstream of the dam. these falls were preferential sites for blackfly breeding, and their permanent submergence eliminated the breeding completely. in west africa, dam construction has led to a significant switch in dominant water-borne diseases. the volta river basin extends over six west african countries ( % in burkina faso, % in ghana, and % in togo, benin, cote d'ivoire, and mali) and covers an area of about km . the volta lake is the largest man-made lake in the world, created after the construction of the akosombo dam in . the primary purpose of the project was to supply cheap electricity to smelt aluminum, other significant uses being transportation, fishery, water supply (commercial and domestic purposes), tourism, and irrigation. construction of the volta lake led to the resettlement of about people from several hundreds of villages. in the other riparian countries of the basin, small and larges dams have been built by governments, nongovernmental organizations (ngos), and local people to secure food produc tion after the severe droughts that occurred in the s and s. in the nakambe sub-basin (burkina faso) alone, more that small dams have been built, mostly during that period (barry et al., ) . there have been serious health issues associated with the volta lake, in particular with two major diseases: schistosomia sis and river blindness. before the creation of the volta lake, schistosomiasis was endemic in ghana; but endemicity was low along the volta river. according to an epidemiological survey done in - before the lake was formed, infection rates of schistosomiasis in the area had been - %, mostly affecting children. in the asukwakwaw area, north of the akosombo dam, the prevalence of onchocerciasis is now almost %. principal public health impacts of the formation of the lake have included reduced prevalence of river blindness, but increased incidence of urinary schistosomiasis and a mas sive increase in malaria, as discussed in the following paragraphs. the dam virtually halted the rate of flow in the volta river, increasing stagnant water conditions and consequently creating ideal breeding grounds for carriers of water-borne diseases. in the period following the construction of the dam at akosombo, there has been a steady decline in agricultural productivity along the lake and the associated tributaries (gyau-boakye, ). the land surrounding lake volta is not nearly as fertile as the for merly cultivated land residing underneath the lake, and intensive agricultural activity has quickly exhausted the already inade quate soils. without the periodic floodings that brought nutrients to the soil, before the natural river flow was halted by the dam, upstream agricultural systems are also losing soil ferti lity (van de giesen et al., ) . the growth of commercially intensive agriculture has produced a rise in fertilizer runoff into the river. this, along with runoff from nearby cattle stocks and sewage pollution, has caused eutrophication of the river waters (gyau-boakye, ) . this nutrient enrichment, in combination with the low water movement, has allowed for the invasion of aquatic weeds (cerratophyllum) (fobil and attaquayefio, ) . these weeds, associated with the aquatic snail, the 'intermediate host', together with mass migration into the fishing commu nities from regions in which the disease was endemic, have led to a great increase in the prevalence of schistosomiasis in many localities around the lake. the presence of aquatic weeds along the lake and within tributaries has resulted in even greater devastation to local human health as they provide an excellent habitat not only for snails but also for mosquitoes (gyau-boakye, ) . before the construction of the akosombo and kpong dams, malaria was not much of a problem along the swift-flowing volta river, but, when it became a stagnant lake, it became a greater public health problem in lakeside villages. by , urinary schisto somiasis had increased to become the most prevalent disease in the area, affecting some % of lakeside residents (gitlitz, ) and reaching a prevalence rate of % among children in certain localities. the problem of schistosomiasis in the lake basin must be seen as embracing both the lake and the volta delta. the migratory habits of the fishermen ensure the spread of the disease from endemic areas to other areas. in particular, resettlement villages have showed an increase in disease pre valence since the establishment of lake volta, and a village's likelihood of infection corresponds to its proximity to the lake. previously, the population in the basin generally lived away from the main watercourses because of the threat from water borne diseases. children and fishermen have been especially hard hit by this rise of disease prevalence (zakhary, ) . additionally, the degradation of aquatic habitat has resulted in the decline of shrimp and clam populations. the physical health of local communities has declined as a result of this loss of shellfish populations, as they provided an essential source of dietary protein (fobil and attaquayefio, ) . conversely, while leading to a dramatic increase in schisto somiasis, the lake has flooded out the riparian forests which constituted a breeding place for a species of tsetse fly, glossina spp. from the palpalis group, the vector of protozoan trypanosoma brucei gambiense, which causes the western african trypanosomiases (sleeping sickness) in people. the lake has also inundated and eliminated the major breeding sites of the onchocerciasis blackfly in rapidly flowing streams and rivers north of the akosombo dam. the construction of the second dam at kpong also eliminated the breeding sites downstream of akosombo and therefore stopped the transmis sion of river blindness in the vicinity. the main benefit to health because of the construction of the akosombo dam in and the kpong dam in is undoubtedly the reduc tion of the incidence of onchocerciasis in the volta basin. about fishermen living mostly in isolated villages around the lake were exposed to the riverine disease and did not have access to health facilities (jobin, ) . increased global demand for biofuels has incentivized sugar cane plantation in brazil, giving rise to several social and environmental modifications (martinelli and filoso, ) . the widespread occurrence of generalist animal species in sugarcane areas has been associated with public health pro blems. for instance, the population increase of the semiaquatic rodent, capybara (hydrochoerus hydrochaeris), in the piracicaba river basin has led to the spread of brazilian spotted fever (bsf) (labruna et al., ) . the bsf is the most important tick-borne disease in brazil and is caused by the bacterium, rickettsia rickettsii, and transmitted by the tick, amblyomma cajennense, its main vector, and capybaras serve as host for the ticks (estrada et al., ) . r. rickettsii infections can cause a wide range of clinical manifestations, ranging from asympto matic or mild febrile illness to overwhelming and fatal disease. failure in diagnosis and delayed therapy have contributed to hidden mortality, frequently a result of atypical fulminant forms of the disease (gonçalves da costa et al., ) and physician's lack of knowledge about the disease, which is exa cerbated by the difficulty of adequate confirmatory laboratory tests during its acute phase. mortality can reach % of the infection cases. the bsf has been known in brazil since . during the period between and the s there was a marked drop in the number of reported cases of bsf in brazil, as well as in the united states (angerami et al., ) . however, since the s an apparent reemergence of the disease has been observed with an increase in the number of reported cases in the southeast of brazil (angerami et al., ) . rickettsial diseases have been considered emerging zoo noses worldwide (raoult and roux, ) and should no longer be classified as rare diseases in brazil. despite the still moderate number of bsf cases, its increasing trend, together with high mortality rates, reflects and calls atten tion to deep changes in land use and habitat structure in brazil. increased fertilizer use, pollution, and soil erosion have caused deterioration of aquatic systems. as colluvium sediments are transported downhill across the landscape from sugarcane fields, they are deposited onto wetlands, and into small streams, rivers, and reservoirs. deposition affects water quality, and ecosystem biodiversity (politano and pissarra, ) and functions. high rates of n export into rivers draining watersheds heavily culti vated with sugarcane in brazil, such as the piracicaba and mogi river basins, have been reported (filoso et al., ) . the indus trial processing of sugarcane for production of sugar and ethanol is another source of pollution for aquatic systems with poten tially harmful effects for human health. waste products (vinasse) are rich in organic matter, and increase the biochemical oxygen demand (bod) of waters receiving these effluents, often causing anoxia (ballester et al., ) . with the boom of ethanol pro duction in brazil in the early s, new legislation was enacted to ban the direct discharge of vinasse into surface waters. since then, nutrient and carbon-rich vinasse has been mixed with wastewater from washing sugarcane and is recycled back to sugarcane fields as organic fertilizer (gunkel et al., ) , although this practice is still far from generalized. as a conse quence, high nutrient concentrations in these effluents also contribute to the problem by enhancing algal blooms and pro moting eutrophication of surface waters (matsumura-tundisi and tundisi, ) . the increase and dispersion of the capybara population and the associated health risk is most likely due to a strong anthro pogenic habitat modification caused by extensive monoculture. paradoxically, this situation could be aggravated by current efforts to restore aquatic ecosystems. typical capybara habitat is com posed of two main components: water and a patch of forest or woodland. in são paulo state, capybaras and ticks share a habitat component, the riparian vegetation called gallery forest. these forests form as corridors along rivers or wetlands and project into landscapes that are otherwise only sparsely wooded, such as savannas, grasslands, or deserts. the boundary between gallery forest and the surrounding woodland or grassland is usually very abrupt, with the ecotone being only a few meters wide. in são paulo state, capybaras shelter in the gallery forest and feed in the sugarcane fields adjacent to the ecotone ( figure ) . thus, the abundant food and lack of predators in this new habitat have led to a strong population increase (labruna, ). in the s, the capybara was considered in danger of extinction in são paulo state, where population can reach densities times higher than in natural environments in some areas, such as the extended wetlands of pantanal (verdade and ferraz, ) . this offers optimum conditions for the increase in the tick and rickettsia population. further, the capybara is a protected species and is gradually adapting to aquatic urban habitats with a larger spectrum of possible vertebrate hosts for ticks ( figure ). capybaras have been observed in the outskirts of são paulo city along the highly polluted pinheiros river (labruna, ) , which flows through the fourth largest metropolitan area worldwide, with ∼ million inhabitants. presently, a substantial cleanup pro gram for this river is underway and an increase of secondary vegetation and fragmented, regenerated systems is expected. with this, a further expansion of capybaras, ticks, and bsf toward anthropogenically modified habitats, including urban and suburban areas close to water sources such as rivers and lakes, is likely. in addition, it has recently been reported (meireles et al., ) that capybaras in the são paulo state were infected by cryptosporidium parvum, which is a protozoan pathogen that causes a diarrheal illness called cryptosporidiosis, an acute short-term infection that can become severe and chronic in children and immunocompromised individuals. despite not being identified until (meisel et al., ; nime et al., ) , it is one of the most common water-borne diseases and is found worldwide, being spread by direct ingestion of con taminated water or food and through recreational water activities. the finding of zoonotic c. parvum infection in this figure aerial photo of a sugarcane plantation and a small water body with highly fragmented remains of riparian forest, brazil. photo credit: geraldo arruda, jr. semiaquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies in brazil may be contaminated with cryptosporidium oocysts from wildlife. cryptosporidiosis is the most significant water-borne dis ease associated with the public water supply in western europe. when contamination occurs, it has the potential to infect very large numbers of people. some notable outbreaks of cryptos poridiosis have been associated with heavy rainfall events. in this chapter and elsewhere (despommier et al., and references therein) , there are indications that the boundaries between ecological systems play a role in some of the most important emerging infectious diseases, with a correspondence between ecotonal processes and the ecological and evolution ary processes responsible for zoonotic and vector-borne infections. terrestrial ecotones include forest-edge habitats, fragmented forest landscapes, and forest-grassland interfaces. terrestrial-aquatic ecotones are found in riparian habitats, riverine landscapes, freshwater and estuarine wetlands, and in the coastal zone. anthropogenic ecotones can include crop land/pasture-natural habitat and settlement-natural habitat, and a combination of these. processes in ecotones can contribute to the shifts or changes in hosts, vectors, or pathogens that produce disease emergence. these dynamics are associated with changes in land cover/use and with the changing nature of the land-water interface. anthropogenic influences can intensify ecotonal processes by increasing their geographic extent and overlap. various ecotone features can contribute to disease emergence. animals congre gate in ecotones. populations of species that normally are members of distinct ecological communities from different habitats or ecosystems overlap in ecotones, facilitating patho gen spillover. host-vector hyperabundance increases the potential for pathogens to achieve critical threshold density. enhanced dispersal conditions facilitate dispersal at a higher rate or over longer distances, along linear habitats defined by habitat edges, such as riverine or gallery forest, and flowing water in streams or rivers themselves. cropland-forest-river transitions appear particularly relevant as sensitive indicators of change in this context. for example, the transition between fragmented riparian habitats such as degraded gallery forests and, for example, sugarcane fields seems to be a priority sector for the control of rickettsia-related diseases since capybaras must cross the ecotone to feed on sugarcane, as described else where in this article. further, nutrient pollution, degradation of riparian habitat, and the loss of ecological functions involving assimilation of nutrients and pathogens, combined with high concentrations of domestic fowl and their waste, are commonly associated with human settlement/aquatic-terrestrial ecotones. the emer gence of ai involved the mixing of three different communities: wild migratory waterfowl (wetlands), wild local birds, and domestic fowl (ponds), and, later, also pigs. like influenza, the emergence of japanese encephalitis has involved transmis sion in the spatial area of overlap of human settlements, agriculture, and natural habitat (despommier et al., ) . while waterbirds and wetland habitat are implicated in influ enza, nonaquatic wild birds and irrigation systems provide the vector habitat for encephalitis. the intensification and expan sion of irrigated rice production systems in southeast asia over the past years have made an important contribution to the spread of this disease, which is produced by a mosquito-borne virus (see table ). the flooding of the fields at the start of each cropping cycle leads to a sizable increase in the mosquito population. domestic pigs and wild birds are reservoirs of the virus and transmission to humans may cause severe symptoms. japanese encephalitis is a leading cause of viral encephalitis in asia with - clinical cases reported annually (who, d) . in general, research in ecotoneparticularly terrestrialaquatic dynamicscan provide vital information about changes in climate, river hydrology, sea level (cohen and lara, ) , and land use (lara et al., ) . a strengthened integration of ecological and biomedical monitoring is essen tial for successfully restoring ecological functions and enhancing environmental and human health. globally, the surveillance of locally and regionally relevant ecotones could provide evidence of disease emergence related to environmen tal change. in this context, restoration of lost ecological functions, such as nutrient sequestration by wetland creation or regeneration of riparian vegetation, must be accompanied by careful monitoring of other changes in the surrounding land scape and in the connectivity between basin processes and land use. created or regenerated aquatic systemsindependently of their purposewill increment existing ecotonal processes or generate new ones. thus, surveillance of the created/recovered/ enhanced system ecological functions should include at least those disease agents (host, vectors, or pathogens) , which according to the present knowledge would have a higher prob ability of proliferation under modified or changing conditions (e.g., mosquito larvae in temperate wetlands and snails in tropical regions). floods and droughts will intensify with climate change and affect health through the spread of disease resulting from habi tat modification, with high risk of rapid increase in diarrheal and other diseases (lipp et al., ; ipcc, ) . there are many pathways through which hydrologically relevant events can affect health; notably when a river or stream bursts its banks producing changes in mosquito abundance (malaria, and dengue), or contamination of surface water with human or animal waste such as, for example, rodent urine (leptospiro sis). flooding may become more intense with climate change and can result in the spread of disease. conversely, droughts can produce changes in vector abundance if, for example, a vector breeds in ponds left in dried-up riverbeds (noji, ; menne et al., ) . coastal ecosystems and their basins are rapidly changing due to anthropogenic pressure and global warming, also inducing changes in patterns of resource use (lara et al., ) . integrative, comparative approaches are needed for the understanding of functional links between basin structure; morphology of different estuaries, marshes, and mangroves; flooding and biogeochemical regimes (lara and cohen, ) ; pathogen life cycles; and disease incidence (wolanski et al., ; lara et al., ) . for improved prediction of the dispersal of inundation waters, formation of drought ponds, or preventive identifica tion of vulnerable locations or sectors that could be used as drainage areas, it is crucial to have a detailed knowledge of the regional and local topography. the elaboration of highresolution topographic models (dem) of basins in connection with hydrology, meteorology, and biogeochemistry data will also allow the assessment of vulnerability descriptors such as soil moisture potential, salinity, or organic matter content, which can be crucial state parameters for the development of microorganisms and/or disease vectors. however, precisely in tropical coastal areas, where the impact of climate change on vector-transmitted diseases is of high concern, there is frequently a lack of topographic informa tion with an adequate resolution for low-lying sectors. in vulnerable regions, the combination of risks to both food and water can exacerbate the impact of even minor weather extremes (floods and droughts) on the households affected (webb and iskandarani, ) . a methodological approach including wetland basin microtopography and its relation with inundation dynamics and estuarine biogeochemistry is necessary for vulnerability assess ment and risk management. the use of geographic information systems (giss) provides an excellent basis for network coopera tion at the interfaces between environmental and biomedical research, adding a critical componenthuman healthto coastal management research. this is a major concern for the who, which also has set a priority on the link between gis and disease surveillance (who, ) . through the joint who/united nations children's fund (unicef) program health map, specific gis software was developed for that purpose, combining a standar dized geographic database, a data manager, and a mapping interface. however, although these concerns are closely linked to the subject matter of ecohydrology, they are not usually included in interdisciplinary research projects dealing with, for example, coastal wetlands. clearly, the only way to concretely reduce vulnerability is to ensure that infrastructure is in place for the removal of solid waste and wastewater and the supply of potable water. no sanitation technology is safe when covered by floodwaters, as fecal matter mixes with floodwaters and is spread wherever the floodwaters run (lara et al., ) . consideration should also be given to the deterioration of groundwater quality caused by salinity intrusion due to climate change and rising sea levels (e.g., sherif and singh, ) . thus, as stated in section . . . , such health issues clearly require a basin approach, that is, considering basins as a natural unit of territorial management. however, a usual shortcoming of gis-derived vulnerability studies is that data sources from official institutions are most frequently based on municipalities or counties as an administrative unit, whose limits do not necessarily coincide with basin boundaries. thus, this vision of the relationship between climate and sealevel change and effect on human health converges with zalewski's ( ) statement that: as a consequence, the issue of water quality at the basin scale cannot be resolved without a profound understanding of the effects of hydrology on biotic processes and of biota on hydrology. the frame work for developing the principles of ecohydrology is logically the water basin scale. (zalewski, : ) creation of wetlands for nutrient sequestration from surface waters requires the inclusion of measures for control of locally major and regionally relevant disease vectors such as snails or mosquitoes. the latter are relevant for disease transmission in several climatic zones besides the tropics, and global warming is widening their habitats with severe consequences for human health (e.g., the dengue outbreak in argentina in ) and will therefore be treated with some detail. early methods of managing salt-marsh mosquitoes have primarily focused on maximizing the reduction of mosquito populations, with mini mizing environmental impact as a secondary consideration. however, in the last few decades, there have been attempts to apply diverse water management models to marsh systems, especially in terms of vector control and habitat modification (dale and hulsman, ; wolfe, ) using programs with minimal environmental impacts. the success of these programs requires a thorough knowledge of mosquito developmental conditions, as well as potential impacts on adjoining ecosys tems. a deep knowledge of the local microtopography and tidal regimes is critical. marsh drainage and hydrological linkage to the tidal source are essential in the determination of what type of wetland occurs where, and for the development of appro priate wetland management measures. elevation of a few centimeters is more critical in the coastal wetlands than that of hundreds of meters in the mountains. in this section we cover not only well-established methods for mosquito control, but a series of successful methods for snail control derived from good agriculture practice, basically from techniques for rice cultivation. ecohydrology can contri bute to and learn from these experiences. water management, molluskicides, and chemotherapy have been the main instruments for preventing or treating schisto somiasis. biological control of snails through predators such as ducks, fish, turtles, crustaceans, water rats, leeches, and aquatic insects have been also used, as yet with very limited success. an interesting lesson on the potential for improved water management to reduce vector proliferation from the s can be drawn from the experience in the baluchi irrigation scheme in then tanganyika (sturrock, ) . this system was remark ably clear of snails, although several species occurred in small numbers. several factors may account for this. first, the water flow in the canal system was very rapid when it was in use, but the canals were completely dried out in the dry season. second, silt and vegetation were dug out of the canals twice a year. third, a complex system of rice husbandry was used in which the rice fields were ploughed, manured, and subjected to a program of alternate drying and flooding. consequently, neither the canal system nor the rice fields contained much in the way of snail habitats. snails were confined to temporary pool sites, filled with rain or seepage water. all these schemes were built on land with an appreciable slope and with relatively porous soils. even when irrigation was in progress, the field canals were not always in use and dried out rapidly. while snails are often able to withstand limited periods of drought, it is unlikely that any large snail populations could develop under these conditions in any one season. furthermore, on five of these schemes, field canals were often re-routed from season to season so that the establishment of suitable snail habitats was rendered even more unlikely. mobarak ( ) reported that after prevalence of urinary schistosomiasis in upper egypt reversed a previous downward trend and began to increase again because of the shift from basin to perennial irrigation. however, in any case, increased use of parenteral antischistosomal therapy (pat, injections of antimony-based drugs) was bringing schistoso miasis under control in egypt. in the northern part of upper egypt, prevalence dropped from . % in to . % in , while further south, prevalence reportedly fell from . % in % in to % in % in (who, . most experts agreed that applying the proper combination of sanitary engi neering, water control management, snail control, infection surveillance, and treatment drugs can avert adverse effects of irrigation on schistosomiasis. moreover, there was an impres sion that even without water control measures and environmental sanitation, chemotherapy with or without treat ing water to kill snails would adequately control schistosomiasis transmission. fenwick ( ) noted that in the newer rahad irrigation scheme, east of the gezira plain in the sudan, because of the use of drugs and snail control, the incidence of schistosomiasis remained very low, despite very poor sanitary conditions. it was predicted, however, that relax ing control measures would cause a surge in schistosomiasis. although oral drugs started to be used in egypt in the s, pat use continued into the mid- s. praziquantel, which also has a high cure rate for s. mansoni, became available in egypt in and has been the treatment of choice there since the late s. although the massive pat campaigns were successful in strongly reducing disease incidence, a study by frank et al. ( ) concludes that the intensity, widespread geographical coverage, and duration of the campaigns, together with unsafe injection practices (inappropriate sterilization pro cedures), have been responsible for the nationwide spread of hepatitis c in egypt in recent decades. the authors state that the enormous dimension of egypt's schistosomiasis problem and the sheer size of the antischistosomiasis effort, combined with the characteristics of pat, provided an effective mechanism for a massive increase and establishment of hepatitis c virus in the egyptian population. according to these authors, this is "the world's largest iatrogenic transmission of blood-borne patho gens known to date." moreover, it is probable that the heavy reliance on an effective chemical treatment also allowed the continuation of water management schemes that were contri buting to the maintenance of large numbers of snails in aquatic environments. the evolution of the schistosomiasis problem in egypt and sudan described above highlights the importance of developing water management programs able to keep vector proliferation under control. this lesson is also highly relevant to the construction of large-scale reservoirs and irrigation facil ities as in the case of the three gorges dam. in israel, biomphalaria alexandrina was eradicated through a combination of factors including chemical applications. as in all cases, snails return some time after the application of mol luskicides. some combined measures used for the control of snail vectors have been successful, such as increasing water currents to over cm s − , rapid emptying and drying up of water reservoirs, and weekly deflection of infested water courses along different routes (saliternik, ) . as stated previously, the success of chemotherapy in treating this disease and of molluskicides for eradicating snails does not imply that preventive measures should not be taken for avoid ing vector proliferation based on knowledge of their ecohydrological setting. all vector snails require water, at least for breeding. the management of water bodies is therefore a potentially powerful control method. for example, in the case of rice cultivation there are opportunities for vector control by changing the aquatic habitat of the snail in a way compatible with maximum crop production. infection of humans mostly takes place not in the rice field itself, but in irrigation canals and surrounding living quarters. different hydrological and rice husbandry approaches have been used in various countries. however, generalizations should be made with care because each snail species has its own preferences and tolerances regard ing shade, water velocity, the steepness of canal walls, and drought tolerance. rice cultivation by itself can be used as an environmental method of snail control in that it brings about ecological changes that can reduce snail habitat. the philippines has promoted more intensive scientific methods of rice cultivation to control schistosomiasis (hairston and santos, ) . snail control is achieved at different stages of rice growing in a number of different ways, for example, by deep plowing, which turns over the soil and buries the snails; or by draining the ricefield at harvest and keeping it dry until the next crop, which kills the snails and prevents them from breeding. in japan, s. japonicum eradication was accomplished by treatment, sanitation, control of animal reservoir host, educa tion, and elimination of most of the snail colonies (garcia, ) . the steepness of the walls of irrigation channels was increased and later they were lined with concrete and main tained clear of silt, vegetation, and debris to supplement the eradication of snails in the ricefields, which resulted from intensive cultivation. extensive rice-growing areas in china's mainland have been cleared of oncomelania snails (garcia, ) . the measures taken have included digging new water channels parallel to the existing snail-infested streams and using the excavated soil to fill the old ones, clearing streambeds, and removing vegetation. where the soil structure permits, the banks have been made steeper. in the philippines, similar measures have been taken as in china; in addition, converting undrainable swampy areas into fishponds and improving agricultural prac tices have successfully controlled snails in limited areas. thus, an integrated approach to drainage problems can result in increased production while reducing health risks. in the ecohydrology approach, it is essential to consider the whole basin in management policies, especially when a disease agent can be transmitted by different species of the same host (in this case a snail) that is differentially distributed along altitude gradients in river basins. both b. truncatus and planorbarius metidjensis are intermediate hosts of s. haematobium in southwestern morocco. a basin investigation (yacoubi et al., ) in five rivers identified sites colonized by these species and compared the habitats in which they were found. the p. metidjensis was observed in the upper valleys of three rivers, whereas b. truncatus was found in sites of lower altitude. a component analysis demonstrated that altitude (from to m), water ph (from . to . ), and electric conductivity (from to μs cm − ) were the main descriptors of environment. the p. metidjensis was associated to high altitude and low electric conductivity. however, b. truncatus was asso ciated to being found in lower altitude sites with medium electric conductivity in water. it has been mentioned above that periodic canal or field drying had been used successfully for snail control. nevertheless, before a control plan is adopted and implemen ted, the effects of drying out have to be monitored and thoroughly understood for each snail species: snails that are capable of undergoing diapause can circumvent unfavorable environmental conditions, including long periods of drought. cooper et al. ( ) found that diapause influenced the sus ceptibility of biomphalaria glabrata snails to s. mansoni infection. juvenile snails exposed just prior to diapause, or immediately following a diapause period of weeks, were highly susceptible to infection by s. mansoni miracidia. however, snails that underwent diapause produced compar able or only slightly fewer cercariae than did nondiapausing snails. these studies indicate that diapause in b. glabrata does little to decrease a snail's ability to act as an intermediate host for s. mansoni or to interrupt the development of the parasite. for these reasons, great attention should be given to diapausing snail populations when planning programs for mollusk control. thus, for snail control, it is important that not only the agricultural field but also irrigation and drainage channels, as well as the water source, be considered part of the agroecosys tem. this parallels the ecohydrology approach that considers the whole basin, from the river source to the wetlands, estuar ine, and coastal zone as an integrated management unit. both agricultural and ecohydrological models should converge in a new synthesis integrating their own tools with practices based on traditional knowledge, socioeconomical cost-benefit analysis of vector eradication, and agri-and/or aquacultural production. the type of wetland management approach, particularly when restoration or creation is planned, will require previous surveys of the mosquito species and their habitat types, locally and at basin level. in the following sections, we present a summary of main species/habitat combinations for different hydrological settings extracted from anonymous ( b) . relatively few mosquito species breed in running waters, such as streams. larvae can be flushed out when stream volume increases, and to remain in the stream requires a large amount of energy. the tropical genus, chagasia, and some anopheles species are stream breeders. stream breeders will find vegeta tion along banks with which to anchor themselves or attempt to remain away from the main flow of the stream by seeking isolated eddies. transient water sources, such as flooded areas and ditches, are used as breeding grounds for species whose eggs can with stand desiccation and whose life cycles require alternating periods of wet and dry, such as aedes and psorophora. the quality of transient water changes with time, which can result in a succession of different species using the same pool. transient waters include woodland pools created by spring rains (aedes stimulans), fresh floodwater (aedes canadensis), and tidal floodwater (aedes sollicitans). permanent or semipermanent waters support characteristic aquatic vegetation. cattail, rushes, and sedges are typical fresh water swamp vegetation. genera associated with permanent water are anopheles, culex, culiseta, coquillettidia, and uranotaenia, whose eggs are not desiccant resistant and must be laid directly on the water. aedes adults will oviposit near the edge of the swamp, or within tussocks of vegetation, requiring later flooding of the eggs for hatching. as with transient waters, there are seasonal changes in the vegetation, water quality, and mosquito species present. permanent waters can include fresh water swamps, such as, for example, tussock (aedes abserratus) or cattail swamps (coquillettidia perturbans), as well as brackish water swamps with salt marshes (culex salinarius). besides nat ural environments, polluted water with floating debris can be a habitat for species such as culex pipiens. container water habitat can be found in both natural set tings, such as water held by plants to artificial settings and water found in tires. container water is characteristically clear and many container species now also use artificial sites as they provide insulation against the weather and are more numerous (aedes aegypti and aedes albopictus). increasing dengue incidence in not only tropical but also subtropical countries requires a thorough elimination of such urban, man-made microhabitats. there are various techniques for mosquito control based on different principles. all involve modification of the hydrologi cal setting, ranging from total wetland drainage to an increase in their tidal flooding. a summary of these methods, including parallel grid ditching, open marsh water management, and runneling, is presented in the following paragraphs. parallel grid ditching consists in the physical removal of water from intertidal marshes and was one of the first largescale forms of mosquito control (lesser ( ) ; figure ). parallel grid ditches were dug in salt marshes, spacing these ditches about m apart to remove standing surface water where mosquitoes might breed. extensive ditching programs for mosquito control in north america were only moderately effective, since many breeding potholes were not drained dry, and there were long-term nega tive effects on wildlife and salt-marsh ecosystems. many nonmosquito breeding wetlands that provided wildlife habitat were unnecessarily drained and salt-marsh vegetation commu nities were changed into fragmented wetlands. birds were particularly affected through the draining of larger natural ponds. by the early s, there was an increasing awareness of wetland values and functions, and the value of parallel grid ditching was questioned. understanding of the drawbacks of the parallel grid-ditching technique led to the development of a new mosquito control source-reduction technique called open marsh water manage ment (omwm). it started in the late s and was further optimized until the early s. the goals of omwm are: ( ) control of salt-marsh mosquitoes; ( ) reduction of insecti cide applications; and ( ) habitat enhancement for salt-marsh fish and wildlife (ferrigno and jobbins, ; hruby et al., ) . the omwm method involves the selective installation of small, shallow ponds and interconnecting ditches superim posed on known mosquito-breeding habitats ( figure ). this aims to eliminate wet-dry-wet cycles necessary for determined species and any newly created permanent water habitats are unattractive for mosquito egg deposition. this simultaneously improves habitats for mosquito-eating larvivorous fishes which can quickly invade, via tidal flooding, any newly created omwm pond or ditch. scattered mosquito breeding depressions and sheetwater habitats are connected through pond and ditch excavations to allow unimpeded water flow and predatory fish movement, while isolated potholes are often filled with natural soils to eliminate these smaller-sized breeding depressions (lesser, ; figure ). the increase of tidal inundation frequency and predation by fish significantly reduce mosquito density. runneling is an effective method for controlling mosqui toes that breed in intertidal salt marshes through a type of habitat modification using shallow channels. this technique is based on omwm principles (wolfe, ) . it increases tidal frequency to a marsh and removes surface sheet water from low-lying areas high on the marsh. runnels are linked to the tidal source, promoting tidal exchange between graded regions of the marsh. they are conceived to allow transport of lowamplitude tides to areas of salt marsh in a way so that pools do not form, even after spring tides. runnels are shallow figure the omwm system, involving the selective installation of small, shallow ponds and interconnecting ditches superimposed on known mosquito-breeding habitats. (< cm deep) spoon-shaped channels constructed along nat ural drainage lines on the salt marsh ( figure ) to a maximum gradient of : (hulsman et al., ) . due to the slight slope, runnels enable slow water movement even during lowamplitude tides. the net result is a reduction in mosquito breeding areas, the modification of pools and edges for egg conditioning (the process involving flooding and drying events that prepares mosquito eggs for hatching), and larval development. there are few apparent negative impacts at the modified site (hulsman et al., ; dale and hulsman, ; dale et al., ; latchford, ) . further, like omwm, they allow water to drain from trap pools and permit predatory fish to gain access to the mosquito larvae, at least during high tide. in comparison to grid ditching and omwm, runneling is an environment-oriented approach to salt-marsh management for mosquito control that aims to alter the salt marsh as little as possible, while causing significant reductions in mosquito numbers. the main difference between the three approaches lies in the magnitude of the habitat modification. ditching involves the greatest alteration to the marsh, and runneling the least. runneling has a lesser effect on the estuarine environ ment as a whole than does either ditching or omwm. . . conclusions . . . some reflections on dams, water scarcity, ecohydrology, and health although the construction of reservoirs is controversial, the rising demand for water by an increasing human population makes more dams inevitable (jobin, ) . in a review, morley ( ) calls attention of the fact that most parasitological studies in relation to reservoir construction have been focused on schistosomiasis and other tropical diseases of humans (stanley and alpers, ; jobin, ) . in comparison, the impact of reservoir construction on indigenous aquatic parasite fauna of wildlife has been a subject largely ignored by the scientific community. however, reservoir formation can have profound effects on the parasite fauna of fish, birds in the reservoir, as well as up-and downstream of it. changes in host-parasite relationships and switches between animal and human hosts can occur (morley, and references therein) . the role of parasites in environmental monitoring is increas ingly recognized (lafferty, ; lafferty and kuris, ) . thus, the surveillance of the effects of reservoirs on parasite fauna of aquatic wildlife may provide important general infor mation on both short-and long-term changes that occur within and downstream of new reservoirs during the maturation pro cess of the reservoir. the above-described cases that show the effect of dam con struction on onchocerciacis and schistosomiasis call attention to the need to evaluate changes in flow power and velocity downstream of proposed dams, in order to assess their likely health impact. ecohydrological measures are required to improve the operation of existing dams to provide more effective control of disease vectors (see section . . . . ). the modeling of the effect of controlled flooding pulses on the plankton dynamics in the guadiana estuary (wolanski et al., ) is an outstanding example of the potential of the appli cation of ecohydrological principles for the control of toxic algal blooms. in arid and semiarid regions, the quality of water in artificial reservoirs is essential to human health, particularly in climati cally unstable regions. for example, recent el niño/la niña-southern oscillation teleconnections have produced a pro longed drought of ∼ years in south argentina. this has interrupted a period of about years of rainfall significantly above the historical average and produced a decline to critical levels in the reserves of drinking water reservoirs, which like other water bodies in the region, have suffered from recurrent summer algal blooms (kopprio et al., ) . however, the perception that cyanobacteria can represent a threat to human health only in connection with acute events such as blooms should be widened to consider the possibility of neurologic damage as consequence of chronic exposition to toxins such as bmaa via long-term ingestion of water or aquatic fauna. this should be taken into account when developing schemes for preventing cyanobacterial blooms by manipulating natural predator communities. the reduction of the nutrient load input to the water reservoir probably remains the best preven tive measure to prevent harmful algal blooms. water treatment should be regularly checked and improved to remove the organisms and their toxins from drinking-water supplies, where appropriate. water treatment by flocculation and sedi mentation, followed by sand filtration, is supposed to remove live cyanobacterial cells and debris. however, there is evidence that plants that are not working properly can actually increase cell counts of algae with potential toxicity (in this case, anabaena circinalis and microcystis aeruginosa) in the treated water (echenique et al., ) . throughout this chapter, examples have been referred to of actual and potential conflicts of interest between alternative uses of aquatic systems, for example, between the desire to restore degraded wetlands and the need to protect health of the human population living nearby. a relevant example of policy conflicts in an industrialized country is the experience of the tennessee valley authority (tva), where health concerns in the past gave rise to management measures that conflict with modern recreational interests (bos, ) . the meeting report of the ninth meeting of the who/fao/united nations environment programme (unep) panel of experts on environmental management for vector control in includes the following passage: as national and regional priorities change, so do policies, and there must therefore always be a provision for their reconsideration and modification. sometimes, though, the acute problems that led to the original priority setting might have become latent rather than have disappeared completely, and while public awareness and political pressure favour a policy change, the original goals of such policies should not be ignored. this was well illustrated by the water man agement policies including mosquito control established by the tva in the s. the standards of mosquito control maintained by tva equalled those maintained in privately owned river impoundments under prevailing public health regulations. the measures included the programmed fluctuation of water levels in the reservoirs, a practice that played a key role in reducing anopheles populations and eradicating malaria transmission from the valley. new uses of the reservoirs, including recreation and the promotion of nature conservation had led to a conflict of interests. for recreation, stable water levels during summer and early autumn were required; con servation of certain fish species and of waterfowl required higher water levels in spring to promote fish spawning and the rapid growth of aquatic vegetation for the fowl. such changes in water manage ment regimes would without doubt result in increased mosquito populations, yet the potential risk for the reintroduction of vectorborne disease was not appreciated after three generations of malaria free experience. in recent years much interest has been directed towards the protection and establishment of wetlands, without pay ing sufficient attention to their mosquito breeding potential. consequently, tva has been faced with a conflict of new policy directives concerning wetlands, existing mosquito control policies and state regulations for impounded water. the use of constructed (artificial) wetlands for the treatment of domestic waste water and its processing for reuse is of particular concern, since these could pro duce large quantities of potential disease vectors and they are often sited close to populated areas (peem/who, : - ). this stresses the necessity of harmonizing policies of reservoir management for vector control with other policies concerned with land use patterns, to ensure that areas of potential risk, such as artificial wetlands, are planned away from areas of human habitation. presently we face a complex environmental situation that seems to be changing at a much faster rate than our current capacity to revise and renew our intellectual schemes or to generate integrated management structures capable to enhance both ecosystem and human health. take, for example, the case of influenza virus. the loss of wetlands around the globe may force many wild birds onto alternative sites like farm ponds and paddy fields, bringing them into direct contact with domestic fowl and humans and providing greater opportu nities for the spread of the h n virus (anonymous, ) . poor planning in response to development pressures has led to the increasing loss or degradation of wild ecosystems which are the natural habitats for wild birds. the displaced wild birds increasingly seek to feed and live in areas populated by domestic poultry and humans. thus, wetland creation must also consider these factors, since they will attract migratory birds if located near their routes. despite this being considered an enrichment of landscape ecology in terms of diversity, recreational value, landscape beauty, etc., the risk of increasing transmission of emerging diseases must be dispassionately considered. an increase in biodiversity does include not only those species humans like, but also the diversity of vectors capable of transmitting diseases. the distance of the wetlands to be created from populated centers, the direction of prevail ing winds, and other transport mechanisms should be carefully taken into account. this issue of 'ecohealth' highlights the interplay between agriculture, animal (domestic and wildlife) and human health, the integral health of aquatic ecosystems, and sociocultural factors. nutrient reduction in wastewaters or surface waters is a priority for reducing risks to human 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coronavirus (covid- ), disease cannot be overemphasized since there is the potential possibility of it wiping out the entire world population within a few months if adequate and quick steps are not taken to curb this menace, and the sub-saharan african (ssan) region is no exception. it is evident that water, as an essential daily commodity, has long been in a state of emergency in ssan nations, which is largely attributed to decades of neglect by the successive governments, because it has not been possible to separate the existing bond between water, health, livelihood and the economy. the laudable millennium development goals (mdgs) proposed by the united nations had yet to achieve the stated objective of improving the standards of living and health conditions of the rural communities in the ssan region before the covid- pandemic outbreak. this failure has been masked by a sort of delusion in which the people of this region are subjected to the hardship of searching for clean and healthy water in their own ponds, rivers, streams and shallow hand-dug local wells on a continuous basis. less than % of the rural population in all the ssan communities can access basic hand-washing facilities and sanitation systems. the total water productivity, as measured by the gross domestic product (gdp) per cubic meter of total freshwater withdrawn, for the people was less than gdp. prior to the outbreak of the covid- pandemic, the world economic forum categorized water scarcity as the third most alarming global risk. sub-saharan africa (ssa) is composed of countries in africa. its erratic and low amount of a good and clean water supply predates the colonial era, and many communities suffer from a lack of access to clean and improved sources of sustainable drinking water. water and sanitation facilities are absent in many of the rural communities despite some laudable water and sanitation policies by successive governments after the colonial era. a majority of the rural communities in this part of the world are conditioned to the seasonal supply of water resources despite the high water availability during the rainy season ( figure ). it is no stretch to say that the majority of the governments at all levels failed to make provisions to establish sustainable rural water supply as a priority project to serve the communities. during the prolonged dry seasons that usually characterize the region, which sometimes last for more than half of the year in many places (figure ), streams and rivers are completely dried up with many of the hand-dug wells having short supplies due to their shallow nature. the number of wells serving a community is more than what the groundwater situation could handle in many of the places, although the united nations has invested heavily in the provision of sustainable, clean and safe drinking water in the whole african continent since the promulgation of the millennium development goals (sachs, , united nations, , hunter, . it is unfortunate that most of the rural dwellers have not yet felt the impact, as many of the installed water systems are not functional, and those that were initially operational were left unmaintained after breaking down. there had been no serious policy to improve the access of rural dwellers sustainable and clean water in all the countries of the ssa. as a matter of urgency, the provision of good and safe drinking water in recent years has experienced consistent decreases due to population explosions in the region (unesco, , smeets, , united nations, . previous works have shown relationships between improved clean and good sources of drinking water and drastic reductions in health risks (bartram and cairncross, , brown, et al., , gsunny, et al., , yang, et al., . the laudable millennium development goals (mdgs) proposed by the united nations had yet to achieve the stated objective of improving the standard of living and health conditions of the rural communities in the ssan region before the covid- pandemic outbreak. hence, the people of the region have been confronted by the menace of the new invisible, yet powerful, coronavirus disease. if the fight against this deadly pandemic global disease is to be successful in the region, quick attention must be given to the provision of good, clean and safe water, especially for the rural communities that have suffered so much neglect from the governments. it is noteworthy that the various restrictions imposed in the aftermath of the covid- outbreak by the centers for disease control (cdc) would be insignificant in the region as long as people have to travel long distances during the february-april dry period in the area in search of clean, safe and drinkable water. hence, exposure to the deadly infectious virus cannot be ruled out. the health of the rural dwellers in ssa is more threatened as the people become from new, et al., . more vulnerable to getting infected by the deadly disease in their search for healthy, clean, and safe water. the situation became worse due to the non-availability of protective devices such as face masks to protect people, even though people, especially women and children, cannot be prevented from gathering in places where water is available, and safe health cannot be guaranteed. conversely, the people are exposed to the danger and challenges posed by the pandemic, which could be a channel through which people innocently get infected. similarly, people are warned to maintain good hygiene through regularly washing their hands with soap, which is the only available means to these rural communities. unfortunately, the issue of the immediate availability of sustainable water will impair this. the more reasonable information of this nature contained in this paper is imperative since it is the medium for disseminating the required knowledge. therefore, the provision of improved clean, potable, and safe water and sanitary facilities in the ssa calls for the declaration of a state of emergency regarding the region's water supplies if the global fight against the covid- pandemic can be won as soon as possible. good health cannot be obtained in the absence of affordable, easily accessible, safe, and dependable potable water for the rural communities of ssa. the respective governments' attention needs to be refocused on the nonfunctional water facilities in many places, such as boreholes and hand-dug wells that were abandoned and left unmanaged in numerous places, either as a result of the lack of constant power supplies, or the lack of maintenance. in the present uncertain situation surrounding the covid- pandemic, it is critical for the people to get access to clean and safe sustainable water for domestic and sanitation uses to guarantee public hygiene and better food and, hence, improve their health conditions (hunter, et al., ). long before the covid- crises started, the provision of clean and safe water for the use of rural communities in ssan nations saw the burden shifted to the poor rural community dwellers by the successive political governments. this burden has been masked by a sort of delusion in which the people are subjected to the hardship of searching for clean and healthy water in their own in ponds, rivers, streams and shallow hand-dug local wells on a continuous basis. it is unfortunate that the depths to the permeable underground water table in the ground areas of the region are too shallow to provide water throughout the year (jha, et al., , kayode et al., , given the complexity of the topography and the effect of the extreme seasonal weather conditions in the region caused by the prolonged dry periods that subject these shallow wells and the streams to a few months of complete dryness. these situations in ssa have a great effect on the provision of sustainable, clean, and safe water for both agricultural and domestic use. . covid- pandemic and the rural communities of sub-saharan africa (ssa) the covid- pandemic has caused global public health crises across entire regions of the world ( figure ) with no exceptions. it was envisaged that the situation in ssa, and africa as whole, would be devastating according to the who regional director for africa (rda) dr. matshidiso moeti in a th april report (who, a) . in the rda's report, the disease was first discovered in the north african country of egypt on th february . as of th april, all african countries had recorded a covid- case because of the influx of the people from the earlier hotspot zones in asian and european countries, as well as the united states, according to dr. moeti. furthermore, the possibility of a pandemic outbreak involving family clusters could not be completely ruled out after the discovery of the first case (guan, et al., , phan, et al., . most reports have shown the increased risks of people with chronic health conditions of getting infected by covid- , particularly adults (guan, et al., , la rosa, et al., , phan, et al., , shereen, et al., , wang, et al., , who, b . the risk of getting infected by individuals with some prior health conditions is very high. if the projected impacts of the covid- pandemic on the people of ssa are to be believed, then the major gains in the sustainability and availability of potable water for these people would suffer a drastic set back, including the areas supposedly known as urban cities. situational reports in earlier studies suggested declaring a state of emergency regarding the provision and supply of potable water to meet the daily needs of the increasing population in the region. the general state of the potable water supply in many of the cities is worrisome. the exponential increase in the cases of covid- recorded so far makes it of utmost importance to research the major impact that the clinical disease would have on the ssan poor rural communities that could not access potable and sustainable water, coupled with the growing population and the increase in the demand for sustainable and potable clean water due to the increasing economic constraints facing the people. as of the end of , the world bank group reported the mortality rates in ssa that are presented in figure . it is alarming to see a whopping percentage of a little less than a % of every , people of the region losing their lives to unsafe water, sanitary systems, and unhygienic conditions. the rapid spread of the covid- pandemic is a serious concern when considering the data from the world bank group's reports on ssa. the results of the mortality rates presented in figure clearly show that the deaths due to unsafe water, the complete absence or improper sanitary systems, and unhygienic conditions are more than double the deaths from every other source put together. the unfortunate situation is the lack of political will on the part of the governments of the countries that make up the ssan region vis-a-vis proactively dealing with the situation of sustainable rural water supplies and the water systems' management over several decades of neglect. there have been concerted efforts to establish national water policies across the region, either by the united nations sanitary projects or by the individual governments in each of the countries that constitute the ssa. however, the absence of sufficient potable water among a larger percentage of the communities is worrisome. figure shows the percentage of ssan people with access to basic hand-washing facilities from - as compiled by the world bank group reports. the results of the data analysis call for urgent attention if the fight against the covid- pandemic is to be globally shared and fully achieved. less than % of the rural population in all the communities can access basic hand-washing facilities. the percentage was less than % in and jumped by over % to the present level of % in and has since then remained at this level. this could be attributed to the concentration of the unicef programs in the aftermath of the deadly ebola virus outbreak. as of the time the data were collected, only < % of the urban population had access to basic hand-washing facilities. again, this level of access was made possible because of the ebola cases. prior to the ebola outbreak in the region, barely % of the urban population was recorded to have access to basic hand-washing facilities from to . the percentage was substantially improved from - to about < %. overall, only approximately % of the entire ssan population had access to basic hand-washing facilities from until , leaving a whopping % of the population of ssan without access to basic hand-washing facilities. due to the necessity of basic hand-washing facilities due to the covid- pandemic, ssan nations would need to seriously brace for the challenges brought about by the emergency created by the deadly pandemic. this report is a total reflection of what is obtainable since many water-constrained factors have led to the low level of accessibility to potable water. these factors together with the population explosion of the region over the past decades have increased the demands for accessible and sustainable good drinking water and proper sanitary systems. there has not been any confirmation so far of people contacting the coronavirus through water bodies, either in underground or surface waters, or spreading it through any contaminated drinking water system (casanova, et al., , shereen, et al., , yang, et al., , despite the virus being detected in all types of water systems, with a reported connection between drinking and recreational water outbreaks. the provision of sustainable, clean, safe, and potable water for domestic and sanitary systems should be pursued vigorously for the ssan people. in the absence of this pursuit, a second wave of the disease could outbreak and spate the area with unforeseen devastating effects that increase global concerns. this paper therefore provides proven knowledge and valuable support for sustainable, hygienic, clean, safe water and good sanitary systems for the rural poor dwellers in the ssa to safeguard the people from unrestricted exposure to possible infection from the covid- disease through water and sanitation systems, with the level of cases recorded as at th april shown in table . people cannot maintain good sanitation and hygienic conditions in situations where the basic water supply required to do so is completely absent. the only avenue for efficiently enhancing public health in rural communities relies solely on the provision of adequate, hygienic, clean and safe water and good quality sanitary systems. the provision of clean and quality domestic water supplies is the key to maintaining good health and, hence, preserving lives. mali tanzania kenya mauritius gabon rwanda liberia ethiopia madagascar togo zambia benin uganda mozambique chad central african republic zimbabwe eritrea malawi angola botswana namibia sao tome and principe gambia burundi seychelles mauritania comoros figure shows the results for the total water productivity as measured by the gross domestic product (gdp) per cubic meter of total freshwater withdrawn using data from to on the ssan region from the world bank group report. ever since the production of approximately gdp of total water per cubic meter in the ssan region, it is worrisome that it has seen a drastic freefall from this level of attainment to just approximately gdp in the following year, with a consistent further plunge to a lower value of approximately gdp in . there was a little improvement in the total water productivity for the people in ssa in the following year (i.e., ) to about < gdp. the next year, , witnessed another sharp drop to < gdp, with a record improvement of < gdp in the year . however, experienced a catastrophic level of water productivity in this region, as shown by the drastic drop again to the lowest total water productivity of < gdp. the years , , and experienced consistent improvements, even though the recorded total water productivity was < gdp, but these levels have since fallen again to < gdp per cubic meter of total freshwater withdrawn in as obtained from the world bank group report. the connections among the major parameters expressed in this study are the following: i) the percentage of people with access to basic hand-washing facilities; ii) the total water productivity, as measured by gdp per cubic meter of total freshwater withdrawn; and iii) the covid- pandemic that has had significant effects on ssan's rural communities in relation to their social economic status. the decades of experience in real daily life with nonexistent accessibility to sustainable, clean, and potable water for domestic and sanitary systems motivate this paper to publicize the major challenges confronting the people of the region that have defied lasting solutions to attract probable lasting solutions in the face of the covid- pandemic outbreak. the present total water productivity, as measured by gdp per cubic meter of total freshwater withdrawn, across the region needs to be boosted by multiple folds due to the pressure of the demands of the covid- pandemic that has further strained the present insufficient water supply systems. although concerted efforts have been made to ward off the deadly virus within a short time frame, attention needs to be focused on the ssan water supply systems. the fight against the covid- pandemic could be a straightforward effort in areas where there are no restrictions to sustainable, hygienic, clean, safe water and sanitary systems. however, it could be much more complicated in the ssan region since the services of this nature are utterly deficient over long decades in the rural areas with many of the urban cities being inclusive. in a situation where the entire total water productivity, as measured by gdp per cubic meter of total freshwater withdrawn, was < gdp in for the region, there are serious concerns for these groups of people due to the level of hygiene required to face the novel coronavirus diseases. the most effectual and clear-cut measures to thwart the transmission of this deadly disease is to maintain high-quality, hygienic conditions through unrestricted access to sustainable, hygienic, clean, safe water and good sanitary systems. . sub-saharan african health care systems and the covid- pandemic. we compare the ssan governments' commitment to the health care systems with what individuals or private people spent on health care across years using data obtained from the world bank group report and presented it in figure . after a close examination of the trends, it is very clear that the governments of the nations in the ssan region paid less attention to the health care systems of the people, particularly the rural dwellers. individuals have been left to provide health care for themselves and possibly their immediate family members. the latest rural community population was , , million as of the end of according to the population data obtained from the world bank group report (figure ) . these people, for a large extent of the ssan rural population, are at the highest risk of being easily exposed to the deadly novel coronavirus disease. this risk stands despite the fact that the region continuously suffers from the effects of other deadly global infectious diseases that were long ago found to be closely connected to the non-availability of a sustainable water supply and hygienic sanitary systems. the communities in the region would definitely experience severe detrimental effects as the covid- pandemic continues to ravage everywhere with no exception for any region. the urban population of , , million people in ssan is not spared from the risks of the covid- pandemic, and most of the cases so far have been reported in urban cities. the percentage of people with access to basic hand-washing facilities, coupled with the available total water productivity, as measured by gdp per cubic meter of total freshwater withdrawn, cannot adequately care for these populations of people. many of the diseases associated with nonaccessibility to hygienic water are very much prevalent in urban cities compared to rural areas. presently, there is no existing known vaccine for covid- worldwide. however, scientists are vigorously working on remedies that will effectively reduce the number of novel coronavirus infections. while the best pathogenesis and treatment alternatives are still been pursued globally (guan, et al., , la rosa, et al., , phan, et al., , shereen, et al., , wang, et al., , who, b , researchers are putting their heads together to find ways of reducing the spread of covid- . the concerns for the rural dwellers in the ssan region is the ability to enact with the solutions defined for the novel coronavirus. the economic conditions of this group of people would definitely subject them to be more vulnerable to the infectious disease, especially due to the nonaccessibility to basic daily needs such as good food; sustainable, hygienic, clean, and safe water; and good sanitary systems. the people are still suffering from the other deadly waterborne infectious diseases that are continuously ravaging the region on a continuous basis. the outbreak of the covid- pandemic would definitely compound the issues of immunities and malnutrition with the people of these communities. figure showed the percentage of the total population and growth data for the ssan region, which was obtained from the world bank group report. rural areas account for % of the entire population, with a growth rate of just % due to the rural-urban migration of the people in search for better living conditions. although the urban population is just % in the region, the growth rate of % is a concern since the mingy available amenities are under stress on a continuous basis as the people scramble for the little available sustainable water supply and hygienic sanitary systems. the large population of people living below $ . per day (i.e., figure ) would not be able to afford the minimum costs of covid- sanitizer that has an average cost of $ . per unit. it is certain that the population of people living below $ . and $ . cannot afford the minimum costs of the basic supplies to fight against covid- . it should pointed out at this juncture that many of the statistics made available by the individual nations in this region may perhaps significantly vary from the world bank group reports since the governments in most of the countries have significant corruption that politically undermines the true realities of the situations. the situations are extremely horrific with no exception for the urban dwellers too. figure shows the proportion of the ssan population in percentages living below the world bank poverty line of $ . a day and the $ . poverty line that accounts for health care expenditures. the fear of this invisible but prevalent coronavirus disease cannot be overemphasized since there is the potential possibility of it wiping out the entire world population within a few months if adequate and quick steps are not taken, and the ssan region is no exception. these enlightening data should focus the attention and concerns of policy formulators on the need to urgently improve and increase the number of new techniques to improve the water supply situation in the region. it is evident that water, as an essential daily commodity, has long been in a state of emergency in ssa, which is largely attributed to decades of neglect by governments, because it has not been possible to separate the existing bond between water, health, livelihood and the economy. all these go pari passu. the actual reality of the day-to-day lives in ssan nations is clearly shown by this study. if the necessary efforts had been made to improve the access to sustainable, good and clean water in these developing countries, the whopping > million deaths due to inappropriate water, sanitation and hygienic conditions that were presented in figure could have been prevented. the actual situations in virtually all the countries are heavily characterized by corruption in all spheres of the economy that politically undermines the true reality of the situation. the people would definitely be subjected to further vulnerability to this infectious disease due to their mingy economic conditions due to the non-accessibility to basic daily needs such as good food; sustainable, hygienic, clean, and safe water; and good sanitary systems. the situations being reported are not limited to rural dwellers, and they exist in all the countries comprising the ssa. challenges of access to clean and safe water together with good sanitary systems predate the colonial regimes when there were far fewer people than what exist today. decadent structural facilities are ubiquitous in this part of the globe. the entire blame should be upon the political class that fails to provide basic social amenities that are crucial to life such as clean and good water supplies. despite the many government policies that address the supply of hygienic, clean, and safe water in most places, the results presented in figure confirm that the water supply systems are quickly deteriorating at astronomical rates. virtually all the countries in this region enjoyed numerous world bank water projects, but the true situation is that the rapid population explosion has jeopardized all these laudable programs. the results shown in figure regarding the population in percentages that lived below $ . a day in imply that over million of the population lived below $ . a day. the figure could be double this when new reports are published. the outbreak of the covid- pandemic could be judged to have originated from poor risk management on the part of health scientists that should have proffered solutions at the early stage. more worrisome is the unprepared research and academic institutions not curbing the astronomical spread of the coronavirus disease. the policies that are responsible for implementing sustainable, hygienic water and sanitary systems in the region were at significant risk for decades before the covid- pandemic issues. for instance, since when the gdp production of total water per cubic meter recorded in the ssan region stood at gdp (i.e., figure ), the measure has experienced a drastic free fall to < gdp in , which is an indication the water supplies have reached a state of emergency. this indication may not be unconnected to the consistent rise in people's health costs per capita, as presented in figure . it could be concluded that this growth was a result of water supply crises coupled with growing and competing demands at the expense of nonexisting sustainable, clean and hygienic water and sanitation resources in several places. with the water situation in ssa having a strong association with the outbreak of many known diseases, such as ebola, malaria, and other tropical infectious diseases, this paper suggests the most important and intensive efforts for improving water and sanitation in order to achieve the total and complete eradication of these deadly diseases as urgently as possible. to be equipped to meet the united nations sustainable development goals (sdgs) regarding targets for the total eradication of these infectious diseases through sustainable water and sanitation programs, a stern obligation of the political governments of each country in the ssan region is to guarantee the people, especially the rural dwellers, unrestricted access to basic daily needs of sustainable, hygienic, clean, and safe water and good sanitary systems. hygiene, sanitation, and water: forgotten foundations of health local drinking water filters reduce diarrheal disease in cambodia: a randomized, controlled trial of the ceramic water purifier. the american journal of tropical medicine and hygiene survival of surrogate coronaviruses in water tracking progress towards global drinking water and sanitation targets: a within and among country analysis clinical characteristics of coronavirus disease in china estimating the impact on health of poor reliability of drinking water interventions in developing countries assessing groundwater quality for drinking water supply using hybrid fuzzy-gis-based water quality index coronavirus in water environments: occurrence, persistence and concentration methods-a scoping review geo-electrical investigation of near surface conductive structures suitable for groundwater accumulation in a resistive crystalline basement environment: a case study of isuada, southwestern nigeria representing twentieth-century space-time climate variability. part ii: development of - monthly grids of terrestrial surface climate importation and human-to-human transmission of a novel coronavirus in vietnam investing in development: a practical plan to achieve the millennium development goals. un development programme covid- infection: origin, transmission, and characteristics of human coronaviruses impact of hydraulic interventions on chronic and acute material loading and discolouration risk in drinking water distribution systems stochastic modelling of drinking water treatment in quantitative microbial risk assessment. water management the world bank group. data on ssan region water: a shared responsibility. the united nations world water development report sars-cov- rna detection of hospital isolation wards hygiene monitoring during the coronavirus disease outbreak in a chinese hospital . covid- cases top in africa: available at water, sanitation, hygiene and waste management for covid- : technical brief united nations. the millennium development goals report assessment of microbial risks by characterization of escherichia coli presence to analyze the public health risks from poor water quality in nepal drinking water and sanitation conditions are associated with the risk of malaria among children under five years old in ssa: a logistic regression model analysis of national survey data we thank the world health organization for the release of the reports where a larger percentage of the data were obtained. thanks are also due to esri for the access to the covid- live tracker. kayode john stephen: conceptualization, investigation, original draft writing, software, formal analysis, manuscript preparation, and resources.asha embrandiri: resources.adijat olubukola olateju: resources. the authors declare that they have no known competing financial interests or personal relationships that have, or could be perceived to have, influenced the work reported in this article. key: cord- - j axjd authors: chen, ying-chu title: system dynamics evaluation of household water use behavior and associated greenhouse gas emissions and environmental costs: a case study of taipei city date: - - journal: journal of water process engineering doi: . /j.jwpe. . sha: doc_id: cord_uid: j axjd abstract taiwan is an island surrounded by sea and only % of its freshwater usage is for domestic applications. a system dynamic model was developed to investigate interactions between household water use behaviors (toilet flushing, clothes washing, bathing/showering, and cleaning) and associated greenhouse gas emissions and environmental costs. six hundred and fifty face-to-face interviews were conducted in districts of taipei. the results showed that the respondents’ individual attributes were not significantly related to water use behaviors. the highest volume of water was used for cleaning ( . %), followed by clothes washing ( . %), bathing/showering ( . %), and toilet flushing ( . %). five water management scenarios with %– % reductions in water volume from different water use behaviors were simulated. the maximum reduction in water use ( . t) was found in the fifth scenario ( % reduction), which reflected the priority the respondents gave to save water if its price increased. . % of respondents had water saving appliances; . % and . % of the appliances were toilets and shower heads, respectively. the environmental cost of ghg emissions associated with water use behavior was us$ . /t, causing an % increase in water price. a better understanding of household water use behaviors is needed to develop bottom-up strategies or measures for sustainable water management. water saving measures or strategies would lead to targets being met in a short time. there are more than , islands in the world, accounting for one sixth of the global land area [ ] and more than million people live on these islands [ ] . small islands comprising over one sixth of the global land area are facing struggles associated with global climate change. taiwan has a population of . million and an average gdp of us$ , /capita in [ ] . the unique topographical and geological characteristics in taiwan affect water availability, water use and conservation. about two thirds of the island is mountainous, which means that water flows into the ocean quickly and cannot easily be stored. total rainfall is hugely different in the dry and wet seasons (november to april and may to october, respectively), leading to water shortages in the dry season. in addition, extreme events such as typhoons increase the deposition of sand and rock in rivers and reservoirs, gradually reducing their storage volumes and increasing water turbidity. furthermore, the groundwater in southern taiwan has been over-pumped for years due to the development of agricultural and aquacultural industries. domestic use accounts for only % of freshwater usage, with industry and agriculture accounting for % and % respectively [ ] . taiwan is already struggling to meet the growing demand for water, particularly in summer. the residential water demand will continue to grow due to urbanization. households are high consumers of water and are therefore important sites for water conservation [ , ] . domestic water consumption in most developed countries is higher than the or l/d specified as being sufficient to fully meet household water requirements [ ] . it ranges from as low as l/capita/d in the netherlands [ ] , to l/ capita/d in the uk [ ] , l/capita/d in taiwan [ ] , and l/ capita/d in the usa [ ] . researchers have investigated household water consumption by types of appliance [ ] , and behavior [ ] . previous findings confirm that attributes including gender [ ] , age [ ] , education [ ] , and income [ ] significantly affect household water consumption. women are more likely to participate in environmental behavior and activism such as water resources conservation. age is inversely related to environmental concern; in other words, younger people are more likely to engage in environmental behaviors [ ] . however, chenoweth et al. [ ] reported that older water users were slightly more likely to consider water saving options. people with a higher level of education often consume more water [ ] . in one study, as income level increased, so did reported participation in water conservation [ ] . household water use can help make water consumption a key indicator of human behavior [ ] . water saving behavior can be sorted into similar groups based on location (indoor or outdoor), and behavior type (efficiency, curtailment or maintenance) [ ] . however, water use behavior has not been fully assessed even though it can lead to considerable reductions in water consumption [ ] . household water use is frequently assessed by questionnaire or established models. yu et al. [ ] used a face-to-face interview method to estimate residential water use associated with three types of behavior: bathing, cooking, and cleaning. they found a strong correlation between water use and energy consumption. chenoweth et al. [ ] investigated the links between household water consumption and wellbeing in southern england via a household survey. the results showed that actual water use appears to be unlinked to willingness to adopt water saving measures. wolter [ ] conducted a survey in oregon and found that environmental concern and sociodemographic attributes predicted water conservation behaviors. daioglou et al. [ ] modeled domestic water demand at the end-use level and indicated that cooking was the main end-use function. other functions, such as space heating and cooling appliances, become more important. ren et al. [ ] developed a tool to predict energy consumption at the end-use level and related greenhouse gas (ghg) emissions of australian households. researchers further compared energy-related ghg emissions from the water systems of various cities without analyzing the behavior of endusers [ ] [ ] [ ] . the pumping, treatment and distribution processes in water systems are the major contributors to ghg emissions [ ] . ghg emissions is a prominent environmental indicator that primarily determines the sustainability of urban water systems. studies have indicated that water conservation at the household level is one of the effective ways to reduce ghg emissions originating from water systems journal of water process engineering ( ) [ ] . system dynamics (sd) modeling has been used to evaluate environmental and water systems at various scales. the sd model, introduced by jay forrester in the s, is a well-established methodology for understanding, visualizing and analyzing complex dynamic feedback systems [ ] . it attempts to quantify qualitative aspects without altering the accuracy of the original statement, providing a much more explicit basis for communication [ ] . sd models are useful for considering dynamic interrelationship among variables within social and economic systems [ ] . water resources (quantity and quality) are integrated with five sectors that drive industrial growth: population, agriculture, economy, nonrenewable resources, and persistent pollution [ ] . an sd model was used to increase public understanding of the value of water conservation in las vegas [ ] . the results of sd model simulations show that water scarcity, unlike other limitations such as nonrenewable resources and persistent pollution, results in severe problems within a short time of its occurrence [ ] . qi and chang [ ] proposed an sd model based on a coupled modeling structure that takes into account the interactions among economic and social dimensions, offering a realistic platform for practical use. a reservoir balance sd model was developed and presented for the pedra 'e othoni reservoir in italy to assess resilience to climate and development changes [ ] . an sd model to evaluate the water-energy-food nexus at the household scale was developed by hussien et al. [ ] . the model successfully estimated a water-energy-food demand and the impact of change in user behavior, diet, income, family size, and climate. more work is needed to develop systematic and detailed approaches to assess household water use in terms of ghg emissions and environmental costs. the aim of this study was to develop an integrated sd model addressing the interactions between household water use and ghg emissions at the household scale. taking taipei city as a case study, the model captures more water use behaviors including toilet flushing, clothes washing, bathing/showering, and cleaning than previous studies. the number of water saving appliances and the priority given to saving water with different appliances were also innovatively evaluated in this study. the potential ghg emissions were internalized into environmental costs to reflect increases in the water price. the price of water has a direct influence on household water use [ , ] unlike tax [ ] . otherwise, water has been considered as a natural resource with little economic and utilization values [ ] . this kind of research is needed to better understand household behaviors regarding the use of potable water. globally, emissions of ghg can be mitigated by taking a bottom-up approach. taipei city, which has a high water quality standard (total coliform < cfu/ml, turbidity < . ntu, heavy metal and nitrite nitrogen are none detected [ ] ), was taken as a case study. the city has districts ( fig. ) and . million residents with us$ , /captia gdp on average [ ] . ghg emissions from the water supply system have been evaluated in several published studies as noted in the introduction. this study focused on ghg emissions caused by different household water use behaviors. respondents were asked to complete a questionnaire about their water use. the results of the questionnaire were incorporated into an sd model. the water quality in households reached the government's quality standard with little variation and therefore is not discussed in this study. face-to-face surveys can be clearly structured, flexible and adaptable [ ] . in this study, at least questionnaires were required when applying a convenience sampling method with a % confidence interval. therefore, six hundred and fifty face-to-face interviews were conducted during the spring vacation (april to april ) of in different districts of taipei city. the questionnaire, which could be completed within min, consisted of three sections. the first section collected information about the houses owned by the respondents, such as age and floor area. it was based on a survey of hong kong residents [ ] . the second section contained seventeen questions about the respondents' water use behavior. the questions were designed with reference to shan et al. [ ] and covered frequencies of bathing/showering, drinking water, and cleaning. the number of water saving appliances (e.g., low-flow taps and/or shower heads, dual-flush toilet) was also recorded. the respondents were also asked about the priority they would give to saving water if the water price increased. the final section elicited the respondents' socio-economic information, such as gender, income, age, and education. an sd model is constructed from building blocks (variables) and includes four main components: stocks, flows, connectors, and converters (fig. ) . stock variables, symbolized by rectangles, are the state variables and they represent the accumulation water in the system. flow variables, symbolized by valves, are the rate of change in stock variables and they represent those activities and decision functions that fill up or drain the stocks. converters, represented by circles, are intermediate variables used for miscellaneous calculations. finally, the connectors, represented by simple arrows, are information links representing the cause and effects within the model. an example relating to household water volume is as follows. the overall volume of water used for clothes washing depends on the water volume used each time and the frequency of clothes washing (fig. ) . the mathematical mapping of an sd stock-flow diagram occurs via a system of differential equations, which are solved numerically in the simulation. the specifications of the sd model are shown in table s . six equations were modeled using stella software in this study. the year was taken as the base time and -year simulations (from to ) with a step size time variable of year were modeled. household water use in taiwan can be divided into five major categories: toilet flushing, clothes washing, bathing/showering, cleaning, and other uses [ ] . the categories of cleaning and other uses were frequently confused by respondents in pretests of the survey and were integrated in this study. the detailed calculations of water volume are described below. the formulae to calculate water use volume was based on yu et al. [ ] and redesigned as follows: where v flush is water volume used for toilet flushing (l/y), f fα and f fβ are the frequency of toilet flushing (times/capita/d), q fα and q fβ are the water volume of toilet flushing (l/times), α is the percentage of households that own water saving toilets, β is the percentage of households without water saving toilets, equal to − α , p is the population of taipei city, equal to , , in [ ] , and is the conversion factor from year to day. where v cloth is water volume used for clothes washing (l/y), f c is the frequency of clothes washing (times/capita/d), and q c is the water volume of clothes washing (l/times). clothes washing is not classified with/without water saving appliances by the government in . where v bath is bath/shower volume (l/y, with or without water saving appliances), f sα and f sβ are the frequency of taking a shower (times/ capita/d). q , sα and q sβ are the water volume of taking a shower (l/ times), f b is the frequency of taking a bath (times/capita/d), and q b is the water volume of taking a bath (l/times). clean clean other ( ) where v clean and v other are water volume for cleaning and other purposes, respectively (l/y). therefore, ghg emissions from household water use can be calculated as follows: where v i is water volume and i can stand for toilet flushing, clothes washing, bathing/showering, or cleaning (l/y). one ton of tap water has been reported to produce . kg co -eq ghgs in [ ] ; therefore, the converting factor is × − . t co -eq/l/y in this study. the ghg calculations are mainly used to represent environmental costs as monetary values. the environmental costs (ec ghg , us$) of ghg emissions can be calculated as follows [ ] : where pi t is the carbon price at year t (us$/t co -eq). for the year of , the carbon price announced by the european union emission trading scheme (eu ets) was . eur/t co -eq (equal to . us$/t co -eq) [ ] . qcf t is the ghg emission from unit water volume each year (t co -eq/y), and r is the discount rate of . % [ ] . therefore, the water price in terms of the environmental costs of ghgs is as follows: where pv is the operating and maintenance costs (us$) and a is the external profits of the water department. currently, the operating and maintenance costs in taiwan are us$ . /t and basic external profits for the water company should be % [ ] . it is known that water saving measures and/or appliances can reduce water usage by %- % [ ] . therefore, this study simulated five different water management scenarios (fig. ) : %, %, % and % reductions in water volume from different water use behaviors, and a scenario to reduce % water volume in which respondents gave priority to saving water if the water price increased. an effective water management system must be accepted by the public [ ] . the randomly sampled people resided in different districts of taipei city. among the questionnaires, ( %) were suitable for analysis; % were excluded due to non-responses. as shown in table , % of the sample was male and % was female; . % of the respondents had four family members (i.e., two children in the family); . % of the respondents were educated to college level; and . % were aged between and . the monthly income of half of the respondents ranged from us$ to us$ , . the respondents' individual attributes (gender, age, education, and income) were cross-analyzed with their water use behaviors. it is interesting that % of the respondents said they took less than s to wash their hands, − min for cleaning, and − min to take a bath/ shower. the unicef [ ] suggests to scrub all surfaces of the hands for at least s to protect against coronavirus (covid- ) which was successfully controlled in taiwan. the promotion of population's awareness of hand washing will still provide the greatest benefit to mitigate the pandemic [ ] . male respondents took shorter showers than female respondents in this study. high school students took longer baths/showers than college students did, confirming the finding of yu et al. [ ] that older people use less bathing water. respondents with an income level between us$ and us$ would take − min shower, versus other respondents ( − min). the water used for clothes washing and shower is greater for lower-income areas [ ] . in other words, higher levels of income demonstrated more engagement in water conservation behaviors [ , ] . rosinger et al. [ ] found that income was linearly associated with tap water consumption among us citizens; however, the individual attributes were not significantly related to water use behaviors in this study. in this study, . % of water volume was used for cleaning, followed by clothes washing ( . %), bathing/showering ( . %), and toilet flushing ( . %) (fig. ) . in the netherlands, a population survey indicated that the main end uses of household water were showering ( %), toilet flushing ( %), and clothes washing ( %) [ ] . an analysis of the north american residential end uses of water study indicated that the top three indoor end uses of water were toilet flushing ( . %), clothes washing ( . %), and showering ( . %) [ ] . the amount of water for lawn and landscape irrigation is rapidly increasing in the us and is estimated at a total of billion gallons per day nationwide [ ] . average water end use consumption in australia has been reported as showering ( %), outdoor uses ( . %), taps ( . %), clothes washing ( . %), and toilet flushing ( . %) [ ] . garden watering and showering are two of the largest contributors to water use in households in australia [ ] . in poland, water used for showering and clothes washing accounts for % and % of total household water use, respectively [ ] . showering is the greatest consumer of domestic water in portugal, accounting for % of total water consumption [ ] . taiwan's water resources agency [ ] found that toilet flushing ( %), clothes washing ( %), and bathing/ showering ( %) were the highest contributors to household water use (fig. ) . in this study, the volume of water used for toilet flushing had reduced to %, perhaps because of an increase in the use of water saving appliances during the past years. other water use behaviors were increased. as shown in table , . % of respondents had water saving appliances; . % and . % of the appliances were toilets and shower heads, respectively. asian countries, like korea, japan and taiwan, had - % water saving toilets; however, other europe countries had higher percentage of using water saving toilet [ ] . a saving of l can be made each time a water saving toilet is used. most respondents reported using l and l each time for clothes washing and bathing, respectively. family size and seasons were positive related to frequency of clothes washing [ ] . taking a shower with or without a water saving shower head would save ca. - % water compared to bathing. showering frequency rather than duration played a dominant role in use of water for bathing [ ] . the participants in this study used . l/d for cleaning. the average volume of water used for cleaning was . l/d in taiwan in the past years [ ] . water use efficiency can be significantly increased by the installation of more efficient water use devices [ ] . for example, solar hot water systems are marketed to households on the basis of reduced energy costs through reduced ghg emissions [ ] . some households adopt water saving appliances and practices to continue their high consumption habits [ , ] . kenway [ ] found that behavioral changes play a vital role in water savings and ghg emissions. different water use behaviors will have their own best water saving scenarios. as shown in fig. , the highest reduction in water volume ( . t) was reached in the fifth scenario, which reflected the priority respondents gave to saving water if the price increased. the - % water volume reduction from different water use behaviors would only reduce water use by . t (first scenario) to . t (fourth scenario). the water volume reduces very fast in the first years ( - ) and stays steady in the following years, showing that water saving measures or strategies would lead to targets being met in a short time (fig. ). the reduction in water used by toilet flushing was highest in the fourth scenario, in which . t was saved during the study period. the reduction in water used for clothes washing was lower in the fifth scenario ( . t) than in the fourth scenario ( . t). the volume of water used for clothes washing may be limited by the size of washing machines that only work when there is a full load. other water use behaviors would be related to the targets for water use reduction. the volume of water used for bathing/showering and cleaning would reduce by . - . and . - . t, respectively, in the different scenarios. shove's [ ] work on changes in showering practices in the uk over time is particularly important. domestic water saving may be increased through economic incentives, technical improvements, or policy instruments and regulations [ ] . in sum, establishing water saving targets for different water use behavior depending on water prices would be more efficient than a single target, which is frequently applied by the government. as water reuse systems are less efficient than conventional water supply systems [ ] this option is not suggested in this study. the environmental cost of ghg emissions from water use behavior was us$ . /t in . the direct and indirect costs of water management in taiwan are ca. us$ . /t and us$ . /t, respectively [ ] . the direct costs are incurred by purifying, treating, supplying, and managing water resources. the indirect costs include employee training, disaster losses, retirement, and interest. the water price would be increased to us$ . /t (according to eq.( )) after monetizing the environmental costs of ghg emissions (us$ . /t). an % increase was accepted by respondents in this study if the government keeps providing clean and safe water resources in taiwan. the majority ( %) of survey respondents are willing to pay - % of the household monthly water bill for reliable water service in banda aceh, indonesia [ ] . houtven et al. [ ] suggested that households are willing to pay approximately us$ - per month for improvements in water access. expenditure on water in taiwan was the third from last when water price was divided by gdp for different countries [ ] . facing a price increase, households will generally adjust their consumption by reducing it. renwick and archibald [ ] found that marginal water price had a strong effect on the number of water-efficient devices in households. however, some people would be willing to pay extra not to compromise their daily showering rituals [ ] . the water price should be increased to reflect environmental costs, not only ghg emissions but also the costs of protection/conservation. the costs of water scarcity could reach % of gdp in some regions [ ] . pricing has been found to be a cost-effective tool for promoting water conservation [ ] . in addition, environmental education and attitudinal change might be more effective in influencing water use [ ] . it is advisable to educate general public the importance/advantage of water and alterative water resources [ ] . taiwan is a small island surrounded by sea. domestic water use accounts for only % of total freshwater usage in taiwan. ghg emissions from water systems have received attention as a prominent environmental indicator in recent years. an integrated sd model addressing the interactions between household water use and ghg emissions was therefore developed in this study. taking taipei city as a case study, the model captures different behaviors including toilet flushing, clothes washing, bathing/showering, and cleaning. potential ghg emissions were represented as environmental costs to reflect an increase in water price. a total of face-to-face interviews were conducted during the spring vacation (april to april ) of in districts of taipei city. the results revealed that the respondents' individual attributes were not significantly related to water use behaviors. the largest volume of water ( . %) was used for cleaning, followed by clothes washing ( . %), bathing/showering ( . %), and toilet flushing ( . %). five different water management scenarios with - % reductions in water volume from different water use behaviors were modeled. it is appropriate to establish water saving targets within years. the maximum reduction in water use ( . t) was found in the fifth scenario, which reflected the priority respondents gave to saving water if the water price increased. the environmental cost of ghg emissions associated with water use behavior was us$ . /t, causing an % cost increase, which was acceptable to the respondents in this study. further research of this kind is needed to better understand household behaviors regarding the application of water saving strategies or measures. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. % res-a challenge for island 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the case of banda aceh, indonesia after ten years since the indian ocean tsunami what are households willing to pay for improved water access? results from a meta-analysis average water price loading in the world demand-side management policies for residential water use: who bares the conservation burden? land econ high and dry: climate change, water, and the economy modeling residential water demand: an approach based on household demand systems analysis of social attitude to the new end use of recycled water for household laundry in australia by the regression models the authors thank the ministry of science and technology of the republic of china for financial support (most - -m- - ). the authors further thank the anonymous reviewers for their invaluable comments and suggestions. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jwpe. . . key: cord- - c authors: gibson, kristen e title: viral pathogens in water: occurrence, public health impact, and available control strategies date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: c the public health impact of the transmission of viruses in water is significant worldwide. waterborne viruses can be introduced into our recreational and finished drinking water sources through a variety of pathways ultimately resulting in the onset of illness in a portion of the exposed population. although there have been advances in both drinking water treatment technologies and source water protection strategies, waterborne disease outbreaks (wbdos) due to viral pathogens still occur each year worldwide. by highlighting the prevalence of viral pathogens in water as well as ( ) the dominant viruses of concern, ( ) wbdos due to viruses, and ( ) available water treatment technologies, the goal of this review is to provide insight into the public health impact of viruses in water. viral pathogens in water: occurrence, public health impact, and available control strategies the public health impact of the transmission of viruses in water is significant worldwide. waterborne viruses can be introduced into our recreational and finished drinking water sources through a variety of pathways ultimately resulting in the onset of illness in a portion of the exposed population. although there have been advances in both drinking water treatment technologies and source water protection strategies, waterborne disease outbreaks (wbdos) due to viral pathogens still occur each year worldwide. by highlighting the prevalence of viral pathogens in water as well as ( ) the dominant viruses of concern, ( ) wbdos due to viruses, and ( ) available water treatment technologies, the goal of this review is to provide insight into the public health impact of viruses in water. waterborne viruses are frequently implicated as the cause of water-related gastrointestinal illness. waterborne disease outbreaks (wbdos) are reported each year and are associated with recreational water (rw), treated drinking water (dw), and ground water (treated and untreated). depending on the water source, the actual source of contamination can vary; however, the two common threads are ( ) the introduction of fecal material into the water source and ( ) inadequate or interrupted treatment of water intended for drinking [ ] [ ] [ ] [ ] . in , the world health organization estimated that worldwide . million deaths each year can be attributed to the water-related (water, sanitation, hygiene) transmission of pathogens (all pathogens, not just enteric viruses) [ ] . for the european union (eu), the european environment and health information system estimated the annual burden of disease due to water-related pathogens at , deaths for children - years old. for the united states, reynolds ( ) estimated million illnesses and more than deaths each year were due to waterborne pathogens though these are based on model simulations and not actual values. unfortunately, the number of illnesses and deaths due specifically to waterborne viruses is difficult to determine and thus basically unknown. the current review ( figure ) focuses on ( ) the occurrence of viral pathogens of primary concern in various water sources; ( ) virus-related wbdos by water type reported worldwide over the past decade (from approximately to ); and ( ) dw treatment options for the inactivation or removal of viruses. finally, this review briefly discusses how we may better understand the public health impact of waterborne viruses as well as potential measures that can be taken to reduce the impact of viral pathogens in water. viruses most often implicated in wbdos include (but are not restricted to) noroviruses (nov), hepatitis a virus (hav), hepatitis e virus (hev), adenovirus (adv), astrovirus, enteroviruses (ev), and rotavirus (rv) ( table ) . although viruses implicated in wbdos are capable of causing a variety of acute illnesses (table ) , acute gastrointestinal illness (agi) is most commonly reported. enteric viruses are host-specific (i.e. in this instance, specific to humans) and are not capable of replicating in the environment outside of its host. in addition, enteric viruses have a presumed low infectious dose (i.e. < - virus particles) [ ] [ ] [ ] [ ] ; prolonged ( - weeks), asymptomatic periods of shedding; and enhanced environmental stability due to their non-enveloped capsid structure [ ] . these characteristics allow enteric viruses to play a significant role in water-related outbreaks. noroviruses have been the largest cause of virus-related wbdos in the u.s. since , and data indicate a similar trend in selected countries (france, japan, sweden, switzerland, the netherlands, uk) [ , , ] . aside from novs, hav, hev, and rv are still of significant concern in low-income countries without adequate water and sanitation. additional viruses of lesser epidemiologic importance though still capable of waterborne transmission include human reovirus, parvovirus, parechovirus, polyomavirus, coronavirus, and torovirus [ , ] . be introduced by land application of municipal biosolids [ , ] ; groundwater impacted by surface water or in proximity to faulty septic systems and leaking sewers [ ] [ ] [ ] [ ] ; and discharge of untreated wastewater [ ] or inadequately treated wastewater effluent [ , , ] . the occurrence of human enteric viruses in water remains largely unknown unless an outbreak is reported and samples are collected since water sources are not routinely tested for viruses. moreover, there are challenges related to sampling studies to determine virus presence due to both differences and limitations in recovery and concentration methods for the detection of viruses in water [ ] . regardless of these challenges, a snapshot of the occurrence of enteric viruses in water sources over the past decade is provided below. in this section, there is a specific focus on dw derived from treated surface water as opposed to treated ground water that is used as dw. keswick et al. ( ) -one of the seminal publications on the prevalence of viruses in dw in the u.s. -reported % of the samples to be positive for either rv or evs [ ] . shortly thereafter, bitton et al. ( ) followed up with a review on viruses in dw both in the u.s. and internationally [ ] . aside from these earlier studies, few studies on virus occurrence in dw in the u.s. have been reported since, and of those, such as , no viruses were detected [ ] . this paucity of available data for viruses in dw can most likely be attributed to the need for very large volumes (> to l) of water to be concentrated followed by subsequent recovery and detection of virus targets -a process that is challenging often viral pathogens in water gibson treatment options specific to removal/inactivation of viruses: -many options available though implementation varies worldwide due to availability of technology. occurrence of viruses in dw, gw, and rw: -dependent on sporadic research studies that vary by region/country. wbdos caused by viruses: -not well understood due to passive or no existent surveillance systems summary of key factors effecting the impact of waterborne viruses on public health. dw: drinking water; rw: recreational water; gw: groundwater; wbdo: waterborne disease outbreak. table viruses of primary concern for waterborne disease outbreaks. virus resulting in low recovery efficiency and detection sensitivity [ ] . in international settings, there is more research related to determining the occurrence of viral pathogens in dw. again, bitton et al. ( ) highlighted several studies out of the e.u. in particular, research from in paris, france was the driving force for the investigation of the occurrence of viruses in dw worldwide for the next years and beyond. as this review is primarily focused on the past decade, data published no later than have been included. lee and kim ( ) reported and % of tested dw samples positive for nov gi and gii, respectively, in south korea [ ] . studies on viruses in dwtps in egypt [ ] [ ] . overall, depending on the type of dw treatment process, source water quality, and sampling and detection methods, there is a wide range of viral pathogen occurrence in dw from around the world. one of the greatest fallacies regarding groundwater (gw) as drinking water is that gw is more likely to be free of pathogenic microorganisms due in part to the presumed natural filtering abilities of subsurface environments [ ] . pathogen contaminated gw can often be attributed to failing or poorly sited septic systems in karst settings that do not allow for proper attenuation of pathogens, especially viruses, due to their rapid movement from surface to aquifer [ , ] . [ ] [ ] [ ] [ ] [ ] . a more recent study by borchardt et al. ( ) reported % of the samples (n = ) positive for human enteric viruses (adv, ev, and nov gi) and also estimated - % of the agi in the study communities (n = ) to be attributable to exposure to viruses in nondisinfected tap water [ ] . one reason for this vulnerability -aside from the complexity of pathogen-subsurface interactions -is that before the ground water rule (gwr) of [ ], public utilities with a gw source were not required to disinfect their water supply; moreover, many individual wells are still used untreated. as reported in fout et al. ( ) , untreated gw was responsible for approximately % of wbdos in the u.s. and this trend continues today [ ] . for an international perspective, a study on nov occurrence in gw sources in south korea reported - % nov positive out of samples collected and analyzed [ ] . another study out of south korea [ ] reported similar levels of gw contamination with %, . %, and . % of samples positive for nov, ev, and adv, respectively. most data available on the occurrence of enteric viruses in rw sources is related to untreated venues such as lakes, rivers, marine beaches, among others as opposed to treated venues (i.e. waterparks, pools); however, virus-related wbdos can occur in both venues as outlined in the next section. moreover, the quantity of data available on human enteric virus occurrence in rw sources is quite significant; therefore, a portion of these data has been compiled in table . overall, rw have a high occurrence of human enteric viruses depending on the water type and virus; however, when considering these studies in table , one should take into account the differences in sampling and detection methodologies which are beyond the scope of this review. [ ] . therefore, in general, outbreak data -such as what is presented here -may not be as comprehensive [ ] . additionally, total morbidity and mortality are uncertain as virus-related illnesses and outbreaks are often unrecognized, unreported, or not even monitored. from to , virus-related wbdos were caused by contaminated dw -all from gw sources ( table ) . reported outbreaks were caused predominantly by novs with two outbreaks caused by hav. the dominance of nov in dw related wbdos may be strictly due to its status as the primary cause of agi in the population or there may be other intrinsic factors involved such as the environmental stability of nov and related viral ecology. of the virus-related wbdos reported from to , more than half ( of ; %) were due to contaminated rw sources -both treated and untreated ( table ). the dominant viral etiologic agents were human novs followed by one outbreak due to an ev, echovirus . as highlighted by sinclair et al. ( ) , the apparent increase in virus etiologies accounting for recreational wbdos is most likely the result of improved detection methods, but may also represent a true increase in incidence related to shifts in viral ecology or changes in population behavior patterns [ ] . other outbreaks that have not yet been reported by the u.s. wbdoss include a nov gi outbreak at lake wazee in jackson county wisconsin that sickened at least people [ ] . as one might expect, estimates on virus-related wbdos in low-income, developing countries are difficult to determine due to the lack of surveillance systems and complexity of pathogen transmission when both adequate drinking water and sanitation are lacking. ashbolt et al. ( ) provides a comprehensive review of the microbial contamination of dw in developing countries and highlights rv as one of the primary etiologic agents in children while hav, hev, and evs are more common in adults [ ] . the options available to treat water for the removal and/or inactivation of enteric viruses range from low-tech (i.e. household water treatment) to high-tech (i.e. dwtps viral pathogens in water gibson table occurrence of human enteric viruses in recreational water sources -u.s. and international combined. ) were positive for adv and rv while % were positive for evs [ ] utilizing advanced membrane filtration processes). treatment options tend to focus solely on removal of bacteria therefore, some of the technologies described below were never initially designed for the removal of viruses. point-of-use (pou), or household water, treatment options are primarily implemented in low-income countries that ( ) do not have a centralized dw treatment and distribution system or ( ) do have a centralized dw distribution system, but one that may inadequately treat water and is unreliable. one of the most recent, comprehensive reviews on pou water treatment options is by sobsey et al. ( ) . sobsey and others systematically evaluated five separate pou options including chlorination with safe storage, coagulant -chlorine disinfection systems (i.e. watermaker and pur packets), sodis (transparent pet bottles filled with water and exposed to sunlight), ceramic filters, and biosand filters [ ] . pointof-use technologies involving the use of chlorine were determined to be the most effective for virus reduction ( to logs) while the available filtration methods were the least effective ( . - log removal). additional research on the efficacy of these various pou water treatment technologies to remove human enteric viruses has been published in the past years [ ] [ ] [ ] [ ] which further support the conclusions of sobsey et al. ( ) . community-based water treatment systems have also been implemented in rural and peri-urban areas of low-income countries over the past decade and are designed to target rural communities with limited opportunity to hook up to a centralized dwtp. opryszko et al. ( ) investigated the impact of water-vending kiosks in rural ghana. the watervending kiosks were designed as 'mini' dwtps including surface water treatment using multi-stage filtration and ultraviolet light disinfection [ ] . a companion study by evaluated the efficacy of the watervending kiosks in ghana to remove human enteric viruses and reported the presence of nov gii and human polyomavirus in of of dw samples analyzed [ ] . additional studies on the efficacy of community-based water treatment systems for the reduction of human enteric viruses are non-existent -most likely due to the methods needed for viral recovery and detection in water. conventional dwtps are designed for reliable physicochemical removal ( - -log ) of microorganisms -bacteria, protozoa, viruses -from public dw supplies during optimal operation [ ] . however, in the past decade, more advanced processes for dw treatment have been implemented including alternative disinfectants (combined chlorine, ozone, uv radiation) and membrane environmental virology filtration (low-pressure microfiltration and ultrafiltration) technologies. however, enteric viruses are more resistant to inactivation by both uv and combined chlorine (monochloramine) when compared to chlorine [ ] . on the other hand, advanced membrane filtration technologies, especially ultrafiltration, allow for the simultaneous removal of all classes of microorganisms from dw sources [ ] . numerous studies highlighted in recent review articles [ , ] have reported on the efficacy of advanced membrane technologies and select alternative disinfectants for the removal and inactivation of enteric viruses in dw sources. viral pathogens in the water environment will continue to adversely impact public health. even though viruses are not the only pathogens present in water that can cause disease, the risk of illness is - , times greater for viruses than bacteria at a similar level of exposure [ ] . because of this increased risk and in consideration of the data presented herein, there are a few points of discussion that can be made. first, human enteric viruses are clearly a concern for both dw and rw microbial water quality; however, we still rely on a bacterial indicator system to alert us if there is a potential contamination issue. the use of bacterial indicators has been debated for decades, and we are still at a stalemate when it comes to actually predicting the presence of human viral pathogens in water. for the protection of public health, a true viral indicator should continue to be pursued. second, in order to move toward an indicator for viruses, we need to harmonize the concentration, recovery, and detection methods employed for the analysis of waterborne viruses. harmonizing steps have been taken by the u.s. environmental protection agency with the introduction of method though this method is specific to evs and novs. last, countries should start investing in both aging dw distribution systems and wastewater infrastructure, especially in large cities, as this would likely decrease a portion of the wbdos that are reported each year, at least in high income countries. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest causes of outbreaks associated with drinking water in the united states from to virus contamination from operation and maintenance events in small drinking water distribution systems inadequately treated wastewater as a source of human enteric viruses in the environment risk of waterborne illness via drinking water in the united states assessing microbial safety of drinking water: improving approaches and methods norwalk virus: how infectious is it human rotavirus studies in volunteers: determination of infectious dose and serological response to infection studies of echovirus- in volunteers: determination of minimal infectious dose and the effect of previous infection on infectious dose hepatitis a transmitted by food survival of human enteric viruses in the environment and food the epidemiology of published norovirus outbreaks: a review of risk factors associated with attack rate and genogroup reports a greater incidence of nov genogroup i in water-related outbreaks even though nov genogroup ii is the dominant circulating group molecular epidemiology of caliciviruses detected in sporadic and outbreak cases of gastroenteritis in france from outbreaks of gastroenteritis due to infections with norovirus in switzerland human enteric viruses in the water environment: a minireview emerging and potentially emerging viruses in water environments quantification of enteric viruses, pathogen indicators, and salmonella bacteria in class b anaerobically digested biosolids by culture and molecular methods leaching of phage from class b biosolids and potential transport through soil massive microbiological groundwater contamination associated with a waterborne outbreak in lake erie groundwater vulnerability to microbial contamination occurrence of viruses in us groundwaters the role of wastewater treatment in protecting water supplies against emerging pathogens municipal wastewater treatment plants as pathogen removal systems and as a contamination source of noroviruses and enterococcus faecalis demonstrates municipal wastewater treatment plants as a source of human enteric viruses in both recreational and drinking water sources presence of human noro-and adenoviruses in river and treated wastewater, a longitudinal study and method comparison challenges in environmental detection of human viral pathogens highlights the challenges and limitations surrounding the recovery and detection of human enteric viruses in environmental water sources detection of enteric viruses in treated drinking water viruses in drinking water tangential-flow ultrafiltration with integrated inhibition detection for recovery of surrogates and human pathogens from large-volume source water and finished drinking water detection of infectious enteroviruses and adenoviruses in tap water in urban areas in korea detection of enteric viruses, giardia and cryptosporidium in two different types of drinking water treatment facilities detection of enteroviruses in treated drinking water analysis of adenoviruses and polyomaviruses quantified by qpcr as indicators of water quality in source and drinking-water treatment plants evaluation of methodology for detection of human adenoviruses in wastewater, drinking water, stream water and recreational waters real-time pcr detection of enteric viruses in source water and treated drinking water in wuhan, china utilizes epidemiological data as well as tracer dye test to determine contamination of a fractured rock aquifer with human norovirus riverbank filtration: comparison of pilot scale transport with theory a multiplex reverse transcription-pcr method for detection of human enteric viruses in groundwater incidence of enteric viruses in groundwater from household wells in wisconsin assessment of sewer source contamination of drinking water wells using tracers and human enteric viruses human enteric viruses in groundwater indicate offshore transport of human sewage to coral reefs of the upper florida keys detection of bacterial indicators and human and bovine enteric viruses in surface water and groundwater sources potentially impacted by animal and human wastes in lower yakima valley viruses in non-disinfected drinking water from municipal wells and community incidence of acute gastrointestinal illness provides evidence that populations served by groundwater-source public water systems producing water without disinfection are exposed to waterborne viruses along with an increased incidence of agi nationwide groundwater surveillance of noroviruses in south korea comprehensive review of groundwater contamination with viruses in a high-income country other than the united states occurrence of norovirus and other enteric viruses in untreated groundwaters of korea us drinking water challenges in the twenty-first century viruses in recreational waterborne disease outbreaks: a review food safety news: ill with norovirus infections after swimming in wi lake who: outbreaks of waterborne diseases norovirus outbreak among primary schoolchildren who had played in a recreational water fountain massive outbreak of viral gastroenteritis associated with consumption of municipal drinking water in a european capital city an outbreak of gastroenteritis caused by norovirus-contaminated groundwater at a waterpark in korea microbial contamination of drinking water and disease outcomes in developing regions point of use household drinking water filtration: a practical, effective solution for providing sustained access to safe drinking water in the developing world using limes and synthetic psoralens to enhance solar disinfection of water (sodis): a laboratory evaluation with norovirus, escherichia coli, and ms microbiological effectiveness of locally produced ceramic filters for drinking water treatment in cambodia the efficacy of simulated solar disinfection (sodis) against coxsackievirus, poliovirus and hepatitis a virus bacterial, viral and turbidity removal by intermittent slow sand filtration for household use in developing countries: experimental investigation and modelling impact of water-vending kiosks and hygiene education on household drinking water quality in rural ghana evaluation of human enteric viruses in surface water and drinking water resources in southern ghana science and technology for water purification in the coming decades micro and ultrafiltration performance specifications based on microbial removal. london, uk: international water association the effect of coupling coagulation and flocculation with membrane filtration in water treatment: a review risk assessment of virus in drinking water pcr detection of pathogenic viruses in southern california urban rivers enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools detection of enteric viruses and bacterial indicators in german environmental waters occurrence of human adenoviruses at two recreational beaches of the great lakes presence of pathogens and indicator microbes at a non-point source subtropical recreational marine beach evaluation of public health risks at recreational beaches in lake michigan via detection of enteric viruses and a human-specific bacteriological marker chemical and microbiological parameters as possible indicators for human enteric viruses in surface water water ingestion during water recreation prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments surveillance of adenoviruses and noroviruses in european recreational waters comprehensive, multi-national study on the occurrence of human enteric viruses (adenoviruses and noroviruses) in recreational waters one-year weekly survey of noroviruses and enteric adenoviruses in the tone river water in tokyo metropolitan area surveillance for waterborne disease and outbreaks associated with recreational water -united states surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking -united states surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking -united states surveillance for waterborne disease and outbreaks associated with recreational water use and other aquatic facility-associated health events -united states surveillance for waterborne disease oubreaks and other health events associated with recreational water -united states surveillance for waterborne disease outbreaks associated with drinking water -united states surveillance for waterborne disease outbreaks associated with drinking water and other nonrecreational water -united states this work was supported by the arkansas biosciences institute and b funding from the arkansas water resources center. key: cord- -oawrnmhy authors: fahimirad, shohreh; fahimirad, zahra; sillanpää, mika title: efficient removal of water bacteria and viruses using electrospun nanofibers date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: oawrnmhy abstract pathogenic contamination has been considered as a significant worldwide water quality concern. due to providing promising opportunities for the production of nanocomposite membranes with tailored porosity, adjustable pore size, and scaled-up ability of biomolecules incorporation, electrospinning has become the center of attention. this review intends to provide a detailed summary of the recent advances in the fabrication of antibacterial and antiviral electrospun nanofibers and discuss their application efficiency as a water filtration membrane. the current review attempts to give a functionalist perspective of the fundamental progress in construction strategies of antibacterial and antiviral electrospun nanofibers. the review provides a list of antibacterial and antiviral agents commonly used as water membrane filters and discusses the challenges in the incorporation process. we have thoroughly studied the recent application of functionalized electrospun nanofibers in the water disinfection process, with an emphasis on their efficiency. moreover, different antibacterial and antiviral assay techniques for membranes are discussed, the gaps and limitations are highlighted and promising strategies to overcome barriers are studies. removing bacteria from water supplies which meet the requirement of effective antimicrobial activity, superior filtration flux with acceptable retention potentials (zodrow et al., ) . nanofiber membranes, because of their high surface area to volume ratio, nano-sized pores, and high porosity, have been illustrated to improve the efficiency of conventional materials employed for the filtration and separation of particulate materials (aussawasathien et al., ) . a number of processing techniques including melt-blown, self-assembly, phase separation, template synthesis, and electrospinning have been employed to prepare nanofibers in recent years. among them, electrospinning is the most promising, efficient method to produce web-like non-woven ultrafine fibers including microfibers (> μm) or nanofibers (< nm) from different kinds of polymers. moreover, incorporation of bioactive, antimicrobial and antiviral agents into nanofiber structure is easily possible through the electrospinning process fahimirad and hatami, ; faccini et al., ) . the present work reviews previous studies on the production and application of electrospun nanofibers as antimicrobial water filtration membranes. the merits and demerits of these novel water microfiltration tools are discussed. moreover, their antibacterial efficiency and disinfection activities are compared with commercial water membrane filters comprehensively. finally, some points are recommended to be noticed as the subsequent future research plans. the objectives of this review were to: (i) introduce the different procedures, which have been applied for incorporation of the various antimicrobial agents into electrospun nanofibers (ii) discuss the different antimicrobial tests used for proving antimicrobial activity of the fabricated electrospun water filters (iii) study the efficiency of the produced antimicrobial electrospun application in the water treatment industry. j o u r n a l p r e -p r o o f the electrospinning approach was invented by cooley in (cooley, . this method is easy, cost-effective, uncomplicated, and has the potential for scale-up production. the flexibility in material selection and additive incorporation to obtain appropriate functionality, as well as its considerable capability to produce fibers in the sub-micron range with the high surface-area (up to m g - based on the fiber diameter), are prominent privileges of electrospinning process for fabrication of nanofibers. in addition, effective porosity of electrospun nanofibers (almost about % with no upper limit) with many small pores, interconnected pore structure directly promote both infiltration rate and contaminant rejection ratio in comparison with conventional materials being used for mf applications (nasreen et al., ; wang et al., ) . in this process, a prepared solution of polymer is loaded into a syringe and fed at a set flow rate to the spinneret. due to the needle connected to a high voltage power supply under the electric field with a specific voltage, a taylor cone is constructed by elongation of the polymer droplet at the end of the syringe into a characteristic conical shape. enhancing the electrical field causes the formation of a steady jet elongated and whipped consecutively by electrostatic repulsion. the solvent evaporates when electrostatic forces prevail over surface tension and the jet gets finer, so electrospun nanofiber in mf application, such as uniform fiber morphology with controllable pore size, interconnected open pore structure, high porosity, and membrane thickness, turn them to a superior substitute to replace the conventional mf membrane such as the millipore gswp mf membrane with an average pore size of . mm (wang et al., ; barhate and ramakrishna, ) . another novel application of electrospun nanofiber in water purification and bacterial rejection is thin-film nanocomposite membrane (tfnc), a major type of reverse osmosis (ro) and nanofiltration (nf) membranes, which compromises of three layers including the first barrier layer of interfacial polymerization, a polyacrylonitrile or poly (vinylidene fluoride) electrospun membrane as the second layer and nonwoven polyethylene terephthalate (pet) as the third layer. the third layer employed as a substructure layer to provide the whole membrane adequate mechanical strength (subramanian and seeram, ; yin et al., ; fig ) . high water flux, great solute rejection, minimum membrane fouling, and perfect mechanical persistence are main properties of an ideal tfc membrane and turn it into an excellent candidate for microfiltration and ultrafiltration applications (li and wang, ) . sato et al., ( ) fabricated a novel composite fibrous membranes, consisting of an ultra-fine cellulose nanofibrous infused into electrospun polyacrylonitrile (pan, with an average diameter of . μm a mean diameter of about μm as the barrier layer ( - μm in thickness) to provide filtration attributes) nanofibrous scaffold on a melt-blown polyethylene terephthalate (pet, with a mean diameter of μm as the support layer (about μm thick) to sustain mechanical strength) non-woven substrate for water purification. the nanostructure showed a retention rate of . % for e. coli filtering and the high percent of the ms virus, with nm sizes, captured in the electrospun pan scaffold infused with m-ufcns (sato et al., ) . recently, taheran et al. ( ) j o u r n a l p r e -p r o o f fabricated a methodical portable water purification instrument using electrospun nanofiber. the device contained three distinct electrospun membranes. the first membrane was made by electrospinning of polyacrylonitrile/chitosan solution at : mass ratio as an antibacterial membrane, the second membrane was produced from laccase ( unit g - ) immobilized onto pan/biochar : % electrospun mat for removal of micro-pollutants and the third layer was fabricated by electrospinning of pan/biochar at : ratio as an adsorptive membrane. the applied technology led to approximately % removal of microorganisms, % of micropollutant removal, and more than % of turbidity decline during less than min contact time (taheran et al., ) . the important characteristics of a nanofiber mat membrane for application as filters for the separation of contaminations and pathogens from a continuous fluid phase are wetting properties, permeability, porosity, fiber size distribution, and fiber structure. for water filtration, a membrane must be wet-table and surface wetting properties are generally specified by the contact angle. a surface with a low contact angle (below degrees) is considered a hydrophilic surface, while a surface illustrating a high contact angle (over degrees) is referred to as a hydrophobic surface. sessile drop and the captive bubble method are two common techniques used for measuring the nanofibers' contact angle (nuraje et al., ) . one of the key parameters in filter design and its performance is porosity. generally, porosity is calculated from the apparent density and bulk density of the membrane. however, other alternative procedures inclusive of image analysis and mercury porosimeter are frequent methods j o u r n a l p r e -p r o o f applied for the evaluation of porosity in the nanofiber membrane (ghasemi-mobarakeh et al., ) . electrospun nanofibers are highly porous with interconnected pores in the size range of just a few times the fiber diameter. the small pore size of the nanofibrous membrane introduces a higher retention rate, the interconnected pores leads to better tolerance against fouling and the high porosity defined a higher permeability capability (homaeigohar et al., ) . clean water permeability (cwp (l/m •h•bar)) illustrates the highest amount of attainable flux dependent on the membrane condition. it can be assayed by calculating the flux at various trans membrane pressures (tmp). the slope of the eventuated curve is regarded as the cwp (bjorge et al., ) . the high cwp grants high flux operation to the membranes, introduces the nanofiber mat as an energy-saving membrane, and means that if fouling does not happen, enormous volumes can be treated (daels et al., ; he et al., ) . the surface charge on membranes is related to affinity corresponding interactions and considered as a significant parameter influencing the disinfection capabilities of the membrane. surface charges can qualify the strength of biomolecular or even pathogen affinity on a material surface. in virus removal, surface charged nanofibers adsorb virus via electrostatic interactivity between the nanofibers and the counter-charges of virus and signify virus remediation improvement (cho et al., ) . a series of studies have confirmed that electrostatic attraction between the cationic membrane and the anionic surface of bacteria may lead to morphological defects in consequence of ros generation and cell membrane destruction. indeed, anionic membranes act as powerful non-adhesive site of bacteria attributable to electrostatic repulsion (mukherjee and de, ; kolewe et al., ) . operating conditions influence the antibacterial activity performance of the nanofibrous membrane. as proved by several experiments, bacterial cells are able to decline their size at higher operating pressure, hence resulting in enhancing permeation through the filter. therefore, less trans membrane pressure (tmp) is usually desired, to retain antibacterial activity during long term application of the membrane. the tmp is described as the mean feed pressure minus the permeate pressure that is essential to push down water through a membrane (mukherjee and de, ; . different factors including surface area, surface roughness, pore diameter, zeta potential, and inclusion of biocides or antibacterial agents determine the antimicrobial performance of membrane (rahaman et al., ; mukherjee and de, ) . accordingly, the employing of electrospun polymeric membranes in bacterial and virus removal from water is performed in two procedures including size exclusion and adsorption (lee et al., ) . in most cases, the diameter of water-borne bacteria is more than . μm. for example, the e. coli size is . - . μm and brevundimonas dimimuta dimension is . - . μm. previous studies have confirmed that using a . μm pore sized mf leads to a log- log bacteria reduction (gómez et al., ; ghayeni et al., ) . thus, based on the degree of exclusion, the electrospun membrane should have an average pore size of fewer than . μm. in addition, the narrow pore size distribution is requisite for achieving a high retention rate (ma et al., ) . there is a direct relationship between the pore size and the fiber diameter of a porous nonwoven structure. the relationship has been confirmed as the average pore size was approximately ± times the mean fiber diameter, and j o u r n a l p r e -p r o o f the greatest pore size was about ± times the mean fiber diameter. thus pore size of electrospun fiber generally grows with increasing fiber diameters (ma et al., ) . various conventionally employed membranes for the application as micro-filters have . μm theoretical pore sizes. the advantage of electrospun nanofiber membranes in comparison to conventionally used membranes are the simplicity of manufacture, adjustable size of the pores and high porosity (saleem et al., ) . in view of the fact that the membrane pore sizes can be controlled by adjusting the electrospinning parameters and besides the fact that the most aquatic bacteria dimensions are more than . mm, electrospun nanofibers can be designed efficiently with smaller pore dimensions suitable for mf applications (wang and hsiao, ) . for instance, accelerating the flow rate raises the pore diameter by enhancing the fiber diameter. moreover, increasing polymer solution concentration and using higher molecular weight polymer increases fiber diameter. employing a secondary ring electrode circling the nozzle cause reducing the fiber deposition and consequently decrease the density of the membrane, the parameter which reduces the pore size. in addition, controlling fiber distribution, post electrospinning modification and using temporary spacers can be utilized for controlling pore size (dong et al., ; haider et al., ) . as discussed above and based on the size exclusion process, microfiltration larger sized bacteria are substantially seized by the membrane but it is not efficient in separating small sized viruses within . - . µm range size (mi et al., a; barhate and ramakrishna, ) . so, rejection of bacteria smaller than membrane pores or viruses needs the incorporation of antiviral or antibacterial agents into the membrane. also, after size-exclusion microbial removal of the membrane, intercepted bacteria can be released and induce membrane biofouling during subsequent filtration. therefore, antimicrobial agents are commonly used to prohibit bacterial j o u r n a l p r e -p r o o f growth and biofoul formation that would decline filter efficiencies (botes and cloete, ; wen et al., ) . various bioactive agents with different fundamental properties may have consequential impacts on bacteria removal. plus, nanofiltration membranes or ultrafiltration membranes with a positive charge on the surface are able to remove viruses selectively (mukherjee and de, ). moreover, incorporating antimicrobial agents into electrospun nanofibers enhance the antimicrobial activity of fabricated nanofibrous membrane (nasreen et al., ; park and kim, ). an ideal bioactive agent incorporated into the functionalized membrane should be nontoxic, water insoluble with no or slight leaching property. also, the functionalization process should not cause adverse influences on the quality and overall performance of the membrane. based on the majority of researches studied in this review, blending and post-modification strategies are two commonly used techniques to incorporate biocide agents into nanofibers aiming for water disinfection application (shalaby et al., ; he et al., ; makaremi et al., ) . blend electrospinning is an easy one-step procedure, mostly used for agents' incorporation into nanofibers (shabafrooz et al., ) . using the same solvent, the bioactive agent is dissolved directly into the polymer solution and a homogeneous blended solution of the incorporating agents in the polymer solution is prepared for the electrospinning step (pillay et al., ; fahimirad and ajalloueian, ). j o u r n a l p r e -p r o o f the agents incorporation into electrospun fibers can be performed after the electrospinning process by physical or chemical treatments. covalent and non-covalent immobilizations are fundamental methods for molecules attached to the fiber surface. non-covalent immobilization is performed by immersion of electrospun mats in a solution compromising the bioactive molecules. by treating with plasma the surface gets activated for subsequent modification using specified ligands like active amine groups. the affinity of incorporated agents to the electrospun nanofiber surface improves by covalent immobilization (wang and windbergs, ; kurusu and demarquette, ) . some commonly used antimicrobial or antiviral agents in electrospun nanofibers are discussed in this section. silver nanoparticles (agnps) are considered the most efficient nanoparticles for biological applications and the most extensively applied antibacterial agent for water disinfection mukherjee and de, ) . agnps are capable to puncture the microorganisms' cell walls, interact with their nucleic acids and attach to their enzymes, which cause the cell membrane destruction and finally growth inhibition. different feasible interactions of ag + ions with various bacterial biomolecules are documented. furthermore, the extended range of antibacterial activities and virulence effects of ag + ions toward several microorganisms (e.g. bacteria, viruses, and fungi) at only a few mg ml - are confirmed in previous studies. thus, silver nanoparticles are recognized as potent disinfection agents (lópez-heras et al., ) . in water purification, nanosilver materials have been mainly applied to prevent the formation of there are three main methods for agnps incorporation into electrospun nanofibers including ) blending of prepared synthesized agnps solutions to the polymer solution, ) agnp synthesis in the polymer solution by employing a precursor, and ) post-treatments of the electrospun nanofibers for agnp synthesis by reduction of the precursor that has been spun along with the electrospinning solution (fahimirad and ajalloueian, ). there are three main approaches to produce iron oxide nanoparticle-nanofiber composites, including ( ) electrospinning of solution containing prepared ionps, ( ) in-situ synthesizing of ionps during the electrospinning process or in the solution to be electrospun and ( ) it has been indicated that due to electrostatic interaction, cunps illustrate antibacterial functions on the bacterial cell through different mechanisms, such as adhesion to the bacterial cell wall, lead to detrimental impacts on protein structure within the cell membrane, denaturation of j o u r n a l p r e -p r o o f proteins in inertial parts of the cell , and adverse effects on phosphorus-and sulfur-containing compounds like dna (raffi et al., ) .recently, cunps have gained considerable interest because of their broad-spectrum and acutely effective antibacterial activity with comparatively low cost and high scalability (taner et al., ) . recently, zinc oxide (zno) has received much attention due to its non-toxic profile, effective antibacterial activity, adsorptive properties, mechanical, chemical, and thermal stability while encountering diverse environmental conditions (tiwari et al., ) . zno particles have illustrated antimicrobial activity against both gram-positive, gram-negative bacteria and even against spores wagner et al., ) . zno nps are considered bio-safe, nontoxic, and biocompatible (hameed et al., ; farrokhi et al., ) . in comparison with bulksized particles, nanoparticles can pass through bacterial cell walls more simply. the release of zn + ions from nps destroy the cell membrane and subsequently enhance cellular internalization of the nanoparticles. it is also confirmed that the antimicrobial function of zno can be ascribed to photocatalytic activity. by receiving uv light which promotes its interaction with bacteria, ros, which has a phototoxic effect on bacteria, will be produced (dimapilis et al., ) . journal pre-proof tio is a biocompatible chemical thermally stable compound with high photocatalytic activity and has shown good antimicrobial activities with wide spectrum function against microorganisms (gram-negative and gram-positive bacteria, fungi, and virus). the generation of reactive oxygen species (ros) is the major mechanism of tio . due to its photocatalytic nature, antimicrobial activity of tio nps enhances by exposing uv light on its surface (de dicastillo et al., ; levchuk et al., b; levchuk and sillanpää, ) . it is proved that lanthanum compounds, such as lanthanum hydroxide (la(oh) ), lanthanum carbonate (la co ), and lanthanum hydroxide (la(oh) ) can attach to phosphate so firmly that they can generate lapo and remove redundant phosphate in a bacterial cell. according to the very significant band to phosphate, nano-lanthanum (la) species represent high effectiveness adsorption and suppress microbial growth by inhibition of the microorganism growth liu et al., ) . carbon is the chemical element with atomic number and six electrons situate s , s , and p atomic orbital. graphene is a one-atom-thick hexagonal structure consisting of a -dimensional bhatnagar et al., ) . furthermore, the existence of these functional groups advances the interactions with biomolecules and leads to bacterial death with no intracellular process. go nano-sheets with sharpe edges hurt the bacterial cell membranes, lead to leakage of the intracellular matrix and eventually cause inactivation of bacteria. plus, go generate oxidative stress by producing ros and lead to dna damage and mitochondrial dysfunction (kumar et al., ) . in addition, the antiviral activity of go is confirmed by several experiments (ye et al., ) . single-walled carbon nanotubes (swnts) are nanometer diameter cylinders fabricated of rolled up graphene sheet in the form of a tube. generally, swcnt length is in the micrometer range and their diameters vary from . to to nm (eatemadi et al., ) . swnts have presented strong and board spectrum antimicrobial activities. the antimicrobial activity of swcnts has been confirmed to be varied by several factors. for instance, longer length nanotubes exhibited superior antimicrobial activity, swcnts having surface groups of -oh and -cooh illustrate more strong antimicrobial activity in comparison with swcnts-nh , also the diameter of nanotubes is an important factor governing their antibacterial effects (dong et al., ) . chitosan [poly-(b- / )- -amino- -deoxy-d-glucopyranose] is quaternary ammonium cations are positively charged polyatomic ions. these ions contain a positively charged nitrogen "head" binding four bonds r including an alkyl group or an aryl group. quaternary ammonium compounds are salts of quaternary ammonium cations (tezel and pavlostathis, ) . because of their positively charged sites, they are able to generate electrostatic bonds with the negatively charged sites on bacterial cell walls, resulting in disruption of a cell wall, defect cell membrane permeability and consequently sever leakage of intracellular low-molecular-weight materials (chen et al., ) . qacs target bacterial cell membranes. therefore, they illustrate extended-spectrum antimicrobial activity and have been widely employed to construct an antibacterial surface (jennings et al., ) . quaternized poly benzalkonium chloride (bac) are some important kinds of qacs (zhu et al., ) . easy release of biocides from the membrane improves their exposure rate to bacterial cell. there is a challenging point since the leaching profile of incorporated biocides determines long term bactericidal efficiency of the membrane. leaching of bactericidal agents resulted in the diminution of the membrane antimicrobial performance over time. gradual leaching of the blended biocides during the filtration process not only declines the antibacterial activity, but may j o u r n a l p r e -p r o o f also lead to secondary pollution (fu et al., ) . besides chemical contamination and cytotoxicity issues, the continuous release of bactericidal agents causes the development of bacterial resistance due to being exposed to sub-inhibitory concentrations of biocides (sile-yuksel et al., ; mukherjee and de, ) . thus, there is a challenge to provide process eluding leaching of toxic materials while illustrating rapid pathogens killing ability. nanofiber coatings based methods which promote contact pathogen-killing capacity are promising and can be obtained by chemical modification with tethered biocides functionalities. these strategies may be successful by regarding the right control over the binding quality between the active agent and the underlying biomaterial surface (zhang et al., ; bazaka et al., ; hilpert et al., ) . despite there are numerous researches on application of antibacterial electrospun nanofiber membrane in water filtration, the leaching pattern and durable bactericidal efficiency of the membranes have not been studied comprehensively. this method is generally used for testing the inherent antibacterial performance of fabricated electrospun nanofibers as a membrane. this assay generally consists of qualitative detection and quantitative measurement techniques (zhu et al., ) . the inhibitory activity of electrospun nanofibers is assayed by the inhibition zone diameter or agar diffusion method toward the considered bacterial sample, based on the clinical and laboratory standards institute (clsi document m -a ) (clsi, ) . for this reason, μl overnight culture of the tested bacteria ( cfu/ml) is spread across the surface of an appropriate agar plate, the electrospun nanofiber is cut to disk with about mm diameters, sterilized under uv light for min and then incubated on the plates for - h at °c. then, the area of bacteria growth is detected, and the diameter of the inhibition zone around the electrospun nanofiber is measured. this procedure modifications are also used (santos et al., ; jatoi and al mamun, ; fig. a) . the antibacterial function of the membrane will lead to changes in the bacteria cell morphology. this method is a shaking flask method. briefly, an appropriate amount of sample sterilized nanofiber is weighted, dipped into a flask containing pbs buffer with a cell concentration of - × cfu ml - . the flask is incubated with continuous shaking at • c for a determined time. after serial dilutions by the phosphate buffer, the bacterial suspensions are plated in the agar plate. the inoculated plates were incubated at • c for h and the viable bacterial cells are counted by a colony counter (kleyi et al., ) . also, the number of bacteria after incubation for a determined time can be indirectly measured by spectrometric optical density at nm . then, the reduction rate is calculated with the following equation: where r is the reduction rate, a is the number of bacteria isolated from the inoculated electrospun nanofibers after defined time contact time, and b is the number of bacteria isolated from the inoculated electrospun nanofibers at zero contact time (yao et al., ) . j o u r n a l p r e -p r o o f the aatcc test method quantitatively evaluates the bacteriostatic (growth inhibition) or bactericidal (killing of bacteria) ability of textiles over a hours contact. for this test, firstly a defined weighted of nanofiber is cut (about mg), get sterilized by uv light, then inoculated with . ml microbial suspension ( - / × cfu ml - ) and finally overnight incubated at • c. over determined contact period, ml pbs buffer is added to the falcon tubes containing the inoculated treated electrospun nanofiber. after min shaking, µl of the solution is cultured on nutrient agar plates and incubated for h (ardekani et al., ). astm e is another antimicrobial standard method used for evaluating the antibacterial function of immobilized antimicrobial nanofibers under dynamic contact conditions. the antibacterial efficiency is evaluated depending on the contact time from several mints to h between the bacterial solution and the sample (ungur and hrůza, ) . for both methods, the percentage of growth reduction is calculated with the r equation, mentioned above. in this method, some known dyes are been used to probe if the entrapped bacteria are inactivated by membranes and quantify surviving bacteria, representing an operative, visual and precise antibacterial assay (zhu et al., ) . for example, a common dye-based method is detecting the optimal analytical parameters for fluorescence measurements from the dyes syto and propidium iodide (pi). the basis of this approach is the attachment of syto to live-cell and propidium iodide (pi) to dead cells or cells with defected membranes. the optimal analytical parameters are used for measurement fluorescence by evaluating the intensity of emissions at - nm for syto and - nm for pi which interpret to quantify of the live cells minimum inhibitory concentration (mic) represents the minimum amount of antibacterial membrane, which could inhibit bacterial growth. in this method, the defined weighted of nanofiber is dissolved in water (or proper dissolvent), µl of this solution is added to the first well and serially diluted by transferring µl of the well pipetted content to the next well containing µl media. thereafter, µl of bacterial cultures ( × cfu ml - ) is poured to each well and plate is incubated at ºc for h. to detect the bacterial growth, resazurin or piodonitrotetrazolium chloride is added to wells. the wells that turned pink (if resazurin used) or purple (if p-iodonitrotetrazolium chloride used) represents the surviving of bacteria, hence no growth inhibition. the nanofiber concentration in the last growth inhibited well is considered as the mic value (nthunya et al., ). the bacteria retention test can also be performed with a dead-end filtration module using a vacuum filtration cell, a syringe filter holder mm, millipore and a dead-end filtration cell j o u r n a l p r e -p r o o f system (jabur et al., ; daels et al., ; son et al., ) . before the experiments, the membrane cut diameter and sterilized. all pieces of filtration equipment are sterilized with an autoclave method for min at °c. the membrane is fitted into the device. after passing sterile water from the filter, the bacterial suspension is filtered through the membranes using a pressure. in this step there are two different techniques for evaluation of bacterial retention: ) the filtrate is serially diluted with sterile distilled water and viable counts are assayed by plate counts. the colony count can be facilitated by staining bacterial cells with syto fluorescent dye and using a fluorescence microscope . then the bacterial retention ratio is calculated in terms of lrv (log reduction value) by the equation: lvr= log (c f /c p ) where r= ( -(c p /c f )) × electrochemical disinfection can destroy bacteria and viruses by electroporation and reactive oxygen species (ros) during a short time. electrochemical treatment devices electrochemical disinfection regarded as an effective portable water disinfectant. fabrication of electrospun j o u r n a l p r e -p r o o f porous membrane filter using agents to provide a conducting bed and a strong electric field, facilitate electroporation and production of ros, which signifies the disinfection process (hong et al., ; huo et al., ) . for testing this ability, an electrochemical filtration device with electrospun nanofiber as a filtration membrane is used. then, a saline solution containing bacterial suspension flows through the nanofiber filter using low voltages at a defined flow rate. the bacterial removal efficiency is calculated by the lvr equation (wen et al., ; tan et al., ; xie et al., ) . in order to determine the deposition of bacteria on a filtrated membrane and the possibility of biofouling, instantly after filtration, the membrane is transferred to an autoclaved beaker filled with pbs buffer and sonicated. the bacteria in the suspended membranes are measured by counting the number of colony cells. moreover, the morphology of trapped bacteria is investigated using sem (xie et al., ; makaremi et al., ; wen et al., ) . different kinds of electrospun nanofibers have been recently fabricated for bacterial removal from water are illustrated in tables . usually, evaluation of the antiviral function of nano-filters is carried out using bacteriophage and polypropylene nonwoven textile as support layer, the structure shows excellent retention of bacteria and fine solids, with - nm pore size and operating pressure < mbar. naked filter is anther novel commercially application of nanofiber in household/bottled water filter with ability to remove . % of the micro-organic contaminants. nanotrap is another commercial household water filter produced by coway company. astrapool, fluidra has introduced nanofiber based product applied in filtration system for residential pools (http://electrospintech.com/products.html#.xvs_nm zbiu). liquidity nanotech corporation has created electrospun nanofiber membrane made water purification cartridge with superior flow rate, about a cup per minute, good microbiological retention, -log bacteria reduction, -log virus reduction and -log cyst reduction and simple usage process (https://product.statnano.com/product/ /liquidity-water-purification-cartridge). pentair company has produced polyethersulfone nanofiber-based cartridge for industrial water purification applications. the cartridge is an absolute barrier to bacteria and viruses: with more than -log reduction rate (https://www.directindustry.com/prod/pentair-x-flow/product- - .html). the researcher's and industry's attention to research and development of electrospun nanofibrous membranes has been growing because of its simplicity, low-cost, scalable molecules incorporation process on the fabricated non-woven mats, production of membranes with the high surface area.. high surface area to volume ratio, uniform pore size, and high pore interconnectivity and adequate antibacterial property improve the performance of the nanofibrous membrane in water disinfection application (subramanian and seeram, ) . however, there are several major concerns to be noticed for the application of electrospun nanofiber in water disinfection. although high surface area and porosity of the electrospun nanofiber are significant advantages, which enhance permeability and selectivity, they also lead to higher mechanical stresses. consequently, the membrane might be compacted or deformed through the filtration process, which causes loss the porosity and subsequently decreases the also, further experimental studies needed to conduct proper control of biomolecules release rate from nanofiber, to ensure a balance between successfully deactivate the bacteria strains and lengthen the period of the function, and minimize contamination. therefore, fabrication of membrane representing inherent self-cleaning, antiviral, and the antibacterial and anti-biofouling feature has gained immense attention for industrial application. recently, focusing on the production of smart antibacterial surfaces has led to a promising "kill−release" strategy. this approach proposed the fabrication of dual-functional antibacterial surfaces by incorporating biocides into non-fouling materials. these membranes are able to maintain their long-term antibacterial activity by killing bacteria attached to their surface and subsequently are potent to release the dead bacteria to reveal a clean surface (wei et al., ) . although these smart membranes are applied for biomedical applications, the strategy can be promising for further j o u r n a l p r e -p r o o f designing of novel electrospun nanofiber with these dual functions and strong long-term functional ability in water disinfection. as it is illustrated in tables and , despite the significant results 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membranes using a controlled-release platform key: cord- -ycdok fc authors: shutler, j.; zaraska, k.; holding, t. m.; machnik, m.; uppuluri, k.; ashton, i.; migdal, l.; dahiya, r. title: risk of sars-cov- infection from contaminated water systems date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ycdok fc following the outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ) in china, airborne water droplets (aerosols) have been identified as the main transmission route, although other transmission routes are likely to exist. we quantify sars-cov- virus survivability within water and the risk of infection posed by faecal contaminated water within countries. we identify that the virus can remain stable within water for up to days, and country specific relative risk of infection posed by faecal contaminated water is related to the environment. faecal contaminated rivers, waterways and water systems within countries with high infection rates can provide infectious doses > copies within ml of water. the implications for freshwater systems, the coastal marine environment and virus resurgence are discussed. the outbreak of the severe acute respiratory syndrome coronavirus (sars-cov- ) began in wuhan province, china in december and has now spread throughout the world with about million cases confirmed globally within countries and territories. water aerosols originating from individuals infected by sars-cov- are considered a major pathway for infection , and the virus has been shown to remain stable in saline solution and under varying environmental conditions . viral shedding in faeces of viable sars- cov- virus is documented (eg ) and sars-cov- ribonucleic acid (rna) has been detected in the shed faeces of both symptomatic and asymptomatic children and adults (eg ); with potentially % of infections being asymptomatic and unreported . human viral pathogens that can be transmitted by water that pose moderate to high health significance as defined by the who include adenovirus, astrovirus, hepatitis a and e, rotavirus, norovirus and other enteroviruses. the survival of the large family of coronavirus in water systems has been highlighted , and viral loads within untreated wastewater, consistent with population infection rates, have been identified . while evidence for sars cov- is limited, other human coronaviruses are documented to survive in wastewater effluent , with colder water temperature likely to increase survival considerably . collectively this evidence suggests that sars-cov- virus can survive within water and the viral loads within untreated sewage effluent are likely high in countries of high infection rates, a portion of which is viable virus, and therefore water contaminated . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . and c) countries where relative risk has been calculated with relative risk as a linear scale; grey signifies a country not included. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . virus copies, the proportion of infectious viruses in sewage must be known. the presence of infectious virus in stool samples has been demonstrated , but there is a lack of quantitative data on this ratio for sars-cov- in stool. we instead used literature on the number of infectious adenovirus copies in sewage (eg ) and wastewater discharge into rivers to select high ( - ) medium ( - ) and low ( - ) estimates for the ratio of infectious virus to genome copies to infectious viruses. we note that adenoviruses are known to be particularly resilient, and therefore likely to represent an upper estimate, but also that our selected range is consistent with the - value used elsewhere for assessing viral risk in water systems (eg ), including one assessment for sars cov- transmission risk to wastewater workers . the temperature dependent survivability means that it is likely that the risk posed by wastewater will increase during winter months as the sewage temperature will be lower enabling longer viral survival, but temperature history and age of the sewage will be needed to fully understand any detected viral loads. sars-cov- infection to, and spread between, domestic cats has occurred due to similarities between human and some animal angiotensin converting enzyme (ace ) gene . increased animal foraging can occur downstream from water treatment facilities, relative to upstream, highlighting possible risk of some riparian wildlife infection if feeding occurs after a spill. it is possible that sars-cov- survivability and transport within rivers could impact drinking water supplies in countries where rivers or reservoirs are the primary drinking . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint water sources and where large populations, with little or no sewage treatment, exist close to the water source, such as within refugee camps or shanty towns. riverine enteric virus transport and catchment accumulation can occur for common viruses (eg ) and under stratified conditions it would be possible for a river plume to enter a reservoir and subsequently exit through the reservoir outlet without mixing with the main body of water. filtering of water, followed by ultraviolet disinfection or chlorination are the recommended approaches for virus removal from drinking water sources . filtering is normally used to remove large particulates. the effective ultraviolet dose for sars-cov- disinfection appears highly variable and dependent upon the surface to which the virus is attached . the upper dosage value of joule (j) cm - to ensure effective ultraviolet disinfection of sars-cov- is an order of magnitude larger than that typically used (~ to mj cm - ) for low volume domestic drinking water treatment. the world health organization (who) guidelines state that effective chlorination disinfection occurs at residual chlorine concentrations of ≥ . mg l - , which matches the minimum needed to deactivate sars-cov- . however, the actual chlorine dosage used for water treatment can vary, based on country, region, water origin and infrastructure (eg uk guidelines are concentrations of . to . mg l - ). collectively this means that if a drinking water source was to become infected with sars-cov- the standard virus removal and disinfection approaches of ultraviolet exposure and chlorination may not reduce the virus below detectable limits. reviewing of regional or countrywide drinking water processing approaches is recommended to reduce the potential for sars-cov- surviving through drinking water processing systems. boiling of drinking water will result in the virus being orca and pilot whales . of particular concern are whales whose throats are exposed to large volumes of water during feeding and who visit coastlines for prey that are known to accumulate around sewage outfalls, such as minke whales feeding on mackerel or orca feeding on chinook salmon. in these instances, the animal could be exposed to a large viral dose, even if the virus is only present within the water in low concentrations. for example, if the riverine viral concentration is low at copie ml - , which is undetectable by pcr (detection limit is > copies ml - ), then a medium sized whale filtering water during feeding could receive repeated doses of . million copies every second (see methods for calculation). a seafood market is among the suspected sources for the origin of the sars-cov- virus, so any viral transmission from land to sea may be a circular process. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint the relative risk of sars cov- from waste water systems is calculated by using a modified version of equations and from , given as where v ww,c is the per capital daily volume of domestic water usage for country c, and df c is the dilution factor downloaded from supplemental table and supplemental table we note that measured wastewater viral counts in paris on the th april were . × genome copies l - with , active cases , whereas using our (albeit country specific) method gives the estimate of . × genome copies l - , which is within the correct order of magnitude (this calculation used the same number of active cases). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint to calculate c faeces we assumed a log-normal distribution and calculated the expected value using the mean and standard deviation from using the standard equation: where µ is the sample mean and σ is the sample standard deviation of the log normal distribution. state that µ of the distribution is . log copies ml - and σ = . log copies ml - which results in an expected c faeces concentration within the sewage effluent of . million copies ml - . v faeces is the mean daily volume of faeces generated per person ( . kg, from table of and assuming faeces has a density approximately equal to water . note we used the 'rich country' value from because the rt-pcr data that we use to estimate c faeces was measured from samples collected in germany. the four orders of magnitude, and as such we selected high ( - ), medium ( - ) and low ( - ) estimates (which equate to %, % and . % proportion of viable versus within the total viral genome counts). the expected number of copies of infectious virus resulting from a sewage spill into a river, lake or coastal region for a given country can therefore be calculated as c spill,c was estimated for may countries that contain large inland water bodies and were known to rely upon reservoirs for drinking water . long-term statistical mean . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint water temperature, needed to calculate virus survivability, was calculated from a climate quality global lake temperature dataset (see below). temperature values for each country were the countrywide mean lake temperature within a rectangular box matching a simplified country outline. the dilution factors reported in can vary by several orders of magnitude and were deemed to provide the major source of uncertainty in the calculation. the concentration of sars-cov- virus needed for infection is not known. provides copies for influenza. the infectious dose for sars-cov- is likely significantly lower because ranks influenza as "very high infective dose" and sars-cov- as "low". we therefore use a value of copies as a concentration that could result in infection. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint number of active cases (± %), mass of faeces generated per capita per day ( . kg, see table of , mean number of viral genome copies in faeces ( . × ) and density of faeces was not included in the uncertainty analysis. this resulted in a combined uncertainty budget of ± % copies ml - . it is important to note that this value does not include uncertainty in the dilution factors or the ratio of viral genome copies to infectious virus. instead, the c inf calculation was repeated for high, medium and low values of these parameters. as reported in , the virus concentration in water follows an exponential decay, with its half-life decreasing with decreasing temperature and the ph control of half life is very small over the ph range of - (which encompasses the range found in natural freshwater and marine systems). based on the in vitro data presented in , the following empirical model was derived to describe virus concentration reduction factor due to the temperature- dependent die-off: = !. !°! !!. ( ) = ! !!" ( ) where c is initial virus concentration (copies ml - ), n(t) is virus concentration after time t (days) and r is hour survival factor due to temperature t driven die off. this model fit to the in vitro data gives a root mean square difference (rmsd) of ± % for water at °c which increases to ± . % at °c. when considering temperature controlled survival in the waste water system, c ww,c becomes the value used for the initial viral load c following a sewage effluent spill. as noted in , , the viral load follows a heavy-tailed distribution with the majority of patients shedding around copies ml - ) but some having viral loads as high as copies ml - . this results in the super-spreader problem where a tiny proportion of the infected population can become responsible for contributing a majority of . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint viral load in the wastewater. for a large infected population, this approach allows robust statistical modeling of viral load. however, in case of smaller communities with low number of infections, the actual viral load could be severely underestimated if a super-spreader is present within the population. the example volume flow rate through the mouth of a medium sized bowhead whale whilst feeding was provided by ) a flow rate of . m s - is given for a m whale (mouth pressure of - pa at a km h - foraging speed, assuming an oral opening of . m with an opening radius = . m). assuming a low viral concentration of copies per ml - , which equates to copies l - . . m s - equates to l s - . the dosage per second as the whale swims during feeding is given by (copies l - ) × (l s - ) = . million copies s - . severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges evaluation of saline, phosphate buffered saline and minimum essential medium as potential alternatives to viral transport media for sars-cov- testing stability of sars-cov- in different environmental conditions infectious sars-cov- in feces of patient with severe covid- virological assessment of hospitalized patients with covid- . suppression of covid- outbreak in the municipality of first detection of sars-cov- in untreated wastewaters in italy first confirmed detection of sars-cov- in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid- in the community potential fecal transmission of sars-cov- : current evidence and implications for public health high levels of sewage contamination released from urban areas after storm events: a quantitative survey with sewage specific bacterial indicators an analysis of sars-cov- viral load by patient age worldwide estimation of river concentrations of any chemical originating from sewage-treatment plants using dilution factors discharge-based qmra for estimation of public health risks from exposure to stormwater-borne pathogens in recreational waters in the united states coronavirus in water environments: occurrence, persistence and concentration methods -a scoping review an improved infectivity assay combining cell culture with real-time pcr for rapid quantification of human adenoviruses and semi-quantification of human adenovirus in sewage quantification of human adenovirus and norovirus in river water in the north-east of france qmra of sars-cov- for workers in wastewater treatment plants evaluation of lockdown impact on sars-cov- dynamics through viral genome quantification in paris wastewaters broad host range of sars-cov- predicted by comparative and structural analysis of ace in vertebrates evidence of viral dissemination and seasonality in a mediterranean river catchment: implications for water pollution management water, sanitation, hygiene, and waste water management for the covid- virus: interim guidance rapid evidence summary on sars-cov- survivorship and disinfection, and a reusable ppe protocol using a double-hit process. medrxiv ( ) study on the resistance of severe acute respiratory syndrome- associated coronavirus freshwater clams as bioconcentrators of avian influenza virus in water. vector-borne zoonotic dis the characterization of feces and urine: a review of the literature to inform advanced treatment technology review of synthetic human faeces and faecal sludge for sanitation and wastewater research world water: resources, usage and the role of man made reservoirs lake surface water temperature (lswt) v . ( - ) influenza virus aerosols in the air and their infectiousness emerging threats from zoonotic coronaviruses-from sars and mers to -ncov models of hydrodynamic flow in the bowhead whale filter feeding apparatus key: cord- - jv em authors: alegbeleye, oluwadara oluwaseun; singleton, ian; sant’ana, anderson s. title: sources and contamination routes of microbial pathogens to fresh produce during field cultivation: a review date: - - journal: food microbiol doi: . /j.fm. . . sha: doc_id: cord_uid: jv em foodborne illness resulting from the consumption of contaminated fresh produce is a common phenomenon and has severe effects on human health together with severe economic and social impacts. the implications of foodborne diseases associated with fresh produce have urged research into the numerous ways and mechanisms through which pathogens may gain access to produce, thereby compromising microbiological safety. this review provides a background on the various sources and pathways through which pathogenic bacteria contaminate fresh produce; the survival and proliferation of pathogens on fresh produce while growing and potential methods to reduce microbial contamination before harvest. some of the established bacterial contamination sources include contaminated manure, irrigation water, soil, livestock/ wildlife, and numerous factors influence the incidence, fate, transport, survival and proliferation of pathogens in the wide variety of sources where they are found. once pathogenic bacteria have been introduced into the growing environment, they can colonize and persist on fresh produce using a variety of mechanisms. overall, microbiological hazards are significant; therefore, ways to reduce sources of contamination and a deeper understanding of pathogen survival and growth on fresh produce in the field are required to reduce risk to human health and the associated economic consequences. foodborne diseases are rife in many regions of the world, with at least in people falling ill yearly from consumption of contaminated food and , deaths occurring as a result, according to the world health organisation (who) ( ) . foodborne diseases have exerted pressure on medical services, contributed to economic and political distress, exacerbated malnutrition and led to human suffering. there are several agents such as chemicals, pathogens, and parasites, which may adulterate food at different points in the food production and preparation process (allos et al., ) . many of these agents have been extensively characterized and investigated by numerous studies (farber and peterkin, ; zhao et al., ; le loir et al., ; ehling-schulz et al., ; adzitey et al., ; botana, ) . strategies and protocols to prevent occurrence (and outbreak) of foodborne diseases have been devised and implemented by many researchers, regulatory bodies, and governments. however, despite the considerable progress achieved scientifically, foodborne diseases continue to occur, representing a significant cause of morbidity and mortality globally (mead et al., ; murray et al., ) . although foodborne diseases are more common in developing countries particularly in africa and south east asia with specific groups of people such as children, the immunocompromised, pregnant and aged being particularly at risk, foodborne diseases are not limited to these regions or groups of people (who, ) . for instance, according to the centres for disease control and prevention (cdc), between and , there were . million episodes of domestically acquired foodborne gastroenteritis caused by unspecified agents in the united states alone (cdc, ) . approximately . million acute gastroenteritis occurred, and there were at least , hospitalizations in the us each year and hospitalizations caused by the known gastroenteritis pathogens. an estimated persons died of acute gastroenteritis each year, of which deaths were caused by the known foodborne pathogens (scallan et al., ) . health canada ( ) estimates that e million cases of foodborne illnesses occur in canada every year. although the conventional notion is that foodborne diseases typically originate from meat and poultry products, vegetables and fruits have been implicated in various foodborne outbreaks (westrell et al., ; lynch et al., ; [european food safety authority (efsa), ] . a significant increase in foodborne disease outbreaks or cases associated with consumption of fresh produce has been reported. this increase has been largely due to a general increase in produce consumption, globalization of the produce industry and more effective surveillance (tauxe et al., ; lederberg et al., ; havelaar et al., ) . increased consumption of fresh produce is likely due to global government efforts to promote healthy eating, the associated health-promoting benefits of consuming fresh produce and ease of access to fresh local produce (pollack, ; regmi, ; berger et al., ; painter, ) . since fresh produce is mostly eaten raw or after minimal processing, pathogen contamination constitutes a potential health risk (callej on et al., ; li et al., ) . there are numerous factors capable of compromising the microbiological integrity of produce along the farm to fork continuum, all of which have potentially fatal outcomes. however, pre-harvest hazards to produce have been recognized as important because usually, once pathogen contamination is established in the field, it can be challenging to decontaminate produce. there are numerous circumstances that can undermine the safety of produce on farms. many of these arise because agriculture has grown more intensive over the years, and produce fields are often located near animal production zones thus entwining the ecological connections between wild animals, livestock and produce (strawn et al., a,b) . this, in many cases, predisposes fruits and vegetables to pre-harvest hazards. some important pre-harvest hazard sources to produce include the use of contaminated soil, irrigation water and manure for produce cultivation. wild animals and insects have also been implicated as vehicles of pathogens to produce. to ensure produce safety on a sustainable scale, it is imperative to correctly understand the routes of entry, fate, transport, establishment, and survival of pathogens in the agricultural environment such as soil, irrigation water and manure. the knowledge gap in this regard is being filled rapidly, as many studies have attempted to explain the behavior of foodborne pathogens in agricultural media and describe the associations among pathogens, produce and the agrarian environment. in this review, the extent of the produce contamination problem is discussed as well as the sources and routes of contamination of soil, irrigation water, fruits, and vegetables. also, the various mechanisms and strategies through which bacterial pathogens become established on fruits and vegetables are briefly examined. the nutritional and health benefits of consuming fruits and vegetables have been recognized and widely publicized. this has elicited changes in human dietary habits, with many consumers incorporating more fruits and vegetables into their meals. consequently, the global production of fruits and vegetables has surged exponentially in recent decades. the increased demand for produce has led to modifications such as increased use of soil amendments, utilization of alternative water sources and increased imports and exports in agriculture-spanning across agronomic practices, processing, preservation, packaging, distribution, and marketing . some of these modifications, however, have great potential to compromise the safety of fruits and vegetables. the biological hazards that are most relevant to fresh produce safety are either zoonotic or human in origin and can be classified into sporeforming bacteria, non-spore forming bacteria, viruses, parasites and prions (james, ) . most studies/surveillance efforts have identified bacterial contaminants in produce-borne illness outbreaks. there is, therefore, a disproportionately higher abundance of information regarding bacterial contamination in the literature. this may be because bacterial species have in fact caused many more outbreaks, but other microbial groups-viruses and parasites have been understudied. the most commonly implicated etiologic agents are presented in table . although data and information available on outbreaks associated with fresh produce are diverse and patchy, the available research evidence indicates that the foodborne illness burden due to contaminated produce has increased, in recent decades. in the united states, between and , approximately % of total foodborne illness outbreaks were produce related (jung et al., ) . in europe, from to , produce was linked with % of the outbreaks, % of the hospitalizations and % of the deaths (efsa, ) . in australia, fresh produce was linked to % of all foodborne disease outbreaks informed from to (lynch et al., . specific produce items are more commonly linked to foodborne illness incidents; for example, leafy greens such as lettuce and spinach, as well as fresh herbs such as parsley and basil are conventional sources of bacterial infections (who, ; berger et al., ; denis et al., ) . berries, green onions, melons, sprouted seeds, and tomatoes are similarly high-level priority produce items (olaimat and holley, ; denis et al., ) . in the us, between and , of multistate foodborne outbreaks were associated with vegetables (cdc, ) . a list of recent produce-related outbreaks is presented in table . most industrialized nations especially the united states have extensive and exhaustive datasets indicating the magnitude of outbreaks, the extent of severity and casualties incurred, the implicated pathogen and produce item as well as documented preventive protocols to avoid future outbreaks. unfortunately, however, the same is not true of many other countries especially african countries, the majority of which are still grappling with other challenges and hence, lack the resources to efficiently track and trace foodborne illness incidents (who, ) . many conventional foodborne detection methods are time consuming and laborious, and advanced techniques have therefore been developed and optimized as alternatives to or for use in combination with these traditional techniques. many of these are rapid, sensitive, reliable and standardized. they can be categorized into nucleic acid based, biosensor-based and immunological based methods (croci et al., ; adzitey et al., ; law et al., ) . typical examples include simple polymerase chain reaction (pcr), multiplex pcr, real-time pcr, nucleic acid sequence-based amplification (nasba), loop-mediated isothermal amplification (lamp) and oligonucleotide dna microarray. other examples are optical, electrochemical and mass-based biosensors, and enzyme-linked immunosorbent assay (elisa) and lateral flow immunoassay (law et al., ; gilchrist et al., ) . these advances in epidemiological investigation approaches and techniques have made it possible to explore the crucial associations between produce and pathogens. in spite of this, however, prompt identification of implicated produce vehicles, location or point of contamination in fresh produce associated outbreaks is still a significant challenge. one prime constraint is the relatively short shelf life of fresh produce, which is often discarded by the time an outbreak is identified (strausbaugh and herwaldt, ; lynch et al., ) . therefore, most of the time, the real source of contamination is not ascertained causing investigators to speculate or assume a source. this is, however, dangerous because, in addition to the possibility of being wrong, there is empirical evidence that once a particular transmission pathway is identified, repeated investigations are bound to be biased in causation (lynch et al., ) . another important consideration is that usually, outbreaks receive widespread attention if the event (i) has severe public health impacts (ii) is unusual or sudden (in that the etiological agent and/produce type are unanticipated; making the circumstances of the outbreak unique and (iii) poses a significant risk of international spread with consequences for international travel or trade. invariably, the smaller, 'less significant' outbreaks are never investigated. more importantly, foodborne illness incidents occur sporadically in populations, and these cannot be captured in routine epidemiological surveillance or outbreak investigations (scallan et al., ) . this means that the data available may not be a valid representation of the problem. it is likely that the foodborne illness burden related to consumption of contaminated produce is still largely underestimated. the possible routes and sources of produce contamination are numerous, and intensive efforts have been made to accurately understand the exact mechanisms through which pathogens are introduced into fresh produce (kotzekidou, ) . sources and routes of produce contamination vary for different production zones. this is because each farm has a distinct combination of environmental risk factors such as topography, land-use interactions, and climate. combinations of these peculiar environmental risk factors influence the frequency and transmission of foodborne pathogens and subsequently impact the risk of produce contamination (strawn et al., b) . primarily, pathogens may contaminate produce 'on-field' via various routes including; atmospheric deposition, uptake from contaminated soils and groundwater (harris et al., ; lynch et al., ; mei soon et al., ) , use of raw (or poorly treated) manure and compost, exposure to contaminated water (irrigation or flooding), transfer by insects, or by fecal contamination generated by livestock or wild table the most commonly implicated etiological agents in fresh produce borne illnesses (brackett, ; buck et al., ; heaton and jones, ; jung et al., ; callej on et al., ) . animals (cooley et al., ; uyttendaele et al., ) . a schematic representation of the main entry points for pathogens to humans via produce is provided in fig. . the use of organic materials such as livestock excreta, slurries, abattoir wastes, sewage sludge as well as municipal and industrial waste treatment residuals as soil amendments is widespread goss et al., ) . although these serve as a costeffective source of nutrients for agricultural purposes, research demonstrates that raw manure as well as contaminated (or improperly treated) manure constitute a significant risk of pathogenic contamination for produce (james, ; manyi-loh et al., ) . public health relevant bacteria, viruses and parasites such as e. coli o :h , salmonella spp., l. monocytogenes, campylobacter spp., porcine enteroviruses, bovine coronavirus, bovine virus diarrhoea cryptosporidium parvum and giardia have been isolated from raw/poorly treated manure (derbyshire, ; derbyshire and brown, ; sellers, ; strauch, ; pell, ; grewal et al., ) . pathogens may be spread through direct interaction of vegetable surfaces with manure, or by splashing of (contaminated) soil/manure particles from the soil on vegetables via rainfall and/ overhead irrigation or by vectors. additionally, manure piles stored next to growing areas may constitute contamination risk due to run-off (james, ; warriner et al., ) . manure application could be by broadcasting as a solid, semisolid or liquid throughout the field or by the introduction of livestock or wildlife feces at distinct locations (jung et al., ) . in many parts of the world, organic cultivation systems use more manure than conventional growers, and chemical treatment against pathogens is prohibited in organic farming. there have thus been some assertions that organic produce represents a more significant safety risk than its non-organic counterpart, although, there is no unequivocal research evidence supporting this claim loncarevic et al., ; warriner et al., ; ivey, ; maffei et al., ) . the survival of pathogens in manure and biosolids depends on factors such as the manure source, production process, and characteristics, treatment technique applied, physicochemical factors like ph and relative humidity, incidence of antagonists or predators, weather conditions, desiccation, aeration, soil type, degree of manure incorporation, amongst others (ingham et al., ; wood, ) (table ) . the manure composition, which is determined in large part, by the feed formulation, dictates the profile of pathogens occurring in manure as well as their ability to persist even posttreatment (franz et al., ) . certain workers have proposed that cattle diet may influence the incidence of representative bacterial species; e. coli o :h and salmonella in manure. these pathogens have been reported to persist longer in manure obtained from cattle fed diets rich in energy but low in fiber content such as high digestible grass silage and maize silage compared to animals that received diets with low energy and higher fiber content such as straw . it has also been suggested that feeding cattle with hay may significantly reduce shedding of acid-resistant e. coli (diez-gonzalez et al., ; . how effective these strategies are in reducing pathogen load in (animal-derived) manure, is however not clear. manure treatment techniques such as composting, aerobic and anaerobic digestion, pelleting, alkaline stabilization, conditioning, dewatering and heat drying have been used to treat manure before application as fertilizer for a long time. while many of them are reasonably efficient, concerns have been raised about their ability to satisfactorily eliminate pathogenic bacteria (day and funk, ; lu et al., ; lorin et al., ) . tailing of pathogen inactivation curves, as well as apparent regrowth or recontamination of bacteria after treatment, have been reported. many pathogens have been shown to be capable of withstanding manure treatment processes, thereby, constituting a major risk of contamination (brackett, ) . composting is a popular manure treatment and composting temperatures that exceed c for three days are considered sufficient to kill most pathogens (grewal et al., ) . however, few studies have demonstrated that the heat-induced death of bacteria in composted materials is a complex phenomenon (ingham et al., ; gupta, ) . bacterial regrowth and recontamination in cooled compost have been reported (hassen et al., ; ingham et al., ) . pelletizing is another common treatment available and is commonly applied to chicken manure (chicken manure pellets). pelletizing the manure reduces the off-odor and facilitates transport and storage. although the process usually involves a thermal procedure, more studies are required to validate whether the process efficiently inactivates clinically relevant pathogens (chen and jiang, ; jung et al., ) . the use of a fish emulsion as fertilizer has raised similar concerns; although most preparation methods available include a thermal process, the ability of this to inactivate enteric bacteria and viruses needs to be rigorously validated (jung et al., ) . due to the diverse range of variables associated with manure composition, treatment, pre-application storage, application and incorporation, regulatory bodies have stipulated minimum manureto-harvest time intervals necessary to ensure microbiological safety. the united states department of agriculture (usda) 'organic production and handling' specifies that unless composted, raw animal manure must be incorporated into the soil not less than days prior to harvest of a product whose edible portion has direct contact with the soil surface or soil particles, or days if there is no direct contact (usda, ) . canadian authorities specify , and months for tree fruits and grapes, small fruits and vegetables respectively as the minimum time delay between manure application and harvest for these crops (olaimat and holley, ) . irrigation water has been identified as a potential source of produce contamination (benjamin et al., ; uyttendaele et al., ; faour-klingbeil et al., ) . being a common and essential requirement for crop production, water must be supplied to plants when necessary, and irrigation water sources are used to supplement limited rainfall in many areas (kirby et al., ) . epidemiological investigations of food poisoning outbreaks, experimental studies examining pathogen contamination of fruits and vegetables as well as observations of increased incidence of disease in areas practicing wastewater irrigation with little or no wastewater treatment indicate that contaminated irrigation water might indeed be a source of foodborne pathogens on fresh produce (norman and kabler, ; hern andez et al., ; steele and odumeru, ) . for example, hepatitis a outbreaks associated with lettuce (seymour and appleton, ) and spring onions (josefson, ) were linked to sewage-contaminated irrigation water (heaton and jones, ) . various factors including irrigation regime (method and timing of irrigation), irrigation water sources, type of crop and land use practices in the farm influence the extent and frequency of pathogenic contamination of produce ( such as pathogen concentration, pathogen strain, weather patterns, plant state, and physiology also have significant implications for produce safety (marvasi et al., ; uyttendaele et al., ; decol et al., ) (table ) . there are several types of irrigation systems available, each of which is typically complex and has its own drawbacks. most irrigation systems create complicated ecological environments with multiple potential sources and routes of pathogenic contamination (pachepsky et al., ) . each irrigation subsystem: collection, replenishment, storage, conveyance, distribution off and on-farm, as well as on-farm application involve processes that have great potential to compromise the microbiological integrity of the irrigation water in unique ways. during transportation from the source to the field, water is susceptible to significant microbiological depreciation (pachepsky et al., ) . the prevailing deterioration dynamic will depend on the transportation mode. for instance, irrigation water transport via irrigation ditches and canals involves interaction with microbial reservoirs of bottom sediments, bank soils, algae and periphyton, whereas water transport via pipes involves interactions with biofilms in the transport pipes pachepsky et al., ) . this sort of contamination is particularly prominent in reclaimed water distribution systems . the method of storage for irrigation water can have a profound effect on pathogen transmission. for example, certain studies have demonstrated that water quality is rapidly degraded in storage ponds and tanks due to inputs from avian species or other wildlife (field and samadpour, ; mclain and williams, ; higgins et al., ) . other storage systems such as check dams, impoundments, inter-basin transfer schemes, abstraction schemes and reservoirs have been identified as places where indicator and pathogenic microorganisms can survive and proliferate (abbasi, ; kirubel, ) . the mode of application also has significant impacts on the risk of microbiological contamination (berger et al., ) . compared with furrow and subsurface drip irrigation systems, sprinkler irrigation poses a higher risk of microbiological contamination (kisluk and yaron, ; pachepsky et al., ) . surface furrow and drip irrigation systems minimize contact between edible portions of certain plants (leafy vegetables provide larger surface area for contact and possible microbial attachment) and irrigation water (directorate, ; fonseca et al., ; mei soon et al., ; uyttendaele et al., ) . hydroponic growing systems also offer this advantage (jung et al., ; allende and monaghan, ) . the irrigation application method has been determined to influence the internalization of some pathogens in produce such as spinach plants. according to some studies, the likelihood of internalizing pathogens increases when the organisms are introduced by water sprinkling systems as opposed to when the water is directly applied to the soil (solomon et al., ; stine et al., ; mitra et al., ; warriner et al., ; erickson et al., a; kisluk and yaron, ; zheng et al., ) . more details on pathogen internalization are provided in section (below). depending on the geographical location, the irrigation regime with respect to time of day, season and harvest time may influence the likelihood of pathogenic contamination. for example, kisluk and yaron ( ) in a study conducted in haifa, israel demonstrated that night-time irrigation and irrigation during the winter season is more likely to contaminate plants with enteric bacteria. contaminated irrigation water poses the most significant risk when crops are irrigated close to harvest time, because harvesting of produce containing viable pathogens is more likely. therefore, an adequate time interval between irrigation and harvest should be conscientiously followed. the microbial quality of irrigation water depends mostly on the source of the water. in order of increasing risk of microbial contamination hazard, irrigation water sources can be ranked as follows: potable or rainwater, deep groundwater, shallow groundwater, wells, surface water and raw or inadequately treated wastewater (james, ; pachepsky et al., ) . the microbial quality of rainwater or rain-harvested water is relatively good. the quality and safety of use, however, depends largely on the collection, transportation and storage means. this can be illustrated with roof-harvested rainwater, which may become contaminated with pathogenic bacteria and protozoan parasites because of the occurrence of animal droppings on roofs, particularly immediately after relatively long periods of drought . groundwater (or borehole water) is usually microbiologically safe, except if it has been contaminated with surface runoff or other sources of contamination close to the aquifer. certain farm operations such as intensive dairying and border-strip irrigation (a type of surface irrigation, which is a hybrid of level basin and furrow irrigation) (valipour et al., ) lead to leaching of pathogens such as e. coli and campylobacter to shallow groundwater, thereby contaminating it (close et al., ) . water from wells that are free from leaks and have sound casing are expected to be microbiologically safe. factors such as the design of wells, nature of the substrata, depth to groundwater and rainfall may affect the microbial quality of good water (james, ; gerba, ) . surface waters; which are the predominant source of irrigation waters in many countries, including open canals, ponds, lakes, rivers and streams are much more susceptible to pathogenic contamination compared to groundwater (allende and monaghan, ; uyttendaele et al., ) . sewage discharges, septic tank contamination, storm drains, wild and livestock defecation, run-off from contaminated fields, industrial and municipal effluents can all potentially contaminate surface waters (steele and odumeru, ; james, ) . wastewater is usually of poor chemical and microbiological quality. therefore, it requires extensive treatment before it can be safely used to irrigate crops. water sources (other than rain) used to irrigate produce is usually only minimally treated or untreated in many cases (steele and odumeru, ; jung et al., ) . it is expensive and time-consuming to treat irrigation water up to drinking water standards, which is the ideal recommendation (crook and surampalli, ; forslund et al., ) . although awareness of the potential dangers of using microbiologically compromised water for irrigation has increased in recent times, scarcity of water resources in certain regions has contributed enormously to the use of sub-optimal supplementary irrigation water sources. in such cases, irrigation water represents a greater microbiological risk to produce (sundstr€ om et al., ) . one of the most frequent pathogens implicated in water-related outbreaks is e. coli o :h (cdc, ; hilborn et al., ) . the organism can survive for a protracted period in water (even in deionized water) depending on temperature conditions (chalmers et al., ; islam et al., a) . it also exhibits a remarkable ability to withstand extreme environmental conditions such as high acidity and extremely low-temperature conditions. the ability of a pathogen to survive (or persist) in the environment (and on produce) is an essential determinant in the risk of human infection. the actual risks associated with pathogens occurring in irrigation water depend on numerous variables including environmental conditions such as temperature, ph and uv light (sant'ana et al., ) . other factors such as the excreted load of the pathogen, its latency period before it becomes infectious, its ability to efficiently multiply outside a mammalian host, its infectious dose for humans, inhibitory competition from the indigenous microflora as well as host response also play a relevant role (steele and odumeru, ) . bacteria and viruses survive for lengthier periods in groundwater compared to surface water because groundwater tends to be cooler, offers protection from sunlight, and has less biological activity (steele and odumeru, ) . these groups of microbes only typically last no longer than and days in surface water and sewage, respectively. conversely, parasites (eggs/cysts) may survive for as long as days or even several months in surface water and wastewater (lefler and kott, ; sagik et al., ; bihn, ) . this suggests that pathogenic microorganisms are capable of surviving for extended periods, which constitutes a profound threat to produce safety. regardless of the source or route of exposure, one potentially fatal consequence of pathogen contamination of irrigation water is the repeated inoculation of plants with the pathogens. the fate and transport of these pathogens once introduced into the produce vary widely (table ). some pathogens are capable of adhesion to surfaces of produce while some others can rapidly internalize into plant tissues under certain conditions, translocate and persist until consumed (wariner et al., ; bernstein et al., a; doyle and erickson, ) . this has rendered many conventional processing and chemical sanitizing methods ineffectual (hong and moorman, ) and is a growing public health concern. although the potential for produce contamination via irrigation water has been identified, it is difficult to estimate the magnitude of the problem (groves et al., ; antwi-agyei et al., ) . despite the fact that numerous studies have linked poor microbiological quality of irrigation water with the incidence of human pathogens on fruits and vegetables, direct evidence of irrigation water causing foodborne disease is relatively rare (harris et al., ) . this is because a substantive "cause-effect" relationship is yet to be established as it is required that the same pathogenic strain is isolated from the patient, produce, and irrigation sources (pachepsky et al., ) . furthermore, there must be a clear sequence of events connecting patient, produce, and irrigation source (steele and odumeru, ) . this is difficult to achieve due to certain limitations such as an inability to promptly identify the locations associated with produce contamination and delays inherent in foodborne outbreak investigations (pachepsky et al., ) . in the absence of direct confirmation, the "cause-effect" relationship can only be deduced based on circumstantial or subjective evidence (pachepsky et al., ) . also, it is apparent that there is no valid link between detected pathogen levels in irrigation waters and disease risk. some studies have demonstrated a lack of correlation between pathogen prevalence in waters used for irrigation and disease incidence due to consumption of irrigated produce (cooley et al., ; mcegan et al., mcegan et al., , . there is an abundance of laboratory studies elucidating potential mechanisms of produce contamination from waterborne pathogens. however, field studies showing the exact process of produce contamination via this medium are relatively scarce. it is thus expedient to generate more field data in this regard. soils typically harbor an abundant consortium of microorganisms, some of which are human pathogens such as b. cereus, clostridium botulinum, c. perfringens, listeria monocytogenes and aeromonas (nicholson et al., ; warriner et al., ; jay, ) . they may, therefore, serve as a medium of plant contamination through seeds, roots or surfaces. many soil resident pathogens have adapted to survival in soil with spores persisting indefinitely. however, since many agricultural soils are predisposed to point and nonpoint sources of pathogenic contamination, allochthonous pathogens may continuously be introduced into soil environments (santamaria and toranzos, ) . some of the primary sources of pathogens into soil include the use of contaminated irrigation water and manure, animal grazing, municipal solid wastes and other effluents (santamaria and toranzos, ; sant'ana et al., ) . the fate, survival and recalcitrance of pathogens in soil depend on factors such as soil type, soil moisture, ph, temperature, nutrient availability, agronomic practices, as well as soil biological interactions (table ) . soil matric potential (moisture levels) is determined by soil properties and water inputs through precipitation and/irrigation and has been demonstrated to be one of the most critical factors influencing microbial transport and survival in soil . cool, moist environments are favorable for the survival of bacteria and viruses. under dry soil conditions, a reduction in bacterial and viral population densities are usually observed (santamaria and toranzos, ; ghorbani et al., ) . escherichia coli survival has been reported to be highest in organic soils under flooded conditions, and peak populations recorded after a rise in the water-table accompanying significant rainfall events (tate, ; rochelle-newall et al., ) . some pathogens such as streptococcus faecalis have been proven to thrive poorly under low soil moisture conditions (kibbey et al., ; jamieson et al., ; cabral, ) . increased rates of virus inactivation at low soil moisture levels have been demonstrated (yeager & o'brien, ) . also, decreased recovery of viral (poliovirus type and coxsackievirus b ) infectivity in dried soils was attributed to evaporation of soil water in the same study by yeager & o'brien ( ) . in addition, experimentation by hurst et al. ( ) correlated inactivation of enteroviruses [echovirus type (strain wallace), coxsackievirus b (strain nancy) and poliovirus type (strain lsc)] in sludge-amended soils with moisture loss in the sludge piles. soil ph influences microbial diversity and the biogeochemical processes, which they mediate (fierer and jackson, ; nicol et al., ) . optimum ph for bacterial survival seems to be neutral, but fungi are known to be more tolerant of acidic conditions, compared to bacteria (leahy and colwell, ) . amino acids (most viruses behave as proteins) have different pk values and so the ratio of positive to negative charges on proteins vary with ph (yates et al., ) . in an experiment that lasted days using poliovirus type , echovirus , echovirus and coxsackie b , viruses were detected up till the th e th day at ph . while at ph . , the viruses died off between the th and th day depending on virus type (bagdasaryan, ) . soil types vary depending on organic matter content, water release characteristics, particle size distribution and moisture retention capacity. these variations significantly influence the survival of enteric pathogens in soil atkinson et al., ) . clay soils support the adsorption of microorganisms onto soil particles, and this reduces microbial die-off rates (reddy et al., ) . clays protect bacterial cells, and possibly viral particles, by creating a barrier against microbial predators and parasites (santamaria and toranzos, ) . viruses, which are mostly large proteins possessing various charges, are capable of forming numerous bonds with clay minerals (stotzky, ) . for example, the survival of e. coli is prolonged in clay soils where adsorption of cells to the soil particles protects it against protozoa (mosaddeghi et al., ) . escherichia coli can persist for up to weeks in clay and loam soils, but for much less ( weeks) in sandy soils (lang and smith, ) . results of a study that compared rotavirus survival in three soil fractions (whole soil, sand and clay) at temperatures , and c for days showed least survival in sand fractions (davidson et al., ) . in the absence of soil particles, rotavirus survived best at c with survival decreasing, with an increase in temperature, except in whole soil, where it survived better over the entire temperature range and for more than a week at c, indicating that whole soil offered some protective effect (davidson et al., ) . conversely, though, there is a report of shorter survival duration of enteroviruses (poliovirus type , echovirus , echovirus and coxsackie b ) in loamy soil than in sandy soil (bagdasaryan, ) . a link between higher organic matter content and enteric pathogen persistence has been established (jamieson et al., ; williamson et al., ; . there is overwhelming research evidence in this regard, seeing that many of the studies that compared the persistence of enteric pathogens in top and sub-soils recorded higher survival rates in topsoil (zhai et al., ; nyberg et al., ) . research has also shown higher pathogen levels in organic soils after manure application compared to sandy soils (tate, ; jamieson et al., ) . therefore, the rates of pathogen survival are lower in sandy soils, which have a low water-holding capacity (mubiru et al., ; erickson et al., a) . lower temperatures are more suitable for bacterial and viral survival. the ultraviolet radiation from the sun inactivates viruses on the surface of the soil, but viruses in deeper soil strata are protected from this (rodríguez-l azaro et al., ; zablocki et al., ) . in loamy soil samples, at ph . , poliovirus and echovirus were recovered after e days at e c compared to recovery e days at e c (bagdasaryan, ) . similarly, cfu/g amended with dairy manure persisted for e d in fallow soils, manure did not seem to affect persistence, clay seemed to improve pathogen persistence and activity gagliardi and karns, (continued on next page) poliovirus type and coxsackievirus b pfu were recovered for up to days at c whereas pfu were recovered from soil for up to days at c (temperature profiles tested were , and c) (yeager & o'brien, ) . the persistence of poliovirus in sludgeamended soil was assessed in a field study where appropriately cultivated and irrigated plots were treated with virus-spiked effluents by flooding. this was done for days spanning through spring, summer and winter seasons. poliovirus survived best during winter (when it was detected after days), but during summer, the longest survival period was days (tierney et al., ) . parasites seem to prefer warm temperature conditions. prevalence of hookworms have been correlated to warm temperatures, relatively high rainfall and low clay content (sandy soils with clay content of less than %) (mabaso et al., ) . nutrient availability is essential for the survival of microbes in the soil. the presence of organic matter promotes the survival, and in many cases, the regrowth of enteric bacteria looney et al., ) . organic matter improves nutrient retention, serves as carbon sources for bacterial species and enhances moisture retention (gerba et al., ; schoonover and crim, ) . apart from environmental stress responses, foreign enteric bacteria must compete with the endogenous microflora to become established in the soil environment (jiang et al., ) . some autochthonous soil organisms have been shown to be resistant to newly introduced microorganisms in their environment (ellis and mccalla, ) . also, certain bacteriophage, some protozoa, nematodes and free-living soil organisms such as bdellovibrio can parasitize non-indigenous pathogens, thereby limiting their survival (klein and cassida, ; goss and richards, ) . additionally, increased pathogen survival, and regrowth in some instances, in sterile soils and soils with relatively low biological activity has been reported (gerba et al., ; tate, ) . there is some research evidence that alien enteric pathogens compete poorly for nutrients and are thereby susceptible to inhibition by soil-borne bacteria (jiang et al., ) . the effects that this has on the persistence of pathogens (especially pathogens introduced via contamination) in soil is however not yet fully understood. the impacts that soil edaphic and biotic conditions have on the occurrence, fate and persistence of microorganisms in soils should not be underestimated. these factors can collectively or independently stifle or encourage foreign pathogens. for instance, members of listeria possess advantageous intrinsic factors such as an extensive repertoire of transport systems (like phosphotransferase system and transcriptional regulators) which makes them capable of successfully persisting in the soil ecosystem (newell et al., ) . however, these species are highly sensitive to extrinsic factors and this affects their ability to survive in soil environments (newell et al., ; locatelli et al., ) . although studies have been conducted on the occurrence of l. monocytogenes in various ecological niches, including soil, more emphasis has been placed on the occurrence of listeria spp. in fresh vegetables under storage conditions, food processing and packaging environments. the expression of genes and induction of proteins such as cold shock and cold acclimation rapid decline in cell numbers with no difference identified for soil type. increase in retention at both rainfall rates. campylobacter jejuni clay loam silt cfu/g cfu/g cfu/g proteins, as well as tolerance for low ph and high salt concentration in these environments have received much research attention. there is however, need for more research to understand the dynamics of listeria survival in soils. agronomic practices such as soil improvement and manure application method influence the survival of pathogens in the soil (table ) . soil improvement strategies (inorganic and organic fertilizer, compost, biosolids and other residuals application), significantly enhance the nutrient loads of soils (diacono and montemurro, ) . in varying degrees, these are important sources of primary nutrients such as n and p as well as secondary nutrients such as ca, mg and s to the soil. a ready supply of essential nutrients encourages the growth of pathogens. compost application modifies the long-term soil conditions by increasing the ph steadily, this, therefore, affects pathogen survival in soil (weller, ; sharma and reynnells, ) . bacteria tend to decline more rapidly when manure is applied superficially as opposed to when incorporated into the soil immediately after application (solomon et al., ; islam et al., a) . this is probably due to the elimination of drying conditions and exposure to uv at the soil surface (schulze-makuch and irwin, ) or because incorporation of manure disrupts macropores and boosts soil-bacteria contact . after manure application on land, if applied manure is contaminated, it is probable that the pathogens will move through the soil matrix, either vertically or horizontally. vertical movement of pathogens through the soil is influenced by the amount and intensity of rainfall, climatic conditions as well as the season of application. horizontal movement is known to be influenced by soil type, moisture levels, temperature, microbial activity, transport through plant roots, rainfall patterns, soil ph amongst other biophysical factors. it is, however, apparent that water flow is the most important dispersal factor for percolation of manure-derived pathogens in soils, regardless of type and structure although more quantitative information regarding this is desirable (mawdsley et al., ; jiang et al., ; jamieson et al., ; islam et al., b; you et al., ; semenov et al., ) . the extent of movement will affect the distribution and eventual fate of the pathogens. some will spread in soil and attach to roots. others may be washed off to surface waters or percolate to aquifers, potentially contaminating irrigation water sources (fig. ) vinten et al., ; avery et al., a, b; islam et al., b) . pathogens occurring in contaminated manure, therefore, can be rapidly transported within soil systems (gagliardi and karns, ; kisluk and yaron, ) . the success of conveyance and distribution, however, further depends on inherent survival capabilities of the pathogen as well as the presence and structure of plant root systems (fig. ) (kemp et al., ; mubiru et al., ; avery et al., a; . there is some evidence that pathogens may indeed survive longer in manure-amended soils than actual manure samples, and this has been illustrated for enteric species such as s. typhimurium and e. coli o :h . salmonella typhimurium, has, however, exhibited superior persistence capabilities compared to e. coli o :h in manure-amended soils (islam et al., b; you et al., ; fremaux et al., ; pornsukarom and thakur, ) . there is a paucity of data on the persistence of pathogens in manure amended-soils in the tropics (ongeng et al., ) . one interesting study provides an insight into the survival of e. coli o :h and salmonella typhimurium under tropical climatic conditions . the study showed that survival periods were mostly shorter than the observed record in temperate regions indicating that biophysical conditions in the tropics may be more injurious to these pathogens. it is, therefore, not prudent to predict the survival of e. coli and s. typhimurium in tropical soils from data obtained in temperate locations. the soil is the most important cultivation medium and represents a relevant risk for produce contamination. a myriad of studies regarding the behavior of pathogens in various kinds of soil ecosystems is available. however, validated consensus protocols for conducting and interpreting experimental studies as well as for evaluating the effects of environmental and soil characteristics on fate of pathogens in soils are not yet available. it is important to further understand the effects of soil types, environmental factors, biological processes and interactions, cultivation and management practices on the behavior of (indigenous and foreign) enteric pathogens in agricultural soils. apart from farm animals, whose roles as reservoirs of enteric pathogens has been established, wild animals such as birds, reptiles, rodents, amphibians, some helminths, and insects like flies and beetles can also serve as vehicles of pathogens to contaminate cultivation media and produce (beuchat, ; lim et al., ) . livestock and wild animals may gain access to cultivation areas either because of adjacent land use (livestock rearing) or by intrusion (jay-russel, ) . birds such as gulls, pigeons, chickens, starlings, canada geese, migratory ducks and sandhill cranes (pacha et al., ; hald et al., ; ekdahl et al., ; humphrey et al., ) have been determined to be carriers of pathogens such as e. coli, salmonella and campylobacter (wallace et al., ; schmidt et al., ; wani et al., ) . insects are typically ubiquitous in cultivation fields, and hence, have unrestricted access to produce. they are usually found in manure piles, feedlots and other habitats near cultivation fields, and so farms practicing mixed farming represent a more significant risk (martínez-vaz et al., ) . many bacterial species have evolved to exploit insects as hosts or vectors. filth flies, fruit flies, cockroaches and other insects act as mechanical and biological vectors to contaminate fruits and vegetables on the field (sasaki et al., ; mpuchane et al., ; alam and zurek, ; humphrey et al., ) . many pathogens use flies as vectors for cross-transmission. for example, the transient survival of pectobacterium carotovorum subsp. carotovorum in the gut of the fruit fly drosophila and subsequent transmission to other plants has been observed (nadarasah and stavrinides, ; lim et al., ) . under laboratory environment, direct bacterial transfer from contaminated flies to fruits or plant leaves was shown to occur (sela et al., ; talley et al., ; lim et al., ) . members of muscidae and calliphoride which are usually abundant in production fields adjacent to cattle rearing lots have been associated with the transmission of e. coli o :h . insects that feed on plants also play significant roles in produce contamination by providing direct routes for internalizing pathogens from manure to plants in the field . insect deterioration creates openings that aid the ingress of pathogens into inner plant tissues, thereby enhancing colonization of spoilage and pathogenic bacteria on produce (warriner and namvar, ; lim et al., ) . a seasonal trend to contamination by insects has been identified. there is increased insect and animal activity during the warmer months of the year. moreover, peak incidences of pathogens have been reported during the warmer months (liang et al., ) . reptiles including snakes, lizards, chameleons, turtles, as well as other ophidians, saurians and chelonians have been found harboring enteric bacteria like salmonella (corrente et al., ; beuchat, ) . many wild rodents are asymptomatic carriers of pathogens like salmonella and campylobacter. the occurrence of rodents on farms are often associated with infrastructural impairment, and although their destructive tendencies have been widely recognized, their zoonotic risks are often primarily underestimated. they are capable of amplifying the number of pathogens in the environment and transferring them to other farm animals and produce (meerburg and kijlstra, ) . commensal rodents (house mice and rats) pose a particular threat because of their ecology (they live close to livestock) and high fecundity (brooks and jackson, ; witmer et al., ) . foodborne illness resulting from the consumption of contaminated produce is dependent on specific factors. first, the produce must be contaminated with a pathogen, which must survive until the time of consumption at levels sufficient to induce illness (harris et al., ) . the dose required to cause illness in many cases, is very low, which indicates that the microorganism needs only to contaminate the food to survive without necessarily reproducing. for instance, pathogenic parasites and viruses are not capable of multiplying outside a human or animal host and only need to survive in sufficient numbers to cause illness (harris et al., ) . the survival and or growth of pathogens is influenced by the kind of organism, produce type, on-field environmental conditions, as well as the physiological state of the plant and pathogen. the possible routes of entry into plant tissues include: natural apertures (such as stomata, lenticels, sites of lateral root emergence), wounds caused by biotic or abiotic circumstances and following the flow of water from roots to leaves, where pathogens can efficiently survive and multiply (steele and odumeru, ; deering et al., ; hirneisen et al., ) . the popular opinion is that pathogens will survive but not thrive on intact (uninjured) outer surfaces of produce, primarily due to the protective effects of natural plant barriers (such as cell walls and wax layers) (mathews, ; heaton and jones, ) . survival and proliferation of enteric pathogens on produce is significantly enhanced if the protective barrier becomes compromised either by physical or biological damage (such as punctures or bruising), insect ruination or through degradation by plant pathogens. it is vital to understand the microbe-microbe and plant-microbe interactions that occur in the phyllosphere and rhizosphere which influence the adaptation, colonization, attachment is pre-requisite for the colonization and subsequent transmission of enteric pathogens throughout plants including the edible portions (berger et al., ) . it is important to note that attachment onto the surface of intact produce is limited in contrast to the attachment on other food commodities such as processed meat tissues . however, the attachment does indeed occur and is facilitated by stomata, lenticels, broken trichomes, as well as bruises and cracks occurring on produce surfaces. the incidence of scars and cracks (which may set in late in the growing season while the fleshy portion is enlarging rapidly) in certain fruits also aids pathogen attachment (bhagat et al., ) . cracks tend to occur in or on the weak areas on plant surfaces such as around lenticels and trichomes, and hence, these areas are more susceptible to invasion by pathogens. cavities within the epidermis may also develop from cuticular cracks as the fruit develops, thereby entrapping pathogens and shielding them from desiccation and disinfection. the initial phase of bacterial attachment is a rapid process initiated once the bacteria establishes contact with the plant surface (phyllosphere) (sant'ana et al., ) . the phyllosphere, also known as the aerial parts of plants pose challenges for microbial survival. exposure to high uv doses, temperature and relative humidity fluctuations sabotage viability (brandl et al., ; heaton and jones, ) . epiphytes that exist within the phyllosphere have, however, evolved specialized mechanisms to improve stress tolerance and nutrient acquisition. for instance, pseudomonas spp. produce pigments to insulate against uv and pectolytic enzymes to gain nutrients (heaton and jones, ) . the ability of the pathogen to persist on the phyllosphere improves the chances of a viable or infectious dose remaining post cultivation (heaton and jones, ) . the successful attachment on the phyllosphere also depends on the crop and pathogen type. a classic illustration is salmonella invasion of lettuce and tomatoes. salmonella contamination of lettuce and tomatoes via soil is usually quite low, implying that salmonella does not readily attach to or grow in the phyllosphere of these crops (critzer and doyle, ) . also, attachment of salmonella and e. coli o :h is observed more frequently with brassicaceae compared to lettuce, carrots, and tomatoes, which has generated the theory of selective attachment, suggesting that certain produce types are more prone to contamination than others (warriner and namvar, ). specific pathogens such as salmonella have surface epitopes that can bind to plant structures such as stomata to aid attachment (warriner and namvar, ). some also have higher capabilities to metabolize nutrients contained within the apoplastic fluid of plants (warriner and namvar, ) . these traits significantly enhance their attachment abilities. finally, hydrophobic interactions between a plants' epidermal layer and microbial cells are believed to play a major role in facilitating this initial phase of attachment (burnett and beuchat, ) . surface colonization is the final phase of attachment during which biofilms may be formed. biofilms are microbial colonies, which form when single microorganisms attach and aggregate on a hydrated surface and undergo a "lifestyle switch," giving up life as a single cell to live on a surface in an adhesive cell matrix with other microorganisms (lemon et al., ) . cells in a biofilm have a better chance of adaptation and survival (especially during periods of stress) as they are protected within the matrix (decho, ) and are usually resistant to antimicrobial agents (lemon et al., ) . naturally occurring biofilms are present in many fruits and vegetables, but the ability of foodborne pathogens to associate with them and persist is not yet fully understood (brackett, ; ferreira et al., ; larsen et al., ) . pathogen serovars that are strong biofilm producers have been shown to attach better to both intact and injured produce surface compared to strains that are weak biofilm producers (lindow and brandl, ; kroupitski et al., ) . the occurrence of biofilms improves the chances of transient occupants of leaf surfaces such as enteropathogens of becoming effectively incorporated into phyllosphere biofilms (heaton and jones, ) . bacterial appendages such as curli, pili, fimbriae, and flagella, as well as proteins in outer membranes and genes, may also facilitate the surface colonization by pathogens. increases in the expression of flic, flagellin-encoding gene have been observed in certain produce contamination studies. after attachment, it becomes very difficult to remove the pathogens from produce by surface washing (beuchat and scoutten, ) . overall, enteric soil pathogens may reach the edible portions of fruits and vegetables via numerous mechanisms and routes and these have been elucidated by several studies (natvig et al., ; johannessen et al., ; barak and liang, ; tyler and triplett, ) . some of these routes include germination of seeds in contaminated soils, which leads to bacterial colonization of roots and edible parts, direct transfer of pathogens within the soil to crops when heavy rain or water gun irrigation causes leaf splash, bacterial infiltration through roots, amongst others. attached pathogens can reach the interior of fruits and vegetables via a variety of pathways. the extent of internalization depends on factors such as the route and mechanism of entry, the type and age of the plant, the aerial and/ or root morphology and exudates, the soil type and biology and the strain and/serovar of bacteria (hirneisen, ; brandl et al., ; lim et al., ) . the mechanism could be either passive or active (sant'ana et al., ) . passive internalization involves the uptake of bacteria mainly through roots and seeds. mechanistically though, enteric pathogens may be internalized via the root system and transported to edible tissues, but the risk of contamination by this route is likely low . this is because in the environment, particularly areas that are not prone to contamination events, the levels of enteric pathogens are likely to be extremely low (cooley et al., ; matthews et al., ) . in contaminated zones, however, human pathogens may indeed invade root tissues and subsequently translocate to edible portions (solomon et al., ; solomon and matthews, ) . depending on the age of the plant, pathogens may invade external root surfaces (main and side roots, as well as root hairs) and subsequently internalize. the developmental stage of plant root systems when contamination occurs influences the capability of pathogens to interact with, penetrate plant roots and migrate to other tissues (mootian et al., ) . the physiological characteristics of the roots may also determine the success of internalization; for example, some root vegetables possess antimicrobial properties, which limits the growth and internalization of enteric bacteria . pathogens like e. coli o :h have been demonstrated to survive longer in the soil in the presence of rye and alfalfa roots (gagliardi and karns, ) . other work has demonstrated that pathogens enter root tissues at sites of lateral root emergence or through damaged roots (mendes et al., ) . salmonella and e. coli o :h have penetrated arabidopisis and lettuce plants' roots, while klebsiella pneumoniae have been detected on numerous plants' roots (tyler and triplett, ) . other examples include the invasion as well as (endophytic and systemic) colonization of barley roots by s. typhimurium, the shoots of black pepper stem cuttings by pseudomonas aeruginosa, as well as roots and shoots of tomato seedlings by p. aeruginosa (kutter et al., ; kumar et al., ) . it is, however, important to note that successful invasion of the root and shoot system may not guarantee translocation to the edible or foliar portions of produce. in some surveys, bacterial pathogens were detected in roots but not leaves of crops examined (watchel et al., ; warriner et al., ; bernstein et al., a; mitra et al., ; sharma et al., ) . a growing body of evidence suggests that seeds may serve as primary inoculum source in produce contamination. in the case of vegetables, seed sprouts have been implicated as the initial inoculum source, severally (warriner et al., ; deering et al., ; kumar et al., ) . in recent time, seeds have been recognized as a significant source of inoculum for foodborne illnesses associated with sprout consumption (mahon et al., ; national advisory committee on microbiological criteria for foods, ; buck et al., ; yang et al., ) . it is possible that enteric bacteria may be transmitted from contaminated seeds to sprout to mature plants, throughout entire plant life cycle up to consumption. the contamination may be transferred from seed again, thus persisting in produce cultivation cycles, for a long time. there is a record of e. coli :h adherence to outer surfaces and subsequent successful internalization of radish sprouts produced from contaminated seed during sprout growth (itoh et al., ) . rate and efficiency of uptake also depends on the type of produce, and the level of internalization varies widely among plants and even among different species of the same crop due to variations in intrinsic factors, which affect pathogen survival and proliferation (golberg et al., ) . for instance, certain produce items, like fully ripe tomatoes are typically in the ph range ( . e . ) which conventionally impedes growth of most enteric bacteria, whereas, the ph of numerous vegetables, melons, and soft fruits is usually . or higher, which is conducive for bacterial growth gagliardi et al., ) . therefore, gram-negative bacteria are more commonly associated with vegetables while molds and certain yeasts mostly occur on fruits, due to the differences in ph requirements of the respective groups of microbes (jay, ) . members of the brassicaceae family (radish, turnip and broccoli) were demonstrated to have a higher prevalence of salmonella contamination than lettuce, tomatoes and carrots when grown in contaminated soil (barak et al., ) . among leafy greens, radicchio and endive may be more likely to be contaminated than lettuce, spinach, parsley or cilantro (barak et al., ) . salmonella typhimurium has been demonstrated to internalize more efficiently in iceberg lettuce and arugula leaves compared to romaine, red lettuce, fresh basil, parsley and tomato leaves, which displayed only marginal internalization. listeria monocytogenes seems to exhibit a selective preference for certain vegetables like radishes and potatoes, as certain studies reported that although l. monocytogenes successfully invaded tissues of a wide variety of vegetables, radishes and potatoes appeared to be more often and severely contaminated (beuchat, ) . it is also apparent that l. monocytogenes does not survive and internalize satisfactorily on fresh carrot, in fact, low doses of raw carrot juice have been demonstrated to inhibit the growth of the pathogen (beuchat et al., ; farber and peterkin, ; oh, ; benkerroum, ) . internalization is believed to be a plant-pathogen specific interaction, and therefore, internalization success varies from pathogen to pathogen. a comparison of the internalization of l. monocytogenes to s. typhimurium on inoculated seeds of cress, radish, spinach, lettuce, mustard, carrots, and tomatoes showed significant variations in the rate and efficiency of internalization by the pathogens. under identical experimental conditions, s. typhimurium internalized into the roots of the vegetables, whereas, l. monocytogenes did not (jablasone et al., ) . similarly, while s. typhimurium was found to be associated with the internal portions of barley sprouts, l. monocytogenes, l. ivanovii and l. innocua were not (kutter et al., ) . furthermore, the degree of internalization is contingent on the serovar/strain (larsen et al., ) . gene expression, metabolic and antimicrobial capacities vary among strains. certain strains manifest up-regulation of peculiar genes like the pdu, cob-cbi, and out which improve carbon source utilization and may confer a competitive edge, thereby enhancing the survival and persistence of these strains (fox et al., ) . some e. coli strains possess metabolic capacities, which foster their survival in certain agroecosystems such as soils (franz et al., ) . in a bid to explain the strain-specific internalization dynamics, a five serovar salmonella cocktail (montevideo, michigan, poona, hartford and enteritidis) was inoculated into hydroponic growth substrates. serotypes montevideo and michigan were most prevalent, while enteritidis, hartford and poona were not detected in any of the tomato tissue samples (guo et al., ) . this is a quintessential illustration of internalization variation among serovars. likewise, salmonella serovars; cubana, infantis and typhimurium exhibited varying capabilities to internalize and colonize alfalfa sprouts when seeds were inoculated under identical environmental conditions (dong et al., ) . some scholars have endeavored to compare the survival of two arguably most prominent foodborne pathogens: e. coli and salmonella. serovars of both can proficiently adapt to environmental stress; -numerous strains are known to become habituated to low ph conditions and subsequently manifest remarkable tolerance to stress conditions. escherichia coli can perpetually evolve new varieties that have neither been previously reported nor characterized and which are capable of exploring and inhabiting previously unrecognized niches (newell et al., ) . both seem to be capable of long-term survival in the agricultural environment and on produce, but it is quite apparent that salmonella survives better than e. coli (brandl, ; mandrell, ; newell et al., ; schikora et al., ; ongeng et al., ) . many salmonella serovars bind to plants significantly better than e. coli strains. escherichia coli's inability to lower its metabolic rate to suit the low availability of accessible organic carbon and to competently cope with low nutrient conditions contributes significantly to its die-out in soils and on produce, and therefore, lowers its competitiveness (survival) compared to salmonella franz et al., , . internalization has been correlated with motility and chemotaxis. flagella mutants (flighi:tn , chey) deficient in motility and chemotaxis respectively have exhibited reduced attachment and penetration of lettuce leaves (kroupitski et al., ; lim et al., ) . it has also been hypothesized that products of photosynthesis serve as nutrients to aid internalization of pathogens (lim et al., ) . active internalization typically involves the penetration of bacteria through natural openings. the ability of foodborne pathogens to internalize in produce represents a significant public health risk because internalized pathogens are protected against optimized disinfection modes (meireles et al., ) except irradiation which seems capable of reasonably eradicating internalized pathogens in produce. the technique penetrates produce tissues to eliminate internalized pathogens, and gram-negative bacteria are very susceptible to even low doses (saroj et al., ; o'bryan et al., ) . however, treatment with irradiation may produce off flavors, colors and odors and may inactivate some of the nutrients (fan and sokorai, ) . it is, therefore, not accepted and endorsed for produce treatment. there are other relatively new technologies such as modified atmosphere packaging, ozone, ultrasound and ultraviolet treatments, which seem promising in ensuring the microbiological safety of fresh fruits and vegetable products (shayanfar and pillai, ) . however, limited commercial applications have been described for most of these new technologies. electron beam technology is another up-and-coming treatment option, which according to experts, can play a pivotal role in mitigating some of the contemporary microbiological risks facing the produce industry (shayanfar and pillai, ; lung et al., ) . it is an environment friendly, cost and time effective decontamination strategy that uses low-dose ionizing radiation to treat crops (-as well as other food items), to eliminate microbial contamination. it is capable of inhibiting the germination of crops and controls the rate of ripening of fruits and vegetables, thereby extending their shelf life (lung et al., ) . it inhibits a variety of enteric pathogens without compromising food sensory and nutritional qualities and can be used in combination with other traditional or non-traditional food processing technologies (lung et al., ) . regulatory authorities such as the us food and drug administration have approved it, but the full import of the safety of use is not yet conclusive. given the amount of evidence indicating that enteric pathogens (that are not plant pathogens) can invade and be internalized into plants, it is important to understand how such microbes establish access to plant tissues, as this may facilitate the development of strategies to reduce internalization. for successful colonization, major interactions take place between pathogens and their plant hosts that determine the success of the pathogenic attack (warriner and namvar, ). many enteric pathogens have devised mechanisms to overcome plants' basal defense mechanisms and innate immune responses (lim et al., ) . plants first line of response to foreign invasion is by the innate immune system. this consists of two main branches: pamp-triggered immunity (pti) and effector-triggered immunity (eti). in the first stage, microorganism associated molecular patterns (pamps or mamps such as flagellin, peptidoglycan, lipopolysaccharide) are identified by plant host receptors popularly known as pattern recognition receptors (prrs) (deering et al., ) . these batteries of receptors deployed by the host are designed to curb the growth and spread of the pathogen (ausubel, ) . pti response is broad-spectrum; sensitive to molecules familiar to many classes of microorganisms including non-pathogens. upon recognition, plant defense signal pathways are activated among which, jasmonate, salicylic acid and ethylene play essential roles. virulent plant pathogens may through diverse strategies, such as the production and secretion of effectors, efficiently override pti, for example, there are some 'effectors' that can overcome pti by interfering with mamp detection and subsequent defense signaling (kazan and lyons, ) . this results in effector-triggered susceptibility (ets). for susceptible interactions, effectors produced and released by the microorganism are transferred into the plant cell through the ttss (type iii secretion system). specific nucleotidebinding leucine-rich-repeat (nb-lrr) proteins encoded by resistance genes, resulting in eti and limitation of pathogen transmission to other tissues, recognize these effectors. while pti is considered the first line of defense against pathogenic infection, eti is an accelerated and amplified response, the outcome of which is often a hyper-sensitive response (hr) (spoel and dong, ) . the ability of pathogenic bacteria to colonize a plant may also be influenced by their interactions with other microorganisms either positively or negatively (deering et al., ) . if other microorganisms supply carbon sources (via degradation of cell wall polymers or induced secretion of sugars), or sequester antimicrobials, this can enhance pathogen colonization (bais et al., ; warriner et al., ; augimeri et al., ) . alternatively, plant pathogens that wound or destroy living tissue may create a microenvironment that is suitable for the survival and/replication of human pathogens (rashid et al., ) . pathogens are often associated more with plants whose tissues have been damaged by soft-rot pathogens compared to those with healthy tissues (brandl, ) . before pathogenic bacteria can colonize the surface or interior of a plant host, they have to contend with the naturally occurring microflora that is already established (deering et al., ) . the ability of the indigenous bacterial community to inhibit the growth of introduced enteric pathogens has been demonstrated by numerous studies (liao and fett, ; matos and garland, ; schuenzel and harrison, ; cooley et al., ; johnston et al., ) . there is direct evidence that the stomata play essential roles in internalization, host immunity and pathogen virulence of pathogens (kroupitski et al., ; zeng et al., ) . some researchers have reported that plant stomata close in response to plant pathogens and some human pathogens (melotto et al., ; roy et al., ) . escherichia coli o :h has been reported to trigger stomatal closure even under high relative humidity, a stressful environmental condition that generally weakens plant defenses against bacteria in field and laboratory conditions (roy et al., ) . stomata closure could be triggered by certain peptides such as flg produced by bacterial flagellin and lipopolysaccharides which are recognized by pamps or mamps in a salicylic acid-dependent manner. in the case of some plant pathogens such as xanthomonas spp. and pseudomonas syringae, virulence factors produced are capable of overcoming this innate immunity and counter stomata defense. for example, pst dc and several other pathovars of pseudomonas syringae, produce coronatine (cor), a phytotoxin which can reverse stomatal closure induced by bacteria or mamps (zeng et al., ) . stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis, resulting in low bacterial titers in the plant apoplast (roy et al., ) . however, plant defense responses induced by pathogens vary and plants may recognize and respond to some human pathogens more effectively than others (roy et al., ) . for example, comparison of plant defense responses induced by e. coli o :h and s. typhimurium sl in arabidopsis thaliana and lettuce (lactuca sativa) revealed some variations. while e. coli o :h triggered stomatal closure, sl only induced a transient stomatal immunity. also, pr gene expression was significantly higher in arabidopsis leaves infected with e. coli o :h compared with sl (roy et al., ) . although, numerous studies have examined the intricacies of internalization in fresh produce, many of these are laboratory based. the few available field studies, which have mostly studied e. coli, indicate that internalization of pathogens may be not be very common in field settings erickson et al., b erickson et al., , erickson et al., , b . more field studies are therefore, required to properly understand the potential/likelihood of enteric pathogens to internalize in fresh produce as well as the actual factors that influence the success of internalization. to successfully achieve an acceptable level of microbiological safety for fresh produce, it is essential to control environmental contamination in the field by taking appropriate pre-harvest precautions. one fundamental factor to consider is the state or quality of the growing fields. fields on which wild or domestic animals have been recently grazed that have been subjected to flooding or may have been previously contaminated with manure constitute an unacceptable microbiological risk (turb e et al., ) . therefore, growers need to scrupulously investigate land history when selecting a location for produce cultivation (islam et al., a, b) . cultivation areas should be safeguarded from flooding, and fecal contamination and manure should be adequately treated (using popular methods like composting and aging) before application as fertilizer. also, suitable buffer zones (physical barriers) such as mounds, diversion berms, vegetative buffers as well as ditches should be erected between animal grazing regions and produce cultivation areas (james, ; olaimat and holley, ) . appropriate livestock waste disposal and farm general waste management should be adopted to ensure safety. numerous experts have highlighted the need for monitoring, regulation and control of the microbiological quality of irrigation water. several regional and international standards exist for irrigation water use and practices to prevent incidence of bacterial contamination. the use of potable water for irrigation (and other cultivation operations) is highly recommended. certainly, this is not economical in many instances and may increase production costs, which will raise prices; it is however, pertinent to public health safety. in developing countries, a myriad of safety regulations exists such as cessation of irrigation prior to harvesting, lowering of watering cans to reduce splashes from (contaminated) soil, adoption of furrow irrigation system over the use of sprayers which expose edible portions of leafy vegetables directly to irrigation water, and so on (keraita et al., ; amoah et al., ; uyttendaele et al., ) . in cases where surface water is the irrigation water source, drainage of contaminated water into the surface water reservoir may be prevented by constructing ditches, buffer strips, as well as retention and drainage systems. potential overflow points should be identified and eliminated. it is also important to determine (potential) points of contamination because control measures are bound to be more effective if focused on eliminating contamination at the source (madramootoo et al., ; pachepsky et al., ) . irrigation wells, functional septic, water and sewage systems should be installed and properly maintained especially during periods of excessive rainfall to prevent pathogen contamination (buck et al., ; olaimat and holley, ) . surface and groundwater resources should be protected from any potential sources of contamination including wildlife, animal waste, agricultural run-off, human activity, sewage, or industrial effluents. other management practices like; removal of riparian areas, erection of fences, and treatment of irrigation water (for example, using uv treatment) can be considered to enhance safety assurance of irrigation water. these precautions will minimize contamination risks on produce farms and should be applicable not just to supposed high-risk crops (such as leafy greens) but all produce (squash, and others) (strawn et al., b) . implementing some of these may, however, be costly and have negative impacts on landscape health. irrigation water sources should be routinely monitored to ensure microbiological safety (brackett, ; islam et al., b) . ideally, there should be more regular reporting on the microbiological quality of irrigation waters in different world regions. such surveys should reflect the true levels of actual pathogens rather than indicators, and bias should be avoided towards contaminated samples by intensively monitoring every irrigation source possible, and not just sites where extensive contamination has been known to occur (stoeckel, ) . as part of a total package of hygiene measures to prevent the transfer of foodborne pathogens, wild animals, birds, flies and rodents should be controlled in cultivation areas. interventions to mitigate wildlife intrusion of a farm may be costly and not entirely effective, especially if not done properly, thereby allowing certain animals direct access to crops. in many cases, it is not economical to fence large farms, but small farms can be fenced to restrict wild animals (jung et al., ) . other mechanical/biological control methods include the use of scarecrows, reflective strips, monitoring of animal tracks and field intrusion as well as gunshots to ward off pests and animals. mechanical traps and baits can be used to control mice and rodents. overall, practical, cost-effective methods should be adopted to mitigate wild sources and routes of produce contamination. considering that, in many important outbreaks, vegetable seed sprouts have been implicated as the initial inoculum source, the elimination of bacteria from seeds before planting has become crucial (buck et al., ) . chemical or physical treatment methods are usually used to decontaminate seeds, in a bid to reduce the risks of sprout borne disease outbreaks. however, this poses some challenges for growers, as the chosen decontamination method has to fulfill certain conditions. one important consideration is the preservation of seed viability. selected treatment dosage should be able to inactivate pathogens without adversely affecting seed viability (buck et al., ) . also, the treatment must be able to penetrate and access bacteria that may be residing in protected seed tissues, and finally, certain treatments may be inactivated by seeds, rendering them less effective (buck et al., ) . nevertheless, the efficacy of chemical seed treatments for sprout seed including chlorine compounds (commonly calcium and sodium hypochlorite), ethanol, hydrogen peroxide, calcium edta, -hydroxybenzoic acid, ozonated water and other commercial disinfectants have been extensively documented. it is also possible to use gaseous chemicals and thermotherapy (e.g., hot water treatment), although excessively high temperatures may affect sprout vigor. another potential issue with hot water treatment is that when treating large batches of seed, it is practically impossible to achieve temperature uniformity throughout the water bath. therefore, while a portion of the seeds receives the appropriate temperature-time exposure, some will still contain viable bacteria after 'treatment.' also, there is a potent risk of cross-contamination with this technique. other viable options include seed treatment with bacteriophage, combinations of thermotherapy with chlorine and the use of ionizing radiation. radiation is particularly appealing because it can penetrate seed tissues and possibly eliminate bacteria localized within protected tissues (buck et al., ) . however, it has been postulated that high levels of irradiation may distort the physiology and organoleptic properties of seedlings, more research is therefore, needed to evaluate the prospects and risks of this approach. other precautionary measures include testing seed lots for purity and germination rate prior to marketing, proper warehouse storage (in metal bins) until bagged, as well as ensuring general facility sanitation and employee hygiene (national advisory committee on microbiological criteria for foods, ). safety criteria and regulations are mostly region specific, it is however, critical to enforce these regulations, ensure that growers adhere to such and there is a need to constantly improve standards; if new information becomes available, regulations should immediately be updated (k€ opke et al., ) . most of the available data is from the developed world mainly from the us and certain parts of europe. it is necessary to develop surveillance and tracking systems and generate robust databases for other regions as well. more studies should be conducted under field conditions, rather than laboratory or greenhouse simulations, as this will provide a better understanding of how enteric pathogens behave in agricultural production environments. finally, and more importantly, it is necessary to ensure producers are mindful of their roles in assuring food safety. growers should be encouraged to adopt the best possible agricultural practices to ensure produce safety. it is also important to enlighten consumers about possible risks and appropriate mitigation strategies. there are wrong notions and misconceptions, which have to be corrected promptly, for example, many people believe it is not necessary to wash organically grown fruits and vegetables (leifert et al., ). it is evident that epidemiologic investigations are worthwhile as public health directives and policies based on investigation output have averted impending foodborne disease crises in many cases. the relevance of epidemiological surveys globally and regionally, therefore, cannot be overemphasized. this means that epidemiological investigation tools and systems need to be objective, updated, precise, flexible and timely. while significant progress has been achieved in the area of epidemiology, there are still certain cracks that need to be addressed. the use of routine, optimized clinical pathogen identification techniques may mean that new pathogens may likely be missed. this is a potentially grave issue, because periodically, since the development of foodborne disease surveillance, the list of foodborne pathogens has continued to expand. care should, therefore, be taken to avoid research bias since it is likely that produce items that have been previously associated with foodborne illness outbreaks and product recalls may receive particular scrutiny. new pathogens emerge due in part, to evolving ecology and technology while already recognized strains continue to evolve, potentially becoming smarter, evading and subverting detection, sanitization and plant host defenses. it is important to further understand the evolution dynamics and emergence of new pathogens, as well as develop and optimize methods to meet the emerging challenges. awareness and surveillance of viral and parasitic enteric pathogens need to be more robustly developed. although noroviruses, hepatitis a, rotaviruses as well as certain emerging viruses such as sars are well known, they are rarely routinely screened for in fresh produce in most countries. also, their ecology in fresh produce is poorly understood, for instance, the knowledge of the stability and persistence of human norovirus in foods has been garnered mostly from the study of surrogate viruses. more importantly, their significance in foodborne disease incidence remains undetermined. parasitic pathogens like ascaris, giardia, entamoeba, cyclospora, cryptosporidia and trichinella are recognized (newell et al., ; robertson et al., ) , but not all are routinely monitored in produce. the roles that livestock and wildlife play in pathogenic contamination of fruits and vegetables as well as their epidemiology through the food chain is poorly understood. it is difficult to compare the available studies because some have used naturally contaminated animals, while others used experimentally inoculated animals. the exact transport/transfer mechanisms of pathogens from animal fecal material or contaminated manure/soil to fruits and vegetables via splash are not yet properly understood. for example, it will be helpful to understand the specific spatial factors that influence the transfer of pathogens from fecal pellets to fruits and vegetables. the survival times for pathogens in fecal contaminants, manure, and manure-amended soils are inconsistent, reflecting the varying conditions under which many of the available studies have been conducted (these variations are demonstrated in tables e ). the fate of pathogens on the soil surface, the relationship between manure-derived pathogens and soil particles, as well as the states in which pathogens occur in soil slurry or manure mixtures, should be further explored. the exact mechanisms of uptake or (transmission) of pathogens from contaminated manure or manure amended soils to plants, particularly in field settings should be studied. this will facilitate the design of scientifically sound produce safety standards. the majority of studies available on pathogen transport in soils have been conducted using homogenized natural soils in laboratory designed soil columns. these may not be a true representation of field conditions and diversifying the experimental conditions will aid the development of efficient, grower-level interventions that will effectively reduce the likelihood of on-field contamination of produce. there are dissenting opinions among experts on a variety of issues pertinent to produce safety. with regards to the factors, mechanisms as well as principles that aid competent internalization and persistence of pathogens on produce, there are many variations. the available studies are difficult to compare largely because they have been conducted under varying physicochemical circumstances, types of microcosms, experimental conditions and used distinct strains (shown in tables e ). most studies were conducted under disparate environmental conditions, and accurate weather data necessary to interpret results from the varying sources is lacking. study results for one crop variety may indeed not hold true for other varieties, for instance, data for apples may not necessarily apply to all pome fruit and data for romaine lettuce may not apply to all leafy greens. when possible, varieties exhibiting greater potential for pathogen survival should be selected for experimental investigations. another relevant consideration for crop selection is preference for varieties that are indigenous to the region in question. some other seemingly trivial controversial issues include whether outer leaves are significantly more likely than inner leaves to become contaminated via splash and whether or not the potential for survival on the abaxial side of leaves is higher than on the adaxial side. the implications of dormant, non-dividing 'persister' cells occurring in certain plant pathogens on the ability to withstand environmental stresses and extensive survival as well as the issues surrounding linked resistance is still an important research debate. also, even though atmospheric deposition seems to be an uncommon route of pathogenic contamination for produce, it has been documented as a potentially important route (beuchat and ryu, ; harris et al., ; mei soon et al., ) . it will be worthwhile exploring how relevant this is for produce safety. while many of the available studies have made stringent efforts to simulate produce cultivation circumstances, it is extremely challenging to create precise/accurate environmental conditions in a laboratory setting. most studies are conducted under controlled laboratory conditions. factors like the biological activity of the soil, manure, water and crops, soil and water chemistries as well as meteorological elements such as wind, uv intensity, temperature, rainfall are simply impossible to replicate under laboratory conditions. laboratory scale model systems may provide important details about the roles of environmental variables on pathogen growth and survival in agricultural environments, but the slightest tweaks in experimental protocols can affect pathogen survival in agroecosystems. unfortunately, actual field-based studies are subject to disruption from unforeseen environmental events such as weather extremes and damage triggered by biological agents including insects or onset of plant diseases. more field studies (where typical agricultural practices and conditions are closely simulated) are therefore, highly desirable to further understand the persistence phenomenon. safety and ethical issues however restrict the use of pathogens in the greenhouse and field-based research. strategies to improve existing biocontainment and decontamination processes should be developed and optimized as soon as possible. another possible solution is to develop and optimize strategies that will cater for the experimental variations in model system development. an assessment and identification of environmental variables that influence the fate of test organisms should be included in experimental designs. despite meticulous planning however, a field trial may fail to yield serviceable results due to factors that are out of the researcher's control. consequently, more replicate trials may need to be conducted. furthermore, agronomic and farm management practices are not uniform in all regions, and production practices significantly differ from region to region depending on seasons and weather patterns within the same region. these often depend on operation scale, type of farming practices et cetera. the risks associated with conventional cropping systems are bound to differ from those of systems that combine intensive livestock farming with arable farming. in addition to general studies, a case-by-case approach should be considered where possible (if financial and technical resources, as well as other circumstances, permit) because farming operations vary widely from farm to farm and this influences the potential for pathogen occurrence, survival, proliferation and dissemination. the potential of fresh produce to harbor pathogens is now well recognized, and fresh produce has been established as a vehicle of foodborne disease. the diverse and complex sources and routes of enteric pathogens to fruits and vegetables have been widely researched. the interplay of land use, water management, weather patterns and specific pathogen properties and sources have been illustrated to have significant consequences for the microbiological safety of fresh fruits and vegetables. attempts have been made to understand the general microbial profile of fresh produce, the behavior, fate and transport of pathogens, as well as their location in and on plant parts. the facts gleaned from these studies have been the subject of many extensive reviews. there is abundant information about the factors that affect the contamination and persistence of pathogens on fresh produce. in light of the available evidence, significant effort must be made to efficiently monitor and illustrate recent trends in the occurrence of foodborne diseases associated with the consumption of fruits and vegetables. partnerships and collaboration among all relevant stakeholders; commercial growers, public health practitioners, veterinary and food safety experts and field biologists is necessary in order to ensure the safety of fruits and vegetables delivered to consumers. on a final note, the need to control all potential pathogen entry pathways has been established and is being continuously stretched by regulators and other specialists. there are numerous other factors along the food production chain that may predispose produce to microbial contamination. however, it is of utmost importance to avoid and control microbial contamination of produce at the preharvest stage. this is because contaminated manure, water and soil have been shown to indeed contaminate produce, and decontamination of produce, polluted arable soil and groundwater has proven to be a very challenging and expensive endeavour. the authors declare no conflicts of interest. . . effect of soil properties and environmental variables on the incidence of pathogens in soils water resources projects and their environmental impacts molecular techniques for detecting and typing of bacteria, advantages and application to foodborne pathogens isolated from ducks kinetics of pathogen destruction during storage of dewatered biosolids association of escherichia coli o : h with houseflies on a cattle farm irrigation water quality for leafy crops: a perspective of risks and potential solutions surveillance for sporadic foodborne disease in the st century: the foodnet perspective inactivation of ms- phage and poliovirus in groundwater lowcost options for reducing consumer health risks from farm to fork where crops are irrigated with a farm to fork risk assessment for the use of wastewater in agriculture in influence of temperature on salmonella survival in hog manure slurry and seasonal temperature profiles in farm manure storage reservoirs potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: a review establishing a role for bacterial cellulose in environmental interactions: lessons learned from diverse biofilm-producing proteobacteria are innate immune signaling pathways in plants and animals conserved? escherichia coli o survival following the surface and sub-surface application of human pathogen contaminated organic waste to soil fate of escherichia coli originating from livestock faeces deposited directly onto pasture survival of e. coli o : h in organic wastes destined for land application persistence of a salmonella enterica serovar typhimurium dt clone in a piggery and in agricultural soil amended with salmonella-contaminated slurry effect of irrigation regimes on persistence of salmonella enterica serovar newport in small experimental pots designed for plant cultivation role of soil, crop debris, and a plant pathogen in salmonella enterica contamination of tomato plants occurrence of generic escherichia coli, e. coli o and salmonella spp. in water and sediment from leafy green produce farms and streams on the central california coast large multistate outbreak of norovirus gastroenteritis associated with frozen strawberries presence and public health implications of listeria monocytogenes on vegetables listeria monocytogenes: incidence on vegetables. food contr. 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the human pathogen escherichia coli o : h in spinach influence of organic waste type and soil structure on the bacterial filtration rates in unsaturated intact soil columns transfer of escherichia coli o : h from soil, water, and manure contaminated with low numbers of the pathogen to lettuce plants impact of soil type, biology and temperature on the survival of non-toxigenic escherichia coli o carriage of microorganisms by domestic cockroaches and implications for food safety mortality of escherichia coli o : h in two soils with different physical and chemical properties experimental studies on the survival of pathogenic and indicator bacteria in aerated and non-aerated cattle and pig slurry disability-adjusted life years (dalys) for diseases and injuries in regions, e : a systematic analysis for the global burden of disease study the influence of soil ph on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria bacteriological study of 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the authors are grateful to the national council for scientific and technological development (cnpq) (grant # / - ) and the coordination for the improvement of higher education personnel (capes) (grant ## p ) for the financial support. oluwadara alegbeleye wishes to acknowledge the financial support of conselho nacional de desenvolvimento cientifico e tecnol ogico (cnpq) through award grant number / - . supplementary data related to this article can be found at https://doi.org/ . /j.fm. . . . key: cord- - g x grl authors: fullick, james; oliver, michael title: “water, water, everywhere”: a challenge to ventilators in the covid- pandemic date: - - journal: br j anaesth doi: . /j.bja. . . sha: doc_id: cord_uid: g x grl nan c o r r e s p o n d e n c e "water, water, everywhere": a challenge to ventilators in the covid- pandemic james fullick , * and michael oliver newport, uk and cardiff, uk *corresponding author. e-mail: james.fullick@nhs.net keywords: covid- ; pandemic; resource management; ventilator; water trap editordwith the spread of coronavirus disease (covid- ), intensive care facilities have been rapidly overwhelmed across the uk and elsewhere. in general, the uk has fewer doctors and fewer icu beds per capita than most of europe. many hospitals have spread into the recovery unit of theatres and are using anaesthetic machines to ventilate patients. we write from a south wales district general hospital that has moved patients into our recovery facility as an outreach icu to discuss some of the challenges and potential solutions of the use of anaesthetic machines in long-term ventilation. the anaesthetic machines used in the expanded icu within newport hospital are the mindray wato ex- (mindray). these use standing gas-driven bellows to provide driving pressure and are connected to a heated circle system, and passive humidification and heating are provided by distal and proximal heat and moisture exchange (hme) filters. one of the main issues we have encountered in ventilating these patients for durations outside the routine scope of anaesthetic machines is water condensation within the mm tubing. this condensation has been sufficient to cause almost complete obstruction and create an oscillating obstructive flow trace. humidification of inspired air is required in the intubated patient to preserve mucociliary function, clearance of secretions, and gas exchange of the respiratory system. disruption of these can cause damage and difficulty in ventilation, even in normal lungs. over h,~ ml of water is lost from the respiratory tract, and a portion of this will collect in the breathing system, which requires multiple disconnections to drain water from the breathing circuit. several methods are proposed to counteract this: increasing fresh gas flow to at least minute ventilation, decreasing breathing circuit length, and introduction of water traps into the breathing circuit. by increasing fresh gas flow, the relative humidity in the circle system is reduced by increasing circuit gas turnover with dry gas. this was partially effective, but this strategy should be discussed with the works and estates team to ensure that the maximal oxygen flow rates are possible. increased numbers of ventilated patients and the introduction of cpap noninvasive ventilation as a viable first-line therapy can exhaust oxygen supplies if all patients require higher flows to match minute ventilation. decreasing the length of the breathing circuit partially helps with this issue but can exhaust the proximal hme more rapidly. it is also difficult to achieve once a patient has been admitted, intubated, and connected to the anaesthetic machine. water traps act as a reservoir for condensed water within the circuit. there are several models available but, as with many supply chains, they are difficult to purchase in a pandemic scenario. they sit between the patient and the anaesthetic machine on the expiratory limb and act to collect condensation via gravity. a further limitation of many of these water traps is that they do not allow the system to be emptied without disconnection. several options were explored including the design of water traps that could be d printed and attached to the circuit, including a luer lock system to extract water using a syringe. this would reduce circuit disconnections and create a large reservoir before ventilation became affected, with the caveat of using an untested medical device. the design of this trap went through several stages, from a sealed reservoir with a large volume (fig a) to a smaller reservoir that could be emptied using a luer lock syringe (fig b) . a final water trap design was created using an hme filter with the filter material removed and connected in the middle of the expiratory limb of the circuit. a needle-free connector was added to the inline sampling port to allow removal of condensation using a syringe without disconnection (fig c) . this design is simple, quick to create using available resources, user friendly, and uses the minimal number of connections. a more complex design using a short in-line suction catheter (fig d) was also proposed, however it requires more complex connections that increase the risk of disconnects, turbulent flow, or leaks. an empty hme filter provides around a ml volume reservoir, with the addition of a e mm connector increasing this to ml. the more complex connection with in-line suction can contain ml. we submit this as a creative solution to a practical problem encountered in the use of anaesthetic machines as long-term ventilators for covid- patients. awareness, consideration, and discussion of this and other issues arising should be encouraged to improve the care and safety of these at-risk patients. the authors declare that they have no conflicts of interest. outbreak of a new coronavirus: what anaesthetists should know the variability of critical care bed numbers in europe humidification in anaesthesia and critical care initial design using large reservoir of > ml. (b) refined design with luer lock port for aspirating condensation. (c) simplified design with available materials using an empty heat and moisture exchange (hme) filter and needle-free connector. (d) modified hme filter with yconnector and in-line suction key: cord- - j ys s authors: ihsanullah, ihsanullah; bilal, muhammad; naushad, mu. title: coronavirus (sars-cov- ) in water environments: current status, challenges and research opportunities date: - - journal: nan doi: . /j.jwpe. . sha: doc_id: cord_uid: j ys s the outbreak of covid- has posed enormous health, social, environmental and economic challenges to the entire human population. nevertheless, it provides an opportunity for extensive research in various fields to evaluate the fate of the crisis and combat it. the apparent need for imperative research in the biological and medical field is the focus of researchers and scientists worldwide. however, there are some new challenges and research opportunities in the field of water and wastewater treatment concerning the novel coronavirus (sars-cov- ). this article briefly summarizes the latest literature reporting the presence of sars-cov- in water and wastewater/sewage. furthermore, it highlights the challenges, potential opportunities and research directions in the water and wastewater treatment field. some of the significant challenges and research opportunities are the development of standard techniques for the detection and quantification of sars-cov- in the water phase, assessment of favorable environments for its survival and decay in water; and development of effective strategies for elimination of the novel virus from water. advancement in research in this domain will help to protect the environment, human health, and managing this type of pandemic in the future. the current global pandemic of covid- caused by severe acute respiratory syndrome coronavirus (sars-cov- ) has been growing briskly [ , ] . although the major transmission routes of sars-cov- are through respiratory droplets and direct contact [ ] [ ] [ ] , recent studies have reported the presence of viral rna of sars-cov- in untreated and treated wastewater and human feces [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in most of the studies, the samples were collected from wastewater treatment plants (wwtps), while one study reported in the presence of sars-cov- in wastewater from a cruise ship and commercial passenger aircraft [ ] . all the reported samples were collected during the period from janruary to july . recent studies conducted in j o u r n a l p r e -p r o o f the netherlands and france confirmed that a reasonably high viral load of sars-cov- rna is found in the sewage/wastewater [ , ] . the presence of sars-cov- in feces and municipal wastewater poses a severe threat to the environment due to its potential spread via these routes [ ] [ ] [ ] [ ] [ ] . therefore, there are serious concerns regarding the spread of sars-cov- through virus-laden aerosols-borne and fecal-oral routes [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is important to take the necessary precautions to limit the spread of the virus in the environment [ ] . nevertheless, it creates some new environmental challenges that demand an imperative need for research. development of effective standard techniques for the detection and quantification of sars-cov- in water, assessment of the existing water purification technologies and development of novel advanced water treatment systems are major challenges and open research opportunities. furthermore, careful surveillance of water and wastewater to be used as an early warning tool for such outbreaks in future, understanding the survival and decay mechanism of the novel virus in water and wastewater, analysis of potential pathways of sars-cov- into water bodies are other potential research opportunities for environmental researchers [ ] [ ] [ ] [ ] [ ] . the major challenges and opportunities in the field of water and wastewater research are presented in fig. . the objective of this article is to highlight the potential opportunities and challenges in the field of water research concerning the novel sars-cov- virus. the directions for future research to safeguard human health, environment, predict and manage this type of pandemic in the future, are also provided. j o u r n a l p r e -p r o o f numerous studies have reported the presence of pathogenic viruses that enter into the water bodies through different sources [ , ] . previous studies have demonstrated that sars-cov- can enter into the water bodies from hospital wastewater and sewage [ , [ ] [ ] [ ] . the waste and wastewater discharged from the quarantine facilities, airports/seaports, residential buildings of infected humans and animals, are the potential sources of sars-cov- than can enter into the water bodies. the sars-cov- may also find its ways to groundwater through possible leaching and infiltrations of effluents from health care facilities, sewage, solid landfill and drainage water. another important source of viral contamination to the water environment is the leakage from sewage pipes. the possible sources and pathways of sars-cov- in water systems are depicted in fig. . due to the fast-growing number of patients worldwide, the virus concentration in wastewater/ sewage is expected to increase rapidly. the potential pathways of sars-cov- into wastewater are via shedding in the stool (for the viable virus), while through urine and stool (for the nonviable virus) [ ] . a careful and extensive investigation of all the potential sources of water contamination by sars-cov- is needed that potentially includes wastewater dispatches from hospitals and greywater from washing personal protective equipment (ppe), surfaces and floors [ ] . table summarizes the literature reporting the presence of sars-cov- rna in wastewater, and untreated water in various parts of the world [ , , , ] . the concentrations of sars-cov- rna in untreated wastewater was in the range of . × to . × copies per liter [ , , , , , ] . some studies also reported the detection of sars-cov- in treated water samples [ , ] . previous reports suggested that sars-cov- survival in an aqueous environment is strongly dependent on the characteristics of water/wastewater [ ] [ ] [ ] . the ph, temperature, presence of antagonistic bacteria, organic matter, sunlight and oxidants might affect the survival of sars-cov- in aqueous environmmnet [ , , ] . a previous study reported that the inactivation of coronaviruses in the water was highly dependent on the level of organic matter, temperature, and presence of antagonistic bacteria [ ] . the suspended solids and organic matter present in water can provide protection for viruses that adsorb to these particles, and they can survive up to several days [ ] . however, some published reports suggested that coronaviruses are very sensitive to high temperature and oxidants, such as chlorine [ ] . studies have reported that surrogate coronaviruses remained infectious in water and sewage for days to weeks [ ] . a recent study estimated that the half-life of sars-cov- in wastewater is in the range of . and . h [ ] , while another study reported that the virus causing covid- could survive in untreated wastewater from hours to days [ ] . it is essential to understand the stability and decay mechanism of sars-cov- in water and wastewater. the significant parameters that can predict the reduction kinetics need to be determined, to establish the favorable and unfavorable conditions for the survival of sars-cov- in aqueous environment. detailed investigations of various water/wastewater samples with different characteristics are required to assess the potential exposure risk of water contaminated with this virus. launching a monitoring program is vital to determine the fate of sars-cov- water cycle [ ] . theoretical and computational analysis might help to provide a basis for experimental research. until now, there is no evidence and enough data to confirm if the water and wastewater containing sars-cov- could be the potential source of its transmission. although some studies predicted a low risk of sars-cov- transmission via wastewater (especially the treated water) [ , ] , still extensive investigations need to be performed to validate these predictions and inial findings. the information regarding the viability of the closely related sars-cov- virus in wastewater may provide useful information about the survival of sars-cov- in an aqueous environment [ ] . as a precaution, the wastewater/sewage treatment plants should be considered as the potential transmission routes. a recent study demonstrated the quantitative microbial risk assessment (qmra) approach to investigate the potential health risks of sars-cov- in sewage to wwtps workers [ ] . results revealed that the viral loads in wastewater/sewage at the entrance of wwtps were above the who benchmark of tolerable risk used for virus infection of - [ , , , , , ] . it is essential to evaluate bioaerosol and airborne particle risks for nearby communities and wwtp workers [ ] . in general, it can be made mandatory for the wwtp workers that perform manual cleaning of screening use face masks and face shields. the aerosol formation during wastewater treatment needs to be carefully examined as a potential source of virus transmission [ ] . water-related exposures may occur in communities with weak sewage infrastructure, or that use wastewater for irrigation [ ] . viral aerosols from a leaking sewage pipe at amoy gardens complex in hong kong were identified as the transmission route for coronaviruses during the sars-cov- outbreak in [ ] . the virus can also make its way into the drinking water distribution systems from the accidental contamination of drinking water with raw sewage and can enter individual homes. the presence j o u r n a l p r e -p r o o f of sars-cov- in water may also affect the water supply system by forming the biofilms that can compromise the water quality and endanger human health. there is also an imperative need for research to investigate the fecal-oral transmission potential of sars-cov- [ , ] . it is crucial to examine if the sars-cov- -contaminated wastewater has adverse impacts on aquatic life, soil, and wildlife. this needs careful examinations, risk analysis and thorough investigations to reach a conclusion. furthermore, monitoring and control measures are essential along the wastewater treatment route to avert coronavirus spread. it is recommended that the wwtp managers and stakeholders develop risk management strategies for the protection of wwtp workers and nearby communities [ ] . the disinfection steps in both wwtps and drinking water plants need to be developed carefully. the quantitative disinfection kinetics need to be established, and an optimum dose for inactivation of sars-cov- need to be determined. decentralized virus inactivation treatment for waster discharged from wwtps and drinking water plants can also be beneficial in reducing the environmental load of sars-cov- . detection and quantification of sars-cov- in water and wastewater is an essential but challenging step to track the infectious disease [ , ] . due to complex nature of wastewater matrix, it is essential to develop new biomarker extraction techniques as well as selective, sensitive, and cost-effective tools for the analysis of wastewater samples containing the novel virus [ ] . to avoid exposure to dangerous virus sars-cov- and to protect the safety of the lab analysts, it is recommended to use virus surrogates instead of harmful sars-cov- [ ] . wastewater-based epidemiology (wbe) has been recognized as a useful tool for evaluating, predicting and managing the disease outbreaks [ , , , ] . the concept is mainly based upon the detection, extraction, and analysis of biological and chemical compounds (referred to as biomarkers). recent studies have reported the successful detection of the novel virus in municipal wastewater and human stool [ , , , , , [ ] [ ] [ ] . surveillance of relative changes in concentrations of sars-cov- rna at the inlet of wwtp over time can serve as a useful tool for early warning for virus spread in the population [ ] . both upstream sampling (i.e., at sewerage maintenance holes) and downstream sampling (i.e., at the wwtp) approach can be used; however, upstream sampling is more appropriate due to variability in downstream samples. a gis-based sewerage map and flow rates would aid in the selection of upstream sampling locations [ ] . quantification of sars-cov- rna in wastewater settled solids in wwtps may also be used as a reliable and sensitive target for wbe [ ] . moreover, monitoring of international airports and seaports sewage would allow very early detection of the entrance of the virus into a country. it could also help the relevant authorities in deciding the implementation or removal of lockdown [ ] . it is essential to develop an integrated wastewater surveillance programs by considering both privacy and inequality concerns of the public [ ] . wurtzer et al. [ ] performed the time-course quantitative analysis of sars-cov- in paris sewage by rt-qpcr to investigate virus circulation in humans. however, these studies are at the initial stage, and a large pool of data from different parts of the world is necessary to develop a reliable and sensitive detection method for sars-cov- detection in wastewater/sewage [ ] . a multidisciplinary research approach, including engineers, microbiologists, chemists, and public health experts, will be productive to nurture more effective techniques for the quantification of sars-cov- in water. since sars-cov- has a presumably short half-life in water, the detection technique must be valid for both viable and non-viable sars-cov- in water. the virus can be degraded into other products; it is essential to develop strategies that could use these degraded products as target materials for detection and quantification [ ] . the lack of standardized and optimized protocol for the detection and quantification of sars-cov- in wastewater is another major challenge [ , , ] . this may lead to discrepancies in the results obtained by different laboratories, as indicated by a recent study [ ] . currently, the reverse transcription-quantitative polymerase chain reaction (rt-qpcr) has been employed widely for detection of sars-cov- in water samples [ ] . there is an imperative need to develop a standard operating procedure for accurate detection and quantification sars-cov- in water and wastewater. furthermore, a standard sampling procedure must be developed to extract/isolate, detect and quantify the virus accurately. this is an essential step for the development of commercial laboratories in various parts of the world that can accurately detect and quantify the virus with reproducible results. advancements in the field of microbiology might play a key role in developing a low cost, efficient method for detection and quantification of viral rna [ ] . recently, various innovative detection techniques have been reported in the literature [ ] [ ] [ ] . novel nanomaterials-based sensors were useful for the detection of waterborne pathogens [ ] . there is a research potential to develop techniques using a similar approach for the detection of sars-cov- by utilizing j o u r n a l p r e -p r o o f different novel nanomaterials. a reliable widely-accepted surveillance system needs to be developed for the accurate quantification of sars-cov- in water samples. the persistence of sars-cov- in the water environment is assumed to be short due to the enveloped nature of the virus [ , , ] . the half-life of sars-cov- in hospital wastewater is in the range of . and . h at °c [ ] . however, little is known about the vitality of sars-cov- in water [ ] . furthermore, the accurate survival period and concentration of sars-cov- in water are still indefinite, and these are open areas for research. it is essential to understand the decay mechanism and breakdown products of sars-cov- in water and wastewater. it will not only help in developing effective techniques for accurate detection and quantification but also will be useful for proposing an efficient disinfection technique. a recent study reported the transcriptomic architecture of sars-cov- [ ] . the sars-cov- may present in the water phase either in a viable state or in the non-viable form of viral debris [ ] . however, the actual degradation of products and accurate decay mechanism of sars-cov- in water phase need to be explored yet. the traditional treatment techniques for the removal of viruses from wastewater include sand filters, chlorine treatment, uv inactivation, ozone treatment, microbial treatment, membranes and pond systems [ ] [ ] [ ] [ ] [ ] [ ] . though it is assumed that the current techniques used for wastewater treatment might be helpful to eliminate the novel virus; due to the global impact of this j o u r n a l p r e -p r o o f pandemic, experimental results are needed to validate this assumption [ , [ ] [ ] [ ] . likewise, experimental shreds of evidence are required to confirm the effectiveness of household filtration systems, chlorination, densification and boiling for the elimination of the novel virus from water. it might be needed to upgrade the existing water and wastewater treatment systems or develop new treatment techniques to treat the sars-cov- -contaminated water. the more stringent treatment above the current level is needed for virus reduction to ensure the safety of recycled water [ , ] . who has highlighted guidelines on the safe management of water in july [ ] . a recent study stated that the current disinfection strategy recommended by who might not be adequate to deactivate sars-cov- in water [ ] . the existing wastewater treatment system might need necessary alternation or additional pretreatment steps to deactivate the sars-cov- . it should also be investigated if the presence of sars-cov- in wastewater affect the treatment process for the removal of other pollutants. recent advancement in nanotechnology, biotechnology and material sciences have opened many doors of applications and exhibited tremendous potential in water purification [ ] [ ] [ ] [ ] [ ] [ ] . the development of effective techniques for the inactivation of sars-cov- is vital to limit its presence in wastewater and lessen its potential adverse effects on human health and the environment. significant treatment steps are essential to upgrade both the wastewater and drinking treatment pants to eradicate sars-cov- or its rna fragments. recent years have witnessed tremendous progress in the applications of various novel materials in water treatment [ ] [ ] [ ] [ ] . nanomaterials and their composites might play a critical role in the development of an efficient method for the eradication of sars-cov- from water [ ] [ ] [ ] [ ] [ ] . advances in membranes systems provide an effective route for the removal of viruses from water [ ] [ ] [ ] [ ] [ ] . for drinking water, a potable water straw or uv based system can be developed to filter/kill the virus and to reduce the risk of waterborne viral infection [ ] . a recent study reported that there is a negative correlation between sunlight uv dose and percent positive of sars-cov- [ ] . other techniques such as oxidation, coagulation and photocatalytic killing of the virus must also be explored to determine the effective treatment option for the deactivation of sars-cov- in water. the efficiency of emerging disinfection technologies for sars-cov- inactivation need to be appraised. the water distribution systems may potentially host the novel virus due to biofilm growth and presence of bacterial colonies. innovations and improvements in the water distribution and plumbing systems are vital to minimize the transmission of sars-cov- through the water [ ] . as a general rule, it is the combination of different techniques in primary, secondary and tertiary treatments that would allow enough virus removal to limit the possibility of environmental contamination. the sars-cov- pandemic should develop a global awareness that solutions have to be found for improving the wastewater treatment even in developing countries, due to the possibility of its spread through the aquatic environment from inadequately treated wastewater. the presence of sars-cov- in wastewater may have consequences for public health in developing countries with poor water and sewage infrastructure, and inadequate institutional treatment/disinfection facilities. future research in these lines is essential for the protection of public health and the environment. in addition, robust policy intervention is essential to ensure reasonable compliance regarding the discharge of wastewater in various parts of the world. j o u r n a l p r e -p r o o f one of the major environmental concern associated with covid- pandemic is the excessive use of disinfectants (such as alcohol-based hand sanitizers and disinfecting soaps). the usage of disinfectants during the covid- pandemic increases environment and energy footprints significantly. it is important to carefully monitor the wastewater for possible commination and mitigate the spread of sars-cov- . a recent report reveals that tons of disinfectants are dispensed at in sewage systems in wuhan city of china from january to february , [ , ] . these discharges not only poses a potential threat to the marine environment but can also contaminate drinking water resources. another study estimated that excessive use of disinfectants and frequent handwashing could increase consumption of drinking water by % and lead to the generation of - % more wastewater [ ] . the alcohol-based hand sanitizer is not only associated with some health issues, but it may also result in adverse impacts on the environment [ , ] . a recent study highlighted that the extensive use of disinfectants poses a significant threat to urban wildlife [ ] . these disinfectants may find their ways to reach 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(february-april) wastewater from wwtpsmilan and rome (italy) [ ] april wastewater from wwtps istanbul (turkey) [ ] june river water quito (ecuador) [ ] (april-may) aircraft and cruise ship wastewater passenger aircraft flight from los angeles -brisbane hongkong -brisbane new delhi-sydney cruise ship (australia) [ ] march wastewater from the treatment facility massachusetts (usa) [ ] (february-march) sewage/wastewater from wwtps netherlands [ ] may wastewater from wwtps gujarat (india) [ ] april raw and treated wastewater samples from wwtpsmilano metropolitan area italy) [ ] (march-april) wastewater from wwtps paris (france) [ ] february sewage from hospital sewage disinfection pool hospital of zhejiang university, china [ ] may wastewater from wwtps, influent pump stations, or interceptor lines new york (usa) [ ] (march-april) wastewater from wwtps different localities in israel [ ] (february-april) wastewater from wwtps valencia (spain) [ ] (march-may) secondary-treated wastewater yamanashi prefecture (japan) [ ] (march-april) wastewater from wwtps ishikawa and toyama prefectures (japan) [ ] april wastewater from wwtps ourense (spain) [ ] (may-june) wastewater from wwtps jaipur (india) [ ] (march-april) wastewater from the drainage of covid- infected areas and quarantine centervarious districts in pakistan [ ] (january-april) wastewater from wwtps southern louisiana (usa) [ ] (april-june ) untreated wastewater from wwtps czech republic [ ] april wastewater from wwtps north-rhine westphalia (germany) [ ] (may-july) wastewater from wwtps montpellier (france) [ ] key: cord- -x amm g authors: rupali, priscilla title: introduction to tropical medicine date: - - journal: infectious disease clinics of north america doi: . /j.idc. . . sha: doc_id: cord_uid: x amm g tropical medicine deals with infectious and noninfectious diseases geographically located between the tropics of cancer and capricorn. it encompasses diseases that result from poverty, poor sanitation, infrastructure, and inadequate health resources. lack of availability of clean water and food made with unhygienic practices add to the morbidity of these diseases. the tropics are reeling under the onslaught of climate change, deforestation, and air, water, and soil pollution, which worsens an already fragile health system. this article provides an overview of the definition, classification, geophysical problems, syndromic approach to common tropical infections, diagnostic challenges in the tropics, and access to medicines. introduction to tropical spanned many decades in china, hong kong, and taiwan (then called formosa). he eventually came back to london where he lectured on tropical diseases at the st. george's hospital. he then became the chief medical officer to the colonial office. at this point he used his considerable influence to found a school for tropical medicine at the albert dock seamen's hospital. the london school of hygiene and tropical medicine was formally inaugurated on october , . he eventually went on to become the first president of the royal society of tropical medicine and hygiene in . he also made numerous contributions to parasitology, such as ( ) the establishment of mosquito as the intermediate host of wuchereria bancrofti, the causative agent of filariasis; ( ) proposed the mosquito-malaria theory, which eventually spurred sir ronald ross to discover that a mosquito was the definitive host for malaria; and ( ) the discovery of a new species of schistosoma (schistosoma mansoni) and a new parasite, spirometra or sparganum mansoni and spirometra mansonoides. a species of mosquito mansonia spp and a filarial worm mansonella spp were so named to honor him. he is hence known as the "father of tropical medicine." the tropics are regions of the earth that lie on either side of the equator between the tropics of cancer and capricorn (fig. ) . the tropics include parts of central and south america, australia, africa, india, and oceania. the tropics account for % of the earth's landmass, and are home to a third of the world's people. the tropics are warm throughout the year with temperatures ranging from c to c ( f- f) because they are close to the equator with direct sunlight most of the year. however, rainfall does vary remarkably from one area to another with the amazon basin recording a high rainfall and north africa being an arid area recording little to no rainfall most of the year. these climatic conditions often influence the flora, fauna, and insect wildlife in these areas. tropical regions are dominated by equatorial climates with a mean temperature greater than c, and arid zones are characterized by general lack of water, which rupali harms plant and animal life. they host % of the world's biodiversity. environmental factors include the following: climate: climate change has a wide-ranging impact on habitats, species distribution, human health, agriculture, sea levels, and the frequency/intensity of extreme weather events. changing rainfall patterns and increased temperatures have also led to increase in vector borne diseases, such as dengue and malaria, because of increased vector distributions and decreased vector and parasite incubation periods. air pollution: among the tropical regions, southeast asia reported the greatest increase in co emissions leading to a decreased air quality followed by south asia and south america, which has significantly impacted the climate and led to higher weather-related mortality and morbidity, infectious disease rates, and respiratory illnesses. land degradation: although land productivity has gone up because of increased livestock and cereal production, land degradation caused by poor agricultural practices and deforestation has led to altered ecosystems and has not changed the fact that more people in the tropics experience undernourishment compared with the rest of the world. water scarcity: the tropics have more than % of the world's renewable water sources, yet more than half of these areas are considered vulnerable to water stress because of the inequality of water distribution, with southeast asia having the highest pollution discharge in the world. overexploitation of wild marine food resources has led to the coral reef systems to be at high or medium risk of damage. biodiversity: tropical biodiversity is being threatened across all taxonomic groups with a rapid loss of primary forests. protection and maintenance of these fragile ecosystems will have a long-term impact on human health and requires cooperation at global, regional, and international levels. health is defined by the world health organization (who) as a "state of complete physical, mental and social well-being not merely the absence of disease or infirmity." however, there are many social determinants of health that affect a person's disease burden or longevity and these are often underemphasized or overlooked because the impact is often unmeasurable. climate and other environmental factors and such social factors as poverty, overcrowding, undernutrition, and limited access to appropriate health care coupled with the lack of education and poor governance contribute to a higher prevalence of communicable and noncommunicable diseases. according to the state of tropics report , globally extreme poverty has declined by almost % since the s but almost two-thirds of the poorest people in the world continue to live in the tropics. poverty reduction has taken place mostly in southeast asia and central america coupled with a rapid increase in urbanization in these areas. the urbanization rate in the tropics has increased disproportionately to almost % by , in turn giving rise to large populations of slum dwellers as compared with the temperate regions, which in turn brings with it diseases of hygiene and sanitation. about % of the world's population and % of children younger than age also live in the tropics. by , this is expected to increase to % and %, respectively. between and , the life expectancy in the tropics increased by . years to . years and infant mortality reduced by %. despite these enormous strides in the improvement of mortality, . million children younger than age died in , of which % were in low-and low-middle-income countries. infectious agents and toxins are transmitted through water, soil, food, vectors, plants, and animals. freshwater constitutes only . % of the total water resources on the planet. of this only . % is available as surface water, whereas the rest is in polar ice sheets, snow cover, and underground aquifers. water scarcity is defined as less than cubic meters of water available per person per year. in the tropics the number of nations with water scarcity has tripled since . this scarcity is most acute in south asia, where % of the population is considered vulnerable, followed by north africa and the middle east at %. statistics gathered by united nations show that million people lack access to safe drinking water and million live without appropriate sanitation. hence access to safe water and sanitation were recognized as human rights in by the united nations general assembly. waterborne or water-related diseases encompass illnesses resulting from indirect and direct exposure to water. the four main routes of transmission include ( ) waterborne, ( ) water washed, ( ) water based/insect vector, and ( ) water related. these diseases are transmitted through the direct drinking of water contaminated with pathogenic microorganisms. contamination of drinking water often occurs through fecal contamination, caused by poor sewage disposal and improper sanitation. if contamination levels are high, the young, the old, and the immunocompromised are at significant risk of diarrheal diseases and some others. some of the pathogenic organisms and diseases they cause are as follows: these are also known as water-scarce diseases, which thrive in water scarcity and poor sanitation. this depends more on water quantity than quality. a. soil-transmitted helminths: diseases of poor sanitation transmitted through contaminated soil. most prevalent helminths include ascaris spp, trichuris trichura, ancylostoma duodenale, or necator americanus. b. acute respiratory infections: they are responsible for % of the total child deaths every year and good hygienic practices including hand washing with soap can significantly reduce the transmission of acute respiratory infections. c. skin and eye diseases: scabies, impetigo, trachoma, yaws, conjunctivitis, and skin ulcers. d. fleas, ticks, and lice: typhus, scabies, relapsing fever. these are infections caused by parasitic pathogens found in aquatic host organisms. humans become infected either through skin penetration (schistosomiasis) or by ingesting the infective forms (dracunculiasis [ingestion of larvae in crustacean], paragonimiasis [metacercariae ingested in crab or crayfish], clonorchiasis [metacercariae ingested in fish]). these are diseases caused by the insect vectors breeding in and around water bodies. malaria is one of the water-related diseases endemic in countries with . billion people at risk. about % cases occur in africa, % in asia, and % in americas. mosquito-borne diseases include malaria, yellow fever, dengue fever, and filariasis. fly-borne diseases include onchocerciasis and loiasis. foodborne illnesses are defined by the who as diseases of infectious or toxic nature caused by the consumption of contaminated food or water. they are classified into two broad groups: intoxication and infection. intoxication is caused by ingestion of toxin produced by pathogens, whereas infection is caused by ingestion of food containing viable pathogens. intoxication is also possible by eating animals that have consumed toxin-producing organisms. foodborne diseases result in considerable morbidity and mortality, and contribute to significant costs in tropical countries. many of these are caused by bacteria, viruses, parasites, chemicals, and prions through contaminated food. the who estimates from to data that million cases were caused by contaminated food resulting in . million dalys (disease adjusted life years). , norovirus is a leading cause of foodborne illness ( million) followed by campylobacter spp with million cases. in addition, nontyphoidal salmonella diarrheal and invasive infections result in a high burden of . million dalys in the african region followed by the southeast asian region in children less than years. the main pathogens of foodborne illnesses are bacteria ( %), viruses ( %), chemicals ( %), and parasites ( %). bacteria cause % of the foodborne illnesses and botulism, clostridium perfringens gastroenteritis, e coli infection, salmonellosis, and staphylococcal food poisoning. the most common clinical symptoms are diarrhea, vomiting, abdominal cramps, headache, and nausea. foodborne parasitic diseases excluding enteric protozoa cause an estimated . million cases and , deaths annually resulting in an estimated . million dalys. among these foodborne ascariasis and toxoplasmosis were common tropical health contributing to . and . million cases respectively. human cysticercosis with . million, foodborne trematodiasis with . million, and toxoplasmosis with , dalys resulted in a high burden of disease. foodborne enteric protozoa resulted in an additional . million illnesses. clinically foodborne pathogens can cause diarrhea, intoxications, and invasive enteric diseases. bacteria producing acute diarrheas include campylobacter, salmonella spp, shigella spp, staphylococcus aureus, nontyphoidal salmonella, enteropathogenic e coli, and v cholerae. bacteria causing intoxications include clostridium botulinum, c perfringens, bacillus cereus, and enterotoxigenic e coli. bacteria causing chronic diarrheas include brucella spp, listeria spp, and mycobacterium tuberculosis in immunocompetent hosts and nontuberculous mycobacteria in immunocompromised hosts. most of the burden of foodborne illness caused by viruses is transmitted by poor hygienic practices either during food handling or food production. they are transmitted by the fecal-oral route infecting their host after ingestion, followed by invasion of cells in the epithelial lining of the gut and replication at the same site or elsewhere in the body. norovirus and hepatitis a are considered priority pathogens by the who and fao (food and agriculture organization of united nations). increasingly hepatitis e is a pathogen that is assuming increasing importance. zoonotic food borne viruses severe acute respiratory syndrome, monkey pox, and nipah virus have been transmitted through various food-related incidents. numerous parasites are transmitted by food including protozoa and helminths. however, some of these can also be transmitted by water, soil, or person to person contact. a wide variety of helminthic roundworms, tapeworms, and flukes are transmitted in foods, such as undercooked fish; crabs and mollusks; meat; raw aquatic plants, such as watercress; and raw vegetables contaminated by human feces. cryptosporidium spp, giardia intestinalis, cyclospora cayetenensis, and toxoplasma gondii trichinella spp and anisakis spp tapeworms diphyllobothrium spp and taenia spp insecticides used in crops, alcoholic beverages containing methanol, poisonous plants (eg, mushrooms, raw cassava roots), oysters, mussels, and clams (which ingest dinoflagellates producing saxitoxin), large reef fish (which ingest marine algae producing ciguatera toxin), finfish spoiled by bacteria leading to scombroid poisoning, and ingestion of puffer fish containing tetrodotoxin all can contribute to significant morbidity. vector-borne diseases impose heavy economic and health burdens leaving many people who survive the infection permanently debilitated, disfigured, maimed, or blind. vectors thrive in conditions where housing is poor, water is unsafe, and environment is contaminated with filth exacting their toll on the poor in developing countries ( table ) . malaria is the vector-borne disease that causes the largest amount of morbidity and mortality. dengue, yellow fever, and zika are diseases that cause large outbreaks paralyzing health systems and contributing to considerable social and economic disruption. onchocerciasis causes blindness, chikungunya severe arthritis, japanese encephalitis permanent neurologic damage, chagas heart failure and early death, and schistosomiasis poor nutritional status and school performance. insect bites can cause problems and are venomous or nonvenomous. venomous insects attack as a defense mechanism injecting painful toxic venom through their stings. nonvenomous insects bite to feed on the blood of mammals (http://www. traveldoctor.co.uk/stings.htm). snakes are common in rural areas of tropical countries. snakebites are a serious occupational hazard for agricultural laborers and fishermen. generally, the two main families with maximum morbidity and mortality include viperidae and elapidae. snakebites have now been included as a neglected tropical disease with one of the highest rates of mortality as demonstrated by the million deaths study. about % of cancers worldwide are caused by infectious agents in the developing world, and the most important cause after tobacco. if infections are controlled up to in cancers in the developing world can be prevented. human papilloma virus is the most common infectious agent, followed by hepatitis b and epstein-barr virus contributing to cancers worldwide. protein energy malnutrition and micronutrient deficiencies of vitamins and minerals are common in tropical countries and these often contribute to delayed childhood growth and poor child health indices. although universal immunization and nutrient supplementation at child care centers through mid-day meal schemes have played a major role in mitigating this problem, these are often encountered in many poverty-stricken or famine-ridden countries. tropical diseases are classified as communicable and noncommunicable diseases. communicable diseases by definition include diseases that are transmitted to humans and in turn are further classified as those caused by bacteria, viruses, protozoa, parasites, and fungi. noncommunicable diseases are diseases caused by genetic and lifestyle factors, such as cancers, cardiovascular diseases, diabetes, and chronic respiratory diseases. in addition, snakebites, scorpion stings, and marine and terrestrial envenomations are also management dilemmas. it is important to note that because of constraints of poverty in the tropics, often unsafe food practices are condoned leading to high morbidity among individuals. air and water pollution can lead to a myriad of respiratory, gastrointestinal, skin, and neurologic disorders along with serving as carriers of infectious pathogens. although it is impossible to list all the causative pathogens of disease in the tropics, a syndromic approach and a region-wise probability of common tropical diseases is most helpful when dealing with a patient in the tropics or even a returned traveler. each area of the world is unique and endemic for specific tropical diseases. there is now through the geosentinel network a large amount of data about the causes of fever in travelers in contrast with data from the tropical regions. studies that are available use inconsistent definitions about "acute undifferentiated febrile illnesses" and diagnoses are not often confirmed. fever in the tropics is often syndromic and in the absence of appropriate and accurate diagnostics, common diseases are vastly overdiagnosed, such as malaria in africa and typhoid fever in south asia. the advent of rapid diagnostic testing has led to the recognition of other important causes of acute febrile illnesses in the tropics ( table ) . neurologic diseases in the tropics, although rare, are a cause of considerable morbidity and mortality. a history of travel including geographic locale and activities indulged in, possible exposures encountered, vaccines, prophylaxis and protective measures taken, along with the immune status of the host could help determine the etiologic organism of a particular neurologic syndrome. the neurologic syndromes are divided into global and focal syndromes ( table ) . skin lesions are a common problem in the tropics and they could be a primary problem or secondary to an underlying systemic condition. it is important to focus on infections that are treatable, transmissible, and have a high morbidity or mortality. the history must include details of previous travel, previous skin lesions, activities indulged in, immune status of the host, vaccinations, and prophylaxis. exposures to fresh or sea water, animals, arthropods, plants, breaks in skin including tattoos, sexual activities, and although tropics are often considered exotic locations skin problems can often be from cosmopolitan causes. sunburn, scabies, and prickly heat are common and chronic skin problems, such as atopic dermatitis, may exacerbate in a tropical environment. tropical biodiversity also results in a wide variety of plants and hence hypersensitivity to plants, plant products, and drugs may also occur. it is important not to forget mundane causes, such as pyoderma and folliculitis. because skin manifestations are myriad a syndromic approach does help to narrow down a definite diagnosis ( table ). diarrhea as a syndrome in the tropics has been described as "montezuma's revenge" and "delhi belly" because of the associated morbidity. it also is the second most important cause of child deaths younger than the age of years contributing to almost , . in the global burden of disease study diarrhea was a leading cause of death among all ages contributing to . million deaths. most of the deaths in children and adults were attributable to rotavirus, shigella spp, and salmonella spp. however, deaths on the whole have been reduced by . % from to . in the global enteric multicenter study done in children younger than age , interventions to reduce deaths should be directed against five pathogens: stable enterotoxigenic e coli, enteropathogenic e coli, cryptosporidium spp, rotavirus, and shigella spp. different clinical syndromes of diarrhea have been defined, each reflecting different etiology and pathogenesis. these are briefly described in tables and . various challenges exist in the diagnosis of tropical infections. the challenges are multiple and are divided as discussed next. the burden of disease and the kind of setting where the disease is being diagnosed often determines the need of an appropriate diagnostic test. in a high-prevalence, low-resource setting a test that is low cost, point of care, requiring little technical expertise, with a high positive predictive value is required. in contrast in a lowprevalence, low-resource setting, additional tests may need to be performed to confirm the diagnosis. subclinical and asymptomatic manifestations of a tropical disease may also make it difficult for a diagnostic test to distinguish between clinical disease and the former. in addition, if a test is serology-based in an endemic setting it may be difficult to establish causality for the clinical manifestations of the disease if baseline antibody titers is high. although there are obvious infrastructural and financial challenges in low-resource settings there are also impediments with the lack of trained laboratory staff and quality assurance of available laboratory diagnostics. in addition, an ideal laboratory test needs to be rapid, point of care, requiring minimal technical expertise, following norms the who constitution dictates, "all people share the right to the highest attainable standard of health." huge advances are being made toward internationally agreed global health targets, some of which include a reduction in child mortality by % between and and a % decline in aids related deaths since the peak of the hiv/aids epidemic in with at least half the infected people being able to access antiretroviral therapy. many international agencies, such as the who and unaid (joint united nations programme on hiv/aids), pharmaceutical companies and combating antibiotic resistant bacteria biopharmaceutical accelerator and the global antibiotic research and development partnership have incessantly campaigned for increased access to medicines leading to an achievement of many of the health-related millennium development goals. controlling, eliminating, and eradicating neglected tropical diseases has been a major focus for the who since as it moved away from specific diseases to the health needs of poor communities. over the years many disease conditions that were believed to require a concerted effort by the who were included. these were identified as follows ( table ) this has led the who to adopt five major strategies to combat these diseases: veterinary public health at the human animal interface: an integrated human and animal health approach is required for such diseases as cysticercosis, echinococcosis, and rabies, which involve vertebrate hosts. . provision of safe water, sanitation and hygiene: better sanitation and improved safe water supply coupled with vector control is being looked at for long-term economic growth and food production. tropical medicine is an amalgamation of infectious and noninfectious diseases and deals with many important issues, such as water, hygiene, and sanitation, which is out of reach for many low-and middle-income tropical countries. as a result, the health indices for these countries often suffer necessitating global and local public health interventions. research, development, global support, and funding along with access to major health interventions has empowered many of these countries to overcome the challenges faced by them while combating tropical diseases. water and waterborne diseases: a review world health organization estimates of the global and regional disease burden of foodborne bacterial, protozoal, and viral diseases, : a data synthesis world health organization estimates of the global and regional disease burden of foodborne parasitic diseases, : a data synthesis a review on major food borne bacterial illnesses improving food safety through a one health approach estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the global burden of disease study burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study access to medicine index -methodology report accelerating work to overcome the global impact of neglected tropical diseases. a roadmap for implementation. available at: www.who.int/neglected_ diseases/en a review of the global epidemiology of scrub typhus world malaria map global morbidity and mortality of leptospirosis: a systematic review burden of typhoid fever in low-income and middle-income countries: a systematic, literature-based update with riskfactor adjustment key: cord- -nxeg xj authors: gerba, charles p.; goyal, sagar m. title: pathogen removal from wastewater during groundwater recharge date: - - journal: artificial recharge of groundwater doi: . /b - - - - . - sha: doc_id: cord_uid: nxeg xj nan solid wastes, and sewage oxidation ponds. additional sources of pathogens in groundwater may involve artificial recharge of groundwater aquifers with renovated wastewater including deep well injection, spray irrigation of crops and landscape, basin recharge, and land application of sewage effluent and sludges. leakage of sewage into the groundwater from septic tanks, treat ment lagoons, and leaky sewers is estimated to be over a trillion gallons a year in the united states [ ] . it should be realized that, as opposed to surface water pollution, con tamination of groundwater is much more persistent and is difficult to erad icate. because restoration of groundwater quality is difficult, time-consuming, and expensive, efforts should be made for the protection of groundwater quality rather than only for its restoration after degradation. secondary sewage treatment including disinfection by chlorination may not be able to remove all of the pathogens present in sewage. thus, intentional or unintentional recharge of groundwater with treated sewage effluent may be potentially hazardous to human and animal life. soil is considered a living filter, capable of removing pathogenic mi croorganisms from applied wastewater. the extent to which soil can remove these microorganisms depends on several factors such as the nature of the soil, the nature of the pathogen concerned, temperature, and antagonism from native microflora. because of their large size, parasitic protozoa and helminths may be efficiently removed by filtration through soil and may not be able to gain entrance into the groundwater. bacterial removal by soils also occurs largely by filtration, although adsorption is also involved. viruses, on the other hand, are thought to be removed by the process of adsorption only [ ] . unfortunately, however, viruses cannot be considered as permanently immobilized because they have been shown to elute and migrate further in soil following rainfall events [ ] [ ] [ ] . several investigators have reported on the isolation of viruses from groundwater [ ] and several outbreaks of viral hepatitis, yersiniosis, ty phoid, and shigellosis have also been attributed to contaminated groundwater [ ] . documented evidence of health problems associated with groundwater recharge is, however, lacking. the paucity of information on health problems associated with groundwater recharge programs may reflect either the absence of a problem, lack of intensive surveillance, or the insensitivity of present epidemiologic tools to detect recurrent small-scale incidents of disease. often the low fecal car riage rates of agents of infectious disease and the low background of enteric disease in the united states has been cited as further evidence that the potential of public health hazard as a result of direct or indirect reuse of wastewater is minimal. it should be realized, however, that levels of enteric disease in the united states are low primarily because of good sanitation, personal hygiene, and a network of sanitary engineering works. as a result of this low exposure to pathogens, the population at large may have become highly susceptible to even small numbers of pathogens. waterborne outbreaks of disease are no longer on the decline in this country (figure . ). a total of waterborne outbreaks occurred during , increasing the annual average of outbreaks to for the -year period from - . this number represents more than a percent increase over the - average of . the -year averages have steadily in creased from an annual average of ten during [ ] [ ] [ ] [ ] [ ] . before that pe riod, the trend was declining [ ] . it should be emphasized that reporting of waterborne disease out breaks, particularly in individual systems, is notoriously poor. according to craun [ ] outbreaks in municipal water systems, which number , and serve about million people, are probably the most likely to be reported. outbreaks in semipublic systems, which number about , and serve numerous transients, are the next most likely to be reported. the least likely to be reported are the outbreaks in individual water systems, which number about , , . in fact, one third of the individual groundwater supplies in a rural neighborhood of oregon were found to be fecally contaminated in a recent survey [ ] . waterborne hypothesis cannot be proved in all instances because epidemiologic investigations are sometimes incomplete or conducted long after the outbreak has subsided. also, the surveillance of waterborne diseases by the centers for disease control (cdc) is largely passive and clearly rep resents a fraction of the total number that occur. according to cdc, "the likelihood of an outbreak coming to the attention of health authorities varies considerably from one locale to another depending largely upon consumer awareness, physician interest, and disease surveillance activities of state and local health and environmental agencies. large interstate-outbreaks and outbreaks of serious illness are more likely to come to the attention of health authorities." of documented outbreaks of waterborne disease from to , ( %) were attributed to illness of probable viral etiology (e.g., hepatitis a, poliomyelitis, gastroenteritis). this number probably represents only a fraction of the actual number of virus-caused outbreaks, because of the difficulties involved in proving a viral etiology of a waterborne outbreak. in fact, direct evidence of virus involvement in waterborne outbreaks is limited to hepatitis a, adenovirus, and recently to norwalk agent and rotavirus [ ] . the lack of documentation of waterborne viral disease outbreaks may be ascribed to limitations in methodology for the detection of viruses in water and relative insensitivity of epidemiologic techniques to detect lowlevel transmission of viral diseases through water. it is easy to recognize the outbreaks of infectious hepatitis by the water route because of their explo sive nature and characteristic symptomatology. most enteric viruses, how ever, cause a wide variety of symptoms so that scattered cases of acute illness would probably have too varied symptoms to be attributed to a single etiologic agent. also, the presence of small numbers of viruses in water may result only in an inapparent infection in a person coming in contact with contaminated water. the virus may then multiply in the respiratory and gastrointestinal tract of that person who may, in turn, act as an effective carrier and transmit the virus to others. the development of acute disease in these contact persons will be epidemiologically classified as "transmitted by direct contact" rather than being waterborne. intensive surveillance is, therefore, necessary to determine the "real" cause of an outbreak. a discussion on epidemiology is incomplete without consideration of minimum infective dosage of various microorganisms. infective doses of most bacterial pathogens are relatively high. for instance, approximately enteropathogenic escherichia coli or vibrio cholerae cells must be con-sumed by healthy male volunteers to produce disease in a significant pro portion of subjects. in case of shigella, however, - cells are enough to cause dysentery. similarly, the infectious dose of protozoan cysts and helminth ova is very low, perhaps . the symptoms of helminth infections are dose-related, however. currently available information suggests that even a single virus particle may produce infection under favorable condi tions. after reviewing infective dose data for various microorganisms in human subjects, akin [ ] reached the conclusion that infective dose for some members of bacterial, viral, and parasitic groups may be as low as =^ detectable units. pathogenic microorganisms such as bacteria, viruses, protozoa, and parasitic worms are almost always present in domestic sewage. the number and types of organisms present in sewage, however, vary from community to com munity depending on urbanization, population density, sanitary habits, sea son of the year, and rates of disease in the contributing community [ ] . the most common bacterial pathogens associated with sewage are salmonella, shigella, vibrio, and campylobacter (table . ). salmonella occurs [ ] . since , however, cases of cholera have been documented along the gulf coast. the strains from all these cases appear essentially identical, suggesting that the toxigenic v. cholerae has persisted in that region for at least years. extra efforts should, therefore, be made to keep track of this potential problem. more than different virus types may be present in raw sewage (table . ). they range in size from about nm for polio virus to nm for rotavirus and up to nm for enteric coronavirus. all virus groups found in sewage contain single-or double-stranded rna except adenoviruses, which consist of double-stranded dna. these viruses are capable of causing a variety of illnesses at very low dosage levels [ ] . the amount of virus present in raw sewage is highly variable but as high as , infec tious virus particles per liter have been detected [ ] . studies indicate that bacteria and viruses are not removed effectively from wastewaters during primary treatment [ ] ; removal of viruses during secondary treatment (active sludge) is dependent largely on virus adsorption to solids. since rotavirus adsorbs poorly to activated sludge floes, it can be speculated that wastewater treatment processes that are highly effective in the removal of enteroviruses may not be as effective in removing rota and reoviruses. even within the enterovirus group, virus adsorption to activated sludge was found to be both type-and strain-dependent [ ] . it stands to reason, therefore, that different viruses will have different removal char acteristics during activated sludge treatment. an average of to percent of the enteric bacteria in sewage are reported to be removed by activated sludge process (table . ). coagulation with alum or lime is considered to be generally efficient for virus removal. in laboratory studies, - log reduction of viruses is common following lime treatment at ph . in field studies, however, viruses were isolated from lime sludge and lime-treated effluent [ ] . other tertiary treatments such as ferric chloride-polyelectrolyte flocculation, sand or granular filtration, re verse osmosis, and carbon adsorption have been found to significantly re duce the level of pathogens. feachem et al. [ ] reviewed the literature on pathogen removal by various sewage treatment processes. removal, but to look at orders of magnitude." they further stated that to talk of percent removal is misleading because a percent removal of path ogens from raw sewage containing pathogens per liter will produce an effluent that still contains pathogens per liter. this level may still be of great public health concern, depending on how the effluent is going to be used. as efficient as it may be, sewage treatment processes cannot be ex pected to remove/inactivate all of the pathogens present. disinfection of treated wastewater is, therefore, practiced to ensure further inactivation of microorganisms. in the united states, chlorination is practically the only process used for disinfection of wastewater. unfortunately, however, there is a great variability in resistance to chlorine among different microorga nisms. it is generally agreed that bacteria are much more susceptible to chlorine than are viruses and protozoan cysts. also, chlorine may be very [ ] . effective against mircoorganisms cultivated in the laboratory under artificial conditions, but it may not be as effective on naturally occurring strains of bacteria and viruses. the fate of pathogenic bacteria and viruses in the subsurface will be deter mined by their survival and their retention by soil particles. both survival and retention are largely determined by the three factors shown in figure . . climate will control two important factors in determining viral and bacterial survival: temperature and rainfall. the survival of microorganisms is greatly prolonged at low temperature; below ° c they can survive for months or even years [ ] . at higher temperatures, inactivation or dieoff is fairly rapid. in the case of bacteria, and probably viruses, the dieoff rate is approximately doubled with each ° c rise in temperature between ° c and ° c [ ] . above ° c temperature is probably the dominant factor determining virus survival time. rainfall mobilizes previously retained bac teria and viruses and greatly promotes their transport in groundwater. sev eral studies have shown that the greatest degree of drinking water well contamination occurs after periods of heavy rainfall [ ] [ ] [ ] . the nature of the soil will also play a major role in determining survival and retention. soil properties influence moisture-holding capacity, ph and organic matter-all of which will control the survival of bacteria and virus in the soil. other soil properties such as particle size, cation exchange ca pacity, and clay content will influence retention. resistance of microorga nisms to environmental factors will vary among different species as well as strains. bacteria are believed to be removed largely by filtration processes while adsorption is the major factor controlling virus retention [ ] . the following sections are a summary of the recent state of knowledge on factors currently believed to influence microbial persistence and transport in the subsurface. the straining or filtration of bacteria at the soil surface is a major limitation in their travel through soils. when suspended particles, including bacteria, accumulate on the soil surface, as water passes through the soil these par ticles themselves become the filter [ ] . such a filter is capable of removing even finer particles, by bridging or sedimentation, before they reach and clog the original soil surface. this phenomenon will in fact largely be dom inant if only a portion of the suspended particles are larger than the pore openings. as soon as a few such particles have accumulated, they become the straining surface for finer particles [ ] . in studies in which e. coli suspended in distilled water was allowed to percolate into sand columns, krone [ ] found that after the first arrival of bacteria the concentration in column effluents continued to rise until a max imum was reached, after which it fell, suggesting that accumulating bacteria at the soil surface enhances the straining removal. this same effect is seen during the land application of domestic sewage when repeated cycles of flooding and drying of infiltration basins is practiced [ ] . for example, at the flushing meadows project near phoenix, arizona, treated sewage effluent is spread into basins underlaid with loamy sand. the greatest numbers of coliforms and fecal coliforms are observed after the start of each new inundation period when newly infiltrated water arrives at the bottom of sampling wells, after which time a general decrease in values occurs. a similar phenomenon occurs when water containing microorga nisms is pumped into recharge wells. studies using sandy soils of various effective porosities indicate removal of bacteria from a liquid percolating through a given depth of soil is inversely proportional to the particle size of the soil. the greatest removal of bacteria occurs on the surface mat (top - mm) that forms on the soil. adsorption is the major factor in the removal of viruses by soil and also plays a role in bacterial removal. factors that reduce the repulsive forces between the two surfaces, such as the presence of cations, would be ex pected to allow closer interaction between them and allow adsorption to proceed. the very small size of clays, their generally platy shapes, the oc currence of large surface area per given volume, make them ideal adsorption sites for bacteria and viruses in soils. thus, adsorption phenomena will play a more important role in the removal of microorganisms in soils that contain clays [ ] . many factors are known to control microbial adsorption to soils and these are listed in tables . and . . soils differ considerably in their textural, chemical, and mineralogical prop erties and hundreds of soil types have been classified in this country [ ] . furthermore, both vertical and horizontal variability is a normal character istic of many soils. it is generally agreed that fine-textured soils retain mi croorganisms more effectively than sandy soils since the soil clay mineral fraction displays a high sorptive capacity toward viruses as a result of its high surface area and ion-exchange capacity. following examination of nine [ ] . soils from arkansas and california, it was shown that virus adsorption in creased with the clay content and the specific surface area of the soil [ ] . iron oxides, particularly magnetite, also display a high affinity toward viruses [ ] . hori et al. [ ] found that polio virus removals from distilled water in -in columns of three hawaiian soils, including two low-humic latosols (lahaina and wahiawa) and a volcanic cinder (tantalus), averaged > , > , and percent, respectively. with the two low-humic latosols, there was a trend of decreased retention over the -day test period. goyal and gerba [ ] noted considerable differences in the abilities of nine different soils to adsorb a number of enteric viruses. statistical analysis indicated that ph was the most important soil characteristic influencing virus retention, with soils having a ph < giving consistently high retention. exchangeable alu minum was another factor that correlated with the adsorption efficiency of in contrast to these findings, wang et al. [ ] and lance et al. [ ] found greater removal of poliovirus in sandy soils than total and fecal coliforms, and fecal streptococcus. but the lowest removal was observed with coliphage f [ ] , indicating that virus type plays a significant role in the extent of virus removal. additional studies are needed on the relative re moval of bacteria and viruses by soil types. moore et al. [ ] recently reported that poliovirus type adsorption to different soil materials suspended in a synthetic freshwater was neg atively correlated with soil organic matter content and with available neg ative surface charge as measured by adsorption capacity for a cationic polyelectrolyte. soil ph, surface area, and elemental composition were not significantly correlated with virus adsorption. furthermore, additional stud-ies by this same group indicated that the two poorest adsorbents for both poliovirus and reovirus among different soil materials were a muck soil and a silt loam, both of which had high organic matter content [ ] . these authors were also able to show a highly negative correlation between virus adsorption and the capacity of soils to bind a cationic polymer, pdadm (polydiallyldimethyl ammonium chloride). it was suggested that the ability to bind the polymer could serve as an indicator of the extent of viral ad sorption [ ] . the results of these studies indicate that soil type greatly influences the extent of virus transport or retention. it may be possible to distinguish soils by general class with respect to virus retention, based on their textural, mineralogic, and chemical properties. however, further studies with a wide range of soil types and viruses are needed to determine if such classifications are possible and to identify the soil characteristics that most influence virus retention. the effects of ph on virus adsorption to soils are explainable on the basis of electrochemical features of virus and soil surfaces. the surface charge of a virus is influenced primarily by ionization of the carboxyl and amino groups on the outer surface of the virion protein capsid; and at neutral ph, most viruses are negatively charged. soils also tend to be generally electronega tive at neutral ph; therefore, virus adsorption is not favored due to repul sion of the two negatively charged surfaces. however, if the ph of the surrounding medium is lowered, protonation causes decreased ionization of virion carboxyl groups and increased ionization of amino groups. as a re sult, viruses become less electronegative or even electropositive at lower ph levels. although soil particles will also tend to become more electropositive at lower ph levels, the isoelectric points of soil particles are generally lower than those of viruses. for example, electrophoretic mobility studies have shown that a common soil clay mineral, montmorillonite, is negatively charged at ph . to . . muck soils also have a high negative charge [ ] . at lower ph levels, the viruses may be electropositive but the soils are still electronegative, thereby resulting in electrostatic attraction and increased adsorption. the relationship between virus adsorption and ph is not clearcut, however, because of many complicating factors. the ph of the soil, as conventionally measured, does not reflect necessarily the ph at the surface of soil colloidal particles such as clays. various soil components (clay, sand, oxides of aluminum and iron) display different isoelectric points. there is also a lack of information on the isoelectric points of more than viruses that occur in wastewater or groundwater. so far, we know that the isoelectric point varies with virus type and strain [ ] [ ] . the results of a number of studies indicate that virus retention by soils generally increases at lower ph levels. in an early report drewry and eliassen [ ] found decreased bacteriophage tl, t , and f adsorption to ar kansas and california soils at higher ph levels. more recently, burge and enkiri [ ] found that the rates of bacteriophage x adsorption to five soils were significantly correlated with soil ph. in batch adsorption studies with a variety of viruses and nine soils by goyal and gerba [ ] , ph was found to be the single most important soil factor influencing adsorption. soils having a saturated ph less than were the best adsorbers. studies by sobsey et al. [ ] showed that poliovirus type and reovirus type adsorp tion to eight different soil materials suspended in settled sewage at ph levels between . and . was generally greater at the lower ph levels. in studies by duboise et al. [ ] with cores of sandy forest soil receiving poliovirus in sewage effluent at various ph levels between . and . , virus retention was best at ph . , and the release and migration of retained viruses by subsequent distilled water applications was lower from the cores that re ceived sewage effluent having lower ph values. similar observations have been made for bacteria [ ] . the types and concentrations of ionizable salts in the soil-water environment greatly influence the extent of bacteria and virus transport. in general, in creasing concentrations of ionic salts and increasing cation valencies enhance virus adsorption. divalent cations (e.g., ca + , mg + ) are very efficient in promoting virus adsorption to a sandy soil [ ] . cations are necessary to reduce the repulsive forces on both the virus and soil particles and allow adsorption to take place. viral and bacterial retention by soils is generally greater in the presence of sewage effluents than in distilled water [ ] [ ] . wastewater effluents have indeed higher conductivity ( - fxmhos/ cm) than distilled water ( - fxmhos/cm) or rainwater ( - |xmhos/cm). rainwater, being of lower conductivity than sewage effluents, may thus lead to reduced viral and bacterial adsorption or to desorption with the subse quent redistribution of these organisms within the soil profile. this phenom enon was well demonstrated via soil core studies under controlled laboratory conditions [ , , , ] . landry et al. [ ] showed that virus penetration was more extensive in rainwater-rinsed cores than in wastewater-rinsed cores. moreover, the desorbed viruses may readsorb at greater depths. heavy rainfall might then remobilize soil-bound viruses with the potential contam ination of groundwater supplies [ ] . however, it now appears that the ability of rainwater to release viruses depends on the soil type, the release being more pronounced in sandy than in clay soils [ ] . the elution pattern also depends on the virus type and strain. for example, poliovirus and echovirus were mobilized by artificial rainwater, whereas echovirus was not affected. the elution pattern of the reference strain of poliovirus differed from that of field and mutant strains [ ] . rainfall will also effect bacterial retention by lowering ionic concen tration and increasing infiltration rates. several surveys have indicated that rainfall and well depth are related to microbial groundwater quality. studies in washington indicated that shallow drinking water wells average medium coliform values of mpn per ml with an average depth of . m ( ft), while deep wells with an average depth of . m ( ft) average mpn per ml [ ] . it was also observed that virtually all bacterial con tamination coincided with the periods of heaviest rainfall. brooks and cech [ ] observed in rural eastern texas that practically all dug wells with depths of ft ( m) or less were positive for either fecal coliforms or fecal strep tococci. while presence of fecal bacteria was much less common in deeper wells, some wells as deep as ft ( m) were positive. increased levels of bacterial contamination of drinking well water after periods of rain have been noted in several studies [ ] [ ] [ ] ] . in one study, it was noted that while an increase in coliform bacteria appears almost immediately after periods of heavy rainfall in shallow wells, in deeper wells the increase did not occur until weeks later [ ] . thus, any satisfactory study of well water quality should include sampling during periods of highest rainfall. soluble organic materials are known to compete with viruses and bacteria for adsorption sites. it may then be possible that organics present in sewage may interfere with virus sorption to soils. however, several studies have shown that viruses are well adsorbed to various types of soils in the presence of secondary and even primary wastewater effluents. as discussed above, wastewater effluents contain enough salts to overcome any interference by soluble organic matter. humic and fulvic acids are highly colored organic compounds that are naturally present in both water and soils. recent studies indicate that these compounds can cause increased virus transport through soils not only by interfering with virus adsorption but also by causing desorption. bitton et al. [ ] found that poliovirus retention by columns of sandy soil was exten sively reduced when applied in highly colored (high concentrations of humic and fulvic acids) cypress dome water compared to its retention from tap water. more recently, scheuerman et al. [ ] reported extensive interfer ence by humic and fulvic acids with poliovirus type retention in columns of organic sediment, muck soil, and brown-red sand. soils that were capable of retaining all or most of the applied virus in the absence of these organics retained considerably less virus in their presence. the extent of virus trans port through the columns correlated with the color of the column effluents. this phenomenon was confirmed by bixby and o'brien [ ] , who re ported that fulvic acids complex ms phage and prevent its adsorption to soil. more recently, such soils were found to display a lower adsorption capacity than other mineral soils [ , ] . the results of a number of studies suggest that organic soils and other soils or waters with high concentrations of humic and fulvic acids may not be suitable for land application of wastewater. the effects of other organics in waters and soils on virus retention remain uncertain. additional studies are needed to further understand and quantify the effects of humic and fulvic acids in water and soil on the infectivity and retention of a variety of viruses in different soils. such studies are also needed for other classes of water, wastewater, and soil organics. hydraulic conditions in soils receiving wastewater appear to have a consid erable effect on virus transport for at least some soils. such conditions as flow rate, hydraulic loading, and application frequency may all influence the extent of virus migration through soils. vaughn et al. [ ] reported that infiltration rate greatly influenced poliovirus removal in a groundwater re charge system where tertiary effluent was applied to a coarse sand-fine gravel soil. recharge at to cm per hour resulted in considerable virus movement into groundwater while at two lower recharge rates, and . - . cm per hour, there was considerably less virus movement. at the lower infiltration rates, the surface mat of sewage solids that formed on the soil surface may have contributed to the greater virus removals observed. lance et al. [ ] found that poliovirus type removal was not affected by infiltration rates in the range of to cm per day. more recently lance and gerba [ ] found that increasing flow rates from . to . m per day resulted in increased movement of viruses down the column. however, there was no further increase in virus movement at flow rates up to m per day. in comparative studies of several soils it was found that by linear regression analyses, the rate of virus removal in soil columns was negatively correlated with the flow rate of the percolating sewage effluent [ ] . the authors suggested that flow rate of water through the soil may be the most important factor in predicting the potential virus movement into groundwater. little virus movement has been observed in unsaturated soil columns [ ] . although the results of at least some studies suggest that virus migra tion increases with increasing hydraulic loads and flow rates and under con ditions of saturated flow, further studies are needed with a wide range of soil types and field conditions to quantify the extent of virus movement through soils under different hydraulic conditions. recent studies have shown that different types and strains of viruses are not equally retained by soils. these virus-specific differences in adsorption to soils are probably related to physicochemical differences in virus capsid surfaces. although all enteric viruses possess outer capsids comprised of polypeptide subunits and generally behave as charged, amphoteric, colloidal particles, the surfaces of the virions differ in the details of their configura tion, charge density and distribution, and other features. in fact, even the same virus can display different surface properties that will influence its physicochemical behavior as a result of conformational changes brought about by ph effects and interactions with soluble chemicals and particulate surfaces [ ] . goyal and gerba [ ] found that different enteric virus types and strains varied in their ability to adsorb to soils. for example, adsorption efficiencies of six different strains of echovirus type in suspensions of sandy soil in deionized water ranged from to . percent. type and strain dependence of enterovirus adsorption to a sandy loam soil suspended in distilled water was also reported in another study from the same laboratory [ ] . adsorp tion efficiencies of ten different virus types and strains ranged from percent for echovirus type , strain v , and coxsackie virus b , strain v , to . percent for echovirus type , wallace strain, and poliovirus type , strain lsc. landry et al. [ ] reported type and strain differences in enter ovirus adsorption to sandy soil cores. vaccine strain poliovirus type (lsc), a widely employed enterovirus model in soil and other environmental stud ies, was efficiently adsorbed but not readily eluted with either distilled water or sewage effluent. some of the other enteroviruses tested, including field strains, were less efficiently adsorbed and more easily eluted. it was con cluded that vaccine strain poliovirus type may be an inappropriate model for studying the nature and extent of virus transport in soils. in contrast to the findings from batch laboratory studies by the same group, hurst et al. [ ] found that under field conditions at a rapid infiltra tion site, echovirus type , farouk strain, did not migrate as far down in the soil as poliovirus type , strain lsc. they suggested that the adsorptive behavior of viruses in laboratory batch studies may not be totally reflective of their behavior under field conditions, possibly because of virus adsorption to soil particles prior to infiltration. it is now agreed that poliovirus type adsorbs well to most soils. it was recently concluded that viruses may be grouped into three categories according to their adsorptive behavior [ ] . category contains the poorly adsorbed viruses (echovirus , echovirus , coxsackie virus b , x , ms ) and category includes the highly adsorbed viruses (poliovirus , echovirus , coxsackie virus b , t , and t ). phage f was placed in a third category exhibiting the lowest adsorption of all viruses tested. at the turn of the century, it was found that the eating of raw vegetables grown on soil fertilized with raw sewage resulted in outbreaks of typhoid fever. as a result, the survival of enteric bacteria in soil systems has been extensively studied. there are several major reviews on the survival of en teric bacteria in soil [ ] [ ] [ ] , and we will only consider herein factors that affect the length of survival of these bacteria. less is known about virus survival. most enteric bacterial pathogens dieoff very rapidly outside of the human gut, whereas indicator bacteria such as e. coli will persist for longer periods of time. survival times among different types of bacteria and viruses vary greatly and are difficult to assess without studying each type individ ually. in most cases, it appears that to months is sufficient for reduction of pathogenic to negligible numbers once they have been applied to the soil, although survival times as long as years have been reported [ ] . factors known to influence bacterial and viral survival in the soil are listed in tables . and . . a major factor determining the survival of bacteria in soil is moisture. young and greenfield [ ] showed that moisture was a factor in the viability of e. coli in soils. beard [ ] stated that moisture was the most important deter mining factor in the survival of salmonella typhosa. bacterial survival was determined in various types of soil exposed outdoors in clay flowerpots. the survival in all types of soil tested was found to be greatest during the rainy season. in sand, where drying was rapid due to its low moisture-retaining power, survival time was short-between and days during dry weather. in soils that retain a high amount of moisture such as loam and adobe peat, the organisms persisted longer than days. bouma et al. [ ] have suggested that survival data for fecal organisms could be compared with soil-moisture characteristic curves, and hence the distance of soil filteration necessary for removal be defined as a function of moisture content. soil moisture also influences virus survival in soil. bagdasar'yan [ ] reported that enteroviruses survived three to six times longer in soils with percent moisture content than in air-dried soils. duboise et al. [ ] found that poliovirus type was inactivated considerably more rapidly in drying soil, as the moisture content decreased from to . percent, than in the same soil type maintained at or percent moisture content. inactivation of percent of the initial viruses occurred within week in drying soil but took - and - weeks in soils with to percent moisture content, respectively. yaeger and o'brien [ ] compared the degree of poliovirus inactiva- ? tion in eight different soils saturated with riverwater, groundwater, or septic wastewater and in the same soils that were allowed to dry out during the course of the experiment. upon drying, none of the initial viruses was de tectable in any of the dried soils (> . % inactivation), but considerable quantities were still present in the same types of saturated soils. in experi ments on the rate of poliovirus inactivation at different soil moisture levels, there was a sharp increase in the inactivation rate at . percent soil moisture compared to that at . percent. hurst et al. [ ] also observed differences in poliovirus inactivation rates at different soil moisture levels, with the greatest inactivation rate at a moisture level of percent. inactivation proceeded more slowly at both higher and lower moisture levels, but the slowest inactivation rates were at and percent. in a field study on virus survival in a rapid-infiltration system for wastewater, hurst et al. [ ] found that virus inactivation rates were greater in more rapidly drying soils. allowing soils in rapid-infiltration systems to pe riodically dry and become aerated between wastewater applications en hances virus inactivation. the effects of both drying and aerobic microbial activity may contribute to virus inactivation under these conditions. in stud ies on the mechanisms of virus inactivation in soils, yeager and o'brien [ ] found that the loss of poliovirus infectivity in moist and dried soils resulted from irreversible damage to the virus particles, including ( ) dis sociation of viral genomes and capsids, and ( ) degradation of viral rna. in both moist and dried nonsterile soils, viral rna was released from cap sids and found in a degraded form. in dried, sterile soils, viral rna was released but remained largely as intact molecules. viral capsid components were not readily recoverable from drying soils due to irreversible binding, but they could be recovered as empty capsids from moist soils. further experiments with dried viruses showed that their capsids became isoelectrically altered. the results of these studies suggest that poliovirus and perhaps other viruses are inactivated by different mechanisms in moist and drying soils. temperature is a major factor in the survival of enteric organisms in soil and other environments. temperature affects chemical and biologic pro cesses in soils, which may indirectly affect the survival of enteric viruses and bacteria. s. typhosa may survive as long as months at freezing temper atures [ ] . mirzoev [ ] pointed out that in areas with prolonged winterse.g., the russian arctic-the processes of soil self-disinfection are slowed down or suspended. he showed that low temperatures (down to - ° c) were very favorable for the survival of dysentery bacilli, which he was able to detect days after it had been added to the soil. van donsel et al. [ ] found that a percent reduction in the number of fecal coliforms took . days in the summer and . days in the winter in exposed soil plots. bagdasar'yan [ ] observed that viruses could survive up to days in soil at to ° c and that survival was higher at to ° c than at to ° c. similar observations were made by lefler and kott [ ] with regard to poliovirus type and bacteriophage f survival in a sandy soil in israel. yeager and o'brien [ ] found that coxsackie virus bl inactivation rates in sandy loam soils suspended in riverwater, groundwater, and septic wastewater increased as temperatures were increased from to ° c. in pilotscale outdoor studies on poliovirus persistence on vegetables and in soils irrigated with sewage effluent in cincinnati, ohio, larkin et al. [ ] and tierney et al. [ ] found that percent inactivation in soils took about months during the winter months and only to days in the warm summer months of june and july. in a field study by hurst et al. [ ] on virus survival and movement in a rapid-infiltration system for wastewater, the rate of inactivation of indigenous viruses was greater in the fall than in the winter, possibly due in part to the effects of higher temperatures in the former season. the direct effects of ionic salts and ph on microbial survival in soils have been less extensively investigated than their effects on virus retention by soils. hurst et al. [ ] determined that virus inactivation in soils correlated with soil levels of resin-extractable phosphorous, exchangeable aluminum, and soil ph. because these same factors also influence virus adsorption to soils, the observed differences in survival rates may be related to changes in the extent of virus adsorption to the soil material and, therefore, changes in the extent of virus protection from inactivation in the adsorbed state. beard [ ] also found that the death of s. typhosa was very rapid in peat soil with a ph between and . kligler [ ] found that moist, slightly alkaline soils were the most favorable for the survival of s. typhosa. cuthbert et al. [ ] inoculated various peat (ph . - . ) and limestone (ph . - . ) soils held in the laboratory with e. coli and strep, faecalis. they found that both organisms could persist for several weeks in the limestone soils, but would die out in a few days in acid peat soils. they felt that the low ph could act to adversely affect not only the viability of the organism but also the availability of nutrients or to interfere with the action of inhibiting agents. the frequent addition of broth culture fluid to soil has been found to in crease the survival of s. typhosa [ ] . under field conditions, it has been found that some aftergrowth of e. coli and strep, faecalis can occur, partic ularly after wet weather [ ] . the survival of fecal coliforms is greatly ex tended in organic soils over that observed in mineral soils [ ] . the extended survival and growth in organic soils may be due not only to the presence of organics but to the high moisture-holding capacity of these soils [ ] . the effects of organic matter on enteric virus survival in soils have not been established, but recent findings suggest that fulvic and humic acids may mask virus infectivity by a reversible process. bixby and o'brien [ ] found that fulvic acid complexation of bacteriophage ms caused consid erable loss of infectivity and prevented adsorption to soil. the infectivity of the complexed phage could be restored by treating with percent beef extract solution at ph . soil moisture, temperature, ph, and the availability of organic matter can also indirectly influence the survival of enteric bacteria by regulating the growth of antagonistic organisms [ ] . bryanskaya [ ] showed that actinomyces in soil were capable of suppressing the growth of salmonella and dysentery bacilli. in addition, the longer survival time of enteric organisms after inoculation into sterilized soil as compared to unsterilized soil found by a number of workers [ ] indicates that antagonism is an important fac tor. tate [ ] observed that the protozoan population of a muck soil in creased dramatically after addition of e. coli and suggested that soil protozoa could play a significant role in the decline of these organisms in these soils. since it is evident that enteric bacteria are capable of utilizing nutrients found in nature, it could be argued that competition by the natural soil microflora is in large part responsible for their eventual disappearance from the soil. bagdasar'yan [ ] noted greater enterovirus inactivation in nonsterile than in sterile sandy and loamy soils, incubated at - and - ° c. in more recent studies by sobsey et al. [ ] on rates of poliovirus and reovirus inactivation in eight different soil suspensions in settled sewage at ° c, the time required for percent inactivation was almost always shorter in nonsterile than in sterile suspensions. hurst et al. [ ] observed increased inactivation of poliovirus and echovirus in nonsterile sandy soil wetted with distilled water and incubated under aerobic conditions at ° and ° c, compared to sterile control samples. however, inactivation rates in sterile and nonsterile samples were similar at ° c under aerobic conditions and at °, °, and ° c under anaerobic conditions. thus, appreciable virus in activation due to microbial activity in soils appears to occur only under aerobic conditions and at moderate to high temperatures. although the mechanisms of microbially mediated antiviral activity in soils have not been fully elucidated, yaeger and o'brien [ ] have reported differences in poliovirus structural changes during inactivation in sterile and nonsterile soils depending on soil moisture level. in both sterile and non sterile soils under moist conditions, viral rna was probably damaged be fore release from capsids. in sterile, dried soils released rna genomes remained largely intact, but in nonsterile, dried soils the released rna was degraded. the role of microbially produced nucleases in these findings is uncertain. data available indicate that viruses survive longer than bacteria in soil (ta ble . ) [ ] . field and laboratory studies using mcfeters's-type survival chambers indicate that enteric bacteria can survive from a few days to more [ ] . than a month [ ] [ ] . it is also possible that under some conditions they could regrow in groundwater if sufficient nutrients are present. e. coli bac teria have been found to survive and even multiply on organic matter filtered out from lake water during underground recharge projects in israel [ ] . in some areas of israel surface water during the rainy season is used to recharge the underground water supply. during those parts of the year when there is an increased need for water the same wells transformed to pumping wells. during such projects it was found that although the water pumped under ground contained less than coliforms per ml after chlorination, the repumped water contained counts as high as - per ml, which persisted for long periods of time after the initiation of pumping. subsequent studies showed that organic matter that had accumulated in the sand around the well casing enabled the regrowth of the few remaining coliforms. also of interest was the finding that so long as recharge continued, the bacteria did not multiply; it was only during the period between recharge and pump ing that growth occurred [ ] . enteroviruses have been detected at the surface of soils irrigated with sewage in the united states [ ] . a field study revealed virus survival for at least days in soil following application of a package treatment plant effluent in a cypress dome in gainesville, florida [ ] . other field studies confirmed the important role played by temperature and soil moisture in virus persistence in soils [ , ] . similarly, it appears that virus survival in sludge-amended soils is controlled primarily by desiccation and soil tem perature [ , ] . during surface application of digested sludge on soils in pensacola, florida, it was shown that indigenous enteroviruses were able to survive only days after sludge application [ ] . a simple conceptual model based on the current state of knowledge on indicator and pathogen dieoff has been described by reddy et al. [ ] . microbial dieoff was described by assuming first-order kinetics. first-order dieoff rate constants (k) were calculated from the literature for enteric mi crobial dieoff in soil-water systems. correction factors were presented to adjust constants for changes in temperature, moisture, and ph of the soil. average dieoff rate constants (log () /day _ ) for selected microorganisms are shown in table . . in the article by reddy et al. [ ] , data on dieoff of viruses during anaerobic digestion were used. only data on virus dieoff in soil systems is shown in table . . these values were obtained from various experiments and represent an average value of several soil and environ mental variables. such an approach could prove useful for estimating mi crobial survival in soil-water systems, but a greater database is needed especially for viruses and other pathogenic bacteria. also, most of our da tabase on microbial survival is in soil-water systems and not in groundwater. even though there have been no reports of disease outbreaks associated with land treatment of wastewater, there are a growing number of studies concerning the detection of viruses in groundwater after wastewater appli cation to land or direct groundwater recharge. these studies are summarized in table . . wellings et al. [ ] demonstrated vertical and lateral movement of virus in secondary effluent discharged into a cyprus dome (a wetland eco system). poliovirus , coxsackievirus b , and echoviruses , , and were recovered from m-deep wells in three of samples, at concentrations ranging from to pfu. viruses migrated to m laterally from the application point and survived at least days. the soil at this site ranged from a top . m layer of black organic soil ( - % clay) to a sandy clay and a solid blue clay with a permeability of x " cm per minute to x ~ cm per minute. thus, the viruses moved horizontally as well as vertically and survived many days under ambient conditions, indicating a necessity to evaluate such sites for their aquifer movement and transmission of viruses to drinking water sources. in an earlier study, wellings et al. [ ] recovered viruses from groundwater after spray irrigation of secondary sewage effluent onto an imolokee sand (little or no silt or clay). of particular interest in this study was that viruses survived chlorination, sunlight, spraying, and percolation through to m of sandy soil; furthermore, after a period of heavy rains, a burst of viruses was detected in samples that had previously been negative. these studies demonstrate that soil type, rainfall, and other factors can affect viral movement into groundwater, and that viruses are capable of surviving long periods-which, when combined with the ability to move long distances laterally, could lead to wide dispersal through an aquifer. vaughn and landry [ ] and vaughn et al. [ ] reported isolations of viruses from four groundwater recharge sites, from a stormwater recharge basin, and from groundwater under a sanitary landfill in new york. these sites have soils of coarse sand, fine gravel, and to percent silt. at the groundwater recharge sites, viruses were recovered at depths up to . m and at distances up to . m from the injection point of secondary or tertiary chlorinated effluent. as much as to percent of the -gal samples at the four sites were positive for viruses, with concentrations of . to . pfu per gallon. in addition, total coliforms were found in these samples. in order to reach the groundwater, viruses moved through . to m of the overlying soil. moreover, at the pines site, viruses were discovered in groundwater under basins where effluent seeded with viruses was applied at rates of to cm per hour. the slower infiltration rates were more effective in re moving the viruses, suggesting that site management is important. both the landfill and stormwater recharge basin also yielded viruses. at the landfill stie, viruses were detected at depths of . m and up to m downstream. coxsackievirus b and other unidentified viruses were de tected. at the stormwater recharge site, samples taken at -m depths directly in the basin were positive for echoviruses and and for coxsackievirus a . this contamination may have originated from runoff from cesspools in the area. schaub and sorber [ ] reported on a study of viruses in groundwater under rapid infiltration cells at ft. devens, massachusetts. the soil consisted of silty sand and gravel underlaid by bedrock. the groundwater contained viruses at depths of m and lateral distances of m, with concentrations of about . percent of the applied effluent. fecal streptococcal bacteria were also found in the . m-deep well. marker f virus was applied at this same site; only about percent of the virus was removed, and it was detectable for at least days. this site was deemed to have poor filtration properties, which points out the need for site-specific evaluation. at the vineland, new jersey, rapid-infiltration site [ ] primary ef fluent was applied to cohansey sand and coarse gravel. viruses were found at . m depths and up to m lateral distances in of samples. polio-, echo-, and coxsackie viruses were identified. total coliforms and fecal coliforms were found consistently at depths up to m beneath the recharge basins. total coliforms also occasionally occurred at greater depths and downstream. in contrast, no fecal coliforms were found in samples taken below . m and coliforms occurred only once in a shallow well m downgradient. thus, viruses penetrated deeper into the ground and moved longer distances than did the coliforms. the potential for viruses to migrate great distances, as in the previous study, was further demonstrated by noonan and mcnabb [ ] , who used the phages x and t to demonstrate lateral movements of m and m, respectively, in new zealand groundwater in just hours. the viruses moved at greater than m per day and survived for at least days. in laboratory studies, . days were necessary for a percent reduc tion in liter; so in this case, the viruses could theoretically travel at least . km in groundwater before a percent reduction could be effected under these conditions. viruses in groundwater at other recharge sites have been studied with varying success. at the flushing meadows site near phoenix, arizona [ ] , it was found that a fine loamy sand over coarse sand and gravel effectively removed viruses. laboratory studies confirmed that this soil was an excellent adsorber. no viruses were detected in any of the samples of renovated water, even though levels of to pfu per liter were detected in the effluent applied. however, coliform organisms were detected in the reno vated water, suggesting that the removal mechanisms must have been dif ferent for viruses and bacteria, and that viruses may have been present. since this site is no longer in existence, these findings cannot be confirmed. however, since then, virus has been detected in a sample from an . mdeep well at a nearby land application site. the isolate was identified as coxsackievirus b . at two land treatment sites where sewage is used to irrigate cropland, both positive and negative virus isolations have been made [ , ] . at the lubbock, texas, site, coxsackievirus b was isolated from a depth of . m; at roswell, new mexico, no virus isolates were detected in samples taken from to m depths. in the latter case, irrigation is seasonal and inter mittent, whereas application at the lubbock site is continuous. at an operational land application site in kerrville, texas [ ] , no viruses were detected in the monitoring wells at depths of . to . m even though viruses could be detected in . m-deep lysimeters. in one often-cited report [ ] on the santee project, no viruses were detected in renovated water. this is not surprising, since the detection meth ods available at that time were not quantitative. these negative results must therefore be considered highly questionable, as should the results obtained at the whittier narrows, california [ ] , projects, which did not employ techniques sensitive enough to detect low levels of virus. this situation reiterates the need for careful evaluation of methods used in any report before negative conclusions are accepted. summaries of data on the soil penetration of bacteria at some of the most important rapid-infiltration systems land treatment sites are presented in table . . the data suggest that bacteria at rapid-infiltration sites may pen etrate about m vertically and variable distances laterally. these distances are, of course, highly site-specific, and the vertical distance may be more than m but is usually much less. to prevent the entry of enteric bacteria into groundwater, it would thus be advisable (unless an underdrain system is installed) not to site land treatment systems where the water table is shallow, particularly if the soil is sandy or gravelly, large cracks or root tunnels are present, or a thin soil mantle overlies rock with solution channels or fissures. this is especially true for rapid-infiltration systems. once in the groundwater, the bacteria may travel long distances in situations where coarse soils or solution channels are present, but normally the filtering action of the matrix should restrict horizontal travel to only a few hundred feet. the actual distance travelled also depends on the rate of movement of the groundwater and the survival time of the bacteria. the rate of movement of groundwater is highly site-specific but often is ex tremely slow. from the foregoing discussion, it is apparent that many factors control the removal of pathogenic bacteria and viruses during the percolation of sewage through the soil. most of this chapter has dealt with the fate of viruses in soil because of their apparent greater potential for health problems associ ated with land treatment. although the presence of viruses in groundwater has been demonstrated, it would appear that with proper site selection and management the presence of viruses could be minimized or eliminated. the key is to define the processes involved in the survival and transport of pathogens in groundwater. with proper design, land treatment could be used as an effective method for reducing the number of pathogens in wastewater. with the proper soil type, viruses and bacteria can be reduced to levels as effectively as by chlorination as currently practiced, after the travel of wastewater through only a few centimeters of soil. as we have shown, high removals by soil can be achieved from even raw wastewater. in the soil natural processes will eventually destroy the pathogens. thus, in groundwater recharge operations, the soil should be considered as part of the treatment process and not simply as a final disposal source. the key to operating such systems for pathogen removal is to gain an understanding of the processes involved and methods by which they can be quantified and controlled. based on both field and laboratory experiments, several potential treatment practices may be useful in enhancing virus removal during land application of sewage, and these are summarized in table . . [ ] . waterborne disease-a status report emphasizing outbreaks in groundwater review of the causes of waterborne disease outbreaks out breaks of waterborne disease in the united states viruses in soil systems magnitude of the groundwater contamination problem wastewater bacteria and viruses in soil virus movement in soil col umns flooded with secondary sewage effluent adsorp tion of enterovirus to soil cores and their subsequent elution by artificial rainwater demon stration of virus in groundwater after effluent discharge into soil viruses in groundwater waterborne disease: occurrence is on the upswing bacterial contamination of drinking water supplies in a mod ern rural neighborhood a review of infective dose data for enteroviruses and other enteric microorganisms in human subjects public health implications of the appli cation of wastewaters to land gastroenteritis association with a sewage leak health effects of land treatment; microbiological viruses in water: the problem, some solutions virus survival in wastewater treatment viruses in sewage: effect of phosphate removal with calcium hydroxide (lime) appropriate technology for water supply and sanitation: health aspects of excreta and sillage management-a state of the art review behavior and transport of microbial pathogens and indicator organisms in soils treated with organic wastes well-water quality deteri oration in central pierce county, washington the relationship between rainfall and well water pollution in a west african (gambian) village the pollution hazard to village water supplies in eastern botswana the movement of disease producing organisms through soils high-rate land treatment. ii. water quality and economic aspects of the flushing meadows project fundamentals of soil science virus movement in groundwater adsorption of viruses onto surfaces in soil and water migration of poliovirus type in percolating water through selected oahu soils comparative adsorption of human enteroviruses, simian rotavirus, and selected bacteriophages to soils virus adsorption by five soils interactions and survival of enteric viruses in soil materials comparative movement of dif ferent enteroviruses in soil poliovirus adsorption by minerals and soils influence of ph and electro lyte composition on adsorption of poliovirus by soils and minerals quantitative assess ment of the adsorptive behavior of viruses to soils poliovirus survival and move ment in a sandy forest soil effect of dissolved salts on the filtration of coliform bacteria in sand dunes virus survival in water and wastewater systems poliovirus retention in -cm soil cores after sewage and rainwater application nitrates and bacterial distribution in rural do mestic water supplies bacterial contamina tion of drinking water supplies in a modern rural neighborhood hohe nitratgehalte in einem landlichen gebiet in nigeria verursacht durch ungeordnete ablagerung hauslicher alofalle and exkremente poliovirus removal from primary and sec ondary sewage effluent by soil filtration effect of secondary treated effluent on the movement of viruses through a cypress dome soil transport of viruses through organic soils and sediments influence of fulvic acid on bacteriophage adsorption and complexation in soil virus re moval during groundwater recharge: effects of infiltration rate on adsorp tion of poliovirus to soil poliovirus movement during high rate land filtration of sewage water effect of soil permeability on virus removal through soil columns enterovirus inactivation in soil type and strain dependence of enterovirus adsorption to activated sludge, soils, and estuarine sediments survival of enteroviruses in rapid-infiltration basins during the land application of wastewater the use of sewage for irrigation: a literature review literature review on the occurrence and survival of enteric, pathogenic and relative organisms in soil, water, sewage and sludges, and on vegetation recycling treated municipal wastewater and sludge through forest and cropland observations on the viability of the bact. coli group under natural and artificial conditions longevity of eberthella tyhosus in various soils university of wiscon sin-extension geological and natural history survey survival of viruses of the enterovirus group (poliomy elitis, echo, coxsackie) in soil and on vegetables infectious disease potential of land application of wastewater effects of environmental variables and soil characteristics on virus survival in soil extent of survival of dysentery bacilli at low temperatures and self-disinfection of soil and water in the far north seasonal variations in survival of indicator bacteria in soil and their contribution to storm-water pollution persistence of virus on sewage-irrigated vegetables persistence of poliovirus in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent investigations of soil pollution and the relation of the various types of privies to the spread of intestinal infections survival of bacterium coli type and streptococcus faecalis in soil cultural and environmental factors affecting the longevity of escherichia coli in histosols antagonistic effect of actinomyces on pathogenic bac teria in soil virus survival in receiving water comparative survival of indicator bacteria and enteric pathogens in well water survival of enteric viruses and indicator bacteria in groundwater water quality aspects of groundwater recharge in israel clogging and contamination processes in re charge wells viruses in groundwater be neath sewage irrigated cropland high levels of microbial contamination of vegetables irrigated with wastewater by the drip method fate of vi ruses following land application of sewage sludge. i. survival and transport patterns in core studies under natural condition survival of enteroviruses and coliform bacteria in a sludge lagoon virus survival following wastewater spray irrigation of sandy soils an assessment of the occurrence of hu man viruses in long island aquatic systems survey of human virus occurrence in wastewater-recharged groundwater on long island virus and bacteria removal from wastewater by rapid infiltration through soil long-term effects of land application of domestic wastewater the quality and movement of groundwater in alluvial aquifers of virus and bacteria removal from wastewater by land treatment viral transport to groundwater at a wastewater land application site virologic assess ment of sewage treatment at water reclamation at whittier nar rows groundwater pollution microbiology virus removal following wastewater spray irrigation of sandy soils long-term recharge of trickling filter effluent into sand dan region project, groundwater recharge with municipal effluent virus removal with land filtration key: cord- - hlatbqe authors: bosch, albert; guix, susana; sano, daisuke; pintó, rosa m title: new tools for the study and direct surveillance of viral pathogens in water date: - - journal: curr opin biotechnol doi: . /j.copbio. . . sha: doc_id: cord_uid: hlatbqe half a century ago scientists attempted the detection of poliovirus in water. since then other enteric viruses responsible for gastroenteritis and hepatitis have replaced enteroviruses as the main target for detection. however, most viral outbreaks are restricted to norovirus and hepatitis a virus, making them the main targets in water. the inclusion of virus analysis in regulatory standards for viruses in water samples must overcome several shortcomings such as the technical difficulties and high costs of virus monitoring, the lack of harmonised and standardised assays and the challenge posed by the ever-changing nature of viruses. however, new tools are nowadays available for the study and direct surveillance of viral pathogens in water that may contribute to fulfil these requirements. as stated by one of the seminal names in water virology, wok grabow, virological analysis of water is required for a number of purposes that include research on the incidence and behaviour of viruses in the water environments, assessment of the presence of viruses and the risk of infection, as well as evaluation of the efficiency of treatment and disinfection processes and routine quality monitoring to test the compliance of water quality with guidelines and specifications [ ] and in essence the public health impact of waterborne viral infections. additionally, identification of strains isolated from the water environment may also be used as a tool for the study of the epidemiology of waterborne viruses providing an overview of all the viruses circulating in the community, including viruses causing both symptomatic and asymptomatic infections [ , , ] . however, the tasks required to fulfil the aforementioned goals are easier said than done. main difficulties to overcome for virus detection and characterisation in water samples encompass viral diversity, occurrence of low particle numbers, particularly in drinking water, and the technical challenges of virus assays. a wide variety of different viruses may be found in human sewage ( table ) . some of these viruses are shed in extremely high numbers, that is, patients suffering from diarrhoea or hepatitis may excrete up to and virus particles, respectively, per gram of stool [ , , ] . since current water treatments do not ensure their complete removal they become contaminants of the water environment in numbers high enough to represent a public health threat, although low enough to pose serious difficulties for their detection. figure illustrates the possible routes of waterborne transmission of enteric viruses. enteric viruses can be transmitted by a variety of routes including person-to-person contact, zoonotic and/or vehicle transmission. poor water quality continues to pose a major threat to human health. billions of cases of gastrointestinal illness occur annually worldwide. the world health organisation (who) declared that diarrhoeal disease alone contributes to an estimated . % of the total daly (disability adjusted life years) global burden of disease and is responsible for the deaths of . million people every year [ ] . it was figured that % of that burden is attributable to unsafe water supply, sanitation and hygiene and it is mostly concentrated on children in developing countries. a significant amount of disease could be prevented especially in developing countries through better access to safe water supply, adequate sanitation facilities and better hygiene practices. viruses are a major cause of water-related disease. it is now well recognised that the most common viral gastrointestinal illness are rotavirus and norovirus diarrhoea in the infantile and adult population, respectively. however, most well documented waterborne outbreaks of viral gastroenteritis are related to noroviruses [ , ] . other gastro enteric viruses, such as rotaviruses [ ] and astroviruses [ ] have also occasionally been implicated in waterborne outbreaks. another major waterborne disease is hepatitis that can be a serious debilitating disease progressing from a non-specific illness with fever, headache, nausea and malaise to vomiting, diarrhoea, abdominal pain and jaundice. hepatitis a represents worldwide around % of the total hepatitis cases and although is self-limiting and rarely causing death may incapacitate patients for several months. the causative agent is the hepatitis a virus that has been linked to several waterborne outbreaks [ ] . hepatitis e, although less frequent than hepatitis a, has a higher mortality rate, particularly in pregnant women. it is the most important or the second most important cause of acute clinical hepatitis in adults throughout asia, the middle east and africa. by contrast, hepatitis e is rare in industrialised countries, but antibody (anti-hev) is found worldwide. hepatitis e is principally the result of a waterborne infection in developing countries and is thought to be spread zoonotically (principally from swine) in industrialised countries [ ] . the significance to human health of many of the nonhuman animal viruses present in environmental samples is less well understood although it is remarkable that zoonotic viruses infecting humans continue to be discov-ered or appear to re-emerge as important human pathogens. one example of an emerging disease is the severe acute respiratory syndrome or sars, reported in november . although several coronaviruses are known to be spread by the faecal-oral route, there is no current evidence that this mode of transmission plays a key role in the transmission of sars in spite of the considerable shedding of the virus in stools [ ] . another important zoonotical issue is the case of influenza viruses. despite human influenza viruses replicate primarily in the respiratory tract, avian influenza viruses, such as the highly pathogenic h n , cause generalised infection in birds with replication in the gastrointestinal duct and virus shedding in faeces. however, their potential waterborne transmission remains controversial [ , ] . water-related diseases are associated with exposure to water environments in many ways. these include not only waters used for drinking and recreation purposes but also those used for agricultural purposes such as crop irrigation, and food processing, eventually resulting in foodborne outbreaks [ , ] . additionally, shellfish grown and harvested in polluted waters is a well-docu- environmental biotechnology table human viruses documented to be found in the water environment [ , ] . while drinking water may not be considered a major public health problem in developed communities, prevention of water-related virus contamination of food remains a perennial challenge both in developing and developed societies owing to its global trade. one of the challenges to overcome in the virological analysis of water is the need to recover the low number of viruses from large volumes of sample. this is particularly important when molecular micro-methods are applied. methods for virus concentration from water samples are depicted in table and reviewed elsewhere [ ] . a good concentration method should fulfil several requirements: it should be technically simple, fast, provide high virus recoveries, be adequate for a wide range of enteric viruses, provide a small volume of concentrate, and be inexpensive. no single method meets all these requests. criteria based on the experience and expertise of the user on a given method should be employed to select the most appropriate system. positively charged filters [ ] and glass wool [ ] based methods are still among the best possibilities. sampling large volumes requires a two-step concentration procedure, with polyethylene glycol precipitation [ ] and ultrafiltration [ ] virus detection in water bosch et al. possible routes of waterborne transmission of enteric viruses. viruses are shed in extremely high numbers in the faeces and vomit of infected individuals. pathogenic viruses are routinely introduced into the environment through the discharge of treated and untreated wastes, since current treatment practices are unable to provide virus-free wastewater effluents. in consequence viral pathogens contaminate the marine environment (a), fresh water (b) and ground water (c). mankind is exposed to enteric viruses through various routes: shellfish grown in polluted waters, contaminated drinking water and food crops grown in land irrigated with sewage contaminated water and/or fertilised with sewage. surface and ground waters are employed for public consumption (e) and have been implicated in waterborne outbreaks of gastroenteritis and hepatitis. foods susceptible to be contaminated at the pre-harvest stage such as bivalve molluscs (d), particularly oysters, clams and mussels; salad crops (f), as lettuce, green onions and other greens; and soft fruits (g), such as raspberries and strawberries have also been implicated in outbreaks of viral diseases. as preferred procedures for reconcentration of the primary eluates. additionally, peg [ ] as well as lyophilisation [ ] may be used for direct virus concentration in heavily polluted medium size samples, for example, sewage, having this latter method the added advantage of removing substances inhibitory to rt-pcr enzymes if this method is employed for virus detection [ ] . when virus detection procedures are mentioned the recurrent issue of detecting infectious or physical particles comes into discussion. whenever possible, infectious assays coupled with identification methods are preferred for direct assessment of human health risk. the detection of infectious enteroviruses and even astroviruses or rotaviruses may be achieved by cell culture techniques with the appropriate cell line. despite recent reports of cell lines allowing the growth of wild-type hepatitis a virus [ ] and norovirus [ ] , issues related to assay complexity, cost-effectiveness and validity for the detection of a broad spectrum of isolates make their use a difficult and unrealistic approach for hepatitis a virus and norovirus detection, respectively. nucleic acid amplification techniques are currently the most widely used methods for detection of viruses in environmental biotechnology water, which also enable to gather information of the virus genotypes occurring in the environment, thus providing most relevant epidemiological information, particularly important for the implementation and follow-up of vaccination programmes [ , , ] . although nucleic acid sequence-based amplification (nasba) and loop-mediated isothermal amplification (lamp) techniques have been reported as highly sensitive and specific, respectively [ , ] , pcr and rt-pcr remain as the current gold standard for virus detection. a further improvement comes from real-time rt-pcr, which enables not only qualitative determination but also, and particularly, quantitative diagnostic assays [ , - ] . however, the significance of a genome copy remains controversial since the virus-specific infectivity or infectious/physical particle ratio is highly variable in environmental samples. several studies show uncoupling between the number of genome copies and infectivity in environmental studies [ ] [ ] [ ] . nevertheless, as stated above, no alternative to molecular detection analysis exists for highly health significant waterborne viruses such as human norovirus and hepatitis a virus. most health-significant waterborne viruses have rna genomes (table ) , and a major challenge in the development of molecular techniques for the diagnostic of rna viruses derives from the facts that they depend on error-prone polymerases that generate high mutation rates and the occurrence of recombination events that altogether lead to complex mutant genome populations or quasispecies [ , - , ] . it is needless to say that this genome variability implies a careful selection for highly conserved sequences targeting primers and probes, particularly when quantification is the objective. rna regions containing complex multidomain structures involved in essential functions such as translation or replication are highly conserved and therefore are good candidates for this purpose. an accurate quantification of genome copies demands the control of crucial steps such as the efficiency of the virus/ nucleic acid extraction procedures and of the enzymes involved in the molecular amplification, particularly reverse transcriptase. while for the quantification of rna viruses in clinical samples the use of an internal control based on the detection of the expression of a housekeeping gene is a clear first choice, this is obviously not valid for water samples leading to compromise in the use of an external control. the best candidate for this latter purpose is the use of an encapsidated rna, such as rna animal viruses, rna bacteriophages or armoured rnas that are pseudoparticles made of target rnas packaged into ms coliphage coat protein [ , ] . however, it is best to avoid the use of viruses that might be present as contaminants in the assayed samples, thus ruling out the use of coliphages or human enteric viruses. the control of a rt-pcr reaction should rely on a ssrna molecule as similar as possible to the viral target and amplifiable with the same pair of primers under exactly the same conditions and with the same efficiency [ , , ] . raw values of genome copies must then be adjusted accordingly to the figured efficiencies for better accuracy of the virus titre. addressing these quality control and quality assurance (qc/qa) issues and harmonising the molecular techniques are required before virus analysis could be included in water quality standards. the inclusion of the control measures above described calls for the development of multiplex approaches with the aim to analyse several pathogens in a single assay without increasing the economic cost. multiplex formats may be based on real-time amplification or pcr-microarray systems [ , ] . a logical step forward is the development of chip/biosensors capable to be used in laboratory as well as in field settings [ ] . viruses will undoubtedly remain a major health threat, and the key scientific issues will have to be addressed to detect and characterise rapidly evolving old and new viral pathogens. one example is the potential emergence of diverse polioviruses from c-cluster coxsackie a viruses [ ] and its implication in a poliovirus-free world with a poliovirus-antibody naïve population. such a situation could be a fertile ground for a poliovirus-like agent to emerge by mutation. environmental virologists should be ready to handle this kind of threat through the rapid identification of the new pathogens by the use, among other tools, of whole-genome amplification and highthroughput sequencing techniques [ , ] . the availability of methods for accurate quantitative virus detection enables a sensible prospective water safety approach based on the identification and prevention of hazards that could cause waterborne illnesses. this is the basis of the hazard analysis and critical control point (haccp) principles that must be applied to ensure the virological safety of the water environment. the actual burden of waterborne viral infections is still hard to figure owing to technical limitations in pathogen detection, scarce data on environmental epidemiology, difficulties in determining the source of infection and occurrence of unapparent infections, among others. these shortcomings may be already hard to overcome for well-known pathogens and awesome for newly emerging agents. the overall picture appears even more complicated owing to issues related with susceptibility to infection of malnourished or immunocompromised hosts and the influence of geographical, socioeconomic and seasonal factors in a global warming situation. overview of health-related water virology a good interdisciplinary synopsis on environmental virology from an expert in the field hepatitis a virus in urban sewage from two mediterranean countries group a rotavirus in sewage samples from barcelona and cairo: emergence of unusual genotypes enteroviruses with special reference to poliovirus and poliomyelitis eradication a review on the significance of the occurrence of enteroviruses in the water environment and its relevance in the present efforts to eradicate poliomyelitis norovirus infections in symptomatic and asymptomatic food handlers in japan persistent gastroenteritis in children infected with astrovirus: association with serotype- strains development, evaluation, and standardization of a real-time taqman reverse transcription-pcr assay for quantification of hepatitis a virus in clinical and shellfish samples an exhaustive analysis for the design and development of standardised molecular assays for virus quantification world health organization: guidelines for drinking-water quality. edn . recommendations. geneva, switzerland: world health organization outbreak of viral gastroenteritis due to drinking water contaminated by norwalk-like viruses gastroenteritis outbreak caused by waterborne norovirus at a new zealand ski resort a large infantile gastroenteritis outbreak in albania caused by multiple emerging rotavirus genotypes are waterborne astrovirus implicated in acute digestive morbidity (e.mi.r.a. study)? enteric hepatitis viruses an updated review on waterborne hepatitis from the molecular biology to the transmission routes hepatitis e: an emerging awareness of an old disease an overview of hepatitis e, an emerging zoonotical agent of concern viral load distribution in sars outbreak evidence of influenza a virus rna in siberian lake ice this and the following article should be considered together and reflect contradictory views on the role of water as an abiotic reservoir for influenza a viruses. a complementary reading on the threat of waterborne transmission of avian influenza virus may be phylogenetic evidence against evolutionary stasis and natural abiotic reservoirs of influenza a virus an outbreak of hepatitis a associated with green onions norovirus and foodborne disease detection of multiple noroviruses associated with an international gastroenteritis outbreak linked to oyster consumption molecular characterization of hepatitis a virus isolates from a transcontinental shellfish-borne outbreak the detection of waterborne viruses an updated review on virus concentration and detection in water samples poliovirus concentration from tap water with electropositive adsorbent filters glass wool for virus concentration at ambient water ph level polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis a virus and human rotavirus, from oyster, water, and sediment samples real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay genotyping of rotaviruses isolated from sewage stable growth of wild-type hepatitis a virus in cell culture vitro cell culture infectivity assay for human noroviruses. emerg infect dis this article throws some light on the search of a cell system to propagate human norovirus that so far has been unfeasible multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr quantitative real-time pcr assays for detection of human adenoviruses and identification of serotypes and a broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection of hepatitis e virus real-time pcr quantification of human adenoviruses in urban rivers indicates genome prevalence but low infectivity detection of murine norovirus by using plaque assay, transfection assay, and real-time reverse transcription-pcr before and after heat exposure removal of astrovirus from water and sewage treatment plants, evaluated by a competitive reverse transcription-pcr viral evolution and its relevance for food-borne virus epidemiology a good review on the variability of human enteric viruses and its impact on the development of molecular detection assays genomic variations in echovirus persistent isolates recovered from a chronically infected immunodeficient child and comparison with the reference strain evidence for quasispecies distributions in the human hepatitis a virus genome evidence for hepatitis e virus quasispecies high throughput sequence analysis reveals hitherto unreported recombination in the genus norovirus armored rna as virus surrogate in a real-time reverse transcriptase pcr assay proficiency panel molecular methods of virus detection in foods a good overview of the existing molecular assays for enteric virus detection development of homologous viral internal controls for use in rt-pcr assays of waterborne enteric viruses multiplex-pcr and oligonucleotide microarray for detection of eight different herpesviruses from clinical specimens combining multiplex reverse transcription-pcr and a diagnostic microarray to detect and differentiate enterovirus and coxsackievirus a porous silicon biosensor for detection of viruses evidence for emergence of diverse polioviruses from ccluster coxsackie a viruses and implications for global poliovirus eradication because of the lack of a zoonotic reservoir, will not re-emerge once eradicated. this report examines the origin of poliovirus from a nonneurovirulent coxsackie a virus ancestor. in a poliovirus-free world without poliovirus neutralising antibodies virus discovery by sequenceindependent genome amplification this article provides another evidence on how new high-tech tools contribute to detect and characterise rapidly evolving emerging viruses. virus detection in water bosch et the authors wish to acknowledge the support from grants food- b- - from the european union, bio - from the ministry of education and science, spain, sgr from the generalitat de catalunya and the xarxa de referència en biotecnologia de catalunya (xerba), generalitat de catalunya. key: cord- -ncjdsbn authors: nazeeri, a. i.; hilburn, i. a.; wu, d.-a.; mohammed, k. a.; badal, d. y.; chan, m. h. w.; kirschvink, j. l. title: an efficient ethanol-vacuum method for the decontamination and restoration of polypropylene microfiber medical masks & respirators date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ncjdsbn a critical shortage of respirators, masks and other personal protective equipment (ppe) exists across all sectors of society afflicted by the covid- pandemic, placing medical staff and service workers at heightened risk and hampering efforts to reduce transmission rates. of particular need are the n medical face respirators that filter % of all airborne particles at and above . um in diameter, many of which use meltblown microfibers of charged polypropylene (e.g, the m ). an intensive search is underway to find reliable methods to lengthen the useful life of these normally disposable units. it is currently believed that these masks and respirators cannot be cleaned with to % alcohol-water solutions, as past wet/dry experiments show that filtration efficiency can drop by ~ % after the first such treatment. this has been interpreted as the liquids disrupting the surface charge on the fibers and has led to a recent cdc/niosh advisory against using alcohol for their decontamination. we have replicated the drop in efficiency after alcohol treatment. however, we find that the efficiency can be recovered by more effective drying, which we achieve with a vacuum chamber. drying at pressures of < ~ mbar ( . kpa) restores the measured filtering efficiency to within % or so of the pre-washing value, which we have sustained for cleaning-drying cycles so far in three models of n masks. the mechanism seems to be the removal of water molecules adsorbed on the fiber surfaces, a hypothesis which is supported by two independent observations: (a) the filtering efficiency increases non-linearly with the weight loss during drying, and (b) filtration efficiency shows an abrupt recovery as the vacuum pressure drops from to mbar, the range physically attributable to the removal of adsorbed water. these results are not compatible with the electrostatic discharge hypothesis, and rather suggest that water molecules adsorbed to the fiber surface are reducing the filtration efficiency via surface tension interactions (e.g., wicking between the fibers and coating their surfaces with a film). such a degradation mechanism has two implications: (a) respirators decontaminated by a soak in % v/v ethanol regain their filtration efficiency once they are fully dry. we employ vacuum chambers in this study, which are inexpensive and commonly available. (b) this mechanism presents the possibility that mask filtration performance may be subject to degradation by other sources of moisture, and that the mask would continue to be compromised even if it appears dry. the mask would need to be vacuum-dried to restore its performance. this study introduces a number of methods which could be developed and validated for use in resource-limited settings. as the pandemic spreads to rural areas and developing nations, these would allow for local efforts to decontaminate, restore, monitor, and test medical masks. the covid- pandemic has created a shortage of masks in hospitals and communities. inexpensive ways of locally disinfecting and testing masks are of great importance, especially as the pandemic spreads to rural areas and developing nations. we have discovered a method to clean hospital-grade face masks by first soaking masks in an ethanol solution, air-drying them, then vacuum-drying them. our study has found that this cleaning process can be used on a mask at least five times without altering the filter rate of the mask by more than one per cent. vacuum-drying the masks removes a thin layer of water that still sticks to the fibers in the mask after air-drying. this layer of water is probably what causes the mask efficiency to drop (that is, how well it filters particles from the air). there is a danger that if the mask gets wet in other ways that it will not filter the air as well. this could also be fixed with the vacuum-drying procedure. our study may lead to inexpensive designs that would allow hospitals to build their own face-mask testing rigs to measure the filtration efficiency of equipment that they have purchased online from unverified sources as well as those being reused. we built a simple mask testing rig from items that are cheap and easy to obtain, though the current design relies on a calibrated $ , laser particle meter. we are working to adapt cheaper laser particle meters into the design. medical respirators and masks are in critically short supply across the globe, in particular the n variety that removes % of particles at and greater than . μm in diameter. most of these masks produced in the us today consist of meltblown microfibers of polypropylene, which are designed to be disposable . during use in clinical settings, the filters may become contaminated with active viral or bacterial particles, necessitating that any efforts to refurbish them should employ decontamination techniques in addition to cleaning. such techniques could ease the supply demand and immediately aid medical personnel treating covid- patients, many of whom currently have no choice but to reuse dirty devices that have unknown filtering efficiency. there is a need for simple methods for using equipment commonly available in most hospital and clinical environments to enable safe reuse of masks. if found, such methods would rapidly ease this critical shortage even in resource-constrained settings. one of the simplest methods for decontamination is to rinse or soak materials in - % ethanol, which is a potent virucidal agent inactivating all of the lipophilic viruses . unfortunately, previous results , found that the performance of n respirators degrades by ~ % to % after a single immersion in a solution of - % alcohol when followed by drying in air. tsai postulated that the mechanism for this deactivation might be the penetration of the alcohol into the polypropylene microfibers, permanently disrupting the electrical charges on the surfaces which trap aerosols. these studies have prompted a warning from the cdc/niosh to avoid using alcohol for their decontamination , . we report here the discovery that decontaminating polypropylene microfiber filters in a % v/v solution of ethanol and deionized (di) water, using standard medical procedures including air drying, does not permanently damage the microfibers as has been suggested , , . instead, this washing procedure appears to leave a film of water molecules adsorbed hygroscopically on the surface that reduces the particulate absorption efficiency; approximately - g of water remains firmly on a typical respirator even after extensive air drying due to the large surface area of the fibers. we found that this layer of adsorbed water can be removed by further drying in a partial vacuum to below mbar ( . kpa), after which we observe the filtration efficiency returning to within % of the original values. as shown in table , we have repeated this on three different types of n respirators for five cycles and see no long-term degradation at this time. we describe the easily constructed experimental setup that we use for measuring filtration efficiency, present results with % v/v ethanol treatment and the effectiveness of vacuum drying on . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint mask efficiency, and discuss our proposed adsorption mechanism of action and two experimental tests of it. in order to measure the efficiency of various medical masks and respirators, it is necessary to monitor the drop in particulate concentration for air that is passing through the filters under conditions that mimic those under natural use. commercial units for doing this that follow formal ashrae (american society of heating, refrigerating and air-conditioning engineers) aerosol standards cost upwards of $ k, with delivery lead times longer than that expected for the covid- pandemic. however, our present analytical needs are more limited as we simply need to measure the filtration efficiency, not achieve formal certification. prior to the pandemic we routinely monitored air quality in a magnetically-shielded, biomagnetic clean lab in the . , . , and μm size bins using a metone tm aerocet s laser particle counter, which is nist-referenced and meets or exceeds ce and iso certifications. these units sample air at ambient pressure with a small pump, running it through the laser detectors and counting particles in the four size bins. in order to use this to sample the air passing through a filter, we constructed a testing facility as shown in fig. . we wrapped a styrofoam bust of a human head (previously used as an eeg blank) in three layers of ordinary kitchen plastic wrap to give surface friction and pliancy, drilled a vent hole "mouth", and placed the n respirators over the opening. this setup was housed inside a plexiglass box, which was then pressurized with 'dirty' pasadena air (the pressurized box reached ~ , , particles > . μm/cf) using an industrial heat gun dryer (a gilson tm ma- f) in non-heat mode, mounted to the side. air passed through the filters and then exited through a pvc pipe, which was sampled by the particle counter at ambient pressure. the air then flowed through a m length of plastic hose to minimize backflow of outside air into the counter from turbulence. the flow rate through the mask was adjusted using the intake manifold on the heat gun to ~ l/min. we found that minor deviations in flow rate had no noticeable effect on the efficiency measure. every two minutes the particle counter inlet was switched between the mask port (post-filtration) and a box port (unfiltered background air in the box), and the ratios of the total particle counts were used to calculate the filtration efficiency. the data were transmitted via usb serial link and stored to file on a local computer. this computer system both collected data from the particle counter and controlled the device via custom-built software in c#, adapted from software developed for monitoring air quality in the aforementioned clean lab . the % v/v ethanol solution was prepared with proof laboratory grade ethanol and deionized (di) water, which is within the cdc guidelines for disinfection . approximately ml of ethanol solution was poured over each mask, such that every part of the mask was saturated. excess liquid was blotted off with a paper towel, and the masks were allowed to air dry for - hours before the vacuum experiments were conducted. the error in the filtration efficiency was split into two different types: error introduced by variations in the fit of the mask and error introduced by the particle counter set up. error from the fit was addressed by refitting the mask - times and calculating the standard deviation from the mean of the measurements. error from the particle counter setup was found by taking - measurements in steady state per mask fitting and calculating the standard deviation from the mean of the measurements. the total error in the filtration efficiency was found through quadrature. the error in the masks' masses in fig. was found through the difference in mask mass before and after the efficiency measurement (this difference is due to some of the adsorbed water being removed by airflow during testing). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint results: table lists the medical masks and respirators that we have examined so far. new n respirators from m tm typically gave filtration readings above % (consistent with their n rating from niosh), and other mask types with lower ratings gave similarly consistent results. although we did not use standard ashrae aerosol procedures, data from the experimental setup, shown in fig. , are in agreement with those procedures and verify the utility of our experimental setup. similarly, the n respirators dropped in efficiency by - % following alcohol cleaning; this is consistent with previously reported results , . table shows the results from five cycles of alcohol decontamination / drying that were conducted on three varieties of m tm n respirators ( m tm , and masks). after these cycles, all masks remained at or above % filtration efficiency. as of this writing have conducted wet/dry/vacuum cycles on different masks, all of which drop in performance after wetting, but return to within % of their initial filtration efficiency after vacuum treatment to < mbar. data for the effect of mass loss on filtration efficiency for two of the m tm n respirators are shown on fig. . the large error in the mass measurements during the main drying interval was due to continued evaporation during the time the masks were on the testing rig with air being forced through them, as shown in fig. . in both cases, the filtration efficiency approached its initial value as the mask weight approached its initial values, indicating that liquid adsorbed in the washing process reduced filtration efficiency. direct measurements of filtration efficiency, as a function of vapor pressure, for these same masks are shown in fig. . in both cases, the major change in filtration efficiency occurred as the pressure dropped between and mbar with the efficiency of both masks returning to close to the initial measurements. this range is compatible with the removal of bound water molecules as considered in the discussion section below. an additional experiment on one m tm respirator soaked in pure di water showed a similar drop in efficiency followed by full recovery to . ± . % after vacuum drying, supporting the bound water hypothesis. two generic masks of unknown composition and filter efficiency, one of which was labelled as "n style" on amazon, were not measured as having filter efficiency comparable to niosh rated n masks. this underscores the need for more distributed mask testing capacity as an increasing number of respirators and masks with untested characteristics are brought into medical and professional use. proposed mechanism of action: previous studies have assumed that the observed loss of filtering efficiency is due to the neutralization of surface charges on the polypropylene microfibers , , . our data point to a mechanism based on surface wetting, which is reversible by drying. it is well known that in order to remove water molecules that are trapped or adsorbed on solid surfaces the pressure of the vacuum chamber must be reduced to a value that is below the saturation vapor pressure of water; lower pressures will thin the adsorbed water layer. we note that the saturated vapor pressure of ethanol and water at ˚c are and mbar respectively. when water molecules are adsorbed to surfaces in vacuum chambers, pressures as low as - mbar are needed to remove them . hence, our data suggest that water bound to the surface of the fibers is responsible for the loss in filtering efficiency, as evidenced by our observation of the same effect in a respirator soaked in di water. this surface wetting mechanism for performance degradation is consistent with the nature of the mask materials. the active material of the m tm n masks is melt-blown, fibrous polypropylene modified by corona discharge to contain embedded charges , . sem indicates that the diameters of the fibrous strands are on the order of ~ μm , giving the filter a huge specific surface area. when the fibers are cleaned and rinsed with the ethanol solution, thin liquid films are introduced onto the material. the surface tension of the liquid film causes the fibers to wick together and form bundles, thereby opening gaps. in addition, the clumping of individual fibers significantly reduces the surface area available for . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint capturing the particles. assuming the diameter of the polypropylene fibers are ~ μm, a monolayer of water (~ . nm thick) would increase the mass of a mask by around % . we have observed this experimentally: when a mask is treated and left to air dry overnight, a residual of several grams is left. hence, it is not surprising that the n face masks become ineffective after even one rinsing process. furthermore, as the % ethanol adsorbs onto the surface of polypropylene fibers and its surface tension should cause fibers to wick together, similar to how the hairs on a fine paint brush stick together when wet. this wicking should decrease the surface area available for particle adsorption and allow pores to form in the filter material: this would then cause the filtration efficiency to drop drastically. when allowed to dry, the mixture evaporates with ethanol preferentially being in the vapor. the result of several hours of air drying is a thin film of primarily water deposited evenly over the surface of the fibers. for fibers that are wicked together, a thin layer of water keeps the fibers bonded. this final layer of water, while not contributing a large amount of mass, decreases the mask efficiency considerably. furthermore, due to the low surface area available for evaporation for water stuck between two fibers, it takes stronger conditions (drying the mask under vacuum) to decrease the partial pressure of water and increase the mean free path of water molecules needed for their removal. our results indicate that mask performance scales with how well the masks are dried, as measured both by the mass of the masks and the vacuum level used to dry them. a mask soaked in % v/v ethanol and dried in air overnight weighs - g more than it did originally and has substantially reduced filtration efficiency (fig. ) . furthermore, our data show that the filtration efficiency scales inversely with the amount of water adsorbed. vacuum treatment of masks at pressures between and mbar restored the filtering efficiency (fig. ) . the mechanism for this would simply be the reverse of the previously stated clumping effects, and the subsequent increase in effective surface area for the capture of particles. we note that a mechanism based on surface wetting raises the possibility that losses in filter efficiency might also be caused by other sources of moisture. spills, accidents, sweat, or moisture from the wearer's breath can become absorbed by the mask. however, due to surface tension, some of the water content might remain as microdroplets harmlessly trapped between the fibers. if some of the water is actually adsorbed onto the fibers, though, then this would cause the wicking and other effects. while absorbed microdroplets may evaporate easily due to their large surface area, the adsorbed water will not dry so easily. thus a mask that seems to have dried may actually be compromised by adsorbed water and require vacuum treatment. it remains to be seen what conditions lend to absorption vs. adsorption. adsorption may also be a propagating effect, such that a single adsorption event may draw neighboring microdroplets onto the surface. liao et al. observed a peculiar feature in that steam cleaning cycles only reduced the efficiency of n masks slightly over the first cycles, but the th cycle abruptly degraded their efficiency by %. our adsorbed water mechanism would explain this result. in the first cycles, moisture would have accumulated harmlessly at lower levels, with the water retained as microdroplets between the fibers. by the fifth cycle, a critical point may have been reached, with droplets merging and adsorbing onto the fibers, causing them to 'wick' together. based on this mechanism, it is likely that the loss of filtration performance stemming from steam cleaning would also be reversed with vacuum-drying. other methods of decontamination that are being suggested include heating in a dry oven at ˚ c for minutes , and hydrogen peroxide vapor treatment . the thermal treatment is well below the ˚c for hours recommended for medical sterilization by dry heat . low heat methods have not been tested on coronavirus outside of solution, nor for effectiveness against covid- specifically . the cdc warns of the uncertainty of the disinfection efficacy of moist heat methods for various pathogens . the effect of such heat levels on the thermoplastic mask seals and other fixtures is unknown. the material data sheet from m tm for their n , and particulate respirator masks cautions against temperatures over ˚c . the hydrogen peroxide vapor treatment requires bulk specialized equipment unlikely to be available at the scale of a local hospital or medical clinic, though it might be practical on a large scale , ; additionally, repeated treatments degrade the elastic bands and plastics commonly used in . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint face masks, impeding their ability to form an adequate face seal [ ] [ ] [ ] . in contrast, the materials used to make the masks and respirators that we tested are inert to % ethanol, water exposure, and vacuum. while several studies have demonstrated how certain methods may minimally damage filter material, we are unaware of any other study that documents the restoration of a mask from a compromised state. the vacuum drying process removes moisture in general, which would be effective not only for the water left from ethanol-based decontamination, but also for moisture that masks might accumulate during extended use. a moisture-based mechanism for efficiency loss and recovery would suggest that performance degradation might accompany normal use, due to water vapor from the users' breathing. if that is the case, then any procedure for processing masks for reuse should include a vacuumbased or other deep drying stage. otherwise, water vapor would continue to accumulate with each reuse. vacuum pumps of sufficient strength to reach the pressure levels needed are common industrial products available from a few hundred dollars, are often used in high-school science demonstrations, and are present in many research labs around the globe. these could be used easily during this pandemic in virtually any clinical environment. such vacuums are also used on industrial scales for a variety of processes, and could be added easily to other major decontamination procedures like the hydrogen peroxide treatment , . our mask testing setup costs around $ k, much lower than the $ k for an industry-standard testing rig with similar functionality but using ashrae aerosol standards. we discuss these limitations in the next section. even our current figure could be pushed down to less than $ if cheaper particle counters can be validated, as our setup uses an expensive particle counter that we happened to have on hand. although our design is less expensive, it is quite accurate within its domain, and has several advantages over established testing methods. in particular, conventional testing rigs do not test the impact of mask fit on performance. the standard procedure is to seal the mask onto the flat surface of the device with beeswax or other adhesive, even deforming the mask if necessary. this is unfortunate because studies have shown that the leakage around the mask, not through the medium, is the main determinant of total mask efficiency . poor fit can reduce an n mask from % to below % efficiency, which is below that of some masks made from commercial fabric , . in contrast, our testing setup uses a head model, upon which the mask is "worn", and is remarkably quick and easy to seal completely against the plastic film wrapped around the head model. we are seeking to improve upon this with d-printed head models and synthetic skin coverings (e.g. ). such an improved version could then be used to validate inexpensive head forms for testing rigs. the issue of fit is of particular concern in the current situation. hospitals are now on their own, searching for masks on the global market. it is difficult to evaluate how a given mask intended for use in one country will fit the range of face shapes present in another. it is also unknown how various disinfection techniques will impact fit. application of heat as suggested by liao et al. , for example, may impact the thermoplastics that line the edges of many masks. use of uv, bleach, or other chemicals may degrade the lining in other ways. all of these fit factors need to be taken into account in determining the best masks to use and the best ways to disinfect them. we avoided the use of tap water in our alcohol mixture because of the previous suggestions of interactions with surface charges on the microfibers. although our findings support surface tension mechanisms instead, we do not know if ions in tap water have an effect. this remains to be tested. it is not known how generalizable these results are across other types of masks or similar decontamination procedures. for example, both ethanol and isopropanol are known to be effective decontaminants, but whether our method extends to isopropanol is unknown. unlike ethanol, isopropyl alcohol damages polyester and may degrade other components of the masks and respirators. the effect of vacuum desiccation on cotton fiber-based masks is also unknown. the procedure outlined here for regenerating mask filtering efficiency has not yet been approved by the fda, niosh, or any other relevant regulatory agencies, although the use of the alcohol solution is . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint a well-vetted technique for disinfection . we believe the methods and techniques used here are simple enough to implement and verify by other research groups. our testing station tests filtering performance on particles in ambient air, rather than ashrae aerosol standards. however, our particle counter directly measures particle sizes, based on calibrations that are nist-referenced and meet or exceed ce and iso certification. the distribution of particle sizes was appropriate, with % of particles falling in the . µm bin, and our analyses were based on data from that bin. further, our tests produced the expected results on certified masks, and replicated the results from other experiments using standard methods. ultimately, we would like to determine the extent to which measurements from inexpensive, accessible designs can be validated as equivalent or correctable to standard. performance degradation in n face respirators is likely caused partially by moisture on the polypropylene fibers. we identify an effective disinfection and restoration method: rinsing the face masks in a % v/v ethanol solution, then air drying, followed by pumping on them in a vacuum chamber to a pressure below mbar. we replicated previous reports that alcohol-based decontamination could result in a decrease in efficiency of ~ % after air-drying. as of this writing, we have conducted wet/dry/vacuum cycles on different masks, all of which drop in performance after wetting, but return to within % of their initial filtration efficiency after vacuum treatment to < mbar. in addition, this restoration has been verified for up to cleaning cycles on the n rated m tm , and masks. we attribute the degradation of mask performance to the presence of adsorbed moisture that causes the micron scale electrostatic polypropylene fibers to clump together, significantly reducing the effective surface area of the filtering material. in addition, water adsorbed on the surface of the microfibers might interfere with their electrostatic interactions. the vacuum treatment fully dries the mask, allowing the fibers to spring back to their original arrangement, and restores the available surface area so that the electrostatic fibers are effective in capturing the micron and submicron size particles. because moisture can accumulate in masks as they are used, mask performance might degrade even absent a decontamination treatment. if so, conditioning processes for extending the usable life of masks, even ones that do not involve water, should completely dry the masks in order to counteract any adsorbed water that may have accumulated during extended use. in addition to the decontamination and restoration treatment, this study suggests that it would be worth exploring a number of simple and accessible procedures that could potentially be employed by institutions of limited means. the filtration testing setup we used can be assembled at low cost, and the decontamination and drying protocols also use low-cost methods. further development may lead to methods for small and resource-limited medical facilities to test, decontaminate, and recondition masks in order to extend the lifespan of existing ppe. further testing is currently underway to reduce costs, optimize accuracy, and validate potential designs which could be implemented quickly and cheaply. tables: figure : left, schematic diagram of the experimental medical filter testing chamber. right: image of the current system with the top removed. the hand-held air blower forces ambient dirty air (~ million particles at and above . μm/cuf) into the chamber, which exits either through the mask/head plumbing system or through the background port. the ratio of the particle counts at and above . μm between the two ports gives a direct measure of the mask efficiency. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint v/v ethanol solution, then allowed to air dry for . hours until the weight had stabilized. the original dry weight of the masks were . g (a) and . g (b). soaking and then air drying increased that weight of the masks to ~ . g (a) and ~ . g (b). the masks were then pumped on and removed at intervals to record the masks' mass and efficiency. as the mass of water in the mask decreased, the mask's efficiency increased. post-rinsing efficiency prior to vacuum treatment for the respirator in (a) was not measured and for (b) was . % at mbar. this confirms the hypothesis that the mass of adsorbed liquid is adversely influencing filtration efficiency. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint masks were soaked with % v/v ethanol solution and progressively dried with a vacuum. the rapid increase in filtering efficiency as pressure drops below ~ mbar is evidence for the removal of water molecules adsorbed to the surface of the microfibers; many studies of moisture removal from vacuum systems have shown that pressures in this range are necessary for this . error bars for pressures below mbar are smaller than the symbol size. these data are consistent with the hypothesis that adsorbed water molecules on the surface of the microfibers are responsible for the loss in filtering efficiency. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint technical specifications sheet m partriclate respirator / (aad), n . m corporation us government can n facial masks be used after disinfection? and for how many times information and faqs on the performance, protection, and sterilization of face mask materials decontamination and reuse of filtering facepiece respirators recommended guidance for extended use and limited reuse of n filtering facepiece respirators in healthcare settings loading and filtration characteristics of filtering facepieces sensory perception of the geomagnetic field in animals & humans. in: blank m, ed. electromagnetic fields: biological interactions and mechanisms removal of water from unbaked vacuum system direct probing of solvent-induced charge degradation in polypropylene electret fibres via electrostatic force microscopy preparation of polypropylene micro and nanofibers by electrostatic-assisted melt blown and their application measuring sub-nm adsorbed water layer thickness and desorption rate using a fused-silica whispering-gallery microresonator cleaning and decontamination of reusable medical equipments, including the use of hydrogen peroxide gas decontamination sterilization by dry heat hydrogen peroxide vapor sterilization of n respirators for reuse. medrxiv preprint doiorg/ / . . final report for bioquell hpv decontamination for reuse of n respirators fit factors for quarter masks and facial size categories fitting characteristics of n filtering-facepiece respirators used widely in china testing the efficacy of homemade masks: would they protect in an influenza pandemic development of an advanced respirator fit-test headform we thank mr. masamoto horikawa, mr. hironori hidaka and dr. atsuko kobayashi of the tokyo institute of technology for the early version of the control software for the laser particle counter. key: cord- - f fizfr authors: valentini, luca; bittolo bon, silvia; giorgi, giacomo title: engineering graphene oxide/water interface from first principles to experiments for electrostatic protective composites date: - - journal: polymers (basel) doi: . /polym sha: doc_id: cord_uid: f fizfr from the global spread of covid- we learned that sars-cov- virus can be transmitted via respiratory liquid droplets. in this study, we performed first-principles calculations suggesting that water molecules once in contact with the graphene oxide (go) layer interact with its functional groups, therefore, developing an electric field induced by the heterostructure formation. experiments on go polymer composite film supports the theoretical findings, showing that the interaction with water aerosol generates a voltage output signal of up to − v. we then developed an electrostatic composite fiber by the coagulation method mixing go with poly(methyl methacrylate) (pmma). these findings could be used to design protective fabrics with antiviral activity against negatively charged spike proteins of airborne viruses. electricity generation from the water-nanocarbon interface has been extensively investigated in the last years for blue energy harvesting purposes [ ] [ ] [ ] [ ] . between nanostructured carbons, graphene oxide (go) is a versatile material due to its surface chemistry, which allows for its dispersion in water and the design and engineering of polymer composites and membranes [ ] [ ] [ ] [ ] . from a first-principles point of view, very recently, it was demonstrated that go in liquid water acquires a negative charge [ , ] . the combination of go sharp edged structure and surface charge when dispersed into nonionic polymers was found to be efficient in antiviral activity [ ] ; thus, aerosols consisting of water droplets with a dimension in the range from nm to µm that cannot be stopped mechanically by the pores of filter fibers could be removed, for example, by the electrostatic interaction with go based polymer fibers, making the face masks more efficient [ ] . in this regard, the surface chemistry of go allows its incorporation into polymer matrices such as a polycarbonate (pc) matrix [ ] , and nylon [ ] to increase the degradation and melting temperatures. moreover, such nanocomposites can be further processed to obtain fabrics with added value in terms of mechanical strength and electrical conductivity. in previous studies, for example, we investigated the mechanical excitation through falling water droplets and acoustic waves on reduced go composites to convert the mechanical energy of the impact to electrical power [ , ] , but the charging of the go surface in contact with water when it is embedded into a polymer matrix, to our best knowledge, has not been investigated yet. the combined provision of what is previously reported [ , ] requires calculation methods dedicated to optimize the surface charge of d materials and deposition techniques, which at first allows for layers of such d materials on polymeric surfaces to be isolated by means of mechanical transfer techniques [ ] . more on the theoretical side, our setup follows those reported in the literature; density functional theory based methodologies are used to predict interfacial electric field within (mono)layers in van der waals heterostructures to evaluate (among the others) the photocatalytic performance enhancement [ , ] , and the storage of alkali atoms (batteries) [ ] , while similar approaches are exploited to compute properties of metal/oxide interfaces in terms of workfunction variation and charge distribution between heterostructure components [ ] . so, a first step will be to verify the confinement and electrical response of go layers deposited on a polymeric surface when in contact with water droplets and then validate the models and results once the go is integrated within a polymeric fiber to also study its mechanical strength for their exploitation in fabrics. in this study, we embedded go flakes obtained from liquid suspension into poly(methyl methacrylate) (pmma)/go fibers by the coagulation method. beyond the superior electrical and mechanical properties with respect to the neat polymer, we observed the segregation on the fiber surface of the go sheets. it was found that water droplet interaction with the go surface generates an electrostatic potential under ambient conditions. on the theoretical side, we confirmed the electrical charge redistribution associated with the approach of water molecules on a go layer. we pave the way so that the combinations of our findings could be used to create an electrostatic nanocomposite fabric as protection against the transmission of aerosol particles. go was purchased from cheaptubes, while poly(methyl methacrylate) (pmma, average mw . ) was purchased from sigma aldrich. the preparation method was reported by us elsewhere [ ] ; briefly go water solution ( mg/ml) was deposited by spin coating ( rpm) on cleaned quartz substrates and left to dry under nitrogen stream. the pmma for the realizations of the films was produced through the direct polymerization of the methyl methacrylate (mma) monomer. it was stirred together with lauroyl peroxide (sigma aldrich, [ch (ch ) co] o , wt%) and n,ndimethyl-m-toluidine (sigma aldrich, ch c h n(ch ) , wt%), for min. the reacting mixture was poured in a petri dish and positioned in a glass vial with an excess of mma. pmma was then spin cast ( rpm for min) on go and annealed for h from • c to • c with a • c/h heating ramp in order to remove residual monomer in the samples; then it was immersed at • c in boiling water until the complete detachment of the pmma film was obtained. metal electrodes (≈ nm) were deposited by vacuum evaporation onto the detached pmma/go film. to prepare the pmma/go composite fiber, g of pmma powder was dissolved in g of dimethylformamide (dmf, sigma aldrich) and g of tetrahydrofuran (thf, sigma aldrich). go was added at . wt% into g of the pmma solution. after stirring the solution for h, it was extruded through a stainless-steel syringe into a coagulation bath with deionized (di) water at a speed of µl/min. commercial beer yeast extract was added to water ( mg/ml) and stirred in a sterilized flask at rpm at • c for h. after that, sugar three times the weight of the medium was added. fermenting yeast was then dropped onto the pmma/go film that was positioned into a circular aluminum mold. after the pmma/go film realization, the current-voltage characterization was performed with a keithley source meter; conductivity values were obtained by applying a linear voltage and monitoring the current across the film. a conductive silver paint was deposited on au thermal evaporated electrodes at the end of rectangular shaped samples with probes that were placed cm apart. the open-circuit voltage (voc) of the pmma/go film was measured after the exposure with a di water aerosol from a distance between and cm at ambient conditions. the aerosol water was left to evaporate in a calm environment or under air flow with a speed of m/s. in the case of the pmma/go fibers, the current-voltage characterization was performed by applying a linear voltage along the fiber and monitoring the current. raman spectra were acquired with labram aramis (horiba jobin yvon, lille, france), where the excitation wavelength used was . nm generated from a he-ne laser with an output power of approximately mw. the diffraction grating used had grooves cm − and the ccd detector peltier-cooled was at~− • c. the acquisition range for each spectrum was to cm − and each spectrum was computed by averaging two successive integrations of s each. the objective lens of the microscope (olympus bx ) used was a × with a long working distance. aperture slit and pinhole were set respectively to µm and µm. kelvin probe force microscopy (kpfm) measurements were carried out on afm instrument from bruker (model: scanasyst) in ambient conditions at % relative humidity. the mechanical properties of the pmma/go fibers were measured by a universal tensile testing machine (lloyd instr. lr k) with a n cell. the strain rate was mm/min, and the gauge length was . ± . mm. five samples for each composition were tested. the morphology of the pmma/go fibers was investigated by field emission scanning electron microscopy (fesem). the optical transparency of the film was monitored by uv-vis spectroscopy, measuring the absorbance between and nm wavelengths. calculations were all performed by means of the density functional theory as implemented in the vienna ab-initio simulation package (vasp) code [ ] [ ] [ ] [ ] . the perdew-burke-ernzerhof (pbe) electron exchange-correlation functional [ ] , along with the dft-d dispersion correction, in order to include the van der waals interactions [ , ] , were adopted in the calculations. the projector augmented wave (paw) potentials [ ] have been similarly employed with a cutoff energy of ev for the plane-wave basis set. optimizations converged when forces were below . ev/Å. the graphene initial atomic structure was taken from the materials project website [ ] . in detail, the graphite primitive cell was considered and "peeled" to obtain a single-layer. a × supercell was then constructed and optimized (adding a very large amount of vacuum,~ Å, along the non-periodic direction to avoid spurious interactions among replicas) and the same number of epoxy and hydroxyl terminations were anchored on the two faces. the choice of the two terminations stems from previous literature that clearly assessed their dominant presence in go [ ] [ ] [ ] . the overall ratio oh:epoxy was : . the so-assembled structure was once more fully optimized (both ionic and lattice parameters), obtaining the geometry shown in figure . we are aware that our model is probably limited and may underestimate the real situation, however, similar findings to that in the previous literature makes us confident of the reliability of our results [ ] . in particular, to validate our results, we calculated the workfunction (Φ) of go, which is the difference between the vacuum level (e vac ) and the fermi energy (e f ): for our analyzed go system, we obtained a value of . ev, while that for pure graphene, as a reference, is . ev, which is in perfect agreement with the value reported by kumar et al. ( . ev) [ ] . to simulate water anchoring on the go layer, we then asymmetrically added, as a test case, a layer of five water molecules on top of the go layer and once more optimized the so-assembled system. from the initially relaxed structure, we constrained molecules of water to progressively get closer to the go layer to evaluate the possible dipole enhancement (charge redistribution) at the interface between the two structures. the final charge analysis was performed by means of the bader code [ ] [ ] [ ] [ ] . to simulate water anchoring on the go layer, we then asymmetrically added, as a test case, a layer of five water molecules on top of the go layer and once more optimized the so-assembled system. from the initially relaxed structure, we constrained molecules of water to progressively get closer to the go layer to evaluate the possible dipole enhancement (charge redistribution) at the interface between the two structures. the final charge analysis was performed by means of the bader code [ ] [ ] [ ] [ ] . on the theoretical side, from the initial modellization, we evaluated the possible charge redistribution associated with the approach of water molecules on the go layer. figures and show the details of the simulation. in details, figure shows the charge distribution induced by the interface formation. kelvin probe force microscopy (kpfm) provides information on the contact potential difference between the tip and the sample surface and is associated with local workfunction [ ] . the results reported elsewhere [ ] indicate a value of nearly . ev, which is within the value range from . to . ev reported for go and is in good agreement with that calculated by our modellization (see figure ) [ ] . on the theoretical side, from the initial modellization, we evaluated the possible charge redistribution associated with the approach of water molecules on the go layer. figures and show the details of the simulation. in details, figure shows the charge distribution induced by the interface formation. kelvin probe force microscopy (kpfm) provides information on the contact potential difference between the tip and the sample surface and is associated with local workfunction [ ] . the results reported elsewhere [ ] indicate a value of nearly . ev, which is within the value range from . to . ev reported for go and is in good agreement with that calculated by our modellization (see figure ) [ ] . inter-and intralayer van der waals forces dominate the heterostructure as one can clearly see from the figure. the physisorbed water layer is expected in this sense not to transfer charge to the go layer because of the formal absence of chemical bonds between the two layers. to prove it, we calculated the total charge distribution in the water layer, finding as expected a small variation (− . ): the o atom charge ranged between − . and − . , and that of h between . and . [ ] . we then focused on the effects of water impact on the go layer and the associated possible inter-and intralayer van der waals forces dominate the heterostructure as one can clearly see from the figure. the physisorbed water layer is expected in this sense not to transfer charge to the go layer because of the formal absence of chemical bonds between the two layers. to prove it, we calculated the total charge distribution in the water layer, finding as expected a small variation (− . ): the o atom charge ranged between − . and − . , and that of h between . and . [ ] . we then focused on the effects of water impact on the go layer and the associated possible charge distribution variation. accordingly, we reduced the distance of water oxygen by . and . Å (closer distances did not lead to any stable heterostructure), keeping the water o positions frozen and allowing all the other ionic positions (and lattice lateral parameters) to relax. the result was an overall change in the net charge of the water molecule layer that in both cases turned to be slightly more negative at − . and − . , respectively. of course, this is not a very sensitive variation, but is still noticeable, and moreover, we could observe a trend indicating an enhanced dipolar redistribution due to the pressure exerted by water on the go layer, which is supportive of the change in the electric field associated with the interface formation. the steps for the transfer method using low molecular weight pmma as the support layer is illustrated in figure . after coating go with the pmma solution (figure a) , the pmma/go film was peeled off in boiling water (figure b) , which was used to weaken the adhesion between the pmma and substrate. the floating pmma/go film was transferred onto the target substrate, heated at • c glass transition to improve the adhesion with the substrate, and then subjected to electrical characterization (figure c ). figure d shows go flakes of micron size on a glass substrate that can be identified by their clear contrast with respect to the substrate. with respect to our previous findings [ , ] , the employment of a pmma support layer with a lower molecular weight reduced the contamination of go; in this regard, the raman spectrum does not show any peak associated with pmma, being the peaks of d (~ cm − ) and g (~ cm − ), the signature of the sp and sp hybridized carbon state, respectively (figure e ) [ ] . the d peak appears due to the disorder and imperfection of the carbon crystallites and the g peak is assigned to one of the two e g modes during the stretching vibration of the graphitic carbon atoms [ ] . the d + g bump is activated by the presence of defects in the go paper [ ] . the absorption of water molecules on nanostructured carbon layers along with fluid evaporation is known to play a crucial role in the generation of electrical potentials [ , ] . after peeling, the pmma/go film is electrically insulating (figure a ) and transparent ( figure b) ; after being in contact with water droplets, it shows an open-circuit voltage (voc) change up to − v and then recovers the initial baseline after an interval between and s (figure c ). by repeating the experiment in dynamic conditions (i.e., by wetting and drying the film with air flow (figure d) ), we observed a change of the open circuit voltage under the water evaporation with a variation that was smaller than that observed in figure c . with respect to our previous findings [ , ] , the employment of a pmma support layer with a lower molecular weight reduced the contamination of go; in this regard, the raman spectrum does not show any peak associated with pmma, being the peaks of d (~ cm − ) and g (~ cm − ), the signature of the sp and sp hybridized carbon state, respectively (figure e ) [ ] . the d peak appears due to the disorder and imperfection of the carbon crystallites and the g peak is assigned to one of the two e g modes during the stretching vibration of the graphitic carbon atoms [ ] . the d + g bump is activated by the presence of defects in the go paper [ ] . the absorption of water molecules on nanostructured carbon layers along with fluid evaporation is known to play a crucial role in the generation of electrical potentials [ , ] . after peeling, the pmma/go film is electrically insulating (figure a ) and transparent ( figure b) ; after being in contact with water droplets, it shows an open-circuit voltage (voc) change up to − v and then recovers the initial baseline after an interval between and s (figure c ). by repeating the experiment in dynamic conditions (i.e., by wetting and drying the film with air flow (figure d) ), we observed a change of the open circuit voltage under the water evaporation with a variation that was smaller than that observed in figure c . figure a ,b). the fesem image revealed that the surface was fairly smooth with the presence of go aggregates (figure a ). in general, the fesem image of the cross section showed hollow pores in the radial direction of the fiber. the formation of this structure is attributed to counter diffusion of solvent/nonsolvent and phase separation during coagulation [ ] . from a mechanical point of view, the addition of go resulted in an improvement of the strength as well as of the elongation at break, leading to a toughness (calculated as the area under the stress-strain curve) variation from . mpa for pmma to . mpa for the pmma/go fibers, respectively (figure b) . contrary to what has been observed for the pmma/go film (figure a ), the addition of go in the fiber generates a considerable increase in electrical conductivity (figure c ). the highest electrical conductivity and the improvement of the mechanical properties of fibers when go was added can be explained by an increase in polymer chain orientation and go orientation along the fiber direction, increasing the electrical current flow along the fiber. the water adsorption in hygroscopic materials was found a green method of converting the evaporation into electric potential [ ] . the water confinement on the pmma/go fiber is shown in figure d . graphene oxide flakes with abundant functional groups on the fiber surface make it hydrophilic, so that water capture occurs along the fiber. after being wetted by an aerosol with deionized water, an open-circuit voltage (voc) of − v was recorded, maintaining this value during the water evaporation in the laboratory environment with the temperature of °c (figure e ). we then repeated the experiment by wetting and drying the fiber with air flow (figure f ). when the fiber was exposed to water aerosol, a variation of the voltage was observed up to − . v. it returned to its original value when we turned on the air flow, promoting the water evaporation. such wetting- figure a ,b). the fesem image revealed that the surface was fairly smooth with the presence of go aggregates (figure a ). in general, the fesem image of the cross section showed hollow pores in the radial direction of the fiber. the formation of this structure is attributed to counter diffusion of solvent/nonsolvent and phase separation during coagulation [ ] . from a mechanical point of view, the addition of go resulted in an improvement of the strength as well as of the elongation at break, leading to a toughness (calculated as the area under the stress-strain curve) variation from . mpa for pmma to . mpa for the pmma/go fibers, respectively (figure b) . contrary to what has been observed for the pmma/go film (figure a ), the addition of go in the fiber generates a considerable increase in electrical conductivity (figure c ). the highest electrical conductivity and the improvement of the mechanical properties of fibers when go was added can be explained by an increase in polymer chain orientation and go orientation along the fiber direction, increasing the electrical current flow along the fiber. the water adsorption in hygroscopic materials was found a green method of converting the evaporation into electric potential [ ] . the water confinement on the pmma/go fiber is shown in figure d . graphene oxide flakes with abundant functional groups on the fiber surface make it hydrophilic, so that water capture occurs along the fiber. after being wetted by an aerosol with deionized water, an open-circuit voltage (voc) of − v was recorded, maintaining this value during the water evaporation in the laboratory environment with the temperature of • c (figure e ). we then repeated the experiment by wetting and drying the fiber with air flow (figure f ). when the fiber was exposed to water aerosol, a variation of the voltage was observed up to − . v. it returned to its original value when we turned on the air flow, promoting the water evaporation. such wetting-and drying-induced voltage change has been observed on several cycles (figure f ). this finding mimics the electrostatic interactions that were observed in natural and synthetic fabrics [ , ] . recently, for instance, a charged polyvinylidene fluoride (pvdf) multilayer nanofiber was exploited in filtering airborne coronavirus under ambient nano-aerosols [ ] . in the absence of novel coronavirus (covid ) to test, we investigated the fermentation process of the beer yeast (s. cerevisiae) on the pmma/go film. we chose beer yeast cells because their surface is negatively charged [ ] and has the spike proteins of coronaviruses [ ] . the effectiveness of the pmma/go film against yeast cells is shown in figure . qualitative and macroscopic observation of yeast cells on the pmma/go substrate revealed that under the water evaporation, namely s, s, and s, led to the presence of transparent regions, indicating fewer cells stained. similar observations on the neat pmma substrate used as the control did not give evidence of transparent regions, suggesting a good adhesion of the biofilm on the substrate ( figure , bottom) . this qualitative investigation of biofilm formation could suggest the efficacy of the composite film against airborne microorganisms. this finding mimics the electrostatic interactions that were observed in natural and synthetic fabrics [ , ] . recently, for instance, a charged polyvinylidene fluoride (pvdf) multilayer nanofiber was exploited in filtering airborne coronavirus under ambient nano-aerosols [ ] . in the absence of novel coronavirus (covid ) to test, we investigated the fermentation process of the beer yeast (s. cerevisiae) on the pmma/go film. we chose beer yeast cells because their surface is negatively charged [ ] and has the spike proteins of coronaviruses [ ] . the effectiveness of the pmma/go film against yeast cells is shown in figure . qualitative and macroscopic observation of yeast cells on the pmma/go substrate revealed that under the water evaporation, namely s, s, and s, led to the presence of transparent regions, indicating fewer cells stained. similar observations on the neat pmma substrate used as the control did not give evidence of transparent regions, suggesting a good adhesion of the biofilm on the substrate (figure , bottom). this qualitative investigation of biofilm formation could suggest the efficacy of the composite film against airborne microorganisms. we exploited a well-known method used to transfer graphene layers to create a composite surface that could generate a negative voltage from water evaporation. first-principles calculations support the idea that water molecules, when approaching a graphene oxide layer, enhance a dipolar redistribution at the interface, thus generating an extra electric field due to the heterostructure formation. experiments on the wet surface revealed a drying-induced open circuit voltage change. a similar effect was observed on pmma/go fibers that were fabricated by the wet spinning method. considering the electrostatic interaction with the spike proteins of coronaviruses, these materials could be considered for integration in personal protection equipment against airborne viruses transported by aerosols. we exploited a well-known method used to transfer graphene layers to create a composite surface that could generate a negative voltage from water evaporation. first-principles calculations support the idea that water molecules, when approaching a graphene oxide layer, enhance a dipolar redistribution at the interface, thus generating an extra electric field due to the heterostructure formation. experiments on the wet surface revealed a drying-induced open circuit voltage change. a similar effect was observed on pmma/go fibers that were fabricated by the wet spinning method. considering the electrostatic interaction with the spike proteins of coronaviruses, these materials could be considered for integration 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stability, work function, and photoluminescence of reduced graphene oxide a grid-based bader analysis algorithm without lattice bias an improved grid-based algorithm for bader charge allocation a fast and robust algorithm for bader decomposition of charge density accurate and efficient algorithm for bader charge integration electronic characterization of organic thin films by kelvin probe force microscopy free-standing hybrid paper of graphene oxide and functionalized carbon nanotube for enhanced electrical and mechanical properties work function engineering of graphene oxide via covalent functionalization for organic field-effect transistors raman spectroscopy in graphene quantifying defects in graphene via raman spectroscopy at different excitation energies surface-energy generator of single-walled carbon nanotubes and usage in a self-powered system all-printed porous carbon film for electricity generation from evaporation-driven water flow effect of jet swell and jet stretch on the structure of wet-spun polyacrylonitrile fiber scaling up nanoscale water-driven energy conversion into evaporation-driven engines and generators characterization of electrostatic discharge properties of woven fabrics filtration and electrostatics-a review of the new technology charged pvdf multilayer nanofiber filter in filtering simulated airborne novel coronavirus (covid- ) using ambient nano-aerosols characterisation of beer particle charges and the role of particle charge in beer processing negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus there are no conflicts of interest to declare. key: cord- -v ay kwg authors: siddiqui, ruqaiyyah; khamis, mustafa; ibrahim, taleb; khan, naveed ahmed title: sars-cov- : the increasing importance of water filtration against highly pathogenic microbes date: - - journal: acs chem neurosci doi: . /acschemneuro. c sha: doc_id: cord_uid: v ay kwg [image: see text] the presence of sars-cov- in human wastewater together with poor quality of public drinking water supplies in developing countries is of concern. additionally, the frequent use of contaminated water for bathing, nasal irrigation, swimming, and ablution can be a risk factor in contracting infectious agents such as the brain-eating amoebae and possibly sars-cov- . the use of appropriate tap water filters should be encouraged to remove pathogenic microbes, together with restrained nasal irrigation (not forcing water inside nostrils vigorously) during ritual ablution or bathing to avoid dangerous consequences for populations residing in developing countries. t he detection of severe acute respiratory syndrome coronavirus- (sars-cov- ) in feces suggested the presence of viral particles in human wastewater. by rt-pcr assay, sars-cov- was detected in wastewater samples accumulated at amsterdam schiphol airport, suggesting the need for surveillance systems for early warning of covid- outbreaks, as well as implementation of effective filtration or disinfection systems to eradicate viral particles at wastewater treatment plants. these findings were strengthened further with the recovery of sars-cov- in wastewater collected near the first covid- case in the community in amsterdam. although the presence of sars-cov- in wastewater may not be a peril to the local community in amsterdam, similar scenarios could be a major hazard for public health in developing countries. the environmental transmission potential of sars-cov- to spread in communities via fecal−nasal route presents an important risk factor in covid- outbreak recurrence, especially for developing countries where diseases with rapid clinical onset can go unnoticed. for example, the observation of primary amoebic meningoencephalitis due to brain-eating amoebae (i.e., naegleria fowleri) is mostly unnoticed, especially in rural areas and disadvantaged communities, and is known to be associated with nasal irrigation for cleansing, ritual ablution, bathing, and swimming. this highlights the need for safe and effective water disinfection systems, as well as personal hygiene, to ensure protection against infectious agents. − given that sars-cov- also uses the nose as a portal of entry and the fact that it has been detected in human wastewater, can there be fecal−nasal transmission as observed with the brain-eating amoebae? while the connection of human wastewater and clean drinking water may be alien to the public in developed nations, it can be common in developing countries. for example, in a city such as lahore (pakistan), thousands of people turn to the canal that passes through the city during summer months ( figure a ). with high temperatures ( − °c), as well as power cuts, while water temperatures are recorded at comfortable − °c, people turn to canals for water-related recreational activities. the troops of people are never ending and are visible for kilometers along the canal, and this "recreational activity" occurs virtually every day, during the summer months ( figure a ). furthermore, there is a dearth of available toilet facilities and often the same canal waters are utilized to defecate in, with no evident warning signs depicting the potential dangers of swimming visible, representing a major risk factor for the community. needless to say, the situation must be more challenging in rural areas (comprising > % of the population) due to limited public awareness of associated risks, absence of effective control measures, and poor health care infrastructure. in remote villages with prolonged hot summer months together with power cuts, the only recreational activity available to the public is swimming in contaminated water without knowing the associated risks ( figure b ,c). our recent interview of selected few members of a small community in india highlighted that covid- infection was considered to be associated with "touching door handles" with no understanding of safe water and nasal irrigation. similarly, religious commemorations like the kumbh mela, where up to million people congregate in the indian metropolis of allahabad to take part in the holy bathing festival in the holy ganges river, can present a major hazard to public health aiding in the transmission of infectious agents like sars-cov- ( figure d ). in supplementation to the diagnosis of covid- cases, there is a clear and urgent need for surveillance systems for monitoring sars-cov- prevalence in such environments to counter potential disease outbreaks. to further exacerbate the situation, developing nations like india and pakistan are challenged with water shortages, and there is disproportionate public dependence on water storage wells or tanks. these water storage tanks are thriving with microbial communities. the drinking water supplies are rarely chlorinated. for example, the sewage management in karachi (pakistan) was built by the british during colonial days and needs urgent restoration. scores of hospitalizations are accredited to breakage of sewage lines and merging with drinking water supplies. thus, the contamination of human waste as well as human wastewater into drinking water supplies highlights a major risk factor in contracting infectious agents such as brain-eating amoeba and possibly covid- , especially for developing countries. although the effective implementation of preventative strategies requires long-term planning and is very costly, the use of simple measures to decontaminate water in household settings can be placed in the interim. notably, n. fowleri goes through the nasal cavity and then migrates along the olfactory neuroepithelial route to reach the brain to produce a fulminating meningoencephalitis. the use of the nose as a portal of entry for sars-cov- suggests that preventative measures can be tailor-made to be collectively effective against both infectious agents. previous studies indicated that nose clips or boiling of water prior to nasal irrigation should be utilized as a preventative measure; however these measures are not always feasible. the use of simple tap water filters in households prior to ablution or nasal irrigation ( figure e ,f) can be effective in eradicating microbial contaminants. water filters have been used traditionally and effectively in eradicating bacteria, fungi, protozoa, and other parasites. however, viruses are considerably smaller, with most viruses ranging in size from to nm, and depending on the size, they may slip through filters with larger pore size. as sars-cov- is approximately nm, the use of simple tap filters with ultrafiltration (pore size of ca. nm) or nanofiltration (pore size of ca. nm) can be effective, but not microfiltration (pore size of ca. nm). thus, the use of nanofilters is highly recommended to eliminate almost all microbes. due to the small size of viruses, microfiltration is not effective for viruses but is reasonably effective for bacteria and protozoa. in addition to pore size, viral surface properties are also important in the design of effective filters. as the viral cell surfaces possess net negative electrostatic charge, adsorption on solid surfaces with modified positive charge centers can be used to capture viral particles. such surfaces are also shown to be effective in eliminating heavy metals that occur in anionic complexes, for example, arsenate or chromate, although negatively charged surfaces, like montmorillonite, the clay mineral, may eradicate heavy metals in their cationic configurations. in this regard, mechanical purifiers with modified solid surfaces with positive charge can be used to physically remove selected contaminants such as sars-cov- . in summary, the poor infrastructure of wastewater management and sanitation in developing countries together with limited public awareness suggests the need for behavioral modification and educational strategies to counter outbreaks of diseases such as covid- . the use of tap water filters should be encouraged to remove pathogenic microbes coupled with restrained or cautious nasal irrigation (not forcing water inside nostrils vigorously) during ritual ablution or bathing to evade dangerous consequences for those communities residing in the developing nations. sars-cov- in wastewater: potential health risk, but also data source primary amoebic meningoencephalitis caused by naegleria fowleri: an old enemy presenting new challenges importance of theranostics in rare brain-eating amoebae infections biology and pathogenesis of naegleria fowleri adsorption of nickel and copper onto natural iron oxide-coated sand from aqueous solutions: study in single and binary systems n.a.k., t.h., and m.k. envisaged the concept amid discussions with r.s. r.s. reviewed literature, and n.a.k. prepared the first draft of the manuscript. r.s. finalized the manuscript. all authors contributed equally to the manuscript and will act as guarantors. the authors declare no competing financial interest. key: cord- -gjrj gvr authors: odei erdiaw-kwasie, michael; abunyewah, matthew; edusei, joseph; buernor alimo, emmanuel title: citizen participation dilemmas in water governance: an empirical case of kumasi, ghana date: - - journal: world development perspectives doi: . /j.wdp. . sha: doc_id: cord_uid: gjrj gvr abstract citizen participation in decision-making on water-related initiatives offers a valuable pathway to move towards a more equitable and sustainable water delivery, now and into the future. however, given the acknowledged impacts of active citizen participation in the water governance process, there is an increase institutional search for ideal approaches to enhance local inputs in water decisions. to enhance institutional knowledge on this quest, this paper adopts a qualitative exploratory method using interviews and focus group discussions ( participants in each group) to present an empirical case of kumasi in ghana. findings from the study show that citizens are willing to participate in decision-making on water-related process through radio programs; public meetings; surveys or polls and telephone. it was also realised that ghana water company focuses more on the technical aspect of water delivery than the social aspect which borders on appropriate engagement strategies that involve customers in the decision-making process. the results imply a significant difference regarding feasibility and outcomes for each currently deployed participatory mechanism. the study recommends a change in current institutional and governance arrangements to influence citizen participation strategies at all levels of water service delivery and governance. water is a scarce resource in most cities in developing countries as a result of its physical non-availability and financial incapacity of cities to supply water for the growing populations (who/unicef, ). the scarcity of water in sub-saharan africa is more of an economic rather than physical (kirono et al., ) . the condition challenges cities to have a governance approach that ensures that water security in all its forms such as availability, accessibility, and quality to citizens (kirono et al., ; munasinghe, ) . previous studies indicate that cities should always have structures in place that involve citizens in decisionmaking on water-related issues to sustain water service delivery (nastar et al., ; richter et al., ) . the idea of participation requires citizens working together with service providers rather than having a defined chain of command whereby citizens are disregarded in decision-making processes erdiaw-kwasie and yamoah, ; okyere, diko, abunyewah, & kita, a) . as a result, ominously, the unesco has demonstrated that the survival of urbanites lies in their ability to respond to the impacts of a 'perfect storm' of its governance system (unece/unesco, ). thus, a strong focus has been put on participatory approaches when it comes to water access and governance over the last decade (ahlers, schwartz, & guida, ; alda-vidal, kooy, & rusca, ; fan, park, & nan, ; okyere, diko, abunyewah, & kita, a) . given the recent increased in water insecurity issues in many of the main cities in ghana (alda-vidal et al., ; harris & morinville, ) , this paper considers it an ideal case for the study. in line with the ghana national water policy that aims to ensure full participation of all stakeholders in decision-making on water-related issues in realization of the ghana water vision for [which states ''promoting an efficient and effective management system and environmentally sound development of all water resources in ghana"], the study's focus is timely and relevant. the paper primarily field-tests how existing citizen participation 'formulas' are delivering on effective water governance promises to meet sustainable development imperatives of urbanites in ghana. in doing this, the paper presents and discusses the study's main findings and demonstrates the key findings with regard to citizen participation mechanisms in water governance. the study is based on the premise that literature related to citizen participation has failed to provide adequate empirical data to help explain how the nature and level of citizen participation impacts waterrelated decision making. this paper, therefore, explores the nature and level of citizen participation in decision-making on water governance process, with a focus on ghana water company in the study area. traditionally, the concept of governance refers to the interactions among structures, processes, and traditions that determine how power and responsibilities are exercised, how decisions are taken, and how citizens or other stakeholders have their say cobbinah and erdiaw-kwasie, ) . however, contemporary perspective on the concept has emerged. many scholars described this new governance as a mode of governing that shows a preference for collaborative approaches among government and non-government actors from the private sector and civil society sithole, ) . in the water governance field, sithole ( ) suggests that higher levels of integration of participants' subjective perspective, emphasis on governance approach that embraces mutual learning, coordination, and an adaptive decision has assumed particular significance. global acknowledgement of the need for citizen participation in development is profound (arnstein, ; erdiaw-kwasie and yamoah, ; okyere et al., b) , particularly in the governance of water in the developing world's context (terry et al., ) . to ensure citizens involvement, 'target b' of sustainable development goals (sdg) number aims to ensure availability and the sustainable governance of water for all and looks forward to supporting and strengthening the participation of local communities in water governance (david et al., ) . again, the international decade for the un-general assembly adopted action ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , also known as the 'water for life', further recognises the full participation of stakeholders, particularly women in water management and supply and related issues (un-water, ) . the united nations world water development report ( ) indicates that vibrant and robust participation in the governance of water is a critical element to help propel sustainable and inclusive development for all citizens (unesco, ) . more recently, there is a general recognition of the need for integrated approaches to water governance (pahl-wostl, ) . given the breadth of the issues and the integral role of many organisations and stakeholders, modes of cooperation and coordination have been widely identified in the research literature as being essential for the improvement in service delivery outcomes (amexo, ; kekez et al., ) . however, a majority of research efforts to date focus on water supply challenges that confront rural and urban regions and impressively articulate the effects of the discrepancies about infrastructure and coverage (jiménez & pérez-foguet, ; munasinghe, ) . also, research that assesses citizen participation 'formulas' in decision-making on water-related issues as a solution to water delivery challenges in urban centres remains scarce in the developing world context (adams et al., ; chitonge et al., ; cobbinah et al., ) . using ghana as a case study, this paper seeks to fill in the knowledge gap by evaluating participatory mechanisms adopted in the governance of water service delivery in kumasi. in doing this, the paper defines the theoretical scope of the article by reviewing relevant citizen participation typologies, collect data on specific citizen participation 'formulas' that are currently deployed by governing water institutions in the kumasi area, presents locals' perspectives on these deployed 'formulas', and consequently analyse the data in line with the theoretical framework adopted for the study. this paper consists of six parts. section reviews the literature on water governance and citizen participation focusing on developing countries. section describes the research area and methods. section presents the study results, whereas section captures the discussion and implications of the research findings. section presents the conclusion of the research. water governance is not a new concept both in literature and practice. over the past decades, water governance has played a pivotal role in sustainable development including poverty reduction especially in developing countries (benson et al., ) . it has transitioned overtime under the principles of transparency, accountability, decentralization, and participation. looking at the debates within the water governance sector, water inequality and insecurity have been identified as transboundary and inter and intra sectoral competition, as well as reliance on a single source or supply network. thus, it is through dissecting the links between control and access to water and social relations of power that scholars demonstrate the ways that urban waterscapes are never socially, nor ecologically, neutral (adams et al., ; swyngedouw and swyngedouw, ) . participation has, therefore, emerged as one of the most advocated and used principles of water governance in an attempt to leverage access to water as well as social relations of power, which significantly impact water demand among a given population (adams et al., ; cobbinah et al., ) . as a result, various forms of participatory approaches to water governance have sufficed (akhmouch and clavreul, ; margerum & robinson, ) . with the ambiguity, confusion and large gaps that exist between policy and practice on participatory water governance, several questions have been raised. critical of these questions is "when do we say water project beneficiaries have participated in decision making and to what extent should government/powerful elite support grassroot members during decision making". arnstein's ladder of participation attempts to provide answers to these questions extensively by conceptualising participation into forms and typology (arnstein, ) . arnstein ( ) highlights the levels of participation and summarises it into eight rungs on a ladder (see fig. ). on the ladder, the movement from one rung to the other corresponds to changes in degrees of citizen engagement and the extent of citizen power in determining a plan. the degree of citizen engagement ranges from noninvolvement through tokenism to citizen power. the ladder illustrates participation as a struggle for power and control over resources between project beneficiaries and the powerful class. according to her, higher rungs of participation create an environment which induces social reforms and enables grassroots members to share in the benefits of the affluent society. lower rungs (manipulation and tokenism) were the commonest in both developed and developing countries in the late s and s, however, in recent times, there has been a strategic shift to shared decision-making concerning water governance at the local level (rogers and hall, ; akhmouch et al, work has been and continues to be an important step in water governance literature and practice. for instance, her work has changed the phase of participation in community development including water governance. in the context of water governance, arnstein's conceptualisation has helped in the redistribution of power and sharing of information to ensure that individuals and groups in the society actively take part in issues relating to water (fig. ) . nevertheless, it has failed to account for institutional participation and its constraints (laskey and nicholls, ) . also, the ladder has been criticised on grounds that, in practice, sometimes the goal of citizens for engaging in decision making is not to take control (collins and ison, ) . choguill in her paper in indicates that 'manipulation' is not always the bottom rung, particularly in a context where there is no government support. in that scenario, she pointed out that citizens resort to self-management which should have been the bottom rung of arnstein's framework. arnstein's conceptualisation shows that participation is linear indicating policy problems remain constant, however, bishop and davis ( ) hold the view that policy problems are unique and require different levels and types of participation. this, therefore, means that participation is a non-linear concept. citizen participation is focused merely on the redistribution of power that enables the have-not citizens, presently excluded from the political and economic processes, to be deliberately included in the future (arnstein, ; erdiaw-kwasie et al., ; okyere, diko, abunyewah, & kita, a) . in other words, citizen participation is perceived as a strategy by which the have-nots join in determining how information is shared, goals and policies are set, tax resources are allocated, programs are operated, and benefits like contracts and patronage are parcelled out. given the heated controversy over the concept in the development space (adams et al., ; wesselink et al., ) , diverse participation typology of citizen participation has been proposed in the context of urban renewal (arnstein , nyseth et al., , anti-poverty programs (kumar et al., ; zimmermann, ) , as well as social programs to encourage a more enlightened dialogue (cornwall, ; theocharis and van deth, ) . adequate clean water is essential to everyone's survival. improvements in water governance and access to water supply and sanitation services are essential to addressing various social and economic inequities (cobbinah et al., ; cobbinah and erdiaw-kwasie, ) . luckily, global organizations are committed to addressing the water-quality crisis. the agenda for sustainable development from the united nations, for example, tackles water inequality within one of its seventeen priority goals, to ensure availability and sustainable management of water and sanitation for all. this initiative is a continuation of the united nations' millennium development goals from the s, which also included goals to reduce the portion of the population that lacked access to infrastructure for quality water and sanitation (who/unicef, ). these goals have resulted in access to improved sources of drinking water across the globe, however, water accessibility risks continue to pose governance challenges. it is officially estimated that three out of ten people do not have access to safe drinking water. the vast majority of those affected are in developing countries -almost half of the people drinking water from unprotected sources live in sub-saharan africa, and the united nations has identified water use as a priority for international aid (who/unicef, ). the world bank's report on sub-saharan africa characterized the crisis in the region as a "crisis of governance", and this has ignited unprecedented interest in governance issues across all sectors in africa (world bank, ) . according to the world bank ( ), a country's ability to pursue sustainable economic and social wellbeing of its people is depended on the quality of its governance system. a significant focus has emerged in the literature on the definition of water governance over the past decade. mancheva ( ) , for example, defined water governance as to how decisions that affect water delivery are made, who is involved in making those decisions, and how power is distributed in the service delivery. elsewhere, bakker and morinville ( ) distinct water governance from water management, while acknowledging the two are closely linked. in their review, water governance refers to the decision-making processes stakeholders follow, and how they make decisions; while water management refers to operational approaches we adopt, and the models, principles, and information we use to make decisions. this paper, therefore, observes water governance to include the structures, processes and actors and the dynamic interactions among them that facilitate and influence decisions affecting water delivery services. in terms of water governance approaches, the global water partnership (gwp) ( ) describes ideal approaches as ones that include the ability to design public policies and institutional frameworks that are socially acceptable and able to mobilize social resources in support of them. to pahl-wostl ( ), understanding governance modes is a critical step towards building effective governance approaches and systems to address the complex societal challenge. the author further argued that the development of such dynamic governance approaches and systems requires a combination of purposeful design and self-organization that are essential for dealing with complex water management challenges (pahl-wostl, ). in related studies on the use and management of water, two broad governance themes are prevalent ( de loe and bjornlund, ; johannessen et al., ; taylor and sonnenfeld, ) . first, a more comprehensive view of the institutional context for water governance that is much more distributed or polycentric across multiple scales and includes informal as well as formal institutional arrangements (adams et al., ; basson et al., ; johannessen et al., ) . the other theme explains a mode of planning that strives to be inclusive, collaborative and participatory, and is grounded in discursive decision making (tlor and sonnenfeld, ). bakker et al. ( ) expanding these themes described distributed and collaborative governance as 'shared water governance'. in their study, they explain that shared water governance works best when governments are committed to educate and empower citizens, build trust and help build collective wisdom. in their study, the importance of sustained capacity was noted and further explained that the benefits of shared water governance would only be realized in situations where financial and other resources are guaranteed and sustained over a long term. the ghanaian water governance trajectory follows a pattern where the tradition of state-led, and command-and-control regimes are giving way to models based on decentralised decision making and participatory planning. this decentralised approach involves state and non-state actors which includes water users, environmental organisations and citizens (acheampong et al., ; erdiaw-kwasie and acheampong, ) . it is acknowledged that decentralised approach helps to overcome the multiple complex problems associated with centralised service provision such as delay in decision making in water provision, compromised local knowledge in water decisions, as well as increased transaction cost of services (acheampong et al., ) . in ghana, there are a number of agencies that provide and regulate water under statute law, namely ghana water company limited, the community water and sanitation agency, and the water resources commission. the ministry of water resources, works and housing (mwrwh) is responsible for formulating water supply policy, overseeing operations of gwcl, sourcing for funding from agencies and coordinating sector investment plans. currently, water governance in ghana is undermined by many challenges which impact on intervention outcomes. these challenges range across issues such as stakeholder participation, pro-poor governance, and democratisation of water services. for example, harris et al. ( ) shared that there is little understanding of the importance of localisation and contextualization in how water governance institutions evolve, how it impacts on the lives of individual citizens, particularly on the lives of poor people. in their study, institutions are not seen merely as ''the rules of the game'' but rather, as embedded in practice where they are reproduced, transformed and subverted through interactions and negotiations between actors. further findings show that approaches adopted by institutions leave no room for less organised, ''informal'' interests, especially poor people, to participate and gain access to water. kumasi metropolis is one of the thirty ( ) districts in ashanti region of ghana. according to the ghana statistical service ( ), kumasi estimated population in was , , , which constituted . % of the region's population size. compared to the national and regional population growth rate of . % and . % respectively, the metropolis is growing at a faster rate of . % indicating the attractiveness of kumasi in the region. also, the unique centrality of the city as a traverse point from all parts of the country makes it a special place for many migrants, hence making it a huge commercial centre. this situation has serious implications for water supply in the metropolis. the metropolis is traversed by a major river (owabi) and streams like subin, wiwi, sisai, aboabo, and nsuben. these water bodies, especially owabi, serve as the primary source of drinking water to residents not only within the metropolis but the region as a whole. notwithstanding the critical role played by these water bodies in the socio-economic wellbeing of residents, human activities have threatened the extinction of some of them. building on watercourses by estate developers and urban agricultural practices are some of the human activities that have polluted these water bodies. this may partly explain the frequent occurrence of water shortage in the metropolis . the study area has been categorised into eight ( ) districts using the existing operational districts of ghana water company limited (gwcl) in the metropolis. out of these eight ( ) districts, four ( ) were purposively selected as shown in table . in sampling these four case districts, the metropolis water reports were examined to gain knowledge about areas where water projects have been initiated and executed by the gwcl in the past years. this constituted the basis for the selection of the four case districts for this study. in each district, a community was again purposively sampled based on the presence of local community committees who are actively involved in water-related issues in the metropolis (table ) . this section presents the demographic characteristics of the customers of ghana water company limited (gwcl) in the study area. it discusses the numbers of years of being a customer of gwcl, educational background, age as well as the marital status of the study respondents. although the researchers intended to use a random sampling approach to select the population for the study, challenges faced on the field resulted in a convenient sampling technique being used. the challenges included that the study participants who were randomly sampled appeared to be new in these communities and as a result had limited knowledge about the existing water project. thus, convenient sampling was adopted to help the researchers recruit members of the community who have experiences with the water project under consideration. the respondents interviewed for information in this study are customers of gwcl in the kumasi metropolis as well as officials of the same institution located in the study area. there were female respondents, representing . %; and male respondents, representing . %. on average, a total of respondents representing . % were between the ages of - years, while respondents were between and years, representing . %. study findings indicate that % of respondents had attained a middle school education. besides these results, it is found that about . % had attained secondary or technical school education, about % had attained tertiary education, and . % had no formal education. in the whole, it became evident that the educational level of respondents is rated low, the majority ( %) of them had not had higher education beyond secondary or technical school level. exploring the number of years respondents have been customers of gwcl, . % had been customers of gwcl for years and above, about . % are within - years, and about . % fall within - years. with regard to this study, at the district/community level, key variables that were adopted in the interview and focus group discussion (fgd) instruments included the following: level of involvement in decision making, governance processes and the effects on citizens' participation. the study data sources included households (customers) and heads of ghana water company limited (gwcl). interviews and focus group discussions were held to collect primary data from the households (customers) and institutional heads. in this study, triangulation was established by comparing the interview results with feedbacks obtained from the focus group discussions. given the workload in the field, interviews were carried with the help of field research assistants (fras) who were carefully recruited and trained to assist in the household data collection. they were made to understand the research objectives and all the questions to be asked. this was done to ensure that the field research assistants have a common understanding of the terms and phrases as well as to enable them to explain the questions in the local language to the respondents. during the interview sessions, a semi-structured interview guide was used to collect data from both the residents and the institutional heads in the kumasi metropolis at the regional and district level respectively. the interview guide was grouped into the following; participation of the citizenry, mechanisms for citizens' participation, obstacles to effective citizens' participation, and benefits of effective citizens' participation in decision-making on water-related issues in the study area. also, two fgds were held. the first fgd was held in suame community, which involved other participants from pankrono. on the other hand, the second fgd was held in atonsu, which also involved participants from bantama. each group for the fgd was made up of participants. participants included in the fgds were selected through recommendations from community leaders and key informants by virtue of their experience and involvements in water projects. while the first fgd session was made up of males and females, the second fgd comprised of males and females. participants selected for the fgds were between and years. fgd was used for this study because of its inherent strengths to help explore issues on the approaches for the involvement of the citizens in decision-making on water-related issues, and the challenges that hinder effective citizens' participation. discussions on the benefits of citizens' participation in decision-making on water-related issues in the study area were also done. research approval was obtained from the knust human research ethics committee. here, consent forms were then presented to the ghana water company as well as to the various respondents involved in the research. the researchers ensured confidentiality and anonymity. respondents were assured that the information provided in aid of the study would be used for academic purposes. right to privacy, willingness, voluntary participation, and informed consent was guaranteed to respondents. data collected from the field were coded and edited immediately research assistants returned from the field each day to ensure that all errors are corrected. first, all interviews were tape-recorded, transcribed verbatim and transferred into the nvivo software which was used for the data analysis. the fgds in each community were also recorded and transcribed. qualitative thematic analysis was performed on the gathered primary data, which included organising the data, finding and organising ideas and concepts, building overarching themes in the data, and ensuring reliability and validity in data analysis mechanisms are instruments used to engage customers in the study area in decision-making in water governance processes. the study revealed a wide range of mechanisms that gwcl uses in making decisions on water governance. a majority of respondents agreed that gwcl uses the following mechanisms in making decisions on participation approaches in water governance issues, radio programs, public meetings, surveys or polls, and telephone. it emerged from the interview that . % of the respondents agreed and preferred gwcl engagement of citizenry through radio programs in the metropolis. data analysis from the interview revealed gwcl radio programs informed customers about on-going and new projects implemented in the metropolis. issues usually discussed during gwcl radio programs included illegal connection by households, tampering with meters, encroachment on intake points and indebtedness of customers. for the purposes of ensuring customers' engagement, the audience are allowed to participate in discussions through phone-in-calls. the gwcl, therefore, compiles all received suggestions and incorporate them into their daily water management and delivery decisions in the form of public memos and notices. cases where issues raised require immediate attention, the gwcl deployed its workers to address such rising concerns. "one day i heard officials of gwcl on nhyiraa fm discussing water issues in the metropolis and the way forward. gwcl also announced the impending mass disconnection of customers owing bills. i called into the program to complain about poor customer care in the district and they promised to attend to it. i can say that the issue has been addressed by gwcl'' [c r ] study findings further reveal that radio as a medium and mechanism for stakeholders' engagement in water supply is fast and has a high coverage concerning the number of people reached. nonetheless, there are inherent weaknesses in the radio mechanism to engage stakeholders in decision making. for instance, information on the day, time and the host radio station where such programs are organised are not usually communicated on time. this situation yields endless frustrations and disappointments among customers who solely depend on the radio as a means to participate in water-related issues. consequently, a majority of stakeholders are relegated during decision making on water supply issues that affect them. "i am always not aware of the day and time for gwcl radio programs. as a result, i do not listen and take part in discussions" [fgd p ] . "during gwcl radio programs, i mostly call in to express my views on issues under discussion. unfortunately, the gwcl officials give a maximum of two minutes for callers to express their opinion which is not enough" [c r ]. a public meeting can be viewed as a forum where individual citizens, local committees and gwcl officials come together to exchange information and opinions on water-related issues. the study showed that % of the interview respondents confirmed their preference for participation in water governance through public forums organised by gwcl. the study made it evident that public forum offers a platform for issues such as poor customer care, complaints not attended to by gwcl officials, billing issues, the extension of pipelines in new areas, poor flow of water and construction of overhead tanks, connection and re-connection of households to the water main (the major pipes that transport water to communities in these regions), indebtedness of customers, tampering of meters and development of guidelines on service delivery standard to be discussed. the public meeting provides a platform for customers to meet gwcl officials on a face-to-face basis to channel their grievances concerning service delivery and cost of water supply. it also offers customers the opportunity to ask for clarification of ambiguous trending issues in the news and social media. in addition, the study revealed that continuous face-to-face public engagement enhanced mutual trust-building among stakeholders. more so, the use of public forum also enabled immediate feedback on prevalent issues of water governance within the region. this study revealed that public forum through face-to-face is the best avenue to resolve equivocal situations and solicit increased public participation in water supply programs. "gwcl public forums present both young and the old, rich and poor the same platform to air every opinion on issues" [fgd p ] . "i love public forums because it offers customers the opportunity to meet gwcl officials face-to-face to channel their discontentment with service delivery" [c r ] . although the face-to-face medium of engagement ranked number one ( st), this participation strategy is still characterised with weaknesses that undermine its outputs. firstly, the public forum is usually considered an excellent platform for opposing groups to share their discontentment about differing viewpoints. however, it is observed that although such disagreements tend to be personal ones, it usually consumes a lot of the meeting time and result in limited time for the discussion of pressing community issues. such tensions undermine the expected outcomes of such meetings. secondly, public forums that are poorly organised and mediated by stakeholders may increase projected time specified to commence and end the meeting. thus, exceeding the timeframe for public forums usually results in crossing over of agenda boundaries as well as minimal time allotted for discussing issues. thirdly, during outbreak of epedemic such as covid- and tuberculosis, participation approach like face-to-face meeting become apparently impossible. a significant proportion of study respondents expressed concerns about how the over dependent on this approach undermine timely updates in service provision as well as effective response to customer requests. lastly, the study revealed that not all people at the forum are given an equal platform to articulate their views. people selected to talk during public forums are mostly those with higher societal status who usually monopolise discussions. "sometimes such public meetings degenerate into arguments among participants. these disagreements sometimes lead to physical fights and uncontrolled misunderstandings…'' [c r ] . "mostly public forums start - hrs behind the scheduled time and a lot of time is spent discussing certain issues outside the meeting agenda. this increases the scheduled closing time" [c r ] . "gwcl officials mostly select community leaders and those termed as vocal to take part in discussions. i have raised my hands on several public forums to take part in discussions, but gwcl officials never called me" [c r ]. surveys or polls are utilised by gwcl to uncover citizens' stance on water-related issues in their communities. the study showed that . % of the interview respondents confirmed that gwcl uses surveys or polls to solicit views on service delivery in the metropolis. respondents indicated that surveys or polls by gwcl, in theory, is expected to present residents the opportunity to express their opinions and provide feedback to gwcl on service delivery in the metropolis. similarly, this participation strategy is also anticipated to give locals the chance to evaluate existing water project or programs being implemented. study findings unveiled that the gwcl survey is a preferred citizen's engagement option in water governance because the survey questions are simple and less time-consuming. adding to this, respondents suggested that this participation mechanism creates platforms where residents can provide feedback on the services offered to them by the gwcl. to many respondents, unlike meetings, the nature of the mail surveys adopted by the gwcl breaks the traditional oneway communication by the simple means of offering residents opportunity to share pressing water concerns, encourages suggestion making, as well as serving as checks on the operation of the gwcl. ''in i had the opportunity to participate in one of gwcl surveys. i found the question much more straightforward and concise. such surveys provide us with the opportunity to offer feedback and opinions on service delivery in our communities'' [c r ] . "i took part in the gwcl survey in , and i loved the simplicity of the questions that were asked. also, the whole survey took less than min to finish" [fgd p ] . despite the potential benefits that mail survey participation strategy stands to offer, prevailing conditions undermine this approach. to many respondents, although this participation 'formula' weighs more in terms of its abilities and potentials to promote citizen involvement in pressing water decisions, its application is faced with challenges within the region. for example, the current poor street and housing address system within the kma pose an enormous challenge to the effective use of the survey as an engagement mechanism for assessing and evaluating water-related programs. many respondents indicated that they are usually sidelined in such surveys given that their areas are hardly located in the radar of the postal service providers. in addition, the low literacy levels, as well as a limited number of people with mailboxes, also impede public participation through a mail survey. many respondents further indicated that their views during surveys are hardly incorporated into gwcl policies and programs, which discourages them from participating in future exercises. "i have taken part in two gwcl surveys and by observation, the survey results have not had any impacts on water supply in the metropolis. because of this, i have decided not to take part in such surveys again" [c r ] . "at the moment, many of us in the region do not have mailboxes where survey instruments can be sent through…and this makes surveys of less interest to us" [c r ]. "our customers' interest in taking part in the survey has dwindled significantly and that gwcl has resorted to other mechanisms for engaging citizens in water governance" [manager at north "b"] it emerged from the study that % of the respondents prefer to participate in water governance issues through the telephone. according to respondents, through the telephone customers lodge complaints and provide feedback on service delivery and water projects through the telephone. the telephone is the second preferred mechanism for participating in water-related programs because the conversation saves customers travel time. similarly, the study showed that engaging citizens through telephone dialogue are one of the easiest and effective mechanisms to solicit views of citizens in decision-making on water-related issues. company representatives further confirmed that they do receive many calls on their telephone lines from the citizens on water-related issues in their districts on a daily basis. according to such officials, most of these calls are to do with service interruptions, broken pipelines, faulty meters, and billing problems. "i usually use the telephone to reach the gwcl about my concerns. i think it is much easier and also saves me time" [c r ] "it is easier to contact the gwcl on the telephone about pressing issues such as bill confirmation, correction of bill mistakes, or even unexpected hazards like broken pipes" [c r ]. though the telephone mechanism was the second preferred choice, still it is characterised by some flaws. the study unveiled that the public did not know the telephone contact of gwcl off the head and this inhibits them from directly contacting the company over emergencies and other related complaints. "although gwcl has call centres, many of us (customers) are not aware of such existing facilities in the districts. this undermines our interest to fully participate in water-related issues within the region" [gwclr ] . "also, the contact number of gwcl is not known by many of the customers and this hinders the timely reporting of water issues that need immediate attention" [gwclr ] . "i usually call gwcl especially if this community has water supply issues. in some cases, the network becomes very poor for me to articulate the issue succinctly" [c r ]. situating the study findings within the arnstein's ladder of participation, the participation level associated with the radio mechanism depicts the very least of arnstein's degree of tokenism -'informing'. study findings indicate that pertinent issues compiled by gwcl are only documented without pragmatic steps towards resolution or any form of citizen's feedback. the situation, therefore, renders the radio participation 'formula' more of a one-way communication mode rather than the dialogic mode that is strongly promoted in participatory models (see jiménez et al., ) . in many instances, respondents indicated that although citizens are sometimes informed about these radio sessions, these programs are usually hijacked by the thoughts of the gwcl than proper agreements with residents. reflections on current participation 'formula' depict that there is the need for proper consultation with residents and timely response to residents' demand. taking from a practical perspective, the study foresees two key benefits to both parties -(i) residents will be more willing to participate in any form of consultation activities by the gwcl where local ideas in terms of needs and aspirations can be properly incorporated into water strategies and policies; (ii) gwcl can restore the community trust and support for their actions and operations since residents will feel that their pertinent demands are being prioritised. aligning this with arnstein's typology of participation, ensuring meaningful collaboration will not only help to strengthen 'media' as a reliable participation 'formula' but also, it can restore some level of satisfaction among residents, which arnstein described as 'placation' in his level of tokenism stage of the participation ladder model (arnstein, ) . with reference to arnstein ladder of participation, study findings revealed that the public forum was tagged as the most preferred strategy due to the sense of partnership among respondents. in many instances, study respondents unveiled how public forum offers a much more reliable platform where residents negotiate with the gwcl to ensuring that decent service delivery is offered to them. deducing from study findings, it is evident that the public forum 'formula' depicts more of a give-and-take style that symbolises what arnstein defined as 'partnership' level of participation in his model. given the high optimism expressed in this participation strategy, study findings support earlier observations by akhmouch et al. ( ) and ricart et al., ( ) that promoting partnership with communities in water governance processes help to enhance the capacity of local people to adapt to change in order to maintain or enhance their wellbeing. although there are a number of weaknesses associated with this participation strategy, evidence indicates that this participation 'formula' requires redistribution of power-relations where citizens feel positive about their input into water-related decisions. this finding is in line with the study of mwihaki ( ) who observed that more attention on power and role distribution in water governance can help address water issues faced by many indigenous communities. our study reveals that such transition into a more active participation level via redistribution of power relations can help restore a sense of citizen support and ownership for water delivery decisions. however, study findings show that invitation for public forum by gwcl only offers local residents a mere voice on issue discussion than any substantial influence on major decisions. others also shared that such public forums are organised by gwcl as a prerequisite for meeting funding conditions, an issue well documented in mukhtarov et al. ( ) . such identified participation style by gwcl reflects the 'camouflage' form of participation well described in arnstein's ladder of participation. also, it is evident that respondents held a high level of confidence in the use of the telephone by gwcl towards reaching out to water institutions taking into account time factor. in this sense, the relationship between gwcl and residents who patronise their services is evident since the majority of interactions benefit all parties and not just the service providers. for example, there is raising concerns about the need for gwcl to modify their telephone contacts into a more standardised digit as used by many service institutions like the ghana police, ambulance and fire service for easy recall during times of emergencies. to this call, gwcl confirmed the relevance of such transition and emphasised that whereas it can help save customers money and time, it stands to offer them timely information from clients during emergencies such as broken pipes in the community. therefore, this study confirms more of a 'placation' style of participationone that supports a participatory model that places the satisfaction of customer concerns at its core, whether at operational or a managerial level. for us, the first step towards addressing the challenges of water insecurity and inequity is, therefore, to acknowledge the connection between participation and water governance systems. this is why the who/unicef ( ) report on this theme is strategically important. some fundamental flaws and important shortcomings in existing water governance approaches is documented and acknowledged -it completely ignores the critical link between participation and water governance, as articulated by pahl-wostl ( ) and mancheva ( ) . a productive next step is to acknowledge the subtle interconnections between citizen participation and water governance, many of which are indirect or occur by stealth. recognizing such subtle and indirect interconnections is a promising step towards forging concrete links between citizen participation and water governance, or what hoogesteger ( ) calls "grassroot water governance"-that is, what true water governance might mean among urbanites in the era of water insecurities. finally, water security and equity thinking should be cognizant of the presence of trade-offs, and these should be a central element in water governance research, particularly in developing countries. it is the (often competing or conflicting) interplay between institutional systems and priorities, citizens involvement, community interests and needs, income, and culture, which makes water governance what it is and how it evolves to address water needs -who gets what water, when and how, and who has the right to water and related services, and their benefits. the fact that several recently published high-profile research works (see kirschke et al., ; romano and akhmouch, ) underplay those trade-offs and instead present the solution as being a search for win-win options is possibly pushing us in the opposite direction of where we need to go. while potential synergies should be looked for and built upon, our analysis suggests that the research agenda on water governance in the developing world needs to focus on better understanding these trade-offs. focusing on these trade-offs can help both institutions and societies to understand their entitled roles in the water governance process and navigate efficiently by adopting a workable citizen participation 'formula' for maximum returns. in this contribution, we have illustrated the real, material link that exists between narratives of citizenship participation and water governance and their concrete effects on water accessibility and availability struggles. such struggles are largely driven by long-standing and powerful policy narratives about water service delivery that are poorly documented and administered empirically. it is, therefore, critical that citizens get opportunities to engage more effectively in decision-making systems on water delivery. thus, this paper explores the citizen participation approaches in the governance process, as well as their effectiveness in decision-making on water accessibility and availability in kumasi metropolis in ghana. the study found that the willingness of citizens to participate in decision-making on the water-related process in the study area is high, but current participation mechanisms by the ghana water company suffers from internal and external challenges. the study revealed that ghana water company is preoccupied with the technical aspect of water delivery (that is the physical infrastructure for water delivery) and neglect the social aspect which borders on appropriate engagement strategies that involve customers in the decision-making process. the study found that citizens primarily engage ghana water company at the district level through the following mechanisms; radio programs; public meetings; surveys or polls and telephone. it emerged from the study that citizens partake more in public forums organised by ghana water company in addressing water-related issues in the metropolis. study results reveal that the public forum is the most preferred medium of engagement due to its face-to-face nature. the study again found that ghana water company informed citizens about programs and activities through public meetings in the various districts in the study area. however, the study revealed that these public meetings together with all other participation mechanisms by the gwcl are undermined by some internal and external factors such as time constraints, and dominance from influential stakeholders, which negatively impact outcomes of such sessions. from a policy perspective, the study has provided sufficient ground on the need for customers to be provided with timely information, feedbacks and also addressing service challenges of customers within reasonable timelines. the study also holds the view that good governance needs to be developed to suit local conditionsand hence incremental improvement and flexibility being key in administering water policies. cases where new reforms are developed to help address existing water accessibility and availability challenges, it is critical to ensure that such policy changes are implemented in a more inclusive and integrative manner. in this way, water policies tend to be workable in practice and likely to generate public and institutional support. the study, therefore, recommends that the ministry of water resources and sanitation, ghana water company and other relevant agencies like district assemblies and non-governmental organisations should ensure functional structures at the local level to serve as a liaison between the citizenry and ghana water company. this study suggests that policy options for promoting grassroot participation can be sustainably effective in strengthening water governance systems and can be used in place of policies that consider residents as passive beneficiaries of the water services. it is important that sustainable options for water security are used to reconcile water governance with citizen participation. as long as underlying principles of citizen participation is adhered to and carefully implemented, strategies for promoting meaningful grassroot participation represent sound policies for addressing water governance challenges. strengthening the information deficit model for disaster preparedness: mediating and moderating effects of community participation sustainable urban water system transitions through management reforms in ghana urban water supply in sub-saharan africa: historical and emerging policies and institutional arrangements the myth of 'healthy'competition in the water sector: the case of small scale water providers mapping operation and maintenance: an everyday urbanism analysis of inequalities within piped water supply in lilongwe 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potential for women's self-help groups to improve access and use of public entitlement schemes in india jumping off the ladder: participation and insurgency in detroit's urban planning complement or competition in water governance? analysing two collaborative water management arrangements in one river basin collaborative partnerships and the challenges for sustainable water management the influence of information and communication technologies on public participation in urban water governance: a review of place-based research water supply and environmental management decentralisation as a tool in improving water governance in kenya the emancipatory promise of participatory water governance for the urban poor: reflections on the transition management approach in the cities of dodowa, ghana and arusha innovative forms of citizen participation at the fringe of the formal planning system toward citizen-led planning for climate change adaptation in urban ghana: hints from japanese "machizukuri"activities. the geography of climate change adaptation in urban africa how a japanese system can help african cities adapt to climate change. last accessed the role of governance modes and meta-governance in the transformation towards sustainable water governance how to improve water governance in multifunctional irrigation systems? balancing stakeholder engagement in hydrosocial territories assessing the sustainability of urban water supply systems effective water governance water governance in cities: current trends and future challenges do urban community gardens matter? the case of bulawayo metropolitan province in zimbabwe social power and the urbanization of water: flows of power water crises and governance: reinventing collaborative institutions in an era of uncertainty improving the effectiveness of ugandan water user committees the continuous expansion of citizen participation: a new taxonomy the united nations world water development report: water for a sustainable world international decade for action 'water for life rationales for public participation in environmental policy and governance: practitioners' perspectives special focus on inequalitiesunited nations children's fund (unicef) and knowledge for development: world development report the dynamic electoral returns of a large anti-poverty program key: cord- -s cb sq authors: ndukwu, m. c.; ikechukwu-edeh, c. e.; nwakuba, n. r; okosa, i.; horsefall, i. t.; orji, f. n. title: nanomaterials application in greenhouse structures, crop processing machinery, packaging materials and agro-biomass conversion date: - - journal: nan doi: . /j.mset. . . sha: doc_id: cord_uid: s cb sq abstract the discovery of nanomaterials has flagged off crucial research and innovations in science and engineering. its unique properties and diverse applications present it as the material for the future. the aim of this study is to presents the relative applications of nanomaterial in some aspects of agriculture production. the study discussed nanotechnology applicability in climate control and photosynthesis in the greenhouse farming, hydroponic systems, solar drying, fabrication of crop processing machine components, oxygen scavengers in crop packaging, and micro-organism stimulant in anaerobic digestion for agro biomass conversion. some highlights from the review revealed that nanotechnology can be applied to increase water surface area to volume ratio and heat transfer in the air moving into a greenhouse farming. water cluster can be changed when treated with nanoparticles through ultraviolet absorption spectrum and nuclear magnetic resonance (nmr) spectroscopy resulting in lower micelles to manipulate water delivery in green house farming. nano-fluids or nano-composites can be used to recombine the reactive parts of thermal storage materials after broken at elevated temperature to recover the stored heat for drying purpose during the off-sunshine periods in solar drying of crops. nanomaterials can be a source of electroluminescence light in hydroponic system and act as coatings and surface hardener in crop processing machinery for post-harvest machines. the reviewed work showed that nanotechnologies has good prospect in adding value in agricultural production in the aspects discussed. nanomaterials are particles in the range of to nanometres with a very large surface to the volume ratio used in manufacturing, measuring, manipulation or observing objects at a nanoscale [ ] [ ] . according to ejeta et al. [ ] , this particular property creates a new quantum mechanical effects and delivers an incredible driving force for diffusion at a high temperature. this creates a high degree of association among particles enhancing activities. nanomaterials consist of nanoparticles made from semiconductors, metals, metallic oxides (cofe o , zncofe o , cufe o , fe o , feoh etc.) or any other particles that possess high physio-chemical and electro-magnetic properties [ ] [ ] [ ] [ ] [ ] . there are also a group of nanomaterials made from allotropes of carbon (graphene) rolled into tubes and spheres or silicon tubes that possess high electro-mechanical strength [ ] . nanomaterials can also generally be classified basically by their constituents make up viz; organics, inorganic and hybrid system [ ] . available literatures have shown that the hybrid system of utilisation of nanoparticles can be widely adopted as an efficient technique which can be employed in advance oxidation processes (aops) to enable catalytic degradation of organic matters [ ] [ ] ] . emphases therefore, is on the increase in the manipulation of these particles for technological developments [ ] . this involves the utilization of these materials in the design and synthesis of new structures or particles control mechanism [ ] , water treatment, waste treatment [ ] [ ] [ ] [ ] etc. for example kakavandi et al. [ ] , examined the use of hybrid nanoparticles in the removal of cyfluthrin from waste water. results demonstrated that these nanomaterials exhibited efficiency rates of . and . % for degradation and mineralisation of the pollutant respectively. similarly, in another study, [ ] applied a hybrid system of magnetic nanoparticles (mnp) coupled with ultrasound and ultraviolet irradiations as a possible mechanism for catalytic degradation and generation of scavengers for treatment of petrochemical wastewater. they reported over % pollutant removal within relatively short period. furthermore, mcintyre [ ] , has observed rapid increase in the application of nanomaterials in commercial products. maynard et al. [ ] reported that in the year , over products on the market possess improved characteristics as a result of integrated nanomaterials; this figure has been increased four times in to . the most common nanomaterial used in a commercial product is silver, followed by carbonbased materials, and oxides of metals [ ] . nanoscience has found usefulness in almost every sphere of human advancement including engineering, medicine, natural sciences and agriculture with a huge market projection of more than . trillion dollars in [ ] . although researchers have reported positive utilization of nanomaterial and nanotechnology as a promising technique but it has its drawbacks [ ] . the magnetic nanoparticles acting alone, possess low surface catalytic activity, records low degradation rates, with the resultant high tendency for them to agglomerate due to lower surface/volume ratio [ ] . there is the possibility of its release into consumables which might cause toxic outbreaks [ ] . these metal-based oxides have been reported to elevate inter-cellular ros that can damage the dna [ ] [ ] [ ] . therefore, various countries have developed framework and policies for research, development and control of nanoscience as it has the capacities of influencing both humanitarian and military applications [ ] . in nigeria, the national agency for science and engineering infrastructure (naseni) established the national centre for nanotechnology with the help of the africa institute of technology, federal institute of industrial research, us-africa materials institute and the european union [ ] [ ] . the major interest of the centre is to participate in the global race to exploit the benefit of nanotechnology, seeing that it has become the future technological direction. despite the interest of the federal ministry of science and technology in nanotechnology, developments in nanomaterial and utilization are still at the infant level [ ] . therefore it can be categorized still as an emerging technology in nigeria and most african countries. all over the globe, research on nanomaterials utilization and nanotechnology has expanded significantly in recent years [ ] [ ] [ ] . the recent coronavirus pandemic has highlighted, the great important role the agriculture and food sector can plays in the society [ ] [ ] [ ] . to enhance the productivity of the agricultural and food sector, applications of the nanomaterials and nanotechnology has been adopted [ , [ ] [ ] [ ] . several kinds of research have adapted nanomaterials in the area of soil bioremediation, biochar enhancement for soil enrichment, crop protection, fertilizer application, renewable energy system application in agriculture, bio-sensors, disease control, and coating of crop processing system against corrosion to achieve high processing efficiency and crop detoxification [ ] [ ] [ ] [ ] [ ] [ ] ] . synthesis mechanism for materials makes use of atomic, fungi, molecular, processing of particulates in vacuum or fluid medium with the fungi method the most widely used due to high cost of adopting other methods [ , [ ] [ ] . despite all this, the real contribution of nano-technology to agricultural production is still questioned [ ] . researchers have synthesized different kinds of nanomaterials ( fig. ) with different research materials for agricultural development but the challenge is in the scaling up of this research to the direct benefits of farmers and agro-processors. generally, amongst most countries, nanomaterial in agricultural productivity is less emphasized than in other areas [ ] . the practice in most nanomaterial applications in agriculture is mostly on the treating agricultural product itself or purification of water delivered for irrigation either to improve vegetative growth or yield [ ] [ ] [ ] , nutritional content [ ] , preservation [ ] [ ] [ ] [ ] [ ] , gene expression [ ] and disease control in plants and animals [ ] [ ] [ ] [ ] [ ] . [ ] [ ] therefore, most reviews on nanomaterials in agriculture focused on this area of seed science, precision farming, disease control in plants and animals, fertilizer application, nutrition and biosensors, feed development, the influence of polymeric nano-carriers in agricultural applications and metal-based nanoparticles interaction and transport in the soil and plants [ , [ ] [ ] [ ] [ ] [ ] [ ] . however, new areas can be exploited in agricultural product delivery using nanomaterial enhanced structures as presented in figure . therefore, the objective of this review is to look at these new areas where nanomaterial applications could be explored in agricultural production, crop processing and biomass conversion to the benefit of farmers. these areas are discussed in the subsequent sections. although greenhouse technology is a practicable route to sustainable crop production in regions with adverse climatic conditions, high summer temperatures impede the successful year crop production [ ] . similarly, greenhouse cultivation in hot climatic regions is usually characterized by the high solar thermal load which forms major problems inside the greenhouse environment and limits plant growth [ ] . this is why climate control in greenhouses is currently one of the major goals of engineering in precision agriculture [ ] . table presents a summary of some possible areas nanomaterials can help in climate control in greenhouse farming. nanomaterial can help the plants in the greenhouse to adjust to a progressive climate change. shang et al. [ ] defined progressive climate change as the change in the baseline of the weather. this can be in the form of modified temperature, water need, ph of the environment and nutrient requirements [ ] . this can be achieved without upstaging the ecosystem as they adjust to environmental stress. while the focus of most research works in this area concern the direct treatment of the seed or material for planting, structure and medium for delivery can also be modified to help in climate control. the application of nanomaterial treatment can be expanded in the medium of delivery, mechanisms or systems component fabrication that can enhance the availability and choice of crops produced by farmers in a country by adapting its mechanisms to develop controlled atmospheric farming where crops will thrive. one of these areas where nature can meet nanotechnology is in the controlled atmospheric greenhouse cooling where temperate crops can be grown in a tropical climate under a controlled climate of evaporative cooling or fogging as shown in the application of evaporative cooling technology utilizes water and cooling pads (porous media) of different materials to cool the air [ ] [ ] [ ] [ ] as shown in fig. . the effectiveness of the evaporative cooling is a function of the condition of the porous media which serves as a water reservoir for cooling the incoming air [ ] . therefore, porous materials should be properly wetted to avoid hotspot. to rapidly move water through and uniformly wet the strands of most of the pads, water molecules need to have a large surface to volume ratio. again, the material must be porous enough to facilitate heat transfer between the air and the fluid with air passage into the structure. despite water delivery to the pads through the spray jets which atomize the water, hot spots can still exist in the porous medium. nanotechnology can be applied to increase water surface area to volume ratio and heat transfer in evaporative cooling of the air moving into the greenhouse. this is because research has shown that water cluster can be changed when treated with nanoparticles through ultraviolet absorption spectrum and nuclear magnetic resonance (nmr) spectroscopy [ ] . it was reported that the o -nmr of water half peak was narrowed, chemically shifted, and decreased resulting in higher activity with smaller micelle when treated with nano-materials [ ] . this is because according to li et al. [ ] the size of half peak of water is directly proportional to the size of the molecules. therefore, the technology can be used to manipulate water delivery to the cooling pads to drive the diffusion force through the cooling pads or water delivery during fog cooling of greenhouse or cold preservation of fruits and vegetables in evaporative coolers. this will create a high degree of association among water and cooling pads particles or contact air in the case of fogging and enhances cooling of the greenhouse. fog cooling is simply based on the dispersion of fine water particles into the air stream to increase the rate of heat exchange between water in the air [ ] . therefore, the smaller the water particles, the greater the degree of association with air and heat transfer between them to lower the air temperature. this is important in greenhouse effectiveness because fogging and misting systems effectively lower temperatures up to - o c below the ambient temperature and also provide more uniform temperature and humidity levels inside the greenhouse when compared to the fan and pad evaporative cooling systems, as reported by ganguly and ghosh [ ] in their report on ventilation and cooling technologies in an agricultural greenhouse application. evaporative fogging systems have been increasingly implemented in arid and semi-arid regions to increase production cycles in very warm seasons and also to achieve near-optimum environments for all-year-round production [ ] . according to misra and ghosh [ ] , a fogging system of cooling sprays water into the airstream as fine droplets in the range of - µm in diameter which direct contact with water in the air takes place. cooling is achieved by the evaporation of the sprayed water droplets, usually with a diameter of - µm [ ] . this water droplets size can be reduced more through nano treatment with increased water to air surface contact area as reported by liu et al. [ ] . again, research has also demonstrated that heat transfer in the porous medium can be improved by a higher percentage of nanomaterial additives in the material [ ] . therefore, the quality of the cooling pad in the evaporative cooling of greenhouse or storage space can be improved with nanomaterials additives. another area that nanomaterial can be made useful in greenhouse farming is in the crop photosynthesis. the speed of photosynthetic activities can be improved in greenhouse cultivated crops by impregnating the greenhouse envelop with nano-materials during fabrication [ ] . it has been reported that tio nanomaterial which is a photocatalyst apart from being antibacterial can hydrolyse light under ultraviolet light into protons, oxygen and electron with the proton and electron going into the reaction chain during photosynthesis at the light reaction stage to speed up the process [ , ] . artificial photosynthesis can be induced through the use of photoexcited nanoparticles in a greenhouse envelop as has been suggested [ ] . other materials like copper and silver which are antibacterial can also be used in building the structural components of a greenhouse. hydroponics is the technology of growing plants in the absence of soil [ ] . many crops especially fruits and vegetables are grown with this method all over the world [ ] . this is an area farmers can exploit the nanotechnology. a nano phosphor-based electroluminescence lighting device can be used as a source of light in this system [ ] . manipulation of the growth process and nutritional content of plant can be easily achieved in a regimented setting like greenhouse and hydroponics. therefore, integration of nano research will be highly effective as a report has shown that green nanomaterials can be grown in plants in this kind of settings [ ] . crop processing is a major challenge to farmers all over the world. researchers are still working to improve the efficiency of all unit operations involved in crop processing ranging from moisture reduction to unit size reductions and packaging equipment [ ] . according to ndukwu et al. [ ] , about % of the crop produced globally was lost in . the situation might not have changed considering the slow pace of technological development in most developing nations. the following subsections will consider possible applications of nanotechnology/materials in crop processing. drying is a major challenge in crop processing. in this era of energy optimization with less environmental impact, solar dryers have been utilized to dry agricultural products instead of fossilbased dryers [ ] [ ] [ ] [ ] [ ] . the challenge for most solar dryers has been the intermittent nature of solar energy available for crop drying, therefore thermal storage materials have been utilized [ ] . again, collector efficiency requires improvement in the increased harnessing of available incident solar radiation. to enhance the efficiency and speed up the mechanism of the energy storage of these thermal energy storage materials, nano-fluids or nano-composites especially phase change materials that suffer incongruent melting or supercooling is being investigated. it has been noted that the intermolecular bonding of most energy storage materials is broken at elevated temperatures to different reactive parts [ ] . these reactive parts can be recombined with the help of nanomaterials to recover the stored heat for drying purpose during the off-sunshine periods in solar drying of crops [ ] . also, sorption heat storage, where the material absorbs or adsorbs hot gas or vapour for heating purpose is being investigated too [ ] . the efficiency of solar crop dryers can be improved on the harnessing of the sun's radiant energy by incorporating nanomaterials as additives to the collector [ ] . due to a large surface area to volume ratios of nanomaterials, more surface of the collector will be exposed to solar radiation [ ] . however, nanofluid in solar drying is very scarce [ ] and presents an exciting research prospect especially nanofluid phase change material [ ] due to its characteristic high energy density. again drying of medicinal, aromatic plants and green vegetables are always a challenge for farmers to preserve the attractive greenish colour of the plants. currently, the best method advocated for drying these plants is shade drying [ ] . therefore, researchers are simulating the shade drying in solar dryers by developing special glass cover for the collector. integration of engineered nanomaterials can be a breakthrough with higher efficiency. dehkordi and keivani [ ] reported an intelligent nano-crystalline glass produced by the method of aero-gel. the glass changes to dark colour when subjected to intense light and brighter at reduced sunlight. with this intelligent glass, the photosynthetic reaction of dried green vegetables can be limited to retain their dry green colour pigment. also, glasses that can reflect proportions of incident solar radiation have been reported by vatan-dolat-xah [ ] . therefore, overheating of solar dryers and subsequent denaturing of sensory and nutritional qualities can be prevented on the drying of medicinal plants and vegetables which require lower drying temperature than other crops. another area of interest in crop processing is the contact between the processed product and the machine components during dehauling or size reduction operation. this raises safety concerns on the processed agro materials due to the oxidation of these machinery surfaces. however, engineered nanomaterial structures show unique properties for high material performances and can be used in the coating of surfaces of machinery for crop processing with high thermal performance and also utilized in the design of nano-sieves for size reduction of crops as has been suggested by researchers in the food industry [ ] . these materials can create additional strength and resistance of machine surface to environmental factors and corrosion. ceramic nanoparticles have been reported to enhance the thermal stability of coated moving parts [ ] . mica and talc as paint have shown to reduce weight losses of equipment and prevented the formation of a hard layer [ ] . the major risk being assessed for nano-materials by an expert in agricultural processing as recommended by codex is on the addition or direct coating of agricultural products. however, they suggested that there may be no need for expert risk assessment but protocols can be developed to measure the extent of the residue of nano-materials if the process machinery is coated with nano-materials [ ] . nano-lubricants exist and can be used to reduce wear and tear in processing machines [ ] . with the improved knowledge on the interaction of sliding layered materials, their friction response has been improved [ ] . the corrugation of interlayer sliding can be reduced with the application of nanomaterials like graphene [ ] . copper nanoparticles and fullerenes additive in lubricants have improved the lubrication properties of lubricants [ ] . in automated systems, for a clearer view of the central display board, engineered nanomaterial can be integrated into the design. magnetite nanoparticles (cationic surfactant coated silica-modified) have been used as an adsorbent for the extraction and prevention of metal reductions [ ] . this can be used in coating and design of materials for fabricating agro-resources machinery. generally, big data approach in information, risk and safety concerns of various nano-based activities have been brought together in a pool by the world health organisation and food and agricultural organization and is accessible through a common dashboard [ ] . nanomaterials have been introduced to improve the flexibility, moisture stability, low volatility, gas barriers and temperature stability in packaging materials for fruits and vegetables [ ] [ ] [ ] [ ] [ ] [ ] . its application has transcended the limits of the packaging of food materials and considers monitoring of stored food material for quality retention, safety, as well as better product packaging and improvement of package biodegradability. nanosensors (especially those from bio-chip dna) are used to detect pathogens and contaminating agents; selection of nanoparticles with good adhesive characteristics for binding and elimination of pathogens. the merits of these nanosensors include but not limited to a high level of sensitivity, compatibility, cost-effective, and proximate real-time detection [ ] . packaging of agro-products using nanomaterials has the following notable potential benefits: improved-performance packaging with greater mechanical and barrier characteristics; packaging with antimicrobial packaging integrated nanoparticles, such as silver nanoparticles; smart-tech packaging that could avert or respond to crop spoilage (for instance applying polymer films that gives out different colour at different stages of product spoilage), dirt repulsive and water impervious packages. however, the barrier ability of the packaging structure can be improved by introducing an oxygen scavenger into the matrix to intercept and scavenge the oxygen through chemical reactions as it passes through the walls [ ] [ ] [ ] [ ] . most of the metal-based nanomaterials are oxygen scavengers especially iron, coatings, extrusion additives and labels, can be used [ ] . again, lightweight packaging materials have been introduced by the integration of nanomaterials coupled with an increased storage life of crops through the production of nano-sensors that respond to environmental changes and detect pathogens [ ] . silver nanoparticles have been used in packaging horticultural crops although not yet approved by the european food safety authority (efsa) but has been approved by the united states for food and drug administration (fda) for packaging [ ] [ ] [ ] [ ] [ ] . nevertheless, the integration of nanoparticles into packaged material for a crop is still a danger due to the migration of this particle into products [ - . this has been reported by echegoyen and nerin [ ] in the investigation of the release of nanoparticle from a container made with nano-silver. nevertheless, research is still ongoing to produce materials that will be able to bridge this migration and douse safety concerns in nanopreservation and packaging. accurate biological monitoring of nano-enhanced materials is very important for global food safety [ ] . generally, one of the major concerns in nigerian agricultural sector is how to dispose of a large amount of agricultural raw materials and residues (biomass) generated either in the farm, poultry houses, livestock pens, or agro-processing industries etc. however, with the increasing energy demand globally and less dependence on fossil-based energy, the emphasis is shifting to bioenergy. therefore, this agrobiomass is now converted to bioenergy for fuel of higher energy densities [ ] . available methods adopted include hydrogenation, pyrolysis, transesterification and gasification [ ] . nanomaterials have been reported in the reforming of this biomass before processing and also in lipid extraction [ ] [ ] [ ] ] . they can help to stimulate microorganism for anaerobic digestion. cuo and mgo nanoparticles have been used as biocatalyst during transesterification [ ] . wastes from nanomaterialengineered crops can increase the bio-oil and biodiesel yield [ ] . therefore, technology can serve as a vehicle for biofuel of the future [ ] . biochar, which is a product of biomass pyrolysis is used to enhance soil nutrients or soil amendments [ ] . it has a very large surface area to volume ratio with a high microstructure [ ] [ ] . research has shown that converting this biochar into micro-nanocomposites has improved its activities in both soil and industrial applications [ ] [ ] . manjunatha et al. [ ] suggested other advanced techniques of exploitation and valorization of agro-wastes which include: ( ) cellulose nanofibers: this is the most common biopolymer obtained from plant materials (lignocellulose), especially wood which is the most vital source of cellulose for industrial use. world production of cellulose of about - t/yr of cellulose has been estimated to be produced through the photosynthetic process [ ] . in-plant tissues, micro and macro fibrils characterize the building units of the structural ranking of cellulose fibres (fig. ). microfibrils also consist of basic fibrils (nanofibres) with a diameter range of - nm based on the cellulose derivative (fig. ) [ ] . [ ] . recently, nanocellulose has been seen as a promising bio-based nanomaterial with outstanding fascinating optical characteristics, high strength and good specific surface area [ ] . nanocellulose can be obtained and chemically improved for different nanocomposites. numerous crop wastes, like soy husk, wheat straw, sugar beet pulp, potato pulp and rutabaga, are potential raw materials for production of cheap and of nanocellulose [ ; ( ) rice husk-based silicon nanomaterials: rice husk is the outer coat of a rice grain which guards the seed during the crop developmental phase. it contains about - % wt. of lignocellulose and - wt % of silica. silicon ranks nd in importance amongst all elements. somanathan et al. [ ] noted that grasses consume enormous quantities of si and store it in phytoliths as amorphous hydrated silica (so nh o); about - % of silica content can be obtained from grass ashes. with the expanding research and innovations in science and engineering, nanomaterials have gained growing interests in agricultural activities. its distinctive ability to diffuse at high temperatures as well as its large surface to volume ratio has aided its projection as a cutting-edge innovation fit for technological advancements. while nanomaterials have found relevance in medicine, natural science and engineering, however, in the agriculture sector, a direct value of nanomaterials to the farmers and agro-processors is yet to be solidly established especially in growing economies like nigeria. this review has explored the areas which the peculiar nanomaterials can be used in agricultural structures. the controlled condition has been a daunting consideration in greenhouse cultivation and nanotechnology can efficiently be employed in the atmospheric control mechanism of the greenhouse structure by increasing the surface area to volume ratio of the coolant for enhanced heat transfer in evaporative or fog cooling system of the greenhouse, thereby helping crops to adjust to a progressive environmental change. it has also been revealed that incorporating photo-catalytic nanomaterials into the material structure of greenhouse during fabrication can present the advantage of quickening photosynthetic process in greenhouse crops which results in faster and healthy crop growth. application of nanotechnology in hydroponic farming is a viable area to be largely explored and techniques to incorporate appropriate nanomaterials into the system further developed. it can be a source of electroluminescence light in hydroponic systems. additionally, nanotechnology has also found application in crop processing machinery for coatings and surface hardener in crop post-harvest machines and in thermal storage and collector for solar systems where nanomaterials have the potential to improve the efficiency of solar crop dryers as a result of their increased surface area to volume ratio, thereby exposing more collector surfaces to solar radiation. also, nano-lubricant can reduce wear and tear in parts of processing machines. nanomaterials have also been found useful in anaerobic digestion and as such can be useful in agro biomass conversion. overall, nanotechnology can promote and drive the next revolutions in agricultural structures but there are scarce data on the health and environmental impacts of these nanomaterial enhanced agricultural structures. in packaging, there are concerns as to the effect of the interactions of these nanomaterials with packaged foods. these health concerns need to be addressed by further research. what are the current possibilities, nano today, ( ), - . 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applications in agriculture:a review silica nanoparticles in drug delivery and biomedical applications synthesis of biogenic silica nanoparticles from rice husks for biomedical applications rice husk silica derived nanomaterials for sustainable applications biogenerated silica nanoparticles synthesized from sticky, red, and brown rice husk ashes by a chemical method graphene oxide synthesis from agro waste nanotubes from carbon nanomaterials for rechargeable lithium batteries carbon nanotubes the covid- pandemic: anticipating its effects on canada's agricultural trade food security and canada's agricultural system challenged by covid- agriculture, transportation, and the covid- crisis nanotechnology in agriculture and food production nanotechnology in sustainable agriculture: present concerns and future aspects nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives preparation, characterization and catalytic potential of γ-fe o @ac mesoporous heterojunction for activation of peroxymonosulfate into degradation of cyfluthrin insecticide efficient treatment of saline recalcitrant petrochemical wastewater using heterogeneous uv-assisted sono-fenton process photo-assisted catalytic degradation of acetaminophen using peroxymonosulfate decomposed by magnetic carbon heterojunction catalyst engineered nanomaterials (enms) and their role at the nexus of food, energy, and water photocatalytic degradation of organic pollutant with nanosized cadmium sulphide production of biodiesel from microalgae via nanocatalyzed transesterification process: a review key: cord- -uarxxalj authors: oluwole, adewumi olufemi; omotola, elizabeth oyinkansola; olatunji, olatunde stephen title: pharmaceuticals and personal care products in water and wastewater: a review of treatment processes and use of photocatalyst immobilized on functionalized carbon in aop degradation date: - - journal: bmc chem doi: . /s - - - sha: doc_id: cord_uid: uarxxalj the presence of emerging contaminants such as pharmaceutical and personal care products in many aqueous matrices have been reported. one of such matrix is streams of wastewater, including wastewater treatment plants inflows and outflows and wastewater flow by-passing wastewater treatment plants. their persistence arises from their resistant to breakdown, hence they may remain in the environment over long time, with a potential to cause adverse effects including endocrine disruption, gene toxicity, the imposition of sex organs, antibiotic resistance and many others in some aquatic organisms exposed to arrays of residues of pharmaceutical and personal care products. among the treatment techniques, advanced oxidation processes have been reported to be a better technique through which these ppcps can be degraded in the wwtps. heterogeneous photocatalysis using various photocatalyst immobilized on solid support such as activated carbon, graphene and carbon nanotubes in aops have been shown to be a viable and efficient method of ppcps degradation. this is because, the performance of most wwtps is limited since they were not designed to degrade toxic and recalcitrant ppcps. this review highlight the occurrence, concentration of ppcps in wastewater and the removal efficiency of heterogeneous photocatalysis of tio( ) immobilized on solid supports. the expansion in the world's population, economic development, the industrial revolution, and climate change has led to an increase in the quantity of waste generation as well as an increase in the introduction of specific categories of compounds referred to as emerging contaminants, into the environments. the u.s. environmental protection agency (epa) described "emerging contaminants" as chemicals or materials which were previously not listed for routine monitoring, characterized by a perceived potential or real threat to human and environmental health, and lacks published health standard, environmental protection laws, and regulations [ ] . according to the u.s. geological survey (usgs), emerging contaminants are "any synthetic or naturally occurring chemical or any microorganism that is not usually investigated in the environment but can find its way into the environment and cause known or suspected adverse ecological and/or human health effects". hence, there is an urgent need to develop methods for the removal of these emerging contaminants from the environment. oluwole et al. bmc chemistry ( ) : emerging contaminants can be classified into several groups, which include; perfluorinated compounds (pfcs), pharmaceuticals and personal care products (ppcps), brominated flame retardants (bfrs), microplastics and toxins from harmful algal blooms, etc. [ , ] . concerns about the potentially adverse effects of these substances on human and ecosystem health are arising from their persistence, environmental accumulation tendency, exposure potential, bioaccumulation/biomagnification potential, and bioactivity. these contaminants may, therefore, be prioritized for regulation, depending on the information pool from monitoring data regarding their occurrence, their toxicity, potential health effects, and public perception. a significant index factor of public and community health is access to good quality water, which is imperatively crucial for all life forms. the pollution of the water system may result in an outbreak of water-borne diseases, which can have acute or long-term health implications and even death. humans and animals are susceptible to these diseases because polluted water is mostly the exposure pathway to infectious pathogens and carcinogenic organic and inorganic contaminants. for instance, while the dissolution of active pharmaceutical ingredients (apis) in aqueous matrices is variable; they are primarily water-soluble and have such properties that allow them to permeate membranes and persist in the biological systems. the inherent properties of apis, however, raise questions about their environmental risk, the potential for accumulation, and bioactivity within an aquatic environment [ ] . as with administered dosages on target organisms, pharmaceuticals are capable of altering biochemical and physiological processes in many non-target organisms. hence, their capacity to induced undesirable consequences on native and peripatetic species of organisms exposed to apis contaminated environment. although the presence of ppcps in the environment has been noticed for some years now, the full extent of their presence and the risk associated with their presence in the environment has not been fully established. also, the removal of these contaminants in wastewater treatment (wwtps), before their discharge into surface water has proven to be difficult due to their low occurrence concentrations, and the challenges arising from analyzing them [ ] . therefore, there is a need to modify and upgrade the existing wwtps to be able to resolve or remove these contaminants. the conventional wastewater treatment methods which include; physical, chemical or combination of physicochemical process, involving the following: filtration, flocculation, coagulation process, sedimentation, biological processes, membrane filtration, chlorination, adsorption process via activated carbon (ac), carbon nanotubes (cnts), graphene oxide (go), ozonation, photo-catalysis ultraviolet irradiation, ultra-sonication, and others. unfortunately, these methods cannot adequately remove organic pollutants from wastewater. in addition to the processes being chemically and operationally intensive, they require large systems, infrastructure, and engineering expertise, which make them burdensome, ineffective, time-consuming, and costly [ , ] . moreover, the use of nano-adsorbents in the removal of ppcps from water matrices has been associated with some challenges, among which are smaller particle size, generation of secondary pollutants, inability to reuse, or regenerate etc. also, the use of semiconductors as catalysts in the degradation of ppcps encounter the following drawbacks such as; requirement for high ultraviolet radiation for their activities due to wide bandgap energy within them; some are carcinogenic, e.g., tio ; there is also the challenge with electron-hole pair recombination after use [ , ] . hence, the need to design materials that are green, economically efficient and effective methods for pollution control and prevention are needed for environmental protection and effluent discharge to have minimal impacts on human health and the biosphere. consequently, the advanced oxidation process (aop) has been reported to be one of the most promising methods that can be employed for mineralization of complex organic pollutants as an effective alternative technique that involves the in-situ generation of strong oxidation radicals sufficient enough to degrade and eliminate organic pollutants within environmental matrices. aop is efficient in the removal of hazardous pollutants and their mineralization into non-toxic inorganic aliphatic acid, co and water as compared to other decontamination techniques like; adsorption, coagulation, flocculation, sedimentation, bio-filtration, etc., as a result of the generation of active oxidizing agents such as hydroxyl radicals, superoxide, ozonide and photo-produced electron-hole pairs [ ] [ ] [ ] . aop can be achieved by direct ozonolysis, catalytic oxidation, homogenous/ heterogeneous catalyzed oxidation, with the more recent advances in photocatalytic oxidation which is one of the green technologies that has attracted scientific interest as viable alternatives for the treatment of wastewater because of its low operating cost, nontoxicity, and effective reduction of contaminants [ , ] . over the years, efforts have been directed towards synthesizing various nanocomposites for photocatalysis purposes. this technique is one of the most preferred aop because it offers the possibility of utilizing naturally available and renewable solar energy as a potential energy source containing visible light in the presence of a suitable photocatalyst prepared from metal oxides or semiconductors as catalyst immobilized on various solid supports for the photodegradation of pharmaceuticals. unfortunately, the photocatalytic performance of most semiconductor photocatalyst is still low due to lack of enough active sites, large band gap, utilization of photons from the sun, and limited fast photoinduced charge carrier recombination [ , ] . hence, the development of efficient and highly stable photocatalysts with increased light absorption in the visible spectrum, reduced band gap, and decreased rapid electron-hole pairs recombination rate is necessary for the photocatalytic degradation of organic pollutants under visible light. hence, the aim of this review is to discuss the occurrence of pharmaceuticals and personal care products in the environment, highlights the failure of the conventional methods in degrading pharmaceuticals and personal care products from the water matrices and why photocatalytic degradation processes of pharmaceuticals and personal care products via the use of various semiconductor photocatalyst immobilized either on other semiconductors, metal oxide or on different carbon sources such as activated carbon, carbon nanotubes, and graphene oxides has been able to degrade these organic pollutants to propose more alternative nanomaterial that is green, efficient, non-toxic, cheap and more effective with a short time for the photocatalytic degradation of different organic pollutants. pharmaceuticals and personal care products (ppcps) consists of apis, moisturizers, lipsticks, shampoos, hair colors, deodorants, toothpaste surfactant, and many household products that are mainly used for improving the quality of daily life [ ] and their degradation products have been inadvertently present in the aquatic environment since the s [ ] . their residues have been detected in all types of surface water, groundwater, and the oceanic environment in the last years [ ] . these compounds find their way into many aquatic environments receiving contaminated water from urban wastewater streams (domestic/industrial discharges, wwtps effluents, etc.), which in turn get transferred into the water cycle, even reaching drinking water, as a result of their hydrophilic character and low removal efficiency of wastewater treatment plants (wwtps). pharmaceuticals generally include prescription or over the counter (otc) veterinary/human drugs and nutraceuticals administered for prophylaxis/therapeutic and health supplements purposes. they are classified as antibiotics, analgesics, blood lipid regulators, natural and synthetic hormones, β-blockers, anti-diabetics, antihypertensive, and many more products that are used for health purposes [ ] . these substances are continuously discharged into the aquatic environment from a point and non-point domestic and industrial sources. a global survey has indicated the presence of several classes of ppcps in most aquatic environments. unfortunately, these data rely primarily on studies conducted in europe, north america, scandinavia, and a few other places. the availability of such data in those parts of the world is because research on ppcps and many endocrine disruption contaminants/pollutants in african countries and especially in the south africa aquatic system is scanty [ , ] . despite the lack of data, the occurrence of some ppcps at concentration levels that sometimes surpassed µg/l level in wwtps effluents and environmental waters have been reported [ ] [ ] [ ] . research studies on remediation of contaminated/polluted aqueous matrices and or the removal of these contaminants in drinking treatment plants and or during wastewater treatment before discharge to surface water is still far behind and probably lacking. commonly detected pharmaceuticals in different environmental matrices and their occurrence levels are as listed in table below. pharmaceuticals can be classified into different active organic groups of compounds [ , ] . these include but not limited to; (i) antibiotics which comprise penicillin, tetracycline, sulfonamides, macrolides, fluoroquinolones, and β lactams; (ii) steroids hormones many of which have been implicated as endocrine-disrupting compounds such as estrogens, estrone, estriol, -β-estradiol, -α-ethinylestradiol, testosterone, etc. (iii) analgesic and non-steroidal anti-inflammatory drugs (nsaids) which are one of the most prescribed groups of pharmaceuticals that include acetaminophen, diclofenac, ibuprofen, and naproxen; (iv) antiepileptic drugs such as carbamazepine, used in reducing the frequency of epileptic seizures; (v) blood lipid regulators, e.g., bezafibrate, gemfibrozil, fenofibric acid and clofibric acid; (vi) β-blockers such as salbutamol, atenolol, sotalol, theophylline and metoprolol used in treating hypertension and cardiac dysfunctions; (vii) antineoplastic such as cytostatic drugs used in cancer therapy; etc. antimicrobial agents, fungicides, disinfectants, synthetic musks, some preservatives, some sunscreen uv filters, etc., may also be referred to as pharmaceuticals depending on the description of use. personal care products (pcps) groups include; (i) pesticides/insect repellants, (ii) musk; widely used as fragrances of many personal care products such as; perfume, body lotion, hair care products, shower bath products facial essence and numerous household products, (iii) soaps and detergents, (iv) sunscreen uv filters, which are chemicals that serve as absorbers and protection of the skin from different ultraviolet radiations from uv-induced damages, (v) triclosan; a lipid-soluble, broad-spectrum, antimicrobial agents which are used as preservatives in personal care products such as hand soap, shampoos, detergents, toothpaste, sunscreen, deodorants, and (vi) antiseptic which serves various purposes in medical devices, household items and as additives in packaging textiles and functional clothing [ ] [ ] [ ] . some of their structures are given in fig. the global annual consumption of ppcps in developed world and countries such as brazil, russia, india, china, and south africa has been reported to have increased over the last decade, due to their medical use for prophylaxis/therapy, and economic use for viability sustenance of commercial aquaculture and livestock agriculture [ ] . as a result, residues of compounds of ppcps are continuously released into wastewater via wash-off, urine, and feces as parent compounds, derivative conjugates, or metabolites [ ] . ppcps can also enter the surface water through direct discharge from industries [ ] , hospitals, household's/domestic wastewater and through surface runoff (as result of the use of contaminated biosolids as manure spread on agricultural land), reaching water bodies and groundwater by leaching or bank filtration. other means through which ppcps enter into the aquatic environment include disposal of unused medicines into landfills, irrigation with wastewater, off-label emissions, and disposal of carcasses of treated animals [ ] , and sewage or wastewater treatment plants [ , ] . brauschet al. [ ] reported that the wwtps are the main route through which ppcps are discharged into the aquatic environment. they have been found in lakes, rivers, groundwater, marine, and coastal area waters and drinking water [ , , ] . aquatic sediments can also hold a substantial amount of ppcps because of their affinity binding/sorption capacity [ ] . thus, excess ppcps do not remain in the water for long, as they partition between water, sediment, and other components of the aquatic ecosystem, via diffusion, seeding, silting out of suspended particle bonded-ppcps and other physical/chemical processes. aside from the application of contaminated biosolids on farmland soils, several ppcps reach the soil and different terrestrial domain through the deposition (air/wet) of short and long-range atmospheric transferred aerodynamic sized ppcps particulates [ ] . a schematic diagram showing potential sources and pathways of pharmaceuticals is shown in fig. . the soluble characteristics and bioactive nature of many pharmaceuticals and some personal care products may facilitate their biotransformation and depuration in biological features. however, this is not the case with most ppcps in the environment, where they do not readily degrade. many pharmaceuticals are discharged from the human and animal bodies in a mixture of their parent compound and conjugate metabolites [ , ] . the extent of body metabolism and pharmacokinetics index is a function of the ratio between the parent compound (unmetabolized) and the conjugate metabolites concentrations measured in urine or feces [ , ] . they may also monitoring metabolites of pharmaceuticals in the environment is, however, limited by the lack of reference standards of environment bound pharmaceutical metabolites, resulting in the scanty information on the occurrence levels, behavior, and half-life of conjugates metabolites in the environment. for instance, and huerta-fontela et al. [ ] reported a concentration range of between and ng/l for carbamazepine epoxide (a carbamazepine metabolite) in influents wastewater, while the concentration of its parent compounds (carbamazepine) range between < . and ng/l. there is, therefore, a need to understand environmental transformation/metabolism, transformation products (metabolites), and analysis of pharmaceutical metabolites. this action is essential because of the expected time-bound decrease in the environmental concentration of parent compounds, with a corresponding increase in metabolites' level, as well as the potential of the metabolites, eliciting probably greater than parent compound adverse effects in the environment. the benefits of the intended use of pharmaceuticals and many personal care products are enormous. the consumption of drugs evokes specific biological responses in the host, after which - % of excess unused doses and some conjugates metabolites are ultimately excreted and passed into the environment [ , ] . their leak and presence in different environmental matrices could cause a risk on non-target organisms, mainly because of their weak degradation, persistence, and biological activities. organisms within the aquatic and terrestrial environments have the potential to be most affected. recent reports indicated that drinking water sources, especially those receiving water input from contaminated sources, contain residues of ppcps, and thus may need further treatment to remove the pollutants before consumers supply [ ] . there has been a growing concern about the environmental impact of pharmaceuticals and personal care products (ppcps) in the aquatic environment [ ] as a result of the fact that they can potentially elicit health and ecological impacts [ ] . furthermore, ppcps may exist as mixtures with other contaminants in the environment and, therefore, can cause synergistic detrimental effects to both aquatic and terrestrial organisms even at low concentrations [ ] . antibiotics usage is common in animal husbandry for prevention, treatment, and growth purposes, making their over-use result in the genetic selection of more harmful bacteria [ ] . continuous exposure to antibiotics was noted as a probable cause of induced drug resistance in many pathogenic organisms, including bacteria. steroid hormones use as sex steroids, contraceptives, and birth control pills can interfere with the endocrine systems, or act as anti-androgenic ligands [ , ] . endocrine disruption leading to adverse effects such as sterilization, the imposition of sex organs (imposex), and feminization of some vertebrate, e.g., fish, mollusks, and other aquatic species have been reported [ , ] . for instance, solé et al. [ ] reported that the presence of the hormone estrogen and progestogens at concentrations as low as approximately . µg/l caused endocrine-disrupting manifesting in fish feminization and decrease infertility. the accumulation of diclofenac on rainbow trout even at a low concentration of µg/l had detrimental endocrine effects. in contrast, exposure to a mixture of acetaminophen, carbamazepine, gemfibrozil, and venlafaxine at concentrations between . and µg/l was noted to cause effects such as tissue degeneration, a decline in embryo production and increase in embryo mortalities in zebrafish [ , ] . exposure to endocrine-disrupting contaminants in the aquatic environment was also reported to lead to the modulation of hypothalamus-pituitarygonad (hpg), hypothalamus-pituitary-thyroid (hpt) and hypothalamus-pituitary-adrenal (hpa), and resulting in the tampering of the activities and functions of various physiological traits in some non-target vertebrate aquatic organisms [ ] . drugs such as carbamazepine, levetiracetam, lamotrigine, and valproate used as antiepilepsy which have been detected in drinking water supply in south africa was fingered to have the capacity to cause a number of several reproductive endocrine system side-effects in certain fish species as well as in humans [ , , ] . nsaids like naproxen and ibuprofen that are often not resolved in wwtps may also induce an adverse effect on the endocrine systems of non-target vertebrate organisms of wwtps effluent receiving waters. for example, japanese medaka fish (oryzias latipes) exposed to . µg/l concentration of ibuprofen manifested delayed hatching while concentration reaching mg/l in humans, can lead to an increase in blood plasma levels. extraneous levels of widely distributed potentially toxics ppcps and edcs have been reported in many aqueous matrices worldwide. a large percentage of south africa waters and aquatic ecosystem receives ppcps contaminants via human excretions and improper discharge of pharmaceuticals into the sewage system [ , , ] . consequently, many non-target marine organisms have magnified drug residues to a varying extent in different organs of their bodies. ppcps such as acetaminophen, caffeine, , -dimethylxanthine, dehydronifedipine, tetracycline, oxytetracycline, sulfonamides, macrolides, and ormetoprim have been reported to be found in fruits, vegetables, fish, meat and even milk and dairy products as a result of uptake or exposure to ppcps contaminated environment [ ] [ ] [ ] . thus, there is a need for the implementation of appropriate and effective environmental remediation to secure the health and safety of biological features, as well as environmental health and safety. several water treatment procedures and technologies have been developed and used for resolving contaminants in the environment, especially water and wastewater. the removal can be achieved through several processes, including; physical, biological, and chemical methods. the physical process may involve filtration where contaminants' removal is achieved by transforming one phase to another, leading to the production of highly concentrated sludge that could be toxic and more difficult to dispose of [ ] . the biological process in water treatment revolves around aerobic and anaerobic degradation/digestion of nutrients and organics, while the chemical process is based on oxidative decomposition. however, many of these stepwise treatment techniques are insufficient, in that they are not able to remove or even degrade residues of ppcps due to their deficient environmental concentrations, and because they were not initially factored in their design for such purposes [ ] . daughton and ternes [ ] reported that ppcps are not entirely removed during wastewater treatment because the wwtps are only designed to remove suspended solids, pathogens, and nutrients from wastewater using a serial link of processes to eliminate impurities, reduce nutrients and organic load and improve oxygen demand levels. petrović et al. [ ] , in a report, noted that the majority of the existing wwtps are not designed to either treat or remove these contaminants and their metabolites. ineffective treatment of wastewater has been listed as a significant source of ppcps in many receiving fresh surface water systems and other aquatic environments, especially those freshwater systems used as domestic water sources. there is, therefore, a need for the development of more advanced, efficient, and powerful water and wastewater treatment technologies for the treatment of ppcps contaminated water, wastewater/effluent from municipal and industrial wastewater discharge streams [ ] . this measure is critical and very important in eliminating the potential toxicity and health implication that may arise as a result of exposure of aquatic organisms and possibly humans to these metabolites. the development of facile, cheap, and green methods that can be employed in the treatment of pollutants has been a significant area of research in the field of environmental science and technology. it is important to note that the ability of certain ppcps to sorb onto bio-solids and siliceous solids, suspended solids confers the need for remediation of contaminated sediment as well as contaminated soil, where they may be ultimately sunk into, or be discharged onto via the application of bio-solids as manure on land. over % of ppcps are released into the environment from the human body in the parent form un-metabolized, i.e., without transformation [ ] . these are fed through into the wastewater treatment plants for removal or decomposition during the water treatment process. many wwtps are, however, unable to wholly eliminate most ppcps, hormones and their metabolites, resulting in their presence as residues in wastewaters, surface waters, sediments, and sludge. this inefficiency of wwtps could be attributed to their conventional integrated treatment uses for biological treatment and microbial decomposition as a process for the breakdown of complex organics, nutrients, and activated sludge for the removal method of contaminants in water/wastewater. unfortunately, residues of ppcps, hormones, and their metabolites are not fully removed as the removal efficiency of most wwtps ranged between and % [ , ] . the removal efficiency of different ppcps differs greatly, and varies with the physical and chemical properties of the parent compounds as well as the environmental conditions such as the configuration of biological reactors and operational parameters such as hydraulic retention time and sludge retention time [ , ] . for example, carbamazepine, one of the contaminants that are most often detected in effluents, is known to be resistant to biodegradation, making it nearly impossible for efficient mineralization and removal in conventional biological treatment [ ] . wu et al. [ ] reported that % removal efficiency for carbamazepine after treatment of wastewater effluent in the conventional treatment process. the removal efficiency of other ppcps such as ibuprofen, naproxen, ketoprofen, diclofenac, bezafibrate, sulfamethoxazole, and trimethoprim ranges from to % [ ] [ ] [ ] [ ] . the differences in the removal efficiency may be due to a number of factors, including the characteristic of the activated sludge, the concentration of the contaminants, the optimal operational condition of the wwtps, and the water composition. thus, the biological treatment process seems to be economically feasible and widely applicable but not effective in degrading many organic pollutants. most wwtps utilizes various method in the conventional treatment systems for the removal of ppcps such as coagulation, flocculation, and sedimentation of raw sewage, activated sludge, membrane bioreactor, sequential batch reactor up-flow anaerobic sludge blanket processes [ , ] . some treatment methods are, however, more efficient and effective in removing pharmaceuticals compounds of interest, compared to another. this might be as a result of the physicochemical properties, the hydrophobicity, and biodegradability of target pharmaceuticals and process operating parameters in the wwtps [ ] . for instance, berset et al. [ ] reported that using the conventional methods involving dioxychlorination and sand filtration processes, cocaine and benzoylecgonine were poorly removed, while they were removed entirely when ozonation process was used. nonsteroidal anti-inflammatory drug removal in wwtps are achieved to a different extent. upon treatment with a mixture of dioxychlorination and sand filtration, diclofenac (over %) was almost completely removed; naproxen was moderately removed ( %), while ibuprofen was poorly removed ( %). when ozonation process was used as a treatment process, more than % ibuprofen, naproxen, and diclofenac were removed by less than %, while dioxychlorination and sand filtration process remove sulfonamides and macrolides efficiently [ ] . thermal destruction of pollutants at high temperatures is also a suggested elimination procedure. however, this technique may lead to undesirable emissions and damage to useful microbes and aquatic life forms, while it is not economically feasible. wastewater treatment plants (wwtps) rely on biodegradation and adsorption methods using conventional adsorbents such as activated sludge for removal of contaminating substances in aqueous matrices, but their use is limited. this limitation is because their removal efficiency changes significantly, and varies with the physicochemical properties of the compounds as well as the environmental conditions and operational parameters [ , ] . the biological, chemical, and physical processes have been studied for the removal of ppcps from contaminated matrices [ ] . among these techniques, biosorptive method is seen as one of the environmentally friendly techniques used in the removal of ppcps from aqueous solutions. bioadsorptive immobilization have been reported to show promising results for the removal of some organic contaminants in laboratory pilot-scale experiments while biodegradation was proven to be inefficient for the removal of ppcps due to poor degradability and low environmental concentration [ , ] . biosorptive processes have the advantages of effectiveness, simplicity, low cost, and sustainability [ ] . despite these advantages, the efficiency of bio-adsorption varies depending on the characteristics, polarity, the activity of the sorbent materials, the chemical properties and behaviours of the adsorbate material, thus suggesting incomplete pollutant removal and thereby passing them out in wwtp effluent. the cost of wwtp operation due to high energy requirements and low output is also a limitation [ ] . there is also a tendency of the transformation of some non-biodegradable contaminants into toxic contaminants that can exert toxicity to micro-organisms under the treatment conditions [ ] . commercial activated carbon as absorbents is one of the most widely studied materials for the removal of organic pollutants. still, its drawback is in the high manufacturing cost and the deficiency of regeneration. hence the preparation of economical adsorbents from agricultural waste, clay materials, biomass, non-living algae, and seafood-processing waste products, which are cheap, have been employed as biosorptive material for the removal of numerous organic pollutants from different water matrices [ ] . li et al. [ ] reported the high removal efficiencies of diclofenac and trimethoprim via the use of agro-waste materials such as biochar, macroalgae, and wood chipping. these biosorptive materials indicated possible high removal at low concentrations, thereby offering a clean, sustainable, and practical water treatment solution for purifying water. alkaline-modified biomass employed for the removal of tramadol achieved a % removal at ph of , biosorbent dose of . g/l, and mg/l contaminant concentration load of tramadol within min. the removal efficiency of the sorbent was possible because of the hydrophilic interaction between amino and carbonyl groups within the pharmaceuticals moieties, and the hydroxyl and carbonyl functional groups generated on the surface of the modified algal biomass [ ] . moreover, different biosorbents such as graphene oxide and carbon nanotubes have been used in the removal of various pharmaceuticals and personal care products [ ] [ ] [ ] . solution ph, temperature, interfering substances, ionic strengths of the matrix are among the environmental factors that play an essential role during biosorptive process for the removal of ppcp. however, their inability to be able to remove a large number of pharmaceuticals from wastewater treatment plants efficiently calls for the development of an efficient technology with acceptable cost and effective for mineralization of pharmaceuticals from the wastewater treatment plants. chemical oxidation method offers a better technology in that the challenges associated with the use of conventional wastewater treatment plants, which include incomplete degradation of organic pollutants, sludge formation, the formation of secondary pollutants, and high cost, are eliminated. chemical oxidation generally overcomes the limitations of other treatment processes, although it may not result in the complete mineralization of the organic pollutants [ ] . chemical oxidation, often referred to as an aop, involves the use of oxidants like ozone, hydrogen peroxide, and chlorine. chemical treatment method involving advanced oxidation processes such as ozonation, peroxidation, ultraviolet light aided ozonation (o /uv), ultraviolet light aided peroxidation (uv/h o ), fenton and fenton-like oxidation, gamma radiolysis, sonolysis as well as electrochemical oxidation process have been reported to be sufficient for the degradation of toxic pollutants in the aqueous matrices [ ] . howbeit, the advanced oxidation process methods have been associated with the disadvantage of high energy demand for various critical devices such as ozonizers, uv lamps, and ultrasonicators, and this leads to high operation costs [ ] . basically, advanced oxidation processes rely on in situ generation of highly potent chemical oxidants in the presence of ozone (o ) or hydrogen peroxide (h o ) and the presence of a catalyst such as the fenton's reagent or uv light [ ] . aops uses reactive species such as the hydroxyl radicals oh· generated in sufficient quantity for water purification, in which both organic and inorganic pollutants are removed through o aided oxidation or h o supported oxidation, in the presence of catalysts such as tio , zno, bio , cuo etc., and uv light as an energy source at ambient temperature and pressure [ ] . aops are classified into two based on photondriven reactions; [ ] . aops excel overall biological and chemical processes in that they are environmentally friendly as they are known not to transfer pollutants from one phase to the other, as seen during chemical precipitation, adsorption, and volatilization [ ] . moreover, they do not produce a large quantity of poisonous sludge during activated sludge processes as with other treatment methods. popular advanced oxidation processes used for the removal of contaminants include tio and photo-fenton processes. generally, photocatalyzed aop is environmentally benign, biocompatible, highly stable with low-cost metal oxide photocatalyst (such as tio ), and can efficiently be used to remove different organic pollutants from water matrices [ , ] . the photocatalytic degradation of organic pollutants within the water matrices are very efficient and effective due to the following factors; (i) good operating conditions at ambient temperature and pressure, (ii) complete mineralization of substrate and intermediate into co and h o, (iii) no problem with disposing of solid waste, (iv) utilization of sunlight, near uv-light or visible light for irradiation, (v) formation of non-toxic by-products and (vi) low cost [ , ] . tio photocatalysis, however, offers better activity during the removal of organic pollutants because of their heterogeneous nature, hence, the possibility of catalyst reuse and their operation at a wide ph range when compared to photo-fenton process. although different aops method has been employed in the degradation of ppcps, however, this review intends to emphasize on the catalytic activity of semiconducting photocatalyst doped on solid supports such as activated carbon, carbon nanotubes, and graphene oxides and their application in the degradation of ppcps. one of such semiconducting photocatalyst is tio , or tio immobilized on other solid materials used in aops processes. photocatalysis occurs upon the absorption of ultraviolet, visible, or infrared radiation by the photocatalyst in such a way that results in a change in the rate of the chemical reaction. the use of catalysts such as tio or tio immobilized on a support material was reported to lead to improved oxidation under solar irradiation, while the radiation also serves as a more sustained and energy-efficient solution for degradation of organic contaminants in the aqueous environment [ , ] . photocatalysis can either be a homogenous or heterogeneous photocatalyst. homogenous photocatalysis occurs when both the photocatalyst and the organic pollutants are soluble and exist in the same phase during the photocatalytic process. an example of homogenous photocatalyzed aop is the photo-fenton process, which involves the iron salt catalyzed h o reaction in the presence of uv-visible radiation as an energy source used for the generation of hydroxyl radicals as shown in the reaction below [ ] . this process is used in the degradation of pharmaceuticals as a result of its simplicity and easy reactor design. photo-fenton processes have been applied for the degradation of organics pollutants in different environmental matrices. de luna et al. [ ] reported the degradation of acetaminophen by fenton oxidation in a fluidizedbed reactor where the effect of operating parameters such as ph, temperature, the concentration of h o and fe + were monitored for the pseudo-second-order kinetic degradation . % of the organic substrate. the fenton process has also been proven to be a viable catalyst for photo-degradation of pharmaceuticals such as hydrocortisone, estradiol, and verapamil. the reaction mixture containing ferric ions as catalyst showed % degradation of the three pharmaceuticals in less than h, compared to the use of tio in a heterogeneous photocatalyzed process which shows a more favorable level of mineralization which in turn prevent the danger involved in the accumulation of harmful organic pollutants in the water body [ ] . the drawbacks in the use of this process include the use of reagents, strict ph control due to the process's ph dependence, and the formation of sludge. to overcome these challenges, the development of heterogeneous catalysts for the oxidation degradation processes was crucial, due to their potential role in facilitating the degradation and complete mineralization of organic contaminants, especially ppcps. current heterogeneous catalyst development studies are directed towards improving the synthesis, and functionality of various sizes and shapes of metal nanoparticles semiconducting photocatalyst, aimed at improving the performance and utilization of nanoparticles in multiple applications, including advanced oxidation processes (aops). in general, the use of ozonolysis and photocatalytic oxidation has proven to be promising as pre-treatment/treatment techniques for the mineralization of organic pollutants when compared with other chemical oxidation processes. the ozonolysis process is an advanced oxidation process that is based on the synergistic interaction between an oxidant such as ozone or abundant oxygen. ozonolysis may be supported by catalyst or radiation or a combination of both resulting in the generation of hydroxyl radicals (ho·), which is a short-lived oxidizing species that reacts with both organic and inorganic pollutants within the water matrices in a non-selective manner [ ] . due to aops ability for selective degradation of recalcitrant organic pollutants, they may function as a pretreatment process prior to biological treatment techniques, thus suggesting integrated treatment as a more efficient and effective means for the removal of organic pollutants from highly contaminated wastewater [ , ] . the preference for the use of ozonolysis in the removal of pollutants is because they do not cause a significant increase in salt concentration. it leaves no presence of chemical residue after usage, as observed with waste activated sludge, where bio-recalcitrant components and low soluble volatile organic compounds, are partially oxidized and solubilized to produce a biodegradable product [ , ] . however, this method is expensive and requires a high operating cost, thereby making its application for pollutants removal limited. the technique is also associated with the generation of intermediates, which could be challenging to eliminate. this process is made up of purely chemical and physical reaction routes in which the photocatalyst is generated and its activity maintained by the absorption of radiant energy [ ] . heterogeneous photocatalysis occurs when the activity of semiconducting photocatalyst, which in most cases is insoluble in the solution that contains the organic substrates, and dependent on the ph of the solution, is initiated by the absorption of radiant energy. the generated photon with energy band gap level equal to or greater than the band gap energy of the photocatalyst which is the difference between the energy of the valence band full of electrons and that of the conduction band that is empty. then, mobile electrons can access the conduction band, leaving positive holes in the valence band [ , ] . hence the catalyst and the substrates exist in different phases. the heterogeneous nature of the oxidative reaction makes for easy and cheap recovery catalyst from the solution after use. the conventional semiconducting nanoparticles used in aops for the treatment of organic pollutants in water matrices include tio in homogenously catalyzed process, while those use as heterogeneous catalysts; zno, sno , al o , in o , zns, fe o , ceo , zro , sio , cuo, mno and cds which function as sensitizers for lightinduced redox processes. the tio photocatalytic process leverage via direct photolysis of photon energy from uv and photocatalysis by producing hydroxyl radicals where tio is photo-activated to generate hydroxyl radicals on the surface of the crystal before reaction with a wide range of organic pollutants which in turn lead to mineralization of the pollutants to co and h o uv radiation [ , ] . the continuous design and development of various photocatalyst by researchers around the world affirmed that heterogeneous photocatalysis presents the best technological opportunities for the degradation of organic pollutants within the environment. nanoparticle photocatalytic reactions can be described as the interaction of light energy with a metallic nanoparticle. this process is used in wastewater treatment as a result of their broad and high photocatalytic activities on the decomposition of various organic pollutants [ ] . nano-photocatalysts consist of nano-size order metal oxides, with semiconducting properties and strong ability to degrade large varieties of persistent organic pollutants such as dyes, detergents, pesticides, and volatile organic compound that wwtps cannot remove [ , ] . quite several semiconductors such as zno, srtio , fe o , sno , cuo , wo , and fe o have been used for the decomposition of organic pollutants from different environmental matrices [ ] [ ] [ ] . they can also be used for degrading halogenated and non-halogenated organic compounds, ppcps, and heavy metals in some specific situations [ ] . the photocatalytic method is a promising technology used in the treatment of contaminants as a result of the ability of the catalyst to use sunlight as the energy source to initiate and facilitate the degradation of organic pollutants [ ] . however, some of the challenges encountered with the use of photosensitive semiconductors as catalysts are low quantum efficiency, rapid recombination of photogenerated carriers, narrow optical response, and a slow transfer rate of electrons to oxygen which hinder their applications in the treatment of organic pollutants [ ] . these limitations could be eliminated by employing strategies such as the modification of the textural properties, heterojunction construction, elemental doping, and morphology control to improve their photocatalytic performance [ ] . the modification of photocatalysts that are highly active via visible light enhances the central part of the solar spectrum to be used. therefore, to exploit the visible light during the photocatalytic process, there is a need for the usage of narrow band gap semiconductors like cu o, wo , cds, bivo etc., for the degradation of different organic pollutants. cheng et al. [ ] reported the photocatalytic activity of cu o-cu composites irradiated under visible light at nm for the degradation of methylene blue, where % removal was achieved after min. the intensity degraded after irradiation at nm as observed by the disappearance of its peak after min. li et al. [ ] synthesized z-scheme heterostructure nanocomposites by loading mesoporous γ-fe o nanosphere on g-c n nanosheet, the synthesized z-scheme γ-fe o /g-c n heterostructure exhibits mesoporous features with the improved specific surface area providing reactive mass sites for tetracycline; also the constructed heterostructure which existed between γ-fe o and g-c n efficiently extends the response range which leads to the quick transfer and separation of photoinduced charge carrier which enhances its photocatalytic activity for mineralization of tetracycline hydrochloride under visible light irradiation. the working mechanisms behind the photo-catalyzed advanced oxidation process are based on the photoexcitation of electron in the catalyst. for instance, if photocatalyst, such as tio , is irradiated with uv light, it causes a generation of holes (h + ) and exits electron (e − ) in the conduction band. in aqueous media, there is an entrapment of water molecules (h o) onto the holes (h + ), leading to the generation of hydroxyl radicals (·oh). these hydroxyl radicals are indiscriminate, highly reactive, non-selective, and powerful oxidizing agents, which in turn, oxidize organic pollutants into water and gaseous degradation products [ ] . tio stands out as an excellent photocatalyst amongst the semiconductors that can be used for the degradation of ppcps, due to its optical and electronic features, photosensitivity, physical and chemical stability, costeffectiveness, resistance to light corrosion, non-toxicity and their high accessibility [ , ] . polymorphs of tio include anatase, rutile, and brookite [ ] , of which anatase tio have been shown to exhibit the highest photocatalytic activity as a result of the extended lifetime of the photoexcited electrons, and the fast migration of photogenerated electrons [ ] . literature reports indicated that the photocatalytic degradation of ppcps using tio as catalysts could result in mineralization reaching close to % within to h under various parameters. for example, the photocatalytic degradation of ofloxacin and atenolol irradiated under a uv lamp was almost complete after min [ , ] . furthermore, photocatalytic mineralization of tetracyclines, paracetamol, caffeine, and atenolol as single/lone pollutants and in mixtures was achieved via tio irradiated under uv and simulated solar irradiation, where the removal of tetracyclines reaches % after min and that of paracetamol, caffeine, and atenolol were between and % after h, while the degradation of the mixture of these pharmaceuticals was achieved at % after h under the same conditions [ ] . he et al. [ ] reported that %, %, %, and % photocatalytic degradation of propranolol, diclofenac, carbamazepine, and ibuprofen, respectively, was achieved after h via the immobilization of tio on the sand. the degradations were made possible as a result of the photo-oxidation of substrates from the single oxygen generated in the reaction mixture [ ] . considerable removal performance can be achieved in the presence of simulated solar light. irradiated tio was also applied for the mineralization of organic molecules such as acetylene in the gas phase. acetylene mineralization/degradation in the gas phase follows a first-order kinetic in which no organic intermediate was detectable, suggesting that no pollutants or any intermediate species interfere in the photocatalytic mineralization [ ] . however, the usage of tio as a photocatalyst has critical limitations such as its wide intrinsic band gap that is . ev and . ev for rutile and anatase, respectively, and this results in achieving an activation at ultraviolet radiation at λ < nm. this wide band gap can be reduced via doping of tio , in order to cause a shift in absorption in the visible light region of λ > nm [ , ] . another challenge of tio as a conventional powdered catalyst for photodegradation is the difficulty of their separation from the slurry of the reactor systems after photocatalysis, and this leads to low efficiency in photocatalytic activities over a range of application [ , ] . due to surface limitations and inadequate adsorption capacity of tio resulting from non-porous nature, current studies are directed towards incorporating photocatalysts on porous supports in order to facilitate the efficient absorption of target environmental pollutants prior to subsequent oxidation aided by the photocatalyst. to achieve a more rapid and efficient removal of ppcps in wwtps, there is a need to modify the photocatalytic process through immobilization of tio onto a suitable solid support such as activated carbon, molecular sieve, graphene, and minerals as a result of their ability to enrich the pollutants and improves their photocatalytic degradation rate due to its stability, mechanical resistance, high surface area and appropriate porosity [ , ] . zno, with strong oxidation ability and a tremendous photocatalytic property, has also been used in the degradation of various organic pollutants due to its direct and broad band-gap energy close to uv spectral region, hence their thermal stability and considerable free-exciton binding energy. it is also relatively cheap when compared to tio , which is said to be non-economical for the treatment of water on a large scale [ , ] . zno nanoparticles have been reported to be more catalytically active compared to tio for degradation of pharmaceuticals, where the photocatalytic degradation of pharmaceuticals was studied using zno and tio nanoparticles [ ] . study results showed that % removal was achieved after min under uva irradiation via zno nanoparticles catalyzed process, whereas only % removal was achieved for tio catalyzed process under the same condition. the photocatalytic activities of different types of zno were studied for the degradation of methylene blue, where the reaction was monitored at ph . for min under uv irradiation of nm. zno nanofibers were noted to be the best among the fabricated zno, as photo-degradation reaches almost % for methylene blue removal [ ] . the photocatalytic efficiency of zno/g-c n composites was investigated by ismael [ ] for the degradation of methyl orange and -chlorophenol under visible light radiation. its photocatalytic performance was due to the synergistic effects between zno and g-c n , resulting in the interfacial transfer of photogenerated electrons and holes hence its effective charge separation resulting in their ability to be able to degrade methyl orange and -chlorophenol. another semiconductor which has been used in the photocatalytic degradation of organic pollutants is wo . wo is an n-type semiconductor with high stability and a wide tunable band gap of around . ev, hence their application in the photocatalytic degradation of various organic pollutants. for instance, complete degradation of rhodamine b was achieved when wo nanoplatelet was employed, with the intensity peak of rhb disappearing after min, when the catalyst reacted with the organic pollutants to form an unstable intermediate under uv light irradiated at nm [ ] . chen et al. [ ] reported the fabrication of wo /g-c n nanocomposites by a simple direct precipitation method, and the z-scheme photocatalyst wo /g-c n was designed and prepared by dispersing wo nanosheets on g-c n nanosheets. the as-prepared wo /g-c n showed enhanced photocatalytic activity toward degrading methyl orange. the improved photocatalytic performance of wo nanosheet doped on g-c n was due to the coupling of effect of g-c n on wo resulting in a slight shifts to lower binding energy, which hints the strong interaction between wo and g-c n . photocatalytic degradation involving tetracyclines reaches . % in min under visible light radiation through the use of ag/agcl/ac composites. the reactive species that aided the degradation are electron holes and superoxide radicals, while the exposed activated carbon surface in the composites photocatalyst enhances the tetracyclines adsorption onto its surface, thereby increasing the contact sites between tetracycline and photocatalyst which lead to a better and efficient photodegradation rate [ ] . the photocatalytic performance of various photocatalysts has been mostly enhanced for the degradation of organic pollutants by combining them with carbonaceous materials. these carbonaceous materials, when compared to single semiconductors, give additional advantages such as chemical stability, thermal stability, high surface area, pore structure, good conductivity, excellent electronic properties, better photocatalytic activity, activity under solar irradiation and easy separation [ ] . activated carbon (ac), carbon nanotubes cnts) and graphene are among the best solid materials for use as catalyst support for photocatalytic purposes, especially those use for the degradation of ppcps in water and wastewater, due to properties such as; large specific surface areas and high charge carrier mobility [ , ] . activated carbon is porous and amorphous solid carbon material that can be derived from coal or plants through carbonization at a temperature between and °c, with an activation process by use of steam or co [ ] . activated carbon is non-photocatalytic in nature but can serve as a means to enhance the photocatalytic reaction between catalyst, e.g., tio and the organic contaminants, by adsorption of pollutants on its surface to facilitate the degradation of organic contaminants [ ] . the use of carbonaceous material in the degradation of ppcps has been studied several times, and it has been proven that they can only adsorb pollutants, but cannot transform them, thus leading to the generation of hazardous products. in a situation where the adsorbents were not regenerated on-site, such adsorbents become hazardous waste and must be disposed of carefully, coming with additional cost. the use of carbonaceous material can, therefore, be expensive [ ] . the affinity of solid materials for ppcps results from the presence of functional groups such as carboxyl, carbonyl, phenol, lactone and quinone, which confers on them surface-active species and specific surface areas that attract solid materials and concentrating them close to the semiconducting active site for improved photodegradation [ ] . this makes the composites serve as effective adsorbent material due to one or more combinations of either hydrophobic interaction, π-π interaction, hydrogen bonding interactions, and electrostatic and dispersion interaction [ ] . the clogging of the pore on adsorbent material is also a common challenge. carbonaceous materials like activated carbon function as electron scavenger because of their enormous electron storage ability, which is known as hoffman mechanism property [ ] . they also act as a sensitizer-providing electron for semiconductor photocatalyst such as tio , g-c n , etc. the electrons are excited by photons of light energy, which produces the superoxide radicals from absorbed molecular oxygen. moreover, the positively charged composite material attracts electron from semiconducting metal catalysts such as tio , to create a hole (h + ) which interact with adsorbed water to generate hydroxyl radicals, in the tio valence band. the presence of carbon-oxygen-titanium linkages within the composite materials leads to a reduction in the band gap and extends the absorption band into the lower energy visible range [ ] . moreover, the interest in the use of activated carbon as solid support for various photocatalyst semiconductor is as a result of its developed pore structures, very large surface area and high adsorption capacity, hence their usage as an adsorbent for both organic and inorganic pollutants [ ] . activated carbon in the tio immobilized on activated carbon photocatalyst, serves as a reaction medium through which organic molecules are absorbed before degradation [ ] . the complete removal of pharmaceuticals such as amoxicillin, ampicillin, acetaminophen, and diclofenac was achieved using ac-tio composite material under sunlight irradiation for min, and this cannot be achieved with bare tio [ ] . this is a result of the transfer of sorbate from activated carbon to photoactive tio due to the presence of a common interface between them [ ] . another solid support that has been of great use owing to its high surface area, chemical and thermal stability is zeolite [ ] . their immobilization on tio has proven to be efficient in the removal of highly volatile organic contaminants due to their adsorptive nature, and this can easily be supplied onto the tio photocatalyst surface [ ] . bare tio samples can rapidly deteriorate, while tio composites can maintain a high adsorption efficiency to remove organic contaminants over a long period [ ] . khraisheh et al. [ ] reported the preparation of activated carbon from coconut shell material, which is an inexpensive source of carbon. ac prepared from many other biomass materials including, plant remains, animal remains, agricultural wastes, decomposing materials, etc., have been reported [ ] [ ] [ ] . carbon sources for the preparation of activated carbon vary from chemical to biological/natural sources, while the method of preparation depends on the nature and characteristics of the carbon source. however, production using biological/natural material sources are relatively cheaper than chemical raw materials because the organic/natural sources are often waste materials, hence the need for the use of plant material as a source for activated carbon. this technique is followed by the immobilization/ doping of activated carbon with a metal oxide or nano metal oxides to form a composite catalyst. different physical and chemical characteristics and attributes are achieved depending on how and which method is used for activated carbon preparation and how the metal oxide is doped/immobilized on the acs. arana et al. [ ] reported that the reaction of tio with activated carbon at different proportions showed that activated carbon not only increase the surface area but there is a modification of the acid-base properties and the uv spectrum of tio . the following methods have been used in the preparation of ac as a carbon source for photocatalytic degradation of organic pollutants: sol-gel, chemical vapor deposition, impregnation, pyrolysis, precipitation, hydrothermal preparation, microwave-assisted synthesis and sonochemical treatment [ ] [ ] [ ] . the mostly used among the above method is the sol-gel method because there is an ultimate mixing or chemical interaction between activated carbon and the corresponding semiconductor photocatalyst. studies proved that doped/immobilized metal oxides composite material possess superior quality and has the highest removal efficiency under uvc intensity. in tio composite material, for example, the composite solid support doped with tio showed higher photocatalytic activity, adsorption capacity, electron scavenging, and sensitization ability and extended visible light adsorption when compared to bare tio [ ] . the photocatalytic activity of prepared nanocomposite zno/ac where the activated carbon was prepared from biomass degrades more than % organic pollutants as a result of the addition of electrolyte, although this decreases the photoactivity of the catalyst [ ] . carbon nanotubes are made up of cylindrical graphene sheets and can be classified either as single-walled carbon nanotubes (swcnts) and multi-walled carbon nanotubes (mwcnts) [ ] . cnts possess a high specific surface area to mass ratio ranging between and m /g coupled with an exceptional sorption capacity [ , , ] . they exhibited hydrophobic interactions, van der waals forces, and π-π stacking, leading to the formation of four different types of adsorption sites such as inner cavities, interstitial channels, external grooves, and outermost surfaces [ ] , making them a suitable solid material for photocatalytic degradation of ppcps. cnts have interesting properties such as large electron storage capacity, superior metallic conductivity, and light adsorption at a broad range wavelength, thereby making them a suitable solid material in the enhancement of the photocatalytic activity of tio [ ] . as monitored on high-performance liquid chromatography, the photocatalytic degradation of oxytetracyclines reaches . % within min when mwcnt/bivo composites were utilized. the photocatalytic enhancement was made possible as a result of the synergistic effect that occurs between mwcnts and bivo , which increased the separation rate of photogenerated electron-hole pairs [ ] . the usage of cnts-tio for the photocatalytic degradation of caffeine was as a result of good contact between cnts and tio , thereby causing a better enhancement in their photocatalytic activity [ ] . cnts-tio -n has shown to exhibit a high photocatalytic activity under visible light irradiation for the degradation of ibuprofen [ ] . this feature can be attributed to the fact that cnts can function as electron scavengers resulting in a good binding between cnts, tio , and n used. modification of cnts on g-c n /bivo was evaluated for the degradation of phenol in the presence of solar light irradiation. the synergistic interaction between the heterostructure was aided by h o as the oxidizing agents for . % removal of phenol after h, where the bivo helps to promote a better optical absorption ability over the well-contacted structure within the heterojunction while the presence of cnts in the heterostructure enhances a higher photocatalytic performance as a result of its ability as an electron bridge mediator which is responsible in facilitating the photogenerated charge carrier separation [ , ] . the methods that are employed during its preparation are as follows; hydrothermal, hydrolysis, restrained hydrolysis, simple evaporation and drying, sol-gel, surfactant wrapping sol-gel, chemical vapour deposition, physical vapour deposition, electrophoretic deposition, electrostatic attraction, solvothermal and impregnation [ , ] . graphene is made up of a two-dimensional sheet of carbon atoms that are connected by an sp bond consisting of an aromatic π electron system [ ] . graphene possesses the following properties; high electron mobility, high mechanical strength, high thermal stability, and high specific surface area [ ] . these unique properties made graphene have a higher density of potential adsorption sites, which are; open-up surface, a longitudinallyparallel external surface, and interstitial channels [ ] . the relevance of graphene includes applications such as in sensors, energy conversion and storage, polymer composite, drug delivery systems, and environmental remediations [ ] [ ] [ ] . there are different type of graphene that is available, which are; graphene oxide, reduced graphene oxide, fullerene, carbon nano-onion, and carbon nanotubes [ ] . graphene oxide (go) and reduced graphene oxide (rgo) are the most commonly used graphene because they show a high efficiency during water treatment. these abilities to adsorb pollutants are due to the presence of several surface functional groups like hydroxyl, carboxyl, and epoxy that function as adsorption sites that aid the removal of contaminants from the water system [ , ] . graphene oxide (go) is prepared via chemical functionalization of graphene through the following method; brodie [ ] , staudenmaier, hoffman, and hummer methods [ ] . graphene oxide (go) is photocatalytically active and can function as an electric insulator as a result of the chemical disruption of the π network. their electrical conductivity can be restored during the conversion of graphene oxide to reduced graphene oxide [ ] . reduced graphene oxide can be prepared using the chemical treatment [ ] , thermal mediation [ ] , and electrochemical treatment [ ] . both graphene oxide and reduced graphene oxide can be used for the photocatalytic degradation of ppcps. go-tio can be prepared via the following method; solution mixing, sol-gel, hydrothermal, electrostatic self-assembly, molecular grafting, chemical exfoliation, liquid phase deposition, and electrospinning [ , , ] . the choice of the method to use will be based on which of them shows a stronger interaction between graphene and the corresponding semiconductor photocatalyst, which in turn will enhance their photocatalytic activity. the degradation of rhodamine reached . % when rgo/g-c n composite was employed. the efficiency of the composites was as a result of the synergistic effect of rgo/g-c n heterostructure formed in the formation of coupled heterointerface and heterojunctions which efficiently and effectively aid in the transfer of photogenerated electron-holes pairs under the influence of visible light where photo-induced electrons can freely migrate along with the conductive network of rgo thereby preventing the recombination of the photogenerated carriers [ ] . there is a presence of strong electronic and physical coupling when tio nanosheet-graphene of d- d composite interact, causing an enhancement of electron transfer between them, thus making the photocatalyst superior when used in the removal of , -dichlorophenol as compared to tio . this is because of the availability of rich oxygen functional groups on the d surface of rgo, which enhances the ability of rgo to interact with other organic/inorganic compounds to form hybrid composites [ ] . shanavas et al. [ ] reported that the advantage in the use of d/ d/ d heterojunction in the photocatalytic degradation of pharmaceuticals such as tetracyclines and ciprofloxacin is the reduction of photoexcited charge carrier migration distance for effective suppression of electron-holes pairs recombination, therefore, creating numerous high-speed photo-induced charge transfer nanochannels in rgo/fe o /g-c n nanocomposite by degrading tetracyclines and ciprofloxacin under visible light irradiation. also, porous d rgo-tio aerogel has been reported to remove over % of carbamazepine. this was made possible because the macro-porous d structure of the aerogel has abundant surface sites, effective charge separation causing an improvement in the mass transportation of the contaminants, thereby leading to easy separation of the catalyst after degrading the contaminants [ ] . the excellent outcome experienced during the degradation of carbamazepine, ibuprofen, and sulfamethoxazole, which is up to % during removal and high durability in h operation, is as a result of the immobilization of rgo on tio on optical fibers [ ] . however, since the photocatalytic activity of solid material immobilized on tio has proved to be efficient in the degradation of ppcps, there is, therefore, a need to develop a standard method with a pilot-scale experiment to ascertain the cost on a large scale use. due to the photocatalytic activity of graphene-tio composite, hence their usage in the degradation of ppcps but their drawback in water treatment arises from the difficulty in separating and recovery of the catalyst after use. the preparation of different photocatalysts can be achieved through various chemical processes. the synthesis methods are usually tailored to suit the size and morphology of the intended materials. some simple synthetic techniques that have been used for the synthesis of functional photocatalyst materials are stated below. the coprecipitation method is a very facile and convenient method used in the synthesis of various photocatalysts. this method involves the dissolution of the salt precursor of interest into an aqueous solution followed by precipitation by addition of a base such as naoh, koh. this procedure is usually followed by washing and drying, then calcination of the precipitate at a desirable temperature to obtain the intended products [ ] . the particle size, morphology, and composition of the synthesized photocatalyst prepared through this method can be controlled by adjusting different experimental parameters, such as the starting material, their ratio, the surface ligand, the reaction temperature with time, and the ph. moreover, the size distribution of the as-prepared photocatalyst can be improved by the addition of ligands, such as surfactants, inorganic molecules, and polymers into the reaction medium, however, size control is generally poor for this method [ ] . this method involves the chemical reaction where the precursors are dissolved in a solvent such as water, organic solvents, or koh. the aqueous mixture will then be subjected to heat at a high temperature around °c in a sealed stainless steel autoclave. consequently, the pressure within the reaction autoclave is drastically increased above atmospheric pressure resulting in the generation of high crystalline materials without the need for further treatments [ ] . the drawback of this method is its low yield of products when compared with the coprecipitation method. thermal decomposition is an innovative method to synthesize stable nanoparticles. this method involves the thermolysis of organometallic complex precursors in high-boiling-point organic solvents in the presence of surfactants to synthesize nanoparticles of various materials. it is faster, clearer, and economical when compared with other methods [ ] . it is also one of the easiest and the most convenient method used to synthesize monodispersed metal nanoparticles. furthermore, it answers the greatest challenge of obtaining a controlled nanometric size and shape in nanotechnology research, which is achieved by controlling the concentrations of the precursors and surfactants; the solvent; and the experimental parameters such as heating rate, heating temperature, and heating time are usually regulated [ ] . however, photocatalyst prepared by this method is usually coated with hydrophobic ligands, thereby making it insoluble in water. therefore, a further surface modification step is required to render these nanoparticles water-soluble for efficient photocatalytic applications. the sol-gel method for the synthesis of nanomaterials is a convenient and versatile reaction technique in the synthesis of numerous functional photocatalyst. the sol-gel process is based on hydrolysis and polycondensation reaction involving the addition of a complexing agent such as; a polymer, citric acid, or other suitable organic entities into a solution of a colloidal dispersion of the metal ions of the target material. these complexing agents function as cross-linkers between the ionic substances in solution followed by gradual removal of the solvent molecules from the mixture to form a thick gel. the gel is then dried and pre-calcined to obtain the as-prepared powder. the as-prepared powders are then annealed at higher temperatures to obtain the finished product. metal oxides can also be used as starting materials in this method [ , ] . several parameters, such as type of precursor, type of solvent, water content, ph, the concentration of precursor and temperature, can influence the structure of the initial gel, which in turn influenced the properties of the resulting materials, including the crystal structure, particle size, shape and crystallinity [ , ] . the solgel method possesses many advantages. for instance, it allows tailoring of both the bulk properties, including phase composition and surface characteristics such as the surface area, the total pore volume distribution, etc., of a material on a nanometer scale from the earliest stages of processing. the main drawbacks are in the possible high cost for the majority of alkoxide precursors, and processing steps are long. the large band gap energy of semiconductor photocatalysts such as tio ( . ev), zno ( . ev), constitute a significant limitation to their photocatalytic usage under visible light spectrum due to its low utilization of solar energy. this limitation makes this method an expensive means for the photocatalytic process; hence the urgent need to research more on visible light semiconductor photocatalyst. g-c n has emerged one of the next generation photocatalysts due to its cheap synthesis, fascinating electronic band structure, non-toxic, graphene-like two-dimensional structure coupled with good visible light adsorption ability and chemical stability for removal of hazardous pollutants from the environment and clean energy production [ , ] . they are made up of two main units to establish their allotropic nature which are; tri-s-trianzine/heptazine (c n ) and s-triazine (c n ) rings (fig. ) ; where the tri-s-triazine based g-c n is highly favoured and the most stable phase of g-c n thermodynamically [ ] . since the discovery of g-c n by wang et al. [ ] as a promising visible light photocatalyst for the evolution of h , much effort has been directed towards its synthesis. g-c n can be prepared by thermal polymerization using the following precursors based on different reaction conditions and differences in the material degrees of condensation; cyanamide, dicyandiamide, melamine, urea, thiourea and ammonium thiocyanate [ ] [ ] [ ] . g-c n has a low energy band gap between . and . ev as a result of the presence of sp -hybridized carbon and nitrogen forming the π-conjugated electronic structure, possess excellent chemical stability, does not dissolve in acid, alkali or organic solvents making it to be environmentally friendly material [ , ] . however, their application in environmental remediations are limited by several obstacles such as low specific surface area, the high recombination rate of charge carrier and low electrical conductivity, hence the need to develop a modified g-c n -based heterojunction photocatalyst with improved physicochemical properties that can be applied in the degradation of organic pollutants in the environment [ , ] . the modification can be achieved through exfoliation of bulk g-c n into nanosheet, nanotubes and quantum dots, deposition of noble metal, doping with metal, coupling with other semiconductors such as tio , zno, ag o, mos , incorporating with carbonaceous materials like; activated carbon, graphene oxides, reduced graphene oxide, carbon nanotubes, and many others to form nanocomposites [ ] [ ] [ ] . to overcome the drawback in g-c n associated with their photocatalytic application, specific surface area, charge separation, and opticals for g-c n have been improved. numerous heterostructure based g-c n photocatalyst has been prepared by many researchers with improved suitable band structures, shaped at the interfaces between g-c n and metal particles, inorganic semiconductors or carbonaceous materials have shown to enhance their charge carrier separation leading to its improvement in their photodegradation activity [ ] [ ] [ ] . hence, the interest in the synthesis of heterostructure based g-c n with suitable band position resulting in the suppression of the recombination rate of the photogenerated electron-hole pairs for improved photocatalytic degradation of hazardous contaminants from the environments. fabrication of g-c n based semiconductor photocatalyst is considered as one of the effective and more feasible strategies to enhance the photocatalytic activity of g-c n where the formation of a heterojunction system improve the photo-generated electron-hole pairs separation leading to the improvement in their photocatalytic activity as seen in the following g-c n based photocatalyst g-c n /tio , g-c n /wo , g-c n /cds, g-c n /cu o, g-c n /cds/cu o for the degradation of numerous organic pollutants in the environment [ ] [ ] [ ] . chen et al. [ ] synthesized a series of visiblelight-driven mgin s /g-c n heterostructures for the degradation of -nitroaniline and methyl orange. the improvement in the photocatalytic activity of mgin s /g-c n can be attributed to the faster charge separation and transport that existed between the interfaces and band structures of mgin s nanoplates and g-c n nanosheets making it an efficient visiblelight-driven composite for the degradation of hazardous pollutants. the photocatalytic performance of z-scheme g-c n /cds composites for the degradation of erythromycin and tetracycline was remarkably enhanced as compared to that of pure g-c n and cds owing to the perfect matching of the band gap, the interface of the close photocatalyst was lower than that of cds which give rise to the formation of heterojunction with g-c n which suppresses the photo corrosion of cds [ ] . table below shows how different heterostructure composites have been used for the degradation of various organic pollutants. the absorption of photon energy by g-c n photocatalyst, which is either equal or greater than the band energy of g-c n leads to the generation of electrons (e − ) in the valence band (vb) to be excited before migrating to the conduction band (cb). the generated electron will be excited and leave the photo-generated holes (h + ) in the valence band. both the photoexcited electron (e − ) and photogenerated holes (h + ) will be trapped at the surface of the catalyst where the h + react with absorbed h o to produce ·oh radicals while e − react with absorbed o to produce ·o − radicals before reacting with organic pollutants and finally degrade them [ , ] . the mechanism is summarized below; however, in the photocatalytic process for the degradation of organic pollutants, it is crucial to understand the transformation of the different intermediates involved in photocatalysis as it will provide information about the toxicity of the intermediates as well as risk assessment. a schematic representation of photocatalysis mechanism is shown in fig. . although many works have been done in fabricating a g-c n based photocatalyst for environmental remediation, however, much effort needs to be directed towards the construction of g-c n -based heterostructure photocatalyst. this act would improve the separation of photogenerated electrons and inhibit the electron-hole pair recombination with an excellent possibility for an efficient and effective photocatalytic activity for the degradation of pharmaceuticals under visible light irradiation. in conclusion, this review highlighted a detailed overview of the detection of organic pollutants such as pharmaceuticals and personal care products, endocrine-disrupting chemicals, organic dyes, and various industrials waste in environmental matrices. additionally, it was revealed that most researches focus on the occurrence of the parent pharmaceutical compound while the transformation products which may even be more toxic than the parent drugs are ignored, hence more focus is needed on how the chronic exposure to these contaminants affect humans and other ecosystems, particularly the nontarget organisms. the environmental impacts of these emerging contaminants led to the need for the development of a technology for its removal from the environment. this problem of pharmaceutical contamination of the environment could be attributed to the inability of the wastewater treatment plants to completely remove the contaminants, making them one of the common · o − + organic pollutants → co + h o · oh + organic pollutants → co + h o. sources through which these pharmaceuticals get into the environment. even though various methods such as activated sludge, wetlands, adsorption, membrane processes have been developed for the removal of these pollutants, unfortunately, many research reports have proven that these methods are ineffective in removing these contaminants. hence, the advanced oxidation process using a photocatalytic method becomes relevant. in this method, various semiconducting photocatalyst can be immobilized on carbonaceous materials, metal oxides, or another semiconductor as the best alternative for the degradation and mineralization of these organic pollutants into harmless products and at the same time, the catalyst can still be recovered and recycled. moreover, it should be noted that from the various reports reviewed, photocatalyst materials emerged as an efficient and effective technology for the degradation of various organic pollutants ranging from pharmaceutical and personal care products to different dyes. furthermore, the engineering of metal-free carbon-based photocatalyst holds a great advantage as an efficient and effective means in degrading organic pollutants because of their tunable band gap, visible light responses, the formation of complex structures, excellent chemical stability, economical, reusability, and ease of fabrication. however, despite the significant progress made in the synthesis of photocatalyst for environmental remediation, their photocatalytic performance for degradation and mineralization of pharmaceutical and personal care products with their intermediates still need additional improvement for practical application. also, their activities are limited by insufficient light absorption, rapid electron-hole pair's recombination rate, difficulty in the separation and recycle of photocatalysts, and photo-corrosion of some semiconductor photocatalysts. the process might be complicated at times, costly, and time-consuming, hence the need to develop a method that is more efficient, easy, affordable, and environmentally friendly for the removal of organic pollutants at the wwtps. thus, the need for the green synthetic routes for more novel based heterostructure photocatalyst. in fig. schematic representation of photocatalysis mechanism (adapted with modification from [ ]) addition, the synergistic effects that existed within the heterostructure photocatalyst, which aid their photocatalytic activity, need more investigation. the mechanism of the degradation pathways, also, needs much attention to be able to verify the most active reactive species in photodegradation of organic pollutants. finally, this review tends to serve as a great advantage for material scientists and nanotechnologists who are interested in seeking materials for environmental degradation of organic pollutants. occurrence, fate, removal and assessment of emerging contaminants in water in the water cycle (from wastewater to drinking water) preliminary assessment on the bioaccessibility of contaminants of emerging concern in raw and cooked seafood occurrence and bioaccumulation of pharmaceuticals in a fish species inhabiting the suquía river basin a review of the effects of emerging contaminants in wastewater and options for their removal photodegradation of ibuprofen by tio co-doping with urea and functionalized cnt irradiated with 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nickel substrate by chemical vapour deposition and application in photoassisted water electrolysis solvent-assisted thermal reduction of graphite oxide facile and controllable electrochemical reduction of graphene oxide and its applications graphene oxide-p photocatalysts for degradation of diphenhydramine pharmaceutical and methyl orange dye the ultra-rapid synthesis of rgo/g-c n composite via microwave heating with enhanced photocatalytic performance a strong electronic coupling between graphene nanosheets and layered titanate nanoplates: a soft-chemical route to highly porous nanocomposites with improved photocatalytic activity two-dimensional interface engineering of a titania-graphene nanosheet composite for improved photocatalytic activity computationally guided synthesis of ( d/ d/ d) rgo/fe o /g-c n nanostructure with improved charge separation and transportation efficiency for degradation of pharmaceutical molecules one-step hydrothermal synthesis of porous d reduced graphene 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visible light irradiation preparation of heterostructure g-c n /zno nanorods for high photocatalytic activity on different pollutants (mb, rhb, cr (vi) and eosin) a novel ag o/g-c n pn heterojunction photocatalysts with enhanced visible and near-infrared light activity distinctive binary g-c n /mos heterojunctions with highly efficient ultrasonic catalytic degradation for levofloxacin and methylene blue enhanced photodegradation performance of rhodamine b with g-c n modified by carbon nanotubes facile synthesis of nitrogen-deficient mesoporous graphitic carbon nitride for highly efficient photocatalytic performance synthesis of mos /g-c n nanosheets as d heterojunction photocatalysts with enhanced visible light activity novel reduced graphene oxide-supported cd . zn . s/g-c n z-scheme heterojunction photocatalyst for enhanced hydrogen evolution novel visible light active graphitic c n -tio composite photocatalyst: synergistic synthesis, growth and photocatalytic treatment of hazardous pollutants synthesis of coral like wo /g-c n nanocomposites for the removal of hazardous dyes under visible light porous-cds/cu o/ graphitic-c n dual pn junctions as highly efficient photo/catalysts for degrading ciprofloxacin and generating hydrogen using solar energy research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over ready to submit your research ? choose bmc and benefit from rational construction of a direct z-scheme g-c n /cds photocatalyst with enhanced visible light photocatalytic activity and degradation of erythromycin and tetracycline performance of the polymeric g-c n photocatalyst through the degradation of pharmaceutical pollutants under uv-vis irradiation batio /tio compositeassisted photocatalytic degradation for removal of acetaminophen from synthetic wastewater under uv-vis irradiation in-situ growth of zno globular on g-c n to fabrication binary heterojunctions and their photocatalytic degradation activity on tetracyclines construction of ag/g-c n photocatalysts with visible-light photocatalytic activity for sulfamethoxazole degradation development of g-c n /tio / fe o @ sio heterojunction via sol-gel route: a magnetically recyclable direct contact z-scheme nanophotocatalyst for enhanced photocatalytic removal of ibuprofen from real sewage effluent under visible light enhanced photocatalytic activity of ternary ag po /go/g-c n photocatalysts for rhodamine b degradation under visible light radiation enhanced photocatalytic degradation performance of organic contaminants by heterojunction photocatalyst bivo /tio /rgo and its compatibility on four different tetracycline antibiotics a novel ag o/ceo heterojunction photocatalysts for photocatalytic degradation of enrofloxacin: possible degradation pathways, mineralization activity and an in depth mechanism insight construction of d porous g-c n /agbr/ rgo composite for excellent visible light photocatalytic activity photocatalytic and electrochemical performance of three-dimensional reduced graphene oxide/ws /mgdoped zno composites recent progress in carbon-based materials as catalysts for electrochemical and photocatalytic water splitting. in: carbon based nanomaterials for advanced thermal and electrochemical energy storage and conversion recent development of graphitic carbon nitride-based photocatalyst for environmental pollution remediation role of nanomaterials and their applications as photocatalyst and senors: a review not applicable. authors' contributions ao conducted desk study and collected and analyzed relevant review information/data, and prepared the first draft of the paper in conjunction with eoo and oso. eoo analyzed review information/data and revised the initial draft of the paper. oo conceived the idea, contributed, wrote some sections, read and edited the manuscript. all authors read and approved the final manuscript. no funding was received for this work. all the information and data cited in this review were generated from literature research and listed in the references. the information, datasets used and analyzed during the current study are available from the corresponding author on reasonable request. we have presented all data in the form of tables and figures. not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -erl jftp authors: hallema, dennis w.; robinne, françois-nicolas; mcnulty, steven g. title: pandemic spotlight on urban water quality date: - - journal: ecol process doi: . /s - - -y sha: doc_id: cord_uid: erl jftp surface water improvements associated with the covid- economic slowdown illustrate environmental resiliency and societal control over urban water quality. available, we can also expect to see examples of temporarily improved surface water quality in urban areas. the nature and extent of these improvements will depend on the level of urbanization (regional and distribution within the watershed), climate (predominant wind direction and precipitation), and physical characteristics of the watershed (topography and infiltration dynamics of the soil). the respective contributions of groundwater and storm drainage to river flow can indicate how reduced input of contaminants affects surface water quality. because upstream portions of watersheds often have a lower level of human disturbance, this is where large basins tend to exhibit a higher level of resilience (hallema et al. ) . under an economic slowdown scenario, headwaters hundreds of kilometers away from urban areas will experience reduced deposition from nonpoint sources of pollution (regionally produced nitrogen dioxide, sulfur dioxide, ammonia, and other gaseous pollutants). for example, given the reduction in nitrogen dioxide concentrations observed over eastern china, europe, the northeastern united states, and india ( fig. ) , this is where urban surface water quality may start showing signs of temporary recovery. downstream surface waters may also receive less input from both point sources of pollution (e.g., industrial sites) and nonpoint sources (e.g., motorized traffic), as the lockdowns continue. under an economic worst-case scenario, input from other major nonpoint sources, like mining and agriculture, can potentially decline due to ripple effects created by a slowdown of the global economy. the effects of reduced point source inputs on surface water quality might become evident within a matter of months while the effect of reduced nonpoint source inputs could take much longer to measure. given the frequent occurrence of urban water crises, it is critical to document how covid- pandemic response management affects natural processes and surface water quality in the short term. it is equally important to determine how we can better optimize the natural function of water supply areas ) once economic recovery is underway. the current challenge, though, is that economic sectors and industries that contribute to pollution (e.g., energy, consumer, pharmaceutical, and other industries) receive little incentive to promote urban water quality beyond what the law requires. they are chiefly evaluated in terms of total returns to the shareholders; however, an important collateral benefit of economic performance is the creation of jobs and public wealth (i.e., taxation revenue). to improve the sustainability of water supplies and cover the associated cost, it will be necessary for the public, by way of governance, to improve laws and direct public wealth toward outcomes that simultaneously support environmental and economic goals (claassen et al. ). there are very few positive aspects to be derived from the current public health emergency. the environmental responses to the economic slowdown emphasize in the first place the negative effect of humans on their environment. however, they are also a reminder that society has the ability to improve urban water quality in normal times, provided we make a conscious decision and concerted effort to create the conditions needed to reach that goal. current and future perspectives on forestwater goods and services water competition between cities and agriculture driven by climate change and urban growth global water resources affected by human interventions and climate change ) fire, forests and city water supply burned forests impact water supplies quantifying urban water supply security under global change fact check: covid- crisis has not created decreased long-term human environmental impact. usa today the urban forest and ecosystem services: impacts on urban water, heat, and pollution cycles at the tree, street, and city scale airborne nitrogen dioxide plummets over china access, visualization, and interoperability of air quality remote sensing data sets via the giovanni online tool ecohydrological processes and ecosystem services service and usda climate hubs. opinions are those of the authors and do key: cord- -weiwksh authors: ramírez-castillo, flor yazmín; loera-muro, abraham; jacques, mario; garneau, philippe; avelar-gonzález, francisco javier; harel, josée; guerrero-barrera, alma lilián title: waterborne pathogens: detection methods and challenges date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: weiwksh waterborne pathogens and related diseases are a major public health concern worldwide, not only by the morbidity and mortality that they cause, but by the high cost that represents their prevention and treatment. these diseases are directly related to environmental deterioration and pollution. despite the continued efforts to maintain water safety, waterborne outbreaks are still reported globally. proper assessment of pathogens on water and water quality monitoring are key factors for decision-making regarding water distribution systems’ infrastructure, the choice of best water treatment and prevention waterborne outbreaks. powerful, sensitive and reproducible diagnostic tools are developed to monitor pathogen contamination in water and be able to detect not only cultivable pathogens but also to detect the occurrence of viable but non-culturable microorganisms as well as the presence of pathogens on biofilms. quantitative microbial risk assessment (qmra) is a helpful tool to evaluate the scenarios for pathogen contamination that involve surveillance, detection methods, analysis and decision-making. this review aims to present a research outlook on waterborne outbreaks that have occurred in recent years. this review also focuses in the main molecular techniques for detection of waterborne pathogens and the use of qmra approach to protect public health. tracking [ , , ] . in developing countries, the lack of financial and technological resources contributes to wbdos [ ] . currently, it is estimated that there are species of pathogens infecting humans, which includes bacteria ( species), viruses ( types), parasitic protozoa ( species), and several fungi and helminths species [ , ] . the development of a disease, when and if infection to the host is produced, depends on factors such as minimal infectious dose (mid), pathogenicity, host susceptibility and environmental characteristics. enteric bacteria have a mid in the range of to cells but it is much lower with some species, such as shigella spp., ( - ), campylobacter spp., (about ), e. coli o :h ( - ), and v. cholera ( ) [ , ] . moreover, protozoan only need a few oocysts ( - ) to produce the disease as well as the viruses which a small number of these are enough to develop a disease [ ] . the protozoa microsporidia, as the bacteria mycobacterium avium intracellulare, helicobacter pylori, tsukamurella, cystoisospora belli and viruses such as adenoviruses, parvoviruses, coronaviruses (sars), and polyomavirus are some examples of the emerging potential waterborne pathogens [ , ] . furthermore, most of these organisms appear to have certain resistance against chlorine such as the microsporidia, enterocytozoon bienusi, encephalitozoon hellem and e. intestinales. m. avium and several viruses have resistance to common disinfectants for drinking water and to inactivation by uv light and heat [ ] , which represents a higher challenge in the treatment to remove these pathogens from water sources. the major pathogens microorganisms in drinking water systems and their related diseases are listed in table . presently, there is no unified method to encompass the collection and analysis of a water sample for all pathogenic microorganisms of interest [ ] . the challenges of the detection methods are the physical differences between the major pathogen groups, low concentration of pathogens in a large volume of water which usually requires enrichment and concentration of the samples prior to detection processing, the presence of inhibitors from the sample (especially if it comes from polluted water), established general protocols for sample collection, culture-independent detection method, as well as detection of the host origin of pathogens [ ] . the most important requirements for reliable analysis include: specificity, sensitivity, reproducibility of results, speed, automation and low cost [ ] . even though culture dependent methods are extensively used for pathogens detection in water, these methods are limited by their low sensitivity and the excessive time needed to obtain reliable results. furthermore, since there is a broad environmental distribution of human pathogens that exist in a viable but non-culturable (vbnc) state such as e. coli, helicobacter pylori and v. cholerae, false negative results may arise from culture dependent methods [ , ] . in both culture and molecular methods, index pathogens for monitoring water quality have been selected in order to indicate the presence of a large amount of pathogens in water. among these, e. coli ( figure ) has been extensively used due to the fact that detection methods for these pathogens are relatively easy and inexpensive; nonetheless, they may have the disadvantage of not providing information on their host origin and, sometimes, they do not correlate with other pathogens present in the water, such as the viruses and protozoa. thus, water characterized as pathogen-free by monitoring e. coli, for example, may be contaminated with viruses or protozoa [ ] . molecular methods can be very specific for particular species and provide further phylogenetic information about pathogens [ ] . these methods allow the use of alternatives indicators which easily relate with the host source. this permits the discrimination between human and animal pathogens and tracking the source of pollution [ , ] . it has been suggested that host-origin libraries, based on a phenotypic methods, may be useful for tracking the pathogen source but the development of such libraries may incur a significant cost [ ] . therefore, molecular methods seem to better suited for health risk assessments. nowadays, there are a number of different molecular methods to detect diverse pathogens. they are used to evaluate the microbiological quality of water, the efficiency of pathogens removal in drinking and wastewater treatment plants, and as microbial source-tracking (mst) tools [ ] . several examples of detection methods and their limits of detection are listed in table . molecular techniques, such as nucleic acid amplification procedures, offer sensitive and analytical tools for detecting a variety of pathogens, including new emerging strains, present the possibility of automation, and real-time analysis to provide information for microbial risk assessment purposes [ ] . immunology-based methods e. coli o :h and s. enterica typhimurium. . × cfu/ml of e. coli and . × cfu/ml of salmonella contaminated food. [ , ] * gc logs are mean values of genome copy logs. polymerase chain reaction (pcr) is one of the most commonly used molecular-based methods for detection of waterborne pathogens [ ] . pcr operates by amplifying a specific target dna sequence in a cyclic three-step process-denaturalization, annealing and extension-in order to achieve exponential amplification of the target sequence [ ] . several variations of pcr such as multiplex pcr (mpcr) allow simultaneous detection of several target organisms by coding specific genes of diverse pathogens in the sample in a single reaction. pcr method has the advantage of quick analysis. however, it necessitates accurate primers and optimal reaction mixture to avoid the risk of false positive and negative results [ , ] . limitations of dna based methods such as pcr include the inability to discriminate between viable from non-viable cells that both contain dna, the low concentration of several pathogens in water such as cryptosporidium, giardia and viruses, and the lack of data to indicate the real infectious risk to a population. challenges of molecular techniques include: the need for water concentration methods (i.e., for virus, ultrafiltration and direct flocculation protocols are used as concentration methods), the presence of inhibitors in water samples including humid acids and metals, to which pcr is sensitive, and the reproducible purification techniques by dna or rna from heterogeneous samples. in addition, result validation is required, since as indicated by hartman and co-workers [ ] ; also, high sensitivity in molecular techniques introduces a high risk of false positive results. an example of mpcr includes the technique developed by omar and barnard [ ] to detect the pathogenic and commensal e. coli from clinical and environmental water sources. to distinguish pathogenic e. coli from commensal, the presence of genes was evaluated in environmental waters. in addition, as control to evaluate the sensitivity of the technique and the false negative due to pcr, inhibitors were controlled using mdh gene (malate dehydrogenase) and gapdh gene (glyceraldehyde- phosphate dehydrogenase). another variation of the pcr is quantitative real-time pcr (qpcr) which enables quantification of dna targets by monitoring amplified products during cycling as indicated by increasing fluorescence [ ] . this method provides high sensitivity and specificity, faster rate of detection, minimizes the risk of cross-contamination, and there is no need for a post-pcr analysis [ ] . the dual-labeled fluorescent probes such as taqman probe and the fluorescent dye sybr green are the most used techniques for detecting pathogens [ ] . qpcr approach has been used to quantify human pathogens, such as e. coli o :h [ ] , and campylobacter spp., [ ] . these qpcr systems can specifically detect and quantify pathogens at concentrations as low as one target molecule per reaction. however, most of these methods can only detect and quantify one pathogen in a single reaction [ ] . ishii and co-workers [ , ] designed a microfluidic qpcr by using taqman probes (hydrolysis probe-based qpcr) labeled with different fluorophores that can detect l. monocytogenes, v. cholerae, v. parahaemolyticus, pseudogulbenkiana sp., s. typhimurium, s. flexneri, c. perfringens and pathogenic e. coli at levels of detection of cells/l. other advances in pcr include the microfluidics and nanobiotechnology field, allowing the construction of high-density and low-volume qpcr platforms, such as the openarray system that accommodates reactions per array [ ] . qpcr protocols have also been developed for the detection and identification of cryptosporidium spp., in river water with a lower quantitation limit of . oocysts/sample [ ] . for detection of g. lamblia and g. ardeae in wastewater samples, qpcr reached a sensitivity of . cysts per reaction [ ] . moreover, because of the importance of biofilm coating pipes in drinking water systems, the detection of pathogens in microbial communities is important. l. monocytogenes has been investigated in biofilms by using qpcr techniques with a number of l. monocytogenes detected growing in biofilm of × cfu/cm [ ] . for rna virus detection, quantitative reverse-transcriptase (qrt)-pcr was developed in order to provide quantitative estimation of the concentration of pathogens in water [ ] . this technique has the advantages of detecting viable cells due to detect messenger rna (mrna), which is present only in viable organisms. however, damaged genomes may fail to be detected with this technique [ , ] . oligonucleotide microarrays are a powerful genomic technology that is widely utilized to monitor gene expression under different cell growth conditions, detecting specific mutations in dna sequences and characterizing microorganisms in environmental samples [ ] . dna microarrays are arrays containing high density immobilized nucleic acids (genomic dna, cdna or oligonucleotides) in an ordered two-dimensional matrix that enables the simultaneous detection of hundreds of genes in a single assay via nucleic acid hybridization [ ] . microarrays are made up of glass slides or chips coated with specific oligonucleotide probes which are chemically synthesized short sequences ( to bp) [ , ] . microarray technology allows the rapid detection of multiple genes of multiple organisms simultaneously in the sample due its capacity for screening large numbers of sequences [ ] , has high throughput capacity and is able to be automated. therefore, large-scale and data-intensive experiments are performed in microarrays [ , ] . microarrays have also the ability to detect antimicrobial resistance to different antibiotics, and the probes could be designed for detecting the host origin of contaminants, which represents another advantage for characterization of contamination in water. however, advanced molecular technologies such as microarrays could experience difficulties distinguishing between viable and non-viable cells, have a relatively high cost, and may have non-specific hybridization resulting in a lower specificity and low sensitivity [ ] . dna microarray coupled with pcr amplification of target genes has been developed for higher sensitivity. through these steps, signal sensitivity increased around -fold [ ] . nonetheless, the pcr-microarray technique might lack sensitivity when the amount of sample is limited, since the technique requires splitting the samples into several pcr reactions to amplify [ ] . dna microarray in a two-step pcr-dna microarray assay that first amplifies the s or s rrna gene, respectively, followed by hybridization of these products onto a low-density dna microarray was developed to detect most of the common waterborne protozoan pathogens including c. parvum, g. intestinalis, acanthamoeba spp., entamoeba histolytica, and naegleria spp. [ , ] . in , wilson and co-workers [ ] were able to identify pathogenic bacteria, eukaryotes and viruses by using species-specific primer sets to amplify multiple diagnostic regions unique to each individual pathogen in the microarray. inoue and co-workers [ ] studied the occurrence of pathogenic bacterial species in groundwater and were able to distinguish between human and animal sources. microarrays for detection of human enteric viruses in community wastewaters using cell culture coupled with multiple targets including dna and rna viruses have also been developed [ ] . the virochip, a pan-virus dna microarray containing thousands of -mer oligonucleotide probes to target all viral families to infect humans, has been described by wang and co-workers [ ] . leski et al. [ ] developed a high-density re-sequencing microarray that has the capability of detecting different types of pathogens ranging from bacteria, protozoa, and viruses, including b. anthracis, francisella tularensis and ebola virus with limits of detection of to copies per test for nearly all the pathogens with high specificity. at present, standard and custom-made dna microarrays are commercially available from companies such as affymetrix, corning and agilent technologies [ , ] . one example of these is the phylogenetic microarray "phylochip" by affymetrix which consists of , oligonucleotide probes capable of identifying strains of bacteria and archaea [ , ] . this technology is very powerful because most known bacteria can be detected in samples without culturing, and the sensitivity allows also the detection of lower-abundance organisms (detection limit of ~ . % of microbial communities) [ ] . pyrosequencing is a dna sequencing technology for short-read dna sequencing that uses enzyme-couple reaction and bioluminescence to monitor the pyrophosphate release accompanying nucleotide incorporation, in real time [ ] . pyrosequencing technology provides a large number of sequences read in a single run [ ] . in the pyrosequencing reaction, the enzymes dna polymerase, atp sulfurylase, luciferase and apyrase are needed. the nucleotides are added to form the complementary strand of the single-stranded template, to which a sequencing primer is annealed. during the nucleotide incorporation, the pyrophosphate (ppi) is released. atp sulfurylase converts the ppi into atp and the atp is then converted to visible light by luciferase and the produced light signal is detected [ , ] . the methodology includes a primary step of concentrating the water pathogens followed by a secondary concentration depending of the type of the pathogen. the third step involves dna extraction (for bacteria and protozoa) and nuclease treatment (viral dna/rna extraction). then, the dna is amplified, the pyrosequencing is taking place and finally the analysis and bioinformatics can be performed [ , ] . in recent years, a new whole genome amplification and sequencing approach called "single virus genomics", which enables the isolation and complete genome sequencing by pyrosequencing of the single virus particle, has been described [ ] . roche pyrosequencing and other commercially available high-throughput sequencing platforms such as solexa/illumina genome analyzer, applied biosystem solid sequencing as well as the most recent, the ion torrent system, have revolutionized the study of microbial diversity [ ] . pyrosequencing could identify novel pathogens associated with water and address multiple etiologies. however, the dna amount present in wastewater samples could limit the sensitivity of this tool as it requires dna templates at picomole level but a much lower amount of dna may be available in water samples. this technology is also limited by the cost, the complexity of analysis, the need for increasing availability of massive computing power and the efficiency of data generation [ ] . pyrosequencing has been used to the analysis of bacterial biofilm communities in water meters of a drinking water distribution system [ ] , mixed urban watershed [ ] , characterization of nontuberculous mycobacterium communities in unchlorinated drinking water [ ] , and the detection of bacteriophages in reclaimed and potable and lake waters [ ] . fish or fluorescence in situ hybridization is based on hybridization of the sample with rrna oligonucleotide probes labeled covalently at one end with fluorescent dye. this technique allows an enumeration of particular microbial cells by the use of confocal microscopy, fluorescence microscopy or flow cytometry in order to obtain qualitative and quantitative results [ ] . fish is commonly used for the detection and identification of different microorganism in mixed populations such as in biofilms ( figure ) and has been used to produce a quantitative description of the microbial community structure in activated sludge and wastewater [ , ] , to study mechanisms of survival, infection at cellular level and detection of emerging pathogens from water, sewage and sludge [ ] . one example is the two-color fish assay, based on species-specific probes for c. parvum (cpar probe) and c. hominis (chom probe), and has been shown to distinguish between the two species of concern to public health [ , ] . several kits of fish are now available in the market, one example of these are the commercial kit label it ® fluorescence in situ hybridization kit cy ® , fluorescein and tm-rhodamine, which optimized the preparation and hybridization of fluorescently labeled dna probes [ ] . viable but non-culturable (vbnc) cells could detected by fish couple with direct viable count (dvc) assay [ ] [ ] [ ] . in the assay, the cells are cultured in reach media with antibiotics which prevent cellular division, increases of intracellular rrna and allow the elongations of viable cells. fish is made with specific probes that target rrna labeled with fluorescent dye. the sample is stained by ,-diamino- -pheynyl indole (dapi) that is bound to double-stranded dna to count the total amount of cells in the samples, and the results are compared to culture methods to measure the proportion of vbnc cells [ ] . members of the family enterobacteriaceae and e. coli in drinking water systems, freshwater and river water have been detected by this tool [ ] . this assay is able to detect viable e. coli/ ml in more than non-e. coli/ ml [ ] . however, the technology is limited by a low sensitivity, and the necessity of pre-enrichment and concentration steps [ ] that also may increase inhibitor concentrations and lead to false negative results [ ] . vbnc cells are living cells, metabolically active, that may still retain or regain their virulence potential upon the resuscitation (return to culturable state) under favourable environmental conditions or when the stress is removed [ ] [ ] [ ] [ ] . due to the fact that vbnc cells cannot be identified by culturable methods, the number of pathogens in this state could be underestimated and, if all bacteria in the sample are in vbnc state, the sample may be regarded as pathogen-free due to non-detection [ ] . the conversion to the vbnc state is supposed to be a response to adverse environmental conditions [ ] [ ] [ ] . resuscitation of these species could be triggered by a variety of stimuli, such as rich medium, an increase in temperature, and the presence of host cells. factors as the strain used, the age of vbnc cells, and the conditions that induced the vbnc state also affect the resuscitation [ , , ] . besides fish, other tools to detect vbnc bacteria includes: immunological techniques; rt-pcr, which target the mrna that indicates the presence of viable cells that carry out transcription; dnase i protection assay, since only the viable cells have intact membranes to protect genomic dna from digestion by exogenous nucleases; enzymatic activity, as viable cells carry out metabolic reactions and respiration; bacteriophage-based assay, using a combination of fluorescence intensity and nutrient uptake analysis; as well as the commercial kit live/dead ® baclight tm assay to distinguish viable cells from dead cells based on the membrane integrity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . immunological detection with antibodies is a technology that has been employed for the detection of multiple pathogens [ , ] . these methods are based on antibody-antigen interaction, whereby a particular antibody will bind to specific antigen which comprise the use of polyclonal and monoclonal antibodies [ , ] . immunological detection includes, for example, serum neutralization tests (snt), immunofluorescence, and enzyme-linked immunosorbent assays (elisa). snt test has been used for the serotyping of viruses and involves mixing a sample, extracted from a plaque assay, with antiserum and then assessing the decrease of infectivity by plaque assay [ ] . assays based on immunofluorescence and immunomagnetic separation (ims) are used to detect protozoan parasites. the us environment protection agent (epa) has established method . for the combined detection of cryptosporidium spp. and giardia spp., or method for cryptosporidium spp., both of which use ims followed by immunostaining and fluorescent microscopy [ , ] . nevertheless, limitation such as low sensitivity, false negative results, cross-reactivity with closely related antigens and the need for a pre-enrichment in order to reduce the cell surface antigens, are present in these techniques [ ] . biosensor based methods have flourished in recent years. biosensor is an analytical device that consists of a bioreceptor that recognizes the target analyte (e.g., enzymes, antibodies, nucleic acids, cell receptors, aptameters, recombinant antibodies, imprinted polymers and synthetic catalyst) and a transducer that converts the biological interactions into a measurable electrical signal (optical, electrochemical, mass-based, thermometric, or micromechanical) [ , , ] , thereby providing selective quantitative or semiquantitative analytical information [ ] . optical biosensors rely on a change in the optical properties of the surface caused by the binding of the analytes used for detection [ ] . electrochemical biosensors are based on measuring changes in conductance, resistance or capacitance of the active surface. in these devices, one of the electrodes is immobilized with a recognition molecule. when analytes bind, a change in electrical properties occurs providing the sensor signal [ , ] . mass-sensitive biosensors include quartz crystal microbalance biosensor, which uses a quartz crystal inserted in two electrodes. as quartz is piezoelectric (used for wave propagation), the crystal can be excited by applying a voltage across the electrodes and will exhibit a resonance frequency [ , ] . biosensor methods have the advantages of automation and miniaturization of biological analytical techniques, have short analysis times, portability, real-time measurements and do not require sample pre-enrichment [ ] . nevertheless, problems such as great sensitivity to ph, change of mass, temperature, etc., represent a great challenge to the use of these methods [ ] . in , taylor and co-workers [ ] used the surface plasmon resonance (spr) detection with pretreatment of the bacteria (heat or ethanol treated or detergent-lysed cells) and the antibodies goat anti-e.coli o :h polyclonal antibody (pab) and mouse anti-e. coli o :h monoclonal antibody (mab) for signal amplification to obtain a limit of detection (lod) of cfu/ml. antibody-based indirect sensor (optical biosensor) for escherichia coli o :h with fluorescent labeling was reported by ho et al., [ ] in to reach a lod of cells/ml. detection and monitoring of pathogens in water continues to be a field with constant improvement and development of new tools that will permit relative low cost assays and rapid identification of multiple pathogens with limits of detection that meet regulatory goals. nevertheless, efforts to standardize techniques in the field have to include sample collection, sample concentration, sample purification, sample processing, analysis, and data collection are still remaining [ ] . sample collection has to be standardizing mainly if the target is on biofilms. validation of new emerging techniques has also to be completed. besides microarrays and biosensors, the integration of molecular technologies is promising. "lab-on-chip" (loc) represents an integration of sensors and microfluidic systems in a miniaturized tool that could archive real-time monitoring of samples [ ] . by this way, loc integrates several laboratory functions on a single chip and reduced sample and reagent consumption is automatized and has fast detection times and low limits of detection. however, there are still challenges related to developing practical loc components, assay procedures and validation of the on-chip detection approaches, and of course, the different scenarios that represent a water sample [ ] . since outbreaks continue to occur despite water quality monitoring, a new approach has been applied to solve the need for a conceptualization of risk and to provide guidance and legislations [ ] . quantitative microbial risk assessment (qmra) is a systematic quantitative assessment process to estimate the health risks or illness rates of human exposure to particular pathogens. the approach combines dose response information for the infectious agent with information on the distribution of exposures routes and is able to demonstrate if the health targets are met in a drinking water distribution system [ ] , which are suggested to be a health risk less than one infection per , individuals per year, or, a disease burden of − disability adjusted life year (daly) per person per year [ ] . this tool helps to predict the burden of waterborne diseases, set tolerable limits of waterborne disease, identify the means to reduce the risk to the consumers and determine measures to protect water safety [ ] . risk assessments involve five steps ( figure ) [ , [ ] [ ] [ ] [ ] . (i) hazard identification: consists in the identification and quality evaluation of the microbial hazard. this step takes into account the potential outcomes (health effects, disease outbreaks), pathogen properties (virulence, adaptation, resistance, and mutation) and hazard pathways; (ii) exposure assessment: is the estimation of the duration of human exposures to pathogens by specific routes, the determination of the size and nature of the population exposed and the barrier reduction and recontamination on water; (iii) dose-response assessment is the characterization of the relationship between dose and incidence of adverse effect in populations exposed to microbial pathogens. it comprises data of illness, secondary transmission and immunity in population. typically, the dose-response is derived from studies of exposure of human volunteers to different doses of the pathogen or is based on previous outbreaks [ ] [ ] [ ] . factors influencing the dose-response are the exposure route, exposure medium pathogen strain host endpoint and the data source. two commonly models are the beta-poisson model and the exponential model, described elsewhere [ , , ] ; (iv) risk characterization is the integration of information from hazard identification, dose-response assessment, and exposure assessment to estimate the magnitude of health effects; (v) risk management and communication is a decision-making process based in all previous steps in risk assessment [ , [ ] [ ] [ ] [ ] . in qmra, the different variants contribute to measuring the health risk but also introduce variability corresponding to dose-response sensitivity, detection methods, temporal and spatial heterogeneities in pathogen densities and uncertainty (i.e., unrepresentative population, modeling pathogen densities and knowledge of dose-response relationships) [ , [ ] [ ] [ ] . the advantage of probabilistic modeling is to distribute the uncertainties through the model [ ] . the qmra approach presents potential advantages and limitations of risk management options that could be evaluate by numerical stimulations to investigate their possible efficacy, and that risk below epidemiologically detectable levels may be estimated under specific circumstances [ ] . however, the greatest limitation on qmra is the lack of available data to carry out this assessment [ ] . qmra also depends on the method used for monitoring pathogens in water. actually, qmra approach is based exclusively on data obtained by cultivation methods, which are not allowing the complete characterization of risk, since as mentioned above, many waterborne microorganisms are not culturable or are inefficiently cultured, thus leading to underestimation of pathogens in water. therefore, selecting the optimal tool for pathogen monitoring in source water that gives the necessary information on occurrence, prevalence, virulence, and even viability of the pathogens influences the results of the qmra approach. polymerase chain reaction methods provide rapid detection of microorganisms but are complicated by the presence of inhibitory compounds in the sample and the detection of viable but non-culturable as well as non-viable pathogens. integrating the culture and pcr methods may help to overcome the weakness of each individual method [ , , ] . as mentioned before, current risk characterization approaches rely on detection of microbial indicators or "index pathogens", which does not accurately predict the occurrence of all pathogens. novel markers that better predict the presence of all pathogens of interest would improve risk assessment and management actions [ , ] . alternative indicators proposed are bacteroidales and lachnospiraceae, which are rich in host-specific bacterial organisms [ , ] , faecal bacteriophages, rotavirus, ascaris [ ] , firmicutes [ ] , bifidobacteria spp. [ ] , and methanobrevibacter smithii [ ] . commonly s rrna gene has been used as a marker to track host-specific organisms [ ] , but other authors such as villemur et al. [ ] and caldwell et al. [ ] suggest the use of dna (mtdna) to target the identification of human and animal origins. qmra has been used to estimate the health risk associated with bathers after surfing and swimming in dry weather and post-storm conditions near beaches [ ] [ ] [ ] , water distribution networks [ ] , recreational waters impacted by agricultural contamination [ , ] , and assessment of the efficiency of the applied treatment processes in drinking water [ ] . such investigations reinforce the need for preventive measures, such as those designed to prevent and take measures to reduce water contamination. a computational tool for quantitative microbial risk assessment, called qmraspot, was developed for rapid and automatic undertaking of qmra for drinking water. qmraspot uses four index pathogens: campylobacter, enterovirus, cryptosporidium, and giardia [ ] . also, a software infrastructure name frames (framework for risk analysis in multimedia environmental systems) have been created to assess the water health risk which allows describing the problem statement, integrating models and data-bases, and provides the infrastructure for performing sensitivity, variability and uncertain analyses [ ] . it is clear that qmra and management of water quality are important tools for governmental regulation and scientific analysis. nevertheless, it is also clear that these tools still are not adopted widely [ ] . efforts to encourage the use of the qmra approach have to be made to support implementation of water safety plans, improve understanding of vulnerabilities of drinking water distribution systems, assess risks associated with extreme events and establish the best management option for the given risk in order to ensure water safety [ ] . waterborne pathogens are a global concern for worldwide public health. since pathogens in water are still a major cause of severe illness and mortality, the control, monitoring and application of regulations for water quality are in urgent need and must incorporate more effective microbiological monitoring, pathogen detection and health risk assessment in order to reach the goal of pathogen-free water. although culture methods for detection of pathogens in water are used routinely, they may underestimate the level of microbial pathogens. molecular techniques improve the characterization of these pathogens; however, several disadvantages such as the lack of standardization of protocols and sample processing are still a challenge. improving available technologies so that they are able to identify causative agents more accurately and in a shorter amount of time, to detect viable microorganisms and characterize them according to microbial communities and that enable the creation of accessible data could enhance the knowledge of waterborne pathogens and the possibilities to predict pathogen contamination and protect public health. pathogen indicators need to be continually improved since a large number of new emerging pathogens are 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contamination the authors would like to thank pathogens editors and the reviewers for their helpful comments. we are gratefully acknowledge the fie grate support of the natural sciences and engineering research council of canada (to j.h, rgpin- ) and from-fonds de la recherche du québec en nature et technologies (centre de recherche en infectiologie porcine et avicole, cripa-regroupements stratégiques rs- ) and from the dfait canada's emerging leaders in the americas program. we thanks to the conacyt mixed scholarship ( / ) of f.y.r.c., and to a.l.g.b., f.j.a.g. and the universidad autónoma de aguascalientes for their funds. josée harel had the original idea for the manuscript and, with all co-authors, carried out the design. flor yazmín ramírez-castillo drafted the manuscript, which was revised by all authors. abraham loera muro and mario jacques included the fluorescence in situ hybridization detection of actinobacillus pleuropneumoniae isolated from water sources. philippe garneau critically revised the manuscript and verified the detection methods. josée harel, mario jacques, alma lilián guerrero-barrera and francisco javier avelar-gonzález revised all the manuscript and profit it. all authors read and approved the final manuscript. the authors declare no conflict of interest. key: cord- -b tduubu authors: sano, daisuke; amarasiri, mohan; hata, akihiko; watanabe, toru; katayama, hiroyuki title: risk management of viral infectious diseases in wastewater reclamation and reuse: review date: - - journal: environ int doi: . /j.envint. . . sha: doc_id: cord_uid: b tduubu inappropriate usage of reclaimed wastewater has caused outbreaks of viral infectious diseases worldwide. international and domestic guidelines for wastewater reuse stipulate that virus infection risks are to be regulated by the multiple-barrier system, in which a wastewater treatment process composed of sequential treatment units is designed based on the pre-determined virus removal efficiency of each unit. the objectives of this review were to calculate representative values of virus removal efficiency in wastewater treatment units based on published datasets, and to identify research topics that should be further addressed for improving implementation of the multiple-barrier system. the removal efficiencies of human noroviruses, rotaviruses and enteroviruses in membrane bioreactor (mbr) and conventional activated sludge (cas) processes were obtained by a systematic review protocol and a meta-analysis approach. the log( ) reduction (lr) of norovirus gii and enterovirus in mbr were . ( % confidence interval: . , . ) and . ( . , . ), respectively. the lr values of rotavirus, norovirus gi and gii in cas processes were . ( . , . ), . ( . , . ) and . ( . , . ), respectively. the systematic review process eliminated a substantial number of articles about virus removal in wastewater treatment because of the lack of information required for the meta-analysis. it is recommended that future publications should explicitly describe their treatment of left-censored datasets. indicators, surrogates and methodologies appropriate for validating virus removal performance during daily operation of wastewater reclamation systems also need to be identified. enteric viruses have caused infectious diseases owing to fecallycontaminated irrigation and environmental water. for example, a large outbreak of norovirus occurred in eastern germany in , which was likely caused by imported frozen strawberries from china (bernard et al., ) . multiple genotypes of norovirus were found environment international ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] from tested strawberries and gastroenteritis patients, which supported the speculation that imported strawberries were cultivated with untreated wastewater (mäde et al., ) . imported foodstuffs contaminated with pathogenic viruses originating from wastewater have also caused outbreaks in the other developed countries (ethelberg et al., ; pérez-sautu et al., ; terio et al., ) , in particular when food products are imported from areas where water and sanitation systems are poorly installed and wastewater is directly used for irrigation. in the worst scenario, pathogens eradicated from developed countries can be imported from endemic areas via foodstuff, after which such neglected pathogens can cause outbreaks of infectious diseases in importing countries without any protective aids such as vaccination. in the future, wastewater will have to be used as irrigation water in arid areas such as australia, india, pakistan, vietnam, the northern part of china, and middle eastern countries including israel (hanjra et al., ) . wastewater is also becoming an important irrigation water source in the usa due to the heavy dependence of agricultural productions on fossil waters from three major over-exploited aquifers (marston et al., ) . the national science foundation/mathematics and physical sciences directorate (nsf/mps) ( ) reported that one opportunity for transformative research can be found in ensuring a sustainable water supply for agriculture, including the expansion of supply through wastewater recycling. in these settings, enteric viruses require special attention because they are excreted to wastewater in larger numbers by infected individuals (up to viruses per gram feces) compared to other enteric pathogens (drexier et al., ; kirby et al., ) , and they are typically so highly infectious that less than particles is often enough to cause an infection in susceptible individuals (thebault et al., ; kirby et al., ) . removal of these viruses by wastewater treatment processes is required if treated wastewater is reclaimed; however, enteric viruses are more resistant than pathogenic bacteria to several water disinfection treatments such as filtration, chlorination, uv irradiation, and ozonation, due to the physicochemical properties of viral particles (who, ) . these features highlight enteric viruses as the most important health issue for the safe use of reclaimed water (atse, ) . in this review article, current guidelines for designing wastewater reclamation and reuse systems from the viewpoint of virus risk management are overviewed, and the efficiency of virus removal from wastewater by currently employed wastewater treatment units are shown by the results of meta-analysis. the regulations for validating the virus removal efficiency of wastewater treatment processes during daily operation are then described in a separate section. finally, the research areas that should be addressed in the future for better application of wastewater reclamation and reuse are identified from the viewpoint of virus risk management. the world health organization (who) published four volumes of guidelines for the safe use of wastewater, excreta, and greywater in . policy and regulatory aspects are compiled in volume , where who employs − disability adjusted life year loss per person per year (daly loss pppy) as a tolerable additional disease burden (who, ) , which is equivalent to rotavirus disease and infection risks of approximately − and − per person per year, respectively (mara, ) . these values of tolerable additional disease burden can be used to calculate the acceptable level of pathogen concentration in water if the dose-response relationship is available for the pathogen of interest; however, the who suggests guideline users set performance targets for wastewater treatment processes in place of the acceptable pathogen concentrations in treated wastewater. the performance target is a value of removal efficiency expressed in common logarithm of the ratio of pathogen concentration in untreated wastewater to that in treated wastewater. for example, the who guideline describes that the health-based target ( − daly loss pppy) for viral pathogens can be achieved by a total virus reduction of - log in reclaimed wastewater for restricted irrigation (the irrigation of all crops except salad crops and vegetables that may be eaten uncooked), while a - log pathogen reduction is needed for unrestricted irrigation (including the irrigation of salad crops and vegetables that may be eaten uncooked) and localized irrigation (e.g., drip irrigation). the total pathogen reduction in log units is obtained by a combination of wastewater treatment including disinfection, natural die-off and health protection measures such as washing produce prior to consumption. the combination of wastewater treatment units is called the multiple-barrier system, consisting of multiple steps where the pathogen removal efficiency (pathogen removal credit) of each step has already been evaluated and determined as a credited log reduction (lr) value (fig. ) . who ( ) recommends guideline users measure reduction/inactivation of adenoviruses, reoviruses, enteroviruses, and hepatitis a virus to validate the efficacy of each treatment when the treatment system starts operation. the who ( ) guidelines also suggest that each country should establish national criteria and procedures that suit its epidemiological, social, and economic needs. the united states environmental protection agency (usepa) ( ) guideline explicitly notes that setting a tolerable virus concentration in reclaimed wastewater (virus limit) is not recommended for the following reasons: ) viruses are well reduced by appropriate wastewater treatments, ) identification and enumeration of viruses is time-and labor-consuming, ) detection of infectious viruses in water is further labor-and time-consuming, ) molecular-based virus detection does not always indicate the presence of infectious viruses, and ) waterborne viral infections due to reclaimed water have not been documented. instead of setting a virus limit, the control of viral infectious diseases caused by contaminated reclaimed wastewater is to be achieved by the multiple-barrier system, which coincides with the who guideline. based on the usepa guidelines for water reuse, several u.s. state governments have set performance targets for virus removal in wastewater reclamation. in the groundwater replenishment reuse project (grrp) of the state of california, where the most stringent and intimate regulations related to recycled water have been established, a lr of viruses along with a lr of both giardia cysts and cryptosporidium oocysts in the wastewater is required when treated wastewater is used for groundwater recharge intended for indirect potable reuse (california state water resources control board, ) . the reductions should be accomplished by three or more successive treatment process units, each of which can reduce - log of viruses. in the state of texas, where direct potable reuse (dpr) of reclaimed water is being implemented, , , and lrs of viruses, cryptosporidium, and giardia, respectively, are recommended as a baseline for dpr when untreated wastewater is used as a source (texas water development board, ) . these required lr values were developed based on the general approach presented in "potable reuse: state of the science report and equivalency criteria for treatment trains", which was prepared as part of the water reuse research foundation project - (wrrf project - ) (texas water development board, ) . the general approach for determining the required pathogen lr used available data obtained from literatures and calculated drinking water concentrations needed to meet a health goal in the usepa surface water treatment rule (swtr), which is a risk of infection per , exposed individuals per year (texas water development board, ) . on another front, texas commission on environmental quality (tceq) has established minimum (or baseline) lr and/or inactivation targets for the dpr, which are , . , and lrs of viruses, cryptosporidium, and giardia, respectively, when wastewater treatment plant effluent is used as a source, although these baseline lr targets are considered a starting point for the tceq approval process and may be revised based on data collected from the wastewater effluent in question (texas water development board, ). in australia, viruses are considered to be the most important pathogens in water and water treatment systems are designed considering virus removal on a preferential basis. the australian guideline is based on a study of quantitative microbial risk assessment (qmra) conducted to determine the required virus reduction level for reclaimed water treatment (nrmmc, ) . in this assessment, rotavirus was selected as a representative virus because of its prevalence, high infectivity, and the availability of a reliable dose-response model. in this analysis, the required reduction level varied from . to . log depending on the degree of human contact. again, the multiple-barrier system is adopted by the australian national guidelines (nrmmc, ) , as with who ( ) and usepa ( ). the advantage of the multiplebarrier system is that the simultaneous failure of multiple process units is not probable and even if one process unit becomes dysfunctional, the remaining barriers should be capable of providing sufficient protection. based on the australian national guidelines (nrmmc, ) , several australian states have set criteria for virus control in wastewater reclamation. for example, the state of victoria has set a criterion that the performance of each process unit is determined by evaluating its virus reductions (the state of victoria, ). a variety of enteric viruses that can cause waterborne infection, e.g., enterovirus, adenovirus, rotavirus, norovirus and other caliciviruses, hepatitis a virus, astrovirus, and coronavirus, are listed as examples of viral contaminants in the guidelines of queensland, australia (the state of queensland, ). in the state of queensland, a . lr of viruses in treated wastewater is necessary to be classified as the highest quality reclaimed water (class a+), which is more stringent than the required reductions for either bacteria ( log ) or protozoa ( log ). if the reclaimed water is used for augmentation of drinking water supplies, a . lr of viruses along with an lr of bacteria and protozoa is required according to qmra studies. some countries in europe, including cyprus, france, greece, hungary, italy, portugal, and spain, have guidelines or regulations for wastewater reuse (raso, ; sanz and gawlik, ) . among them, only france has set a regulation regarding viral contamination, in which a to lr of f-specific bacteriophage by reclamation processes is required. in the netherlands, a unique qmra-based viral regulation in drinking water is implemented, but viruses in reclaimed water are not regulated (smeets et al., ) because fresh water resources are rich and the use of reclaimed water is mostly limited to industrial purposes (rietveld et al., ; paranychianakisa et al., ) . in singapore, approximately % of the water supply is provided by a reclaimed water program called newater. secondary treated wastewater is sequentially treated by microfiltration, reverse osmosis (ro), and uv for producing newater (pub, ) . no specific regulation for reclaimed water quality is present in singapore, but newater has been shown to meet who and usepa standards for drinking water and to be negative for enteroviruses (pub, ) . newater is not supplied directly as drinking water but is thought to be safe for direct potable use. these examples indicate that the multiple-barrier system serves as a global basic concept for the regulation of pathogenic viruses in wastewater reclamation and reuse, in which these viruses are not being regulated by virus concentration limits in the treated wastewater, but rather are managed by the sequential use of technological units for wastewater treatment and disinfection where the virus removal efficiency of each treatment unit has previously been determined. the required virus removal efficiency (performance target) is determined by qmra, and subsequently it is required to combine sequential wastewater treatment and disinfection practices in such a way that the sum of pre-determined virus removal efficiencies exceeds the performance target. under this concept of the multiple-barrier system, two important issues must be addressed: how to determine the virus removal efficiency of treatment steps prior to operation and how to ensure that the pre-determined performance targets are met or exceeded in daily operation. these two issues are discussed in the following sections. solid removal techniques such as filtration, flocculation, coagulation, and sedimentation, and disinfection techniques such as uv irradiation and ozonation are regarded as effective for reducing viruses in wastewater and producing virologically safe reclaimed water; however, the values of virus removal efficiency for these processes are heavily dependent on operation conditions, such that efficiencies can vary greatly even within the same reactor for a given treatment (teunis et al., ) . it is necessary for water engineers to know the "average" virus removal efficiency of each wastewater treatment unit as a virus lr credit in order to design efficient wastewater reclamation systems under the concept of multiple-barrier system (fig. ) . in this section, we employed a meta-analysis approach to obtain the representative values of virus removal efficiency for wastewater fig. . schematic representation of the multiple-barrier system for the pathogen risk management in wastewater reclamation. the multiple-barrier system consists of multiple steps where the pathogen log reduction (lr) credit of each step has already been determined. the total pathogen lr in the system is compared with the performance target with respect to the usage purpose of reclaimed wastewater. treatment process units. we selected norovirus (genogroup i and ii), rotavirus, and enterovirus as enteric viruses of interest and papers investigating the removal efficiency of these viruses in wastewater treatment processes were collected using a systematic review protocol. google scholar, pubmed, and web of knowledge were used as search engines in this study. relevant articles published between and were found in all three databases using the search terms "removal efficiency" and "wastewater treatment" combined with each viral genus name of norovirus, rotavirus and enterovirus. all articles obtained from pubmed and web of knowledge coincided with those from google scholar, therefore all the article numbers indicated below are the results obtained from google scholar. norovirus, rotavirus and enterovirus combined with the above-mentioned terms gave , , and retrievals, respectively (table s ) . a meta-analysis methodology was employed to estimate how effectively viruses are reduced by two widely-employed wastewater treatment technologies: membrane bioreactors (mbrs) and activated sludge (as). data obtained from google scholar was processed according to the prisma flow diagram used in clinical medicine with some modifications (moher et al., ) . the prisma flow diagram explains the process of selection of relevant articles among the records obtained via web search ( figure s ). after identification of records, they were screened to eliminate non-english records, articles without virus data or quantitative virus data, doctoral dissertations, book chapters, review papers, posters, conference proceedings, and other technical reports. the number of articles selected by the initial screening were , , and for norovirus, rotavirus, and enterovirus, respectively (table s ) . after the screening, information required for the calculation of averaged virus lr values was extracted from the full text articles, including quantitative data of virus reductions (average and standard deviation) and the size of samples used in the calculation of virus reductions. table s summarizes the articles selected for the meta-analysis stage. additionally the data in one article was directly obtained from one of the authors (katayama et al., ) . arithmetic mean and standard deviation values of lrs were calculated from the available data when these values were not specified explicitly in the articles. the imagej program developed by the national institutes of health was used for data extraction from figures if necessary. extracted data of lr were separated into that of mbr and as processes. meta-analysis calculation and forest plot construction were performed using a microsoft excel software package originally developed by neyeloff et al. ( ) , with some modifications. the step-by-step calculation procedure is described in the supplementary materials. sample numbers considered for calculation, data extraction, and calculation methods are summarized in table s . the forest plot of lr values of norovirus gii by mbr with % confidence interval (ci) values is shown in fig. . four articles were selected based on the availability of quantified lr data (sima et al., ; simmons et al., ; chaudhry et al., ; miura et al., ) ( table s ). these studies investigated virus concentrations in the influent and effluent of an mbr process by quantitative reverse-transcription pcr (rt-qpcr) and lr values, each calculated as a ratio of virus concentration in influent to that in effluent at each sampling event, were presented. lr values were extracted from these studies only when viruses were detected in both influent and effluent samples. we excluded paired data of virus concentrations in influent and effluent from the calculation when either the influent or effluent sample was negative (nondetect) for virus quantification. since viruses are not detected from an effluent sample when highly efficient virus removal is achieved, this practice may lead to the exclusion of superior lr values (higher virus removal efficiencies) from the calculation. the reasonable treatment of datasets including non-detects is discussed in the following section. the mean values of norovirus gii lr varied between . (miura et al., ) and . (chaudhry et al., ) , which may reflect differences between these mbr configurations and their operational conditions (table s ). since the pcr for norovirus does not discriminate genotypes within a genogroup, the fluctuation of virus removal efficiency between studies may also reflect that different genotypes or strains of norovirus gii were removed dissimilarly, although genotype-or strain-dependent removal properties have not been thoroughly investigated (miura et al., ) . chaudhry et al. ( ) reported the gradual increase of lr when the time after membrane wash was increased from to h, which implies that the level of membrane fouling is one of the determinant factors of virus lr. the hydraulic retention time (hrt) is another influential factor for virus reduction in mbr (campos et al., ) , but not all of the selected publications specified the details of operational conditions including hrt. among the retrieved papers, sima et al. ( ) and chaudhry et al. ( ) did not specify hrt values of the reactors, because these studies did not focus on the relationship between hrt and virus removal efficiency. since these determinant factors of virus removal efficiency in wastewater treatment units may be beneficial information for wastewater engineers, it is recommended that future publications with regards to virus lr include detailed operational conditions (hrt, time after membrane wash, etc.) of the wastewater treatment units. fig. . removal efficiency of norovirus genogroup ii by membrane bioreactors. black square is the mean value of virus log reduction (lr) obtained in each study, and a bar indicates % confidence interval (ci). the sample number in each study is indicated in a parenthesis next to % ci values. the total lr value is obtained by the calculation procedure indicated in supplementary materials. since heterogeneity was detected via q-statistics (eq. (s )) between studies of norovirus gii reduction in mbr, a random effect model utilized by neyeloff et al. ( ) was applied (see eqs. (s ) to (s )) in order to calculate a representative value of . lr ( % ci between . and . , fig. ). some articles were excluded from this representative value calculation; for example, zhou et al. ( ) investigated the concentrations of multiple virus types including norovirus gii before and after an mbr process by rt-qpcr, but no information about sample size in the calculation of average lr was described, thereby making it impossible to include the presented lr data in the meta-analysis. da silva et al. ( ) also analyzed the removal efficiency of norovirus gii in mbr, but all effluent samples were norovirus gii-negative and could not be included in the meta-analysis. all manuscripts excluded in this meta-analysis because of the lack of required information are likely trustworthy and useful as case studies for virus removal in wastewater, but without key experimental information it is not possible to utilize such data for calculating the average value of virus lrs by meta-analysis in the present study. it is strongly recommended that arithmetic mean and standard deviation values of lr as well as sample size, which are necessary for meta-analysis calculations, be clearly described in the publication, otherwise lr datasets cannot be included in the calculation of representative values of virus removal efficiency. we retrieved three articles related to the reduction of enteroviruses in mbr processes (ottoson et al., ; simmons et al., ; miura et al., ) (table s ). q-statistics detected heterogeneity between these studies as with norovirus gii; therefore, the random effect model was again employed to calculate a representative virus removal value of . lr with a % ci between . and . (fig. ) . the broad ci may be attributable to different operational conditions (table s ) , and/or different removal efficiencies among viral species within the genus enterovirus. the rt-qpcr assay used for enterovirus in these studies targets the ′ non-coding region, which is highly conservative among enteroviruses and includes more than serotypes. this means that the virus removal efficiency obtained in each study is the representative removal efficiency of multiple enterovirus types in each mbr process. zhou et al. ( ) was again not involved in the meta-analysis, because of the lack of sample size information. francy et al. ( ) reported the removal efficiency of enterovirus in mbr, but there was only one enterovirus-positive sample in the mbr effluent, therefore this study was not included in the meta-analysis. with the exception of norovirus gii and enterovirus, we could not retrieve a sufficient number of publications regarding the removal of human enteric viruses (rotavirus, adenovirus, aichivirus, astrovirus, hepatitis a virus and sapovirus) in mbr that met the described systematic review criteria. it may be possible to supplement this lack of datasets for human enteric viruses by providing additional phage removal information; however, we found that the majority of publications regarding phage removal in wastewater treatment units were based on the infectious phage concentration in wastewater samples measured by cell culture methods (e.g., purnell et al., ) . it is not common to directly compare the lr values calculated based on genome copy to those calculated from infectious units, because the reduction of infectious units can be caused by both the physical removal and disinfection of viruses. for this reason, we chose not to include phage reduction datasets in this study. four articles were selected for rotavirus reduction in as processes (li et al., ; prado et al., ; el-senousy et al., ; qiu et al., ) ( table s ). the mean values obtained in each study ranged between − . (prado et al., ) and . (el-senousy et al., ) (fig. ) . q-statistics detected heterogeneity between these studies as well, and a representative removal efficiency of . lr with % ci between . and . was calculated. kitajima et al. ( ) reported the removal efficiency of rotavirus in two wastewater treatment plants employing as processes, but the removal efficiency was obtained for the combined process of as and chlorine treatment. since this study aims to calculate the virus lr in each treatment unit, we did not use the datasets in kitajima et al. ( ) in the meta-analysis. four articles were also obtained pertaining to norovirus gi and gii removal in as processes (da silva et al., ; katayama et al., ; nordgren et al., ; flannery et al., ) (table s ). the representative removal efficiency of norovirus gi in as processes was . with % ci between . and . (fig. ) , while that of norovirus gii was . with % ci between . and . (fig. ) . these results indicate that the removal efficiencies of gastroenteritis viruses (rotavirus, norovirus gi and gii) by as are very comparative, and about to . lrs are expected on average. the systematic review process eliminated some articles about norovirus reduction in as processes. for example, campos et al. ( ) reported the norovirus concentration in influent and effluent samples of as processes, but did not provide the sample number and sd values of the concentration, which are indispensable for the meta-analysis. la rosa et al. ( ) also reported the norovirus concentration in influent and effluent samples of as processes, but did not provide sd values. with the exception of rotavirus and noroviruses, we could not retrieve a sufficient number of publications regarding the reduction of human enteric viruses (adenovirus, astrovirus, aichivirus, hepatitis a fig. . removal efficiency of enteroviruses by membrane bioreactors. black square is the mean value of virus log reduction (lr) obtained in each study, and a bar indicates % confidence interval (ci). the sample number in each study is indicated in a parenthesis next to % ci values. the total lr value is obtained by the calculation procedure indicated in supplementary materials. virus, and sapovirus) in as processes that met the described systematic review criteria. although we did not include information regarding the removal efficiencies of bacteriophages from wastewater in the present study due to the reason described above for mbr, the meta-analysis of phage lr values in wastewater treatment processes may be beneficial in the discussion of the human health impact from wastewater reuse and should be addressed in future publications. the examples of meta-analysis in this section help to define the shape of the dataset that needs to be taken to facilitate calculating virus removal efficiency of a wastewater treatment unit. one of the essential qualifications is to explicitly describe the treatment of nondetects. since any pathogenic viruses can be removed by wastewater treatment to some extent, the quantification datasets of viruses in treated wastewater commonly include a significant number of nondetects. even for untreated wastewater samples, pathogenic viruses are frequently not detected because of diel and annual variability. for example, noroviruses are frequently detected in untreated wastewater during the epidemic season, but not during other times of year (pérez-sautu et al., ) . these non-detects do not allow us to calculate the virus removal efficiency and, therefore, the meta-analysis examples above employed only datasets where viruses in untreated and treated wastewater samples were both quantified and could be used to calculate the log-ratio of virus concentrations before and after a given treatment. this approach, in general, may underestimate the true virus removal efficiency value because viruses are not detected from an effluent sample when highly efficient virus removal is achieved as described above. substitution of the non-detect data with specific values such as the limit of quantification, the half value of quantification limit, or zero has been used as a classical approach for dealing with non-detects, but the substitution gives inaccurate estimation of distribution parameters when the distribution of concentration is predicted (helsel, ) . it has been proposed that datasets including non-detects or so called "left-censored" datasets can be analyzed by bayesian estimation of the posterior predictive distribution of pollutant concentrations in environmental samples (paulo et al., ) . we have extended the bayesian model to estimate the posterior predictive distribution of virus- fig. . removal efficiency of rotaviruses by activated sludge processes. black square is the mean value of virus log reduction (lr) obtained in each study, and a bar indicates % confidence interval (ci). the sample number in each study is indicated in a parenthesis next to % ci values. the total lr value is obtained by the calculation procedure indicated in supplementary materials. fig. . removal efficiency of norovirus genogroup i by activated sludge processes. black square is the mean value of virus log reduction (lr) obtained in each study, and a bar indicates % confidence interval (ci). the sample number in each study is indicated in a parenthesis next to % ci values. the total lr value is obtained by the calculation procedure indicated in supplementary materials. removal efficiency (kato et al., ; ito et al., ) . these models employ a parametric probabilistic distribution such as a log-normal distribution for expressing pollutant concentrations, which may give inaccurate estimations when the observed data is skewed (shoari et al., ) . it is necessary to test the robustness of these statistical models and their applicability to environmental survey data. while datasets were collected for human enteric virus removal in mbr and as in this study, under the systematic review criteria employed in this study we found that there were a limited number of publications, if any, about other wastewater treatment processes that fulfilled our prerequisites for meta-analysis. in the multiple-barrier system, wastewater engineers have to connect wastewater treatment units to establish a wastewater reclamation system that can achieve the target lr values, which means that the accumulation of virus lr information in other potential wastewater treatment units (e.g. reverse osmosis (ro), disinfection practices, etc.) that include all of the required information for meta-analysis is very critical and mandatory in the future study. wastewater engineers can design a system for wastewater reclamation that can achieve the required levels of virus removal by combining virus lr credit of each wastewater treatment unit under the concept of multiple-barrier system. in this section we discuss the issue of how to validate that the employed wastewater treatment processes are working correctly and achieving the pre-evaluated performance targets for the safe management of wastewater reuse. although usepa ( ) does not require the monitoring of viruses in wastewater effluent, some states in the u.s. classify reclaimed water into several classes based on their degree of treatment and quality of the final product; virus monitoring is included in these regulations. the state of california has established sophisticated regulations related to recycled water, and stipulated how to validate each of the treatment processes used to meet the requirements. according to the california code of regulations (title ), a grrp sponsor must prepare an operation optimization plan that identifies and describes the approaches for operations, maintenance, analytical methods, and monitoring necessary for the grrp to meet the requirements using pathogens of concern or microbial surrogate parameters that verify the performance of each treatment process to achieve its credited lr, as well as report monitoring results to the regulatory agency (state water resource control board, ) . to meet these regulatory requirements, the first option for a grrp sponsor may be to monitor microbial indicators or surrogate parameters in effluent. there is a long history of usage of conventional indicator bacteria, including total coliforms and escherichia coli, for such monitoring; however, the absence of indicator bacteria in treated wastewater does not necessarily indicate the absence of viral pathogens because viruses are typically more resistant to wastewater treatments (kitajima et al., ) . the selection of appropriate indicators or surrogates is thus important to ascertain the reliability of the treatment. who ( ) and atse ( ) suggested selecting bacteriophages, especially somatic coliphages and f-specific bacteriophages, as viral process efficiency indicators. recent studies reported that the frequent and abundant appearance of pepper mild mottle virus in wastewater (rosario et al., ) , and used this virus as an enteric virus surrogate for evaluating the virus lr (symonds et al., ) . other viruses, such as adenoviruses and aichi virus have been also proposed as surrogates for wastewater treatment removal of enteric viruses (hata et al., ; kitajima et al., ) . the issue of proper use of indicators or surrogates for monitoring viral lrs in wastewater treatment processes is still in open discussion, and further investigation is needed to select the most appropriate and affordable indicators or surrogates for validating virus removal efficiency in wastewater reclamation systems. since multiple viruses should be compared to test their appropriateness as viral lr monitoring indicators, the micro-and nano-fluidic qpcr system for virus quantification may be a powerful tool because of its costeffective and time-saving features (ishii et al., ; coudray-meunier et al., ) . the state of arizona requires virus monitoring for class a + reclaimed water, which can be used for any purpose excluding ) direct potable reuse, ) when the water is not treated by designated techniques (filtration, denitrification, and disinfection after secondary treatment), or ) when the class a+ reclaimed water is used after mixing with lower classes of water. arizona's regulations stipulate that virus concentrations should be less than the detection limit value for sampling events during a -month monitoring period (arizona department of environmental quality, ) . in the state of florida, the acceptable virus concentration in reclaimed water varies between . and per litters depending on virus type, with rotavirus corresponding to the lowest concentration (usepa, ) . to meet these regulatory requirements, the monitoring of viruses in reclaimed wastewater has to be performed constantly in the daily operation of wastewater reclamation and reuse systems, and the first choice mean in the monitoring may be the molecular biology techniques including pcr. although time-and cost-saving pcr systems have been recently proposed (ishii et al., ; coudray-meunier et al., ) , a highly frequent monitoring of viruses in reclaimed wastewater, such as on a daily basis, is not affordable and realistic. further technical innovation in the virus fig. . removal efficiency of norovirus genogroup ii by activated sludge processes. black square is the mean value of virus log reduction (lr) obtained in each study, and a bar indicates % confidence interval (ci). the sample number in each study is indicated in a parenthesis next to % ci values. the total lr value is obtained by the calculation procedure indicated in supplementary materials. detection from reclaimed wastewater, including the virus recovery procedures from large volume of water, is needed to optimize the monitoring system (water examination frequency, sample volume, etc.) that can manage virus infection risks through reclaimed wastewater. another important issue in the validation of virus removal performance is how to assess the infectivity reduction by disinfection. cell culture assays are available for some enteric viruses, but cell culture-based approaches for testing infectious titer, such as plaque assay, are timeconsuming and are not appropriate options to ensure the significant reduction of viral infectivity in daily operation of wastewater reclamation systems. furthermore, there are epidemiologically important viruses that cannot yet be cultivated by cell culture. norovirus is one of the most important waterborne gastroenteritis viruses, but in-vitro cultivation of this virus using cultivated cell culture has not been established (herbst-kralovetz et al., ) . this means that the infectivity reduction of human norovirus by any disinfection practices cannot be evaluated at the moment. recently, jones et al. ( ) reported that co-cultivation with an enteric bacterium strain enhanced the norovirus infection of b cells, and the applicability of this methodology to the other norovirus strains is currently being tested. culture-independent approaches for evaluating the inactivation of non-cultivatable viruses have been proposed (hamza et al., ) , and some of them may be available in some specific settings ; however, there is no consensus as to which methodology for culture-independent testing is the most appropriate for the evaluation of infectivity reduction of noncultivatable viruses. one of the possible solutions for the issues caused by the insufficient technological innovation in the monitoring of virus presence and infectivity can be seen in the regulation of the state of california, where novel and unique regulations have been established also in various applications of reclaimed wastewater other than grrp. the california code of regulations (title ) stipulate three levels of recycled water quality, which are ) disinfected secondary- . recycled water, ) disinfected secondary- recycled water, and ) disinfected tertiary recycled water (table s ). the disinfected tertiary recycled water has the highest hygiene quality, and can be used for food crops, parks and playgrounds, school yards, residential landscaping, unrestricted golf courses, and so forth. the disinfected tertiary recycled water that has not received conventional treatment can be used also for non-restricted recreational impoundments. as indicated in table s , the disinfected tertiary treated water is a filtered and subsequently disinfected wastewater with either chlorine at a ct (the product of total chlorine residual and modal contact time measured at the same point) value of not less than mg-minutes per liter at all times with a modal contact time of at least min, based on peak dry weather design flow, or a disinfection process that has been demonstrated to reduce the infectious titer of the f-specific bacteriophage ms or poliovirus by log in the combination with the filtration process. the use of other viral indicators that show comparable resistance against chlorination to poliovirus is also possible. recent publication reported that free chlorine treatment at a ct value of mg-minutes per liter was enough to inactivate bacteriophage ms by log at a full-scale wastewater treatment plant (adelman et al., ) . the enacted ct value in the title ( mg-minutes per liter with a minimum contact time of min), which is more than tentimes larger than that are sufficient for log reduction of infectivity of bacteriophage ms , gives a sufficient oxidative stress to destroy virus particles and ensures the safety of disinfected water in terms of virus infection risk, and may exempt the obligation of virus monitoring in reclaimed wastewater that is obliged in several state regulations. non-microbe parameters can also be potential indicators for virus reduction. for example, turbidity can be an indicator for protozoan and viral reductions by filtration processes (usepa, ) . usepa also suggests that the removal of suspended matter is related to virus removal because many viruses are particulate-associated, and that such particulate matter can shield viruses from disinfectants such as chlorine and uv. fluorescent dyes such as rhodamine wt are potentially useful to assess the efficiency of viral removal by ro membranes; however, the assay sensitivity of rhodamine wt analysis should be improved for this application. some of these non-microbe indicators can be monitored using real-time sensors, thus very compatible with water management systems based on hazard analysis and critical control point (haccp), in which multiple parameters are determined as the important monitoring points for ensuring the water safety. the who sanitation safety plan (ssp) employs the concept of haccp, and is one of the strong options for governments that do not have any wastewater reuse guidelines in operation. another haccp-based who plan, the water safety plan (wsp), can also be applicable when the reclaimed wastewater is used as a drinking water source either directly or indirectly. the water, sanitation & hygiene (wash) safety plans proposed by the united nations children's fund (unicef) are a congregative approach for public health, which also employs a haccp-like system (sanderson and mckenzie, ) . the haccp concept was developed in the food industry, and control parameters for pathogen inactivation such as d-values (decimal reduction time, which is the time required to reduce lr of the heat-exposed microorganisms at a given temperature) and z-values (temperature required for achieving lr more in dvalue) are investigated in the study field of predictive microbiology, one of the disciplines within food microbiology (mcmeekin et al., ) . because of the increased complexity of wastewater matrixes compared to food, the approaches of predictive microbiology have not been fully applied to water virology. for the full introduction of ssp, wsp or wash safety plans for the safe usage of reclaimed wastewater, predictive microbiology-like approaches need to be introduced for identifying appropriate indicators of virus lr in the haccp-based systems for wastewater reclamation. current guidelines for wastewater reclamation operations have proposed that virus infection risks for any type of reclaimed wastewater usage are to be regulated by the multiple-barrier system, in which sequential wastewater treatment processes are designed in a way that the sum of virus removal efficiencies for each process exceeds a predetermined performance target, which is a total virus removal efficiency calculated by virological risk assessment. a meta-analysis approach is useful for obtaining the averaged value of virus lr in each wastewater treatment unit; however, there are several academic issues that should be further addressed including ) how to treat left-censored datasets in the calculation of virus removal efficiency, and ) what indicators and methodologies are appropriate for validating virus removal and disinfection performance in the daily operation of wastewater reclamation systems. the frameworks of ssp, wsp and wash safety plan, employing haccp along with the multiple-barrier system, must enhance the efficacy of the risk management of virus infection through reclaimed wastewater. the authors declare no competing financial interests. required c × t value for -log virus inactivation at full scale water quality standards drinking water through recycling. the benefits and costs of supplying direct to the distribution system large multistate outbreak of norovirus gastroenteritis associated with frozen strawberry levels of norovirus and e. coli in untreated, biologically treated and uv-disinfected sewage effluent discharged to a shellfish water mechanisms of pathogenic virus removal in a full-scale membrane bioreactor a novel high-throughput method for 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regulations related to recycled water a case study of enteric virus removal and insights into the associated risk of water reuse for two wastewater treatment pond systems in bolivia outbreak of hepatitis a in italy associated with frozen redcurrants imported from poland: a case study characterization of drinking water treatment for virus risk assessment infectivity of gi and gii noroviruses established from oyster related outbreaks guide for the completion of a recycled water quality management plan. for class a water recycling schemes guidelines for water reuse who guidelines for the safe use of wastewater guidelines for drinking-water quality source identification of bacterial and viral pathogens and their survival/fading in the process of wastewater treatment, reclamation, and environmental reuse we really appreciate dr. jason torrey (university of tokyo, japan), dr. masaaki kitajima (singapore-mit alliance for research and technology, singapore), dr. yoshifumi masago (united nations university, japan), and dr. eiji haramoto (yamanashi university, japan) for their fruitful comments on this work. this study was supported by the japan science and technology agency (jst) through core research for evolutionary science and technology (crest), and the japan society for the promotion of science through grant-in-aid for scientific research (a) ( ). key: cord- -wrgg met authors: campling, paul; joris, ingeborg; calliera, maura; capri, ettore; marchis, alexandru; kuczyńska, anna; vereijken, tom; majewska, zuzanna; belmans, els; borremans, lieve; dupon, elien; pauwelyn, ellen; mellander, per-erik; fennell, christopher; fenton, owen; burgess, edward; puscas, alexandra; gil, elena isla; de alda, miren lopez; tudel, gemma francès; andersen, erling; højber, anker lajer; nowakowska, marzena; suciu, nicoleta title: a multi-actor, participatory approach to identify policy and technical barriers to better farming practices that protect our drinking water sources date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: wrgg met despite an improvement of water quality across europe there are many pollution hotspots for both nitrates and ppps, mainly due to agricultural activities. the bmps and mms to reduce pollution from agriculture are well known, and there are policy instruments in place to ensure drinking water standards, but the current approach has not been efficient enough. within the h water protect project the premise was that there is a need for a multi-actor, participatory approach to tackle the issue from a new angle, and to assess why the uptake of known bmps and mms was not better among farmers. seven “action labs” were selected that represent major physical, socio-economical, cultural and farming settings across europe. a methodology of multi-actor engagement was chosen but with different approaches due to the local context. initially the level of farmers' awareness about water quality issues was matched to the observed uptake rates of bmps and mms. in a second survey barriers hindering the uptake of measures were identified. the first survey revealed a low general awareness on the potential pollution to drinking water sources. despite this, between % to % of the surveyed farmers per action lab were already voluntarily adopting one quarter of the selected bmps and mms. the second survey demonstrated the need to address organisational, legislative, sociological and technical barriers. the lack of coordination between different institutional bodies promoting measures and the financial incentives needed to invest and operate these often-costly measures need to be considered. the multi-actor, participatory approach with its improved awareness and collaboration made it possible to identify the crucial factors for improvement - to build a social acceptance among all actors and communicate the issues and solutions from the start. high-quality, safe, and sufficient drinking water is essential for life: we use it for drinking, food preparation and cleaning. however, more than half of the river and lake water bodies in europe are reported to be in less than good ecological status. the main environmental pressures are point ( %) and diffuse source pollution ( %) and about % of groundwater across europe is in poor chemical status (eea, ) . the wider research community recognises that management of water in a sustainable way "is the key for the future of food and agriculture" (oecd, a) . the agenda for sustainable development acknowledges the importance of water quality and includes a specific water quality objective in sustainable development goal (sdgs, ) . farming accounts for between % and % of local water used across europe and contributes to water pollution sourced from nutrient, pesticide and other pollutant discharges (e.g. tractor oils) (oecd, b) . through pollution from nitrates and plant protection and policy-makers in designing cost-effective measures for addressing water pollution in agriculture (mateo-sagasta, j., et. al., ) . water quality in surface and ground waters has generally improved across europe thanks to the adherence to waste water treatment standards for point emissions and the uptake of bmps and mms by the farming sector for diffuse emissions. however, there are still a number of pollution hotspots across europe for both nitrates and ppps, largely due to agricultural activities. the bmps and the mms to reduce pollution from nitrates and ppps are well known, there are also policy instruments in place to ensure that drinking water standards are met, but drinking water companies are still being required to invest and operate drinking water treatment facilities to deliver drinking water that meet the required standardsso in essence the current approach is not working and more should be done to reduce costly end of pipe drinking water treatment solutions. therefore, the h water protect project started with the premise that a different multi-actor, participatory approach was needed to tackle the issue from a new angle and to get to the bottom of why farmers are not sufficiently taking up known bmps and mms to protect drinking water sources. we selected seven action labs across europe that represent some of the major differences in physical, socio-economical, cultural and farming systems across europe. best management practices (bmps) and mitigation measures (mms) are key elements to limit or prevent water and environmental pollution from agricultural activities. the analysis of their efficacy has thus raised several scientific projects and multiple exchanges between regulatory authorities. a number of initiatives have been undertaken across member states to stimulate their uptake (topps http://www.topps-life.org, magpie https://www.setac.org/magpie, compass https://balticcompass.org/). this has resulted in the genesis of a wide variety of approaches, implemented at national levels such as good agricultural practices or legislative measures. the increased uptake of measures to reduce nitrogen pollution from agriculture and improvements in urban waste water treatment have led to a steady reduction in average nitrogen concentrations in rivers from . to . mg no /l ( to ) and average concentrations in groundwater are well below the groundwater quality standard of mg no /l (european commission, quality standard (eqs). the reduction of the pollution of drinking water sources by ppps and fertilisers used by the agricultural sector remains the biggest challenge. despite the increased integration of policies to deliver clean and safe drinking water over the last years there is clearly a need to increase the engagement between interdependent actors and stakeholders (european environment agency, ) . efficiency and innovation do not seem to be the only parameters that can solve the challenge of sustainable and low-impact agriculture, which instead requires a much more complex analysis with an integrated approach. an integrated approach involves an understanding of technological problems, social behaviour of rural communities, economic constraints, the legal and institutional framework and contextual agricultural practices (chartzoulakiset and bertaki, ) . belmans ( ) suggests that a move towards a more "horizontal" and "participatory" water governance between the various actors and stakeholders would be more productive: water companies, farmers, nature conservation ngos, plant protection product producers, fertilizer producers, food and retail businesses, consumer organisations, environment agencies and ministries. there are other approaches, such as stewardship programmes, financed by food and drink companies to protect the quality of local water supplies (newborne and dalton, ) . in a similar vein certification schemes such as the european water stewardship (ews) certification scheme (https://ews.info/) have the same goal to organize companies to improve the environmental footprint of their value chain and protect water resources (jones et. al., ) . in this framework, the h funded project waterprotect was established with the aims to: ( ) analyse whether the solutions offered by the existing governance systems can adequately cover the impact by the agriculture; ( ) demonstrate how selected mitigation measures have an impact on improving water quality; and ( ) contribute to the effective uptake and realisation of best management practices to deliver good water quality. a pan-european, multi-actor approach was adapted in the context of seven rural or mixed rural/urban action labs (also known as living labs) to determine barriers and factors that hinder the uptake of bmps and mms to protect drinking water sources from nitrates and ppps and that have an impact on farmers' decision making and strategies. the hypothesis is that water and agriculture policy reforms to support farmers and policy makers in their decision-making requires an in-depth understanding of the local context and of the policy-related driving factors that impact on water quality. on the basis of this understanding, recommendations were provided to improve awareness, collaboration and in some case the uptake of bmps j o u r n a l p r e -p r o o f journal pre-proof and mms in the action labs. the seven action labs are located in six different environmental zones (wur, ) which cover % of the area relevant for agriculture ( figure , table ). the main farming systems in the eu are considered: mixed farming (two action labs), field crops (three action labs), and permanent crops (two action labs). among the seven action labs, there are three rural land uses (romania, ireland, denmark) and one mixed urban and rural land uses (belgium) with small water supplies. larger action labs, including two mixed urban and rural land uses (italy, spain) and one rural land use (poland), are delivering drinking water to hundreds of thousands of inhabitants. in five of the action labs the focus is on groundwater that is used either in public supply (denmark), or both public and private supply (ireland) or used locally from private wells as drinking water or for agricultural use ( spain, italy, romania). in the action labs, the water quality of water resources is under pressure from manuring and agro-chemicals, but different action labs chose specific targeted pollutants: four action labs focussed mainly on ppps (belgium, italy, ireland, spain), whereas three worked mainly on nitrates (denmark, poland, romania). sustainable agriculture is the result of complex "systemic interactions" between different actors involved in various ways, such as researchers, farmers, entrepreneurs, regional and national organizations, etc. indeed, a complex socio-ecological issue such as water quality related to agriculture cannot be solved by just one actor but rather from a multi-actor approach perspective (belmans, ). all of them have different forms of knowledge (practical, scientific, policy based, etc.) and there is the need to create conditions for interaction between them and combine their knowledge, perspectives, resources, and experiences, to identify and discuss solutions and new ideas. therefore, all the actors identified in the seven action labs that were considered to have an influence on or to be influenced by the environment and the farming systems, were invited to engage in the study development and activities. since it is recognised that at the context level there is not a "one fits all approach", in each action lab the stakeholder involvement was conceptualised as an "active engagement" that followed a stepwise approach. the approach included both j o u r n a l p r e -p r o o f water quality analysis (e.g. participatory monitoring) and stakeholders' analysis, with different levels of participation based on actors' roles that ranged from specific consultations to active involvement in the project (e.g. hosting demonstrations, facilitating meetings, field visits etc…). based on the initial knowledge status of each action lab, the level of collaboration and the specific local objectives, the approach led to a range of different strategies throughout the overall process. a detailed description of each strategy adopted is provided by calliera et al. ( , submitted to this vsi) . in summary, the strategies included: (i) face-to-face meetings (such as seminars, workshops, community events, or site tours), an exchange/informing qualitative "dialogue-based method" that allows greater spontaneity and interaction between the researchers and participants; (ii) surveys, exchange questionnairebased quantitative tools, where stakeholders are requested to individually answer questions by choosing from a limited number of provided answers; (iii) participatory monitoring, the engagement of farmers, farmers associations and farmer's consultant organisation in the design and the setup of water monitoring for the catchment; (iv) participatory training approach and demonstration farm, exchange/informing activities able to identify and bridge "the gap between what is and what should be in terms of incumbents' knowledge, skills, attitudes, and behaviour for a particular situation at one point in time" (farm path project, ) . the collation of information underpinning the multi-actor engagements allows us to summarise the level of stakeholder awareness at the beginning of the project in each action lab, list the stakeholders involved and define the strategies for engagement (table ). to better define the context of the study, it was necessary to carry out an analysis of the water and agriculture related policies, so as to identify the policy-related driving factors that impact on water quality in the action labs. the analysis considers the critical success factors that enhance the effective integration of environmental water concerns into agricultural practices, including the contribution of agricultural and environmental policies and regulatory frameworks. agriculture and water management go hand in hand, and within the eu policies related to these two sectors there are many opportunities for synergies and reinforcements (european commission, ) . however, j o u r n a l p r e -p r o o f the eu water and agriculture policies also have individual objectives and different implementation mechanisms, which creates differences in the depth and coherence of their coordination. the first element worth noting is that the common agricultural policy (cap) for the period - is a fully integrated policy, meaning that standards are set at eu level with member states having little room for flexibility in the implementation, except for the rural development pillar measures. consequently, the eu legal framework is built on directly applicable regulations, so there is no requirement for transpositions. on the other hand, environmental policy, including water policy, has been founded completely on the subsidiarity principle. this means that major policy goals are agreed at the eu level, while member states are given the powers to implement and enforce by transposing the directives into national legislation. the main environmental policy instruments related to water that are to be considered here are: directive (wfd) and sets groundwater quality standards, introducing measures to prevent or limit inputs of pollutants into groundwater (european parliament and council, ) ; and, for eu agriculture policies, the cross-compliance requirements (set of minimum agricultural production standards) and the measures included in the rural development plans are those that define the framework for the farmers, in order to benefit from eu subsidies. being part of cross-compliance, directive / for the sustainable use of pesticides (sud) (european parliament and council, ) is an important instrument to help achieve good water status, although broader in scope, it includes relevant measures aimed at protecting the water resources by restricting the use in certain areas and by implementing buffer zones and other measures to reduce run-off and leaching. also included in cross-compliance is the nitrates directive (council of the european union, ) that aims at the reduction of pollution from agricultural nitrogen. several other eu policies have a rather indirect impact on water and will not be considered in this analysis. for example, the recent eu circular economy package includes provisions like the rules for water reuse or the new rules on fertilisers that open the door to a different approach in water and nutrient management. another piece of legislation that can play a role in water management and agriculture is the habitats directive. the policy analysis focuses on two fundamental questions: . what are the interactions between the various policies as well as the coherence, exchange of information and coordination at the implementation level? . how are the guidelines, requirements and rules in various policy instruments translated at farmer level in the action labs? the choice of methodology was to structure the evaluation around the main stages of the policy cycle. the policy cycle-based assessment has at the centre the policy instruments, hence facilitating the formulation of specific policy suggestions and recommendations ( figure ). the evaluation of the water and agriculture policies coherence, coordination and interaction has included an extensive literature review on existing relevant reports and policy documents produced at eu level and/or commissioned by eu institutions. to answer the first fundamental question the analysis has established a framework for all potential and possible interactions within the intervention logic for water and agriculture policies of the eu. the realities of implementation in the seven action labs areas were judged against this framework (see results . .). for the second fundamental question, the analysis has included a structured questionnaire collecting expert opinions from the action labs (see results . ). the seven action labs (case studies) cover different climatic conditions, different types of farming systems, different legal frameworks, larger and smaller water collection areas. each action lab was chosen, based on the local specificities and pressures on water resources, to focus on nitrates and/or ppps (table ) . hence, each action lab gave a different weight on what is considered relevant in the policy framework, based on the pollutant (s) of concern. the action lab leaders were asked to evaluate the policy implementation realities in their study area using the following criteria:  effectiveness of the exchange of information and interaction;  coherence of the requirements, rules and guidelines (as perceived by the farmer);  relevance of the implementation mechanism in relation to the objectives; and,  added value of coordination and synergies between policy areas. due to non-availability of information or data on the efficiency and effectiveness of individual policy instruments in the action labs the assessment was based on expert opinions on the integration of water and agriculture policies using a structured set of key questions (s ). the first step in the analysis of the uptake of bmps and mms to prevent the pollution of drinking water sources from agriculture was to match the wealth of information from previous european projects that assess mitigation measures with the local knowledge of the farming systems and the focus issues in the seven action labs. the three projects consulted were topps, magpie and compass: the bmps selected were chosen on the basis of protecting drinking water sources from nitrates and/or plant protection products. while some technical measures provide solutions for a wide range of pollution problems, such as grass buffer zones or constructed wetlands, others are more problem specific, such as phytase supplementation or drift reducing nozzles. non-technical measures focus on changes in behaviour rather than the use of technology or techniques, such as only spraying when weather and field conditions allow safe ppps use. other bmps require new or improved technologies or infrastructure, such as the management of polluted water derived from cleaning of sprayers. many of these measures are well known across europe but are not all fully implemented. in addition, these measures may simply not be appropriate for a given farming system, due to the physical characteristics of the area and / or the prevailing socioeconomics of the farming sector in an action lab. therefore, the action lab leaders did a first assessment of the relevance of selected bmps in the action labs, including a discussion with stakeholders in the field (farmers, farm advisors, government and non-government officials). this resulted in an extension of the basket of bmp measures from to (s ). these are grouped into seven categories of bmps (table ) reflecting the pollutant focusnitrates and / or ppps and whether the measures are related to soil management, farm operations, or animal husbandry. one category of measures focuses on practices in animal husbandry and manure management (n manure) and contains farm practices such as e.g. reducing the dietary nitrogen and phosphorus intake or adopting phase feeding of livestock. also, in this category are management practices on the field such as incorporating manure immediately after application on cultivated land. another category of bmps & mms focusing on nitrate is related to soil management plant production practices (n soil). included here are e.g. making a nutrient balance on farm and/or field level, using treated urea or liming. a specific subgroup of soil management practices is related to mitigating runoff and is equally relevant for nitrates and ppps (n&ppp). this category contains runoff control measures such as grass buffer zones, crop rotation to preserve organic matter or establishing retention structures. a small category of measures is related to control of diffuse losses specifically for ppps (ppp soil): permanent grassing in the interrow and weeding the row, considering alternative systems for pest control. for ppps, three more categories are delineated focusing on point source prevention (ppp point, e.g. use a safe filling and cleaning place for the spraying equipment), on spray drift mitigation (ppp drift, e.g. use drift reducing nozzles) and general precautionary measures (ppp general, e.g. ensure adequate training for sprayer operator). in each of the action labs a questionnaire was developed to take to the farmers. the objective of the survey was to gather information regarding the uptake of bmps and mms within the action labs (table ), and to assess the willingness of farmers to implement additional, innovative measures, depending on costs and benefits. each action lab selected a number of bmps and mms from the basket of measures based on the relevance for the area (targeted type of pollutant, well established and novel approaches, critical pollutant routes in the area). this resulted in different questionnaires that started from the same objective but contained different lists of bmps and mms. the coverage of the survey in the different action labs varies but is considered good: for bollaertbeek the farmers represent % of the farmland and % of all farmers, with a relatively higher proportion ( %) of farmers that spray themselves compared to farmers using a contract sprayer. in wexford, % of the farmers responded to the survey. in val tidone the survey was conducted in two stages with farmers ( %) taking part in the general survey including bmps on point pollution ( bmps) and farmers ( %) in the second survey including bmps on diffuse pollution ( bmps). in gowienica % of the farmers took part in the survey covering % of the farmland. in mara river % of the farmers j o u r n a l p r e -p r o o f responded representing . % of the catchment. in lower llobregat river the survey was taken by farmers that are members of a plant protection association (receiving training and advice) and by farmers that are not. non-professional farmers could not be reached but the overall response rate was %. in vester hjerk out of farmers in the capture area were reached (representing % of farmland in the capture area) and some additional farmers in the supply zone. the majority of the action labs also included obligatory bmps and mms in the questionnaire (between % of the list in gowienica up to % in lower llobregat) while two action labs only focused on the uptake of voluntary measures (val tidone, vester hjerk) ( table ). obligatory bmps and mms are either mandatory by national law (evaluated in each action lab separately) or, in the case of bollaertbeek, mandatory for certification to be allowed to sell the crop (vegetables) on the market. voluntary measures are not mandatory by law and can in some cases be eligible for funding from rural development funds (e.g. as erosion-control or biodiversity promoting measures). follow up work was carried out after the questionnaire surveys, in line with the multi-actor engagement techniques (described in section . ), with the specific objective to establish the barriers to the uptake of bmps and mms, so as to understand what motivates farmers to adopt voluntary measures and what holds them back. an iterative process was adopted by the action lab leaders (table ) from which a check list of possible barriers was establishedthis was used as a template to interview the farmers again to understand the underlying barriers that influence decision making. the checklist of barriers was further organised into the following categories: organizational, legislative, sociological, political and technical. the basket of collaborative tools and results were further used to reflect on how the policy instruments could be better used to improve awareness, collaboration and in some cases the uptake of bmps and mms in the action labs in the future. the intervention logic describes the way various elements of a policy aim at influencing the target groups towards achieving common objectives. the eu policies are developed in an intervention logic that seeks to combine various policy instruments and secure synergies to achieve a multiplier effect on the ground. journal pre-proof however, the reality is much more complex in the action labs where various specific conditions, administrative organisation or local culture and ways of working, can facilitate or hinder such policy interactions. this enabled us to develop an evaluation framework that maps out the potential interactions that exist between policy implementation instruments (figure ), leading to an assessment of what the intensity and effectiveness of policies are in each action lab. the general assessment is that the rural development plans do make use of the information collected in the implementation of the water related policy instruments. in general, this is done at a higher integration level and contextualising the information into the specific objectives of rural development. the type of data used is not homogenous in the eu, some countries make use of general statistical data, some explore the monitoring data in detail while others integrate such data with specific assessments and evaluations done by recognized national or local organisations. when it comes to evaluating the coherence between rural development and rbmps, again, the level of integration varies between countries. however, in general, the two programming instruments seem to be generally correlated. national action plans adopted, as required by the sustainable use of pesticides directive / /ec (european parliament and council, ) , include quantitative objectives, targets, measurements and timetables to reduce the risks and impacts of pesticide use. water resources and recommendations are made j o u r n a l p r e -p r o o f for taking appropriate risk mitigation measures on the territory to avoid pesticides contamination of water resources. farmers receive information on the standards to be followed through many channels, mainly from the farmer associations and farmer advisory centres. they issue guidelines and management practices to make requirements clear and feasible in practice, which is not always possible since the legislation is not always coherent in definitions and concepts. through the expert analysis we can conclude that there is a certain level of complexity for the implementation of the eu legislation at national level, which is often influenced by the political context, administrative or organisational specificities, history or culture of participatory policy making and public consultation. provisions and national level requirements on water protection are considered to be generally coherent and no contradictions between objectives or measures for their implementation were identified by experts. however, the complexity of the implementation arrangements translates into a difficult communication on the objectives and the related means or measures in place for their achievement. the bmps which are currently implemented and those that were deemed to be implementable mostly are small and simple measures that do not require big investments or big adaptations in the farming system and/or offer clear benefits to farmers; examples of these are keeping soil cover in autumn and winter (high uptake in wexford, bollaertbeek, gowienica and mara river) or not spraying non-target areas (high uptake in val tidone, gowienica, wexford (mandatory) and bollaertbeek). increase in productivity is a strong incentive for farmers to implement bmps (e.g. crop rotation to preserve soil organic matter) while environmental aspects such as need for minimization of the risk of drift of ppps or nutrient losses to the environment are poorly recognized by farmers in some cases, depending on the awareness of the water quality issues. knowledge on and perception of effectiveness and usefulness of different bmps varies between countries. for example, results of the survey in wexford suggest poor performance of constructed wetlands and therefore low potential for implementation of this measure. in contrast to that, in vester hjerk landscape level bmps such as constructed wetlands, set aside and afforestation seem to have a relatively high potential. larger and more expensive measures are more difficult to implement; nonetheless measures perceived as being immediately beneficial for farmers (i.e. giving long term financial benefits) have relatively high potential for implementation. for example, the use of gps technology in farming shows high potential for implementation in wexford, gowienica and vester hjerk. other measures recognized as effective but not giving direct benefits to farmers, e.g. anti-hail nets are not considered interesting or applicable, mainly due to the excessive costs. for these measures, financial incentives would be necessary to increase their implementation. implementation of measures that require land area, such as for example vegetated buffer strips at the edge of a field or within a field, are not welcomed by farmers due to loss of land for agricultural production. however, farmers in gowienica indicated that they would be in favor of them if given land tax exemptions for these areas and/or state/commune support in preventing weed infestations. in some cases (vester hjerk, bollaertbeek, gowienica) there is a positive approach to collaborative solutions, where more farmers and stakeholders are involved. for example, farmers indicated they would welcome a solution where a common, public cleaning place for cleaning sprayers was provided. organising these solutions needs facilitation of dedicated institutions and/or a leader with good communication skills, j o u r n a l p r e -p r o o f as making a consensus among farmers themselves to work together has also been identified as a potential barrier. some cases (val tidone, bollaertbeek) highlighted that farmers showed to be open for cooperation and expressed their interest in obtaining more information about specific bmps or how to mitigate defined problems. in another action lab (gowienica) farmers admitted to participating in many trainings related to bmps, however these were "too theoretical" and hence were not effective since they did not sufficiently contribute to understanding the problem. the follow up survey after the formal questionnaire to determine the rate of uptake of and potential interest in voluntary bmps and mms revealed that there are a wide range of barriers that can be categorised under: organisational, legislative, sociological, political and technical (table ), which are fully described in s based on accompanying field notes. the barriers that occur in three or more of the action labs provide guidance in terms of commonality and provide policy makers the required focus on the issues that are troubling farmers. the barrier that was most commonly pointed out (by out of participating action labs) was too complex organizational set up of institutions responsible for implementation and execution of water management policies. this was highlighted by poland, italy, spain, romania, ireland and belgium. the general conclusion is that there are too many institutions involved in water management at national and regional/local levels, which causes roles and responsibilities to be unclear and sometimes overlapping. many countries noted a definite dispersion of competences and a large variability in the stakeholders' perception of the water governance structure and the stakeholders' roles. this confuses farmers and discourages them to contact specific authorities and inhibits uptake of measures. little cooperation between stakeholders at local level was pointed out by of out action labs (poland, italy, spain, belgium and romania) as another important factor hindering the effectiveness of measures. by lack of cooperation it is understood that there is not only little integrated effort in defining and implementing measures (which relates to point ), but also a lack of communication and exchange of information. this further implies that the voices of some stakeholders are not heard by others and that actions taken by the government officials favour specific groups of stakeholders. this was highlighted by farmers during multiple workshop meetings in poland, belgium, italy, romania and spain. regulations from different policy areas, such as groundwater, surface water, drinking water, agriculture and nature conservation are poorly coordinated. this was indicated by action labs: poland, italy, belgium and denmark. regulations developed by different governmental departments are only focused on their own interests. this creates situations where finding practical solutions at a local level is very difficult, as requirements of one regulation often contradict requirements of the other. on the other hand, the implementation of one measure can often fulfill requirements of more than one regulation and this could significantly boost their effectiveness, reduce costs and allow for more measures to be introduced. this requires good coordination and an integrated water management at local levels. in addition, low awareness of farmers regarding impacts they may cause on the environment has also been identified as an important barrier, noted by out of action labs (poland, italy, romania, belgium). the field assessments revealed that farmers in general still have a problem with linking how their everyday activities may affect the environment. it has been noted that the economic sustainability of the activity prevails over environmental sustainability. the family economy and personal goals influence the transition to sustainable agriculture. nonetheless, this links closely to the problem of little transparency of environmental monitoring programmes which do not inform farmers about their findings. farmers are not aware about water quality results from national/regional water monitoring campaigns in their areas and as such are not aware of impacts they make. another common barrier highlighted by out of action labs (poland, italy and belgium) was the multiplicity of regulations, which often are unclear. farmers need to be aware of multiple regulations regarding nitrates, ammonia, ppp, erosion control, etc. some of which are very long and complex. this causes regulations to be difficult to apply and to control in practice not only by farmers, but also by civil servants. three out of seven action labs (poland, romania and belgium) indicated inefficient control mechanisms to be factors inhibiting implementation of measures. there are two aspects in here to be considered. first of all, lack of actions taken towards those that do not fulfill legal requirements makes farmers to feel above the law and do not motivate them to take actions. another aspect that has been highlighted by farmers themselves is that the lack of control and actions towards those who break the law discourages farmers that take actions and do things according to legal protocols. an additional problem highlighted by farmers in poland is that the level of environmental fines is too low for large scale farmers to respect. farmers often j o u r n a l p r e -p r o o f journal pre-proof admitted themselves that breaking laws and paying fines was more worthwhile to their business than taking up mitigation measures required by law. farmers in poland, spain and belgium pointed out that the lack of long-term vision for environmental protection with respect to water and agriculture is an important problem. this relates to frequent changes in regulations and lack of continuity in approaches taken. the environment needs time to respond to changes that have been introduced. belgium highlighted that regulations change too often and become stricter and stricter every few years. farmers, who already implement measures and try to do their best are often 'penalised' when stricter rules are imposed. as a result, farmers lose their faith in legislations; they become suspicious and refuse to implement measures on a voluntary basis. in the water protect workshops, farmers asked for a clear and long-term vision from the government. the typical -years duration of governments does not allow to advance in questions that require more time for its implementation. in italy, spain and ireland farmers noted that time is needed for stakeholders to adapt to changes. this regards not only logistical issues such as time needed for utilization of older products that have been made prohibited but may still be stored by farmers, but also mental ability of people to adapt to changes such as the implementation of new measures. in poland, spain and ireland farmers think that standards and recommendations from applicable law and action programs are not fully adapted to climate changes (e.g. mild winters and earlier start of the growing season, and periods of allowed fertilization). farmers in poland, romania and belgium advocated that systems of support incentives for best practices in relation to water management as well as for agro-ecological approaches are too little. in poland, there are premises that cultivation on a particular land is more beneficial than applying new voluntary bmps and receiving subsides for that action. this is especially visible in areas with rich soils that can be cultivated intensively with profitable results. as a result, little interest is given for new more environmentally friendly initiatives on a voluntary basis. in italy, spain and ireland farmers highlighted that there is too little financial support for implementation of more advanced measures that are expensive. these measures can be attractive to implement but their cost and complexity can hinder straightforward implementation. too much bureaucracy is a common complaint expressed by the farmers. filling of paperwork required from farmers causes additional costs and confusion (poland, italy and spain). three countries, namely, italy, poland and ireland noted the lack of knowledge transfer from science to policy. policy and science are not commonly integrated causing knowledge gaps where decisions are made. important research findings are not efficiently disseminated to the right stakeholders or are not acknowledged enough in order to support decisions on the right measure, in the right places and at the right time. for example, the action lab studied in poland has a very long-lasting history of research, yet their findings have not led to changes in local policies and regulations. lack of interest in participation in the process of law creation. polish, spanish and romanian partners informed that consultation (in general, and specifically with respect to water management) is still a process that needs to be learnt and few people are interested to participate in the consultation, mainly specialized ngo's or directly interested stakeholders. farmers have little confidence that their opinions will be incorporated, so they have little motivation to participate. as a team we set out a methodology of multi-actor engagement that started at the same point but needed to be adapted to the local context, meaning that the execution of the approach was different. the strength of this method meant that the individual teams in the action labs could ensure that farmers and other actors were properly engaged in a meaningful and relevant way, with a consistent policy and field assessment framework. the weakness was that standard statistical comparisons concerning the uptake of bmps and mms to protect drinking water sources from nitrates and ppps cannot be inferred. we therefore discuss our results in a qualitative manner to guide our recommendations and conclusions. we look to match the level of awareness that farmers expressed about water quality issues at the start of the process (table ) to the uptake rates of bmps and mms observed at the action lab level (figure ). this is further discussed in light of the barriers to taking up measures identified during the follow-up surveys (table ), accounting at the same time for the policy evaluation framework that identifies potential interactions between policy instruments ( figure ). a herbicide) is a potential problem for drinking water supplies further down the catchment, although there was inadequate knowledge about the pollutant transfer pathways. at the lower llobregat (spain) basically all stakeholders, including farmers and citizens, were aware of the general bad quality of the water, but due to the complexity of the area it is difficult to know which activities are the most polluting. on the whole farmers believe that agriculture is not the main source of pollution. in val tidone (italy) there was also very little awareness from the farmers that ppps might have a deleterious effect on the local groundwater. participatory monitoring in all action labs was therefore essential to show farmers and other stakeholders mms in all action labs were complaints about the complexity of the legislation and follow-up, including the inherent bureaucracy involved. in addition, farmers in bollaertbeek also found that some of the mitigation measures, such as buffer zones, were technically confusing and difficult to implement, whereas on the other hand they are looking for measures that could reduce spray drift better. it was felt that there were also not enough control mechanisms to deal with non-compliant farmers, which was a complaint. in addition, farmers in these action labs indicated that there was insufficient financial support now to encourage them to step into the voluntary measures requiring major investments. the farmers in val tidone also found that some of the measures being promoted were not even suitable for their particular landscape and setting. action labs that focussed mainly on nitrates (gowienica -poland, mara -romania and vester at gowienica (poland), even though the environmental agency has been operating a nitrate vulnerable zone for more than years, the farmers in the area had very little awareness that nitrates are polluting the surface and ground waters. in mara (romania) this lack of awareness was also apparent. in vester hjerk being implemented by % of the farmers surveyed, but almost % of the farmers were considering implementing additional selected measures in the future. in addition to low awareness the other typical barriers to the uptake of bmps and mms in all action labs were the complex and poorly coordinated legislation and actions from the different government agencies, the lack of specialised personnel to provide professional guidance on measures, and the perception that there were insufficient control mechanisms for compliance. in gowienica and maraformerly eastern bloc countriesthe farmers found the approach of authorities too centralised with a lack of information. in vester hjerk the hydrogeology is very complicated so that it was questionable whether there is a direct link between surface measures carried out by farmers and the impact on groundwater water quality. in terms of using the policy evaluation framework that identifies interactions between policy instruments it is clear that policy instruments have to begin to address the organisational, legislative, sociological and technical barriers to the uptake of bmps and mms. at the farmer level the most important barrier after low awareness is that the process for them to take up bmps and mms is too complex and uncoordinated. this calls for legislation and regulations from both the ministries (or departments) of agriculture and environment to be simplified and integrated. clear and unambiguous messages on production standards and requirements is needed. the measures should also be selected in an open and constructive dialogue, which j o u r n a l p r e -p r o o f journal pre-proof take into account aspirations but also limitations and difficulties of the different actors. furthermore, the measures should be feasible in practice and if the measure involves extra costs without any return on investment for the farmer, it is clear that improved financial incentives need to be provided. with limited public finances it is therefore necessary that cooperation and synergies between the various institutional actors funding measures to be improved. while the purpose and technical aspects are usually quite clear and straight forward, the desired transition towards a 'new normal', including standardization and education of best management practices is almost never completed towards satisfying and long-lasting levels. matters of organizational, financial or cultural origins, which are inevitable occurrences of life seem to keep us away from what is sensible to do. the long value chains that we have built for our food production dilute both the impact and responsibility which the involved people experience. there is however, a huge common success factor: bottom up societal support. this creates peer pressure to continue performing, when times are getting tough. sometimes, extreme events like water quality issues, drought, heavy rain, covid- , trigger the justification of rapid implementation of bmps, and addressing the barriers for uptake is an important outcome of waterprotect. this can go very fast, whereas the usual uptake is often a long and difficult process. a conclusion is that the building of societal acceptance, the communication of issues and solutions before the start, are the crucial success factors for a multi-actor, participatory approach to improve farm management practices and protect our drinking water sources. the eu policy architecture that governs the water and agriculture areas is complex, partially due to the historical evolution of the two eu policy areas, but also partially due to the complexity of the challenges these need to address. coherence of water and agriculture policies at eu level is recognized as an area where improvement is needed. several actions have been taken at political and technical level, but there are also further opportunities for improvement. we have first taken the step to assess the situation by embarking on a multi-actor engagement strategy in the seven action labs. from this assessment we can conclude that the general awareness of farmers about the potential pollution problems caused to drinking water sources by the applications of nitrates and ppps is low. despite this low awareness between % to % of the surveyed farmers per action lab were already voluntarily adopting one quarter of the selected bmps and mms.information about the potential uptake of voluntary measures in the action lab ranges j o u r n a l p r e -p r o o f journal pre-proof from % to % of the farmers being willing to adopt additional selected measures. the follow up surveys provided us a better understanding of what is preventing farmers from taking the step to incorporate voluntary bmps and mms in their farm management. therefore, we can conclude that the next steps are to address the complexity of the process (administrative and technical), the lack of coordination between the different institutional bodies promoting measures, and the financial incentives needed to invest and operate these often-costly measures will increase the likelihood of more farmers taking up bmps and mms. with these points in mind and using the analysis on the coherence of water and agricultural policies and cross-referencing those with the implementation realities, we can make the following policy recommendations: . the eu should exploit the cycles of policy revisions to better integrate objectives and create mechanisms and structures of coordination. needs to be fully exploited to ensure coherence of objectives for water management and agriculture at national level, given that the new policy implementation arrangements give more flexibility to eu member states in deciding agricultural policy priorities, and to allocate much needed resources for farmers to make a positive contribution to sustainable water management. . member states should strive to streamline the implementation structures and procedures based on sound governance concepts that ensure the involvement of all concerned stakeholders in sustainable management of water resources & agriculture. . future policy implementation approaches should state the need for further exchange of information and data between the various programmatic and enforcement instruments and structures. results of controls over agricultural activities will have to influence priorities in water management and, equally, information on water quality and quantity issues, should be better transferred to the farmers. promoting multi-actor, participatory water governance models are recommended due to their capacity to: easily transfer information on the water management challenges, collaborative development of solutions, capacity to address local specificities and limitations and can create synergies with other action areas. j o u r n a l p r e -p r o o f . proactive provision of information on the challenges in water quality and their potential cause are essential to ensure awareness at farm level and understanding of the positive contribution farmers can make. currently, information is often unclear, scattered or not easily accessible. in many cases farmers rely on informal channels (farmer associations, media, extension consultants, etc) to obtain such information. . the positive contribution to sustainable water management of agriculture, including through implementing bmps and mms should be evaluated, recognized and communicated. a set of indicators that highlight the contribution agriculture has into water management (able to capture positive and/or negative trends) will help with farmers' involvement of and will stimulate ownership of the process. impact on the willingness of farmers to implement them. hence, direct information, know-how and as well as support for actual investments needed for implementation of will play a key role in the future uptake of such measures by farmers. finally, on the basis of the approach to improve awareness, collaboration and in some cases the uptake of bmps and mms in the action labs the crucial success factors to achieve this are to build social acceptance among all actors and to communicate the issues and potential solutions right from the start. directive / /ec of the european parliament and of the council of october establishing a framework for community action to achieve the sustainable use of pesticides directive / /eu of the european parliament and of the council of august amending directives / /ec and / /ec as regards priority substances in the field of water policy transition pathways towards sustainability in agriculture: case studies from water stewardship and corporate sustainability: a case study of reputation management in the food and drinks industry water pollution from agriculture: a global review executive summary agriculture and the environment: better policies to improve the environmental performance of the agriculture sector water and agriculture: managing water sustainably is key to the future of food and agriculture corporate water management and stewardship: signs of evolution towards sustainability. london, overseas development institute this research is part of the waterprotect project and has received funding from the european union's key: cord- - h mld x authors: butler, m. title: virus removal by disinfection of effluents date: - - journal: viruses and wastewater treatment doi: . /b - - - - . - sha: doc_id: cord_uid: h mld x the safe disposal of effluents can present a major problem to large urban communities because of their inevitable content of potentially pathogenic enteric viruses. at least one hundred types of virus may be present although many of these are difficult or even impossible to characterise under these conditions. wastewater treatment does not greatly effect the survival of many enteric viruses and some survive well even after effluent disposal. the use of disinfectants for the inactivation of virus in effluent is practicable but requires careful manipulation in order to avoid the disemination of byproducts toxic to man or capable of interferring with the ecology of the receiving waters or soils. no one system is likely to be either universally acceptable because of the variable quality of effluents and much research remains to be done before guidelines can be recommended or established. is disinfection of effluents necessary? seven years ago berg ( ) made the point that although it was not easy to remove all viruses from sewage, the technology to do so existed and we should be prepared to pay the cost. it is the object of this brief review to re-examine the practicability of achieving virus-free effluents, a subject recently reviewed by several others in particular, grabow ( ) , white ( ) and bitton ( ) . the safe disposal of effluents and the associated sludges can present a major problem to urban communities where the volumes involved may exceed millions of litres each day. although these products of wastewater treatment are potentially valuable for irrigation and fertilisation of land, they are usually discarded because it is simpler and cheaper . sludges are frequently dumped in the sea, but may be spread on land where the treatment works are too far from the coast for transportation to be economical. effluents typically pour into rivers, estuaries or the sea depending on the location of the treatment works, but, where water is scarce,they may be used as an irrigant or even recycled for Μ. butler potable supply (grabow, ) . however, apart from the technical and engineering problems involved in such productive distribution of effluent, serious consequences may result from their use because of their load of chemical contamination and the presence of pathogenic microorganisms including viruses. the degree of chemical and biological contamination reflects on the origin of the wastewater which may range from purely domestic sewage to principally industrial effluents. furthermore, the quality of products of wastewater treatment depends on the type of process used which range very widely indeed. the final processing or finishing of effluents and sludges is necessarily different because of their different constitution and this is particularly obvious so far as the removal of viruses is concerned. the removal of viruses from sludges has only relatively recently attracted serious attention (cliver, ; berg, ; osborn and hattingh, ) but for effluents, various procedures have been adopted for some time, particularly disinfection with chlorine, a treatment now under critical review. the problems associated with the removal of viruses from effluents are complex and it is essential to pay close attention to the following interdependent questions if a proper evaluation of suitable methods of treatment is to be reached: . how many types of viruses are present in effluent, how numerous are they and what is their pathogenic potential? . how reproducible and sensitive are the methods for the isolation and characterisation of viruses in effluent and is there a representative virus. how does wastewater treatment affect the distribution and fate of viruses and how well do viruses survive after the disposal of untreated effulent? what methods are available and practicable for the removal of viruses from effluents, how do these work and is there a recommended procedure? . viruses get into wastewater with the faecal solids within which they may be present at levels of up to '^/g as judged by electron microscopy (flewett, ) . however, levels of infectious virus in faeces may be much lower (madely, ) and since faecal solids represent only a minute fraction of wastewater, the predicted input of infectious virus may be no more than vlitre (melnick, gerba and wallis, ) . it is well known that at least a hundred different types of enteric viruses (table ) may be found in human faeces (melnick, gerba and wallis, ) , of which at least some could be expected to be in final effluents. it'is also possible that certain viruses excreted with the urine could also be present (utz, ) but in general, it is likely that the frequency and pathogenic potential of enteric viruses will be influenced by geographic, seasonal,as well as socioeconomic factors. most enteric virus infections in developed countries are sporadic and episodic but in developing regions they may well be seriously epidemic. the range of symptoms they may cause include not only gastroenteritis but such divergent clinical features as meningitis, exanthema and even respiratory symptoms (andrews, perlera and wildy, ) . some enteric viruses, for instance poliovirus vaccine strains, may for obvious reasons be regularly isolated from effluent or contaminated waters although apparently not necessarily so even during a vaccination campaign (katzenelson & kedmi, ) . of the other common enteric viruses, the echoviruses, the coxsackieviruses, the reoviruses and the adenoviruses many may be isolated throughout the year, but the serotypes may vary from year to year and valuable corroborative data for this has been provided in the u.k., by the public health authority through its weekly and quarterly reports (cdsc, i o). unfortunately, several important enteric viruses such as hepatitis a, the rotaviruses and several so-called 'small round viruses' cannot be readily cultivated in the laboratory (table ) and evidence for them depends wholly on clinical or immunological data, or on their visualisation in the electron microscope. although many different enteric viruses are likely to be present in wastewater, the risks of transmission of infection via contaminated water in developed countries by various routes (fig. ) is thought to be slight although probably increasing, but elsewhere the risks may be very great indeed (who, ) . undoubtedly, outbreaks of gastroenteritis have been associated with effluentpolluted lake water (cdc, ; denis et al., ) and bathing beaches (cabelli et al., ) and many cases of hepatitis a as well as other enteric virus infections have resulted from the consumption of shell fish harvested from contaminated waters . in general, where epidemiological evidence for gastroenteritis and diarrhoeal diseases has been assembled, it accounts for about half the recorded outbreaks of waterborne diseases (craun, mccabe and hughes, ) , although in relatively few cases is the aetiological agent identified either by isolation or by serological means. it is however believed that a substantial proportion of these infections could be of viral origin because of the development of symptoms between one and two days after infection and the recovery of the subject within two to three days without significant sequelae. although clinically characteristic infections such as hepatitis a may well occur, it must be borne in mind that only a small proportion of infected subjects, especially children, develop symptoms (evans, ) and this is also true of virtually all enteric virus infections even those which may be very serious, such as poliomyelitis. the significance of this observation is that infected symptomless individuals may well represent foci for further infection in the community, and should this happen, then disease of epidemic proportion could subsequently develop. of course, in these circumstances, it would be very difficult to relate such an epidemic to an original waterborne infection. incidentally, it is suggested by some that low levels of infection through the water route may be advantageous to a community by providing it with a relatively harmless mechanism for the circulation of viruses which could otherwise become dangerously epidemic in a largely susceptible population (mosley, ) . it has also been argued (gamble, ) that even if the elimination of such low-level transmission of viruses were possible it would be unlikely to have much effect on endemic infection which is commonly by the direct person to person method. neverhteless, it is thought that viruses in water represent an underestimated problem (mahdy, ) . . an assessment of the extent of the hazards of viral contamination of effluents and the effectiveness of any treatment for its removal depends critically on the sensitivity and reliability of the procedures for the recovery and characterisation of the viruses. successful characterisation of viruses depends essentially on the infectivity test because not only is this the paramount property under scrutiny but the levels likely to be present are very small indeed. certainly they would not be sufficient, without impractical levels of concentration, for detection by electron microscopy or serological techniques, both of which require at least ^ particles/ml. although infectivity assays may be quite sensitive, it must be borne in mind it is possible that only one infectious particle is sufficient to infect a .pa susceptible subject and in this regard, it is important to note the difficulty which exists in defining an infectious particle (floyd and sharp, ) which may be an aggregate of particles, or even particles embedded in organic floe. another impoortant limitation on the interpretation of infectivity assays relates to the sensitivity of the selected cell culture. the choice of this is often dictated by such pragmatic considerations as cost and practicability, for instance, it is claimed that one of the most sensitive cell systems for a wide range of enteric viruses is the primary rhesus kidney cell culture but this is now virtually unobtainable and one or more monkey kidney cell lines like vero and bgm have become popular alternatives . none of these cell cultures is equally sensitive to all the enteric viruses, furthermore, some viruses replicate in them much more slowly than others so that overgrowth of the culture by one virus may occur, a not unlikely event, from an effluent sample which could well be expected to be contaminated with several different viruses. this problem would be less troublesome where the assay for infectivity was the plaque test, but it would be intractable where a quantal assay was employed based on degeneration of the whole culture. even greater problems occur when the virus is noncytopathogenic, and this could be compounded if such a virus interfered with the growth of a cytopathogenic virus. another limitation which must be applied to the interpretation of infectivity assays is that cell sensitivity to laboratoryadapted virus and to fresh viral isolates is known to differ, especially to isolates from faecal samples (madeley, ) with which virus in effluents are comparable. these various points highlight the difficulties in developing viral standards for effluent quality where the assay of infectivity is only effective for some, uncertain for others and totally unavailable for those in which we are particularly interested another constraint on the sensitivity of the infectivity assay relates to the small number of infectious particles which may be present, such that concentration of the sample before isolation may well be essential. many methods for concentration of viruses from water have been recommended, such as filtration, flocculation or two phase liquid separation, but their value for effluent treatment will be greatly influenced by the effluent quality especially its content of suspended solids. (seeley and primrose, ) . one particular problem is the concentration of cytotoxic substances which may be difficult to characterise and remove (glass, sluis and yanko, ; schmidt et al., ) . having isolated and cultured a virus from effluent, there remains its characterisation and identificationwhich is a major task. this, to a large extent, depends on the development of a characteristic cytopathology and the application of serological tests, particularly the neutralisation test. other serological tests like immunoflourescence for non-cytopathogenic viruses may have to be used and characterisation by electron microscopy may also be necessary. clearly, the complexity of this situation makes the testing for the presence of all possible enteric virus out of the question, yet it is equally true that the monitoring for the effectiveness of effluent treatment is essential, so the selection of an indicator virus would be useful. for bacteria it is generally agreed that certain coliform bacteria may be regarded as representative, but with viruses it is not at all obvious which, if any one type, could be selected. the idea that enteric bacteria could be used as models for enteric viral contamination has been rejected, not only because there is little correlation between the levels found of the two groups, but because many viruses are more resistant to disinfection, which is the usual object of the study (berg, et al., ) . furthermore, the range of types of enteric viruses which may be present vary widely because, unlike many enteric bacteria, they do not appear to form part of the natural gut flora but occur sporadically. however, it has been frequently suggested that a coliphage,which would be expected to be part of the natural flora, would be suitable indicator of enteric virus pollution (scarpino, ; kott et al ) . the coliphages certainly fulfil a number of recommended prerequisites (haas, ) that they are present whenever their pathogenic hosts are present, they are incapable of regrowth in the effluent, but are at least equally resistant to environmental stress, including disinfection. furthermore, they are present in large numbers and are readily enumerated. however the selection of a particular coliphage poses a problem, for instance, an obvious contender like the f coliphage recommended by shah and mccabe ( ) is not the only phage to replicate in its specific host escherichia coli (k-, hfr). the use of the less selective £^ coli Β strain would isolate even more types of coliphages (kott, et al., ) . the deliberate introduction of a phage type unlikely to occur naturally, for example, a phage of serratia m^rc^seng (castens and coetzee, ) would be impractical for routine monitoring of effluent treatment, even supposing it had the right properties, like high resistance to disinfectant. to take a phage dependent on a host thought to be commonly present in wastewater treatment like a cyanophage (smedberg and cannon, ) would only be useful if it occurred reliably and was also very resistant to effluent treatment. nearly all these studies, as well as those in which a model enterovirus was propsed, suffer from the disadvantages peculiar to such laboratory studies, in that it is not certain how far results can be extrapolated to field conditions. so how worried need we be about viruses in effluent? the answer depends essentially on the nature of the wastewater treatment providing the effluent and, of course, on the fate of the effluent and the survival of the viruses in the receiving waters or soil. the nature of the wastewater treatment will inevitably influence the behaviour and fate of viruses, for instance, it is well known that much more virus is found in effluents after percolating filtration than those from the activated sludge treatment (berg, ) . the degritting and settling procedures carried out during the early stages of sewage treatment lead to an unpredictable dispersion of the virus originally present in the faecal solids. for instance, although a substantial proportion of the solids settle out into the primary sludges there is little apparent loss of infectivity in the settled sewage effluent (kollins, ) presumably due to a break up of vvviral aggregates and floes to release individual infectious virions and the fact that viruses have a strong predeliction to adsorp to solids (bitton, ) accounts for their effective removal during the activated sludge aeration. indeed, good quality effluents from such treatment should have a very low suspended solids content and a good correlation between low solids content and low viral infectivity of effluents has been observed (balluz and butler, ) . the absence of solids from effluent is also important because they provide a mechanism for the protection of adsorbed virus against disinfection (boardman and sproul, ; hajkal, et al, ) virus in untreated final effluents is ultimately subject to various forms of environmental stress in the receiving waters or soil (bitton, ) , but the fact that virus is readily isolated from effluent polluted river water obviously implies reasonable survival and experimental studies in different types of water provide additional evidence of good survival (gerba, wallis and melnick, ) especially in clean (mehnel et al., ) or heavily polluted waters but surprisingly and inexplicably, less so in moderately dirty water (clarke, et al ) . virus apparently survives less well in sea water (gerba and schaiberger, b) indeed there is some evidence that it is specifically inactivated (fujioka, loh and lau, ) . disposal of effluent to land may result in percolation of virus through the soil to contaminate ground water supplies (schaub and sorber, ) but normally viruses eventually disappear, due to adsorption and ultimately inactivation. .pa the distribution of viruses in soil is known to be strongly influenced by the degree of hydration, the ph, the ionic strength and the organic content of the medium (akin et al., ) . it is clear from all this that the ecology of viruses during wastewater treatment and after the disposal of the products is evidently very complex. furthermore, it is also worth noting that virions enter the sewage plant discontinuously and in various states of aggregation so that no one sample at any one stage in the process can be taken as representative of anything other than what is present at that time in that sample. . the methods available for the inactivation of viruses in effluent differ little in principle from those applied to potable water, but are distinct from the disinfection of viruses contaminating, laboratory or medical equipment, where highly toxic chemicals like detergents, phenols, formaldehyde or permanganate may be used (spalding et al ) . for effluents, the choice of treatment is limited by the requirement for a high quality final effluent free from harmful by-products. the treatment must be cheap, easy to produce. the disinfection methods available range from the purely physical (table ) , to purely chemical (table ) like the application of the halogen disinfectants. in addition, there is the more complex and interesting application of a combination of one or more of these basic methods. indeed, the distinction between the categories is by no means clear cut, especially in effluents where, because of the impurities, a number of mechanisms may operate antagonistically or synergistically. for example, ionising irradiation although able to act directly on the virus, may also function chemically in so far as it may induce the production of toxic free radicals. in contrast, a chemical disinfectant like chlorine, may react preferentially with organic and inorganic contaminants so that its full potential is lost. thus, the effectiveness of any one of these treatment systems may be much influenced by the presence of chemical and physical impurities and the pretreatment of effluents to remove these may be essential for the effective, economic and safe disinfection of viruses (guy and mclver, ) . culp ( ) questioned the need for disinfection after effective per treatment but this very much depends on the fate of the effluent and a number of procedures have been adopted for the improvement of effluent quality before disinfection. of the most commonly applied methods, flocculation with a variety of salts or synthetic polyelectrolytes is probably the most useful, but not all viruses behave in the same way, for instance rotavirus is less efficiently adsorbed to aluminium hydroxide than poliovirus, (farah et al., ) , but in practice such differences might not be important. filtration of effluents by slow sand fliberation (poynter and slade, ) results in the removal of virus by adsorbtion to the complex microbial population which grows in the upper layers,but rapid sand filtration although removing solids from effluent and therefore effecting some useful clarification does not remove suspended virus well (guy and mclver ) furthermore its function in this regard is greatly influenced by ph, ionic concentration and organic contamination. adsorption of virus to such substances as activated charcoal is efficient from clean water (oza and chang, ) but such substances are rapidly blocked by organic matter (sproul, ) . incidently all these transport and store. it must be potent at low dosage and readily decompose, either spontaneously or by the application of a neutralising agent, into harmless by-products. furthermore, it must be simply and reliably assayed and unreactive with other chemical or physical constituents of the effluent. in this regard, the characterisation of effluent is an important consideration because wide variations in physical and chemical quality are known to occur and are likely to influence the effectiveness of disinfection (tonelli, ) . treatments which result in the removal of virus merely defer the problem of its inactivation, to the treatment of the resultant sludge. excess lime treatment has the greatest promise for the treatment of effluent with resultant inactivation of virus (grabow, middendorf and basson, ) it is thought that the single-stranded rna viruses are particularily susceptible to the high ph values obtained with the genome as the prime target although sproul ( ) thought that inactivation was the result of denaturation of the capsid. in general, enteric viruses have a wide ph tolerance which is, presumably, a reflection on their natural history as gut parasites transmitted by the faecal oral route and, therefore, exposed to the acidity of the stomach and compensating alkaline secretions of the small intestine. inactivation of virus present in good quality effluents by physical of physicochemical methods has attracted increasing attention because disinfection by chlorine has come increasingly under attack. the most promising developments use ionising (sinsky, ) and non-ionising irradiation (vajdic, ) not only alone but in conjunction with chemical methods. the main disadvantages in these methods is that ionising irradiation by gamma rays or high energy electrons, like that with ultraviolet, has poor penetrating power and its effect is greatly diminished by turbidity. however, although expensive in comparison to the application of conventional disinfectants they may, as a result of improved, technology become competitive and acceptable (singer and nash , ) heat also would work well but remains impractical for effluents, although applicable to sludges (ward and ashley, ) where anaerobic digestion results in the development of elevated temperature. however, studies of heat inactivation of viruses in water have, incidently, provided a useful basis for understanding viral inactivation in general. in particular, of the characteristic biphasic inactivation where after treatment there remains a residual of apparently unaffected virus. the influence of ph, divalent cations and redox potential on heat inactivation may also provide some insight into the influence of these factors on chemical disinfection, indeed the action of ph, cations or redox potential alone on viruses should not be ignored (poynter, slade & jones ) because both redox potential and ph are both believed to act on viral capsid proteins (mandel, ) which may either alter their sensitivity to disinfectants or their ability to adsorb to sensitive cells. the most practical and successful disinfectants of wastewater effluents are the oxidising agents like the halogens, ozone and peroxides, although it is likely that the action of these chemicals is not exclusively through oxidation. the general principles of disinfection were established by chick (i o ) who formulated a law which stated that the ratio of a given population decreased exponentially with time, that is that the reaction obeyed 'first order* kinetics. however this only applies if the disinfectant is in excess (hiatt, ) , if the system is homogeneous and if the interaction between virus and the disinfectant is direct. such conditions do not, of course, apply in sewage effluent which has a complex and variable constitution. deviations from first order reactions have been commonly observed and they mainly fall into two categories, those which show an initial lag before maximum or optimum rates of inactivation develop and those where, after an initial period of rapid inactivation, a plateau develops representing a persistant infectious fraction. sometimes both features are observed in the same system (fujoka and ackerman, ) . various explanations have been sought for these phenomena. for instance, an initial shoulder would appear when the multihit response occurred as would be expected with most suspensions of infectious virions which, of course, include viral aggregates of various size (floyd & sharp ) . there would be, in such examples, a delay in the measured loss even though much virus was actually inactivated. aggregation could equally explain residual infectivity where infectious particals remained inaccessible to the disinfectant (broardman and sproul, ) it is also possible that viral populations are genetically heterogenous with respect to sensitivity to disinfection, indeed some studies have resulted in the selection of population with increased resistance (bates, shaffer and sutherland, ) but it is also possible that some residual infectivity is the result of multiplicity reactivation (young and sharp, ) that is the restoration of the complete replication mechanism due to the multiple infection of a cell with virions with differntly but only slightly damaged genomes. such doubts re-emphasise the neeed for reliable and reproducable infectivity assays and in such situations each virus particle must be assumed to be infectious and its infectivity determinable. there are infact few examples where the total number of virions present corresponds to the number of infectious units and usually there is a considerable difference. this also emphasises that a disinfection activity may have to be determined empirically for each virus in question if precise data is required (hajenion and butler, ) . certainly, assay of its efficacy depends not only on effective assay of infectivity but also on that of the disinfectant. the mechanism by which the oxidative disinfectants work is probably complex and a basic understanding is required. so far as the halogens are concerned they are known to react with viral proteins and nucleic acids ( ivieri et al., ; »brien and newan, ) and in this debate it is worth noting the essential structural features of the enteric viruses which have a proteinaceous capsid enclosing the single or double stranded nucleic acid genome. the capsid proteins may be organised as specific receptors, the integrity of which is vital to the infectious process but damage to the receptors may not result in de naturation of the genome which, of course, leaves the possibility that viral replication could ultimately occur. furthermore, as noted above, partial denaturation of the genome may not inevitably result in failure to replicate. a disinfectant may dissociate or denature viral proteins or react with the genome or both and one which reacts specifically with the genome must be able to penetrate the capsid. of the halogen disinfectants, chlorine has a long history as a successful disinfectant (white, ) . it is most commonly used for finished potable waters and swimming pools but is widely used for treatment of effluents, particularly storm waters and even for oxidation ponds (kott, ) . its chemial properties have been exhaustively examined and described. basically, when a halogen is dissolved in water it hydrolizes to form the hypohalous acid. x + h o >-hox + x" + h+ and the acid ionises to the hypohalite ion. ox" + Η·*-hydrolysis and ionisation are ph, temperature and concentration dependant and the three halogens chlorine, bromine and iodine behave differently. it is important to know this because it is the hypohalous acid which is the most active molecule. for instance at low ph the chlorine molecule is predominant whereas above ph the hypochlorite ion is present. chlorine functions best against viruses at about ph when optimum levels of the acid are formed (kott, nupen and ross, ) and it is worth noting, here, that the ph of many effluents is about ph . halogens react with other nitrogenous matter to form similar derivatives but the chemistry of such compounds is complex. halogens also react with inorganic matter to produce stable but non-disinfecting compounds and the loss of halogen this way is referred to as the halogen demand of the system, which is usually high in effluents. in the case of chlorine it is clear that it has some remarkably useful characteristics especially in water with slight nitrogenous contamination when it forms stable, persistent and disinfecting chloramines. these are, however, less active against viruses than against bacteria (shah and mccamish ; hart, ) . in heavily polluted effluents the loss of chlorine, its conversion into chloramines and many even less acceptable byproducts may be so great as to render its highly unsuitable (ward and degraeve, ; smith, mccall and chen, ) mainly because of their toxicity to the natural flora and fauna of the receiving waters and their carcinogenic potential. furthermore, it may be necessary to acidify effluent to obtain optimum conditions for chlorine disinfection (mills, ) . the disinfection potential of chlorine against viruses has been demonstrated by many people with experimental model systems as well as in the field situation. it is important to note that viruses range in their sensitivity (lund ) for instance a laboratory strain of coxsackievirus was the most resistant enterovirus tested and some other enteritis viruses like reovirus and adenovirus were more sensitive than than any of the tested enteroviruses. more interesting is the observation that fresh isolates of enterovirus appeared to be more resistant than laboratory adapted strains (kelly and sanderson, , lui et al ) . furthermore, certain viruses may have resistance selectively induced by cultivation in the presence of chlorine (bates, shaffer and sutherland, ) . this observation raised objections to inadequate chlorination (nupen and morgan, ) because of the possibility that such resistant viruses may, when released in effluent, ultimately replicate in susceptible people. the need for thorough mixing of disinfectant to ensure optimal activity is stressed (longley, ) and this is especially important where virus is another interesting and important phenomenon is that hypohalous acids react with ammonia to form mono, di and tri halomines: hox + nh nh x + h o. hox + nh nhx + hox + nhx >-nx + h o these reactions are concentration dependant such that, for instance, when the ratio of chlorine to ammonia is greater than : , free chlorine is again available for hydrolysis, a phenomenon well known as break-point chlorination (palin, ) . the reaction is also ph dependant with the highly substituted derivatives being found in acid conditions. the mono and dihalomines decompose to release nitrogen and those formed with chlorine are the most stable: nh x + hox >-n + hx + h o nhx n + hx + x adsorbed to particulate matter by which it is protected (boardman and sproul, ; hijkal et al, ) so the design of efficient chlorinating systems is .pa important (tickhe, ) . furthermore, its meaningful assay in effluent has been closely questioned (morrow and martin, ) . of these methods chemical assay, especially by the dpd method (palin, ) , is the most accurate and reproducible but for automatic monitoring the known relationship between redox potential and disinfection potential (victorian, hellstrom and rylander, ) has led to the development of assay based on the electrical charge however, the value of this has been questioned (rosenblalt, ) especially its application to water heavily contaminated with nitrogenous compounds (johnson, edwards and keeslan, ) when an electrode responding directly to hypochlorous acid was used. no one method is wholly satisfactory because the chemical property of a disinfectant may not correspond with its disinfecting potential. the problems resulting from the excessive use of chlorine for effluent disinfection (comp. general, ) has led to quite extensive searches for alternative. furthermore, the disontinuance of the production and distribution of liquid chlorine, which some critise (humphrey, ) ,has precitated even more active interest in alternative disinfectants. of the other halogens the one least likely to be useful alone is iodine. it is both poorly soluble in aqueous solution and not very reactive. however, it is easily stored and transported and is therefore useful for emergency sterilisation of water. in aqueous solution it forms hypoiodous acid and hydrogen iodide, the acid being the most active molecule: hoi + hi it experiences considerable demand but it persists for longer in effluent than chlorine (cramer et al., ) and is a more effective virucide than chlorine both at neutral ph and, especially, at higher ph values. bromine, although known to be a powerful virocide (taylor and johnson, , floyd, johnson and sharp, ; hajenian and butler, i o) is poorly soluable in water and is a highly corrosive liquid. although, as such, it is not suitable for effluent treatment its chlorine derivative, bromine chloride has great promise. indeed this has been found to be more effective against viruses than chlorine on a weight for weight basis (keswick et al., ; kawata et al., ) and experienced less interference from added ammonia or glycine in sewage effluent. however,.its potential against a wide spectrum of enteric viruses has yet to be evaluated as has the effect of its residual on the ecology of the receiving waters (mills, ) . it is readily soluable in water and in a aqueous solution hypobromous acid is formed which is readily ionised: brcl + h o hobr + hcl if some hypochlorous acid is also formed it is unstable in the presence of the bromide ion forming further hypobromous acid, the most active molecule hocl + br >-hobr + cl" these reactions are ph dependant and the greatest disinfecting potential is between ph and which would mean that for use in effluent no acidification would be required. the disinfectant reacts, of course, with nitrogenous compounds to form bromamines which are claimed to be highly virucidal (mills, ) . however, they are short lived which although a disadvantage for clean water treatment would therefore be a valuable property for effluents treatment. the excellent potential for disinfection possessed by chlorine dioxide was recognised over thirty years ago (ridenour and ingols, ) and it has been .pa shown to be very useful against viruses (dowling, ; kawata et al., ) .in aqueous solution it produces chlorous and chloric acids the normal method of production of chlorine dioxide which is from chlorine and sodium chlorite means that a residual of chlorine remains which in the form of hypochlorous acid reacts with chlorine dioxide to produce the highly reactive chloride ion (tifft et al., ) : some of the early reports on the disinfecting activity of chlorine dioxide failed to take into account that chlorine was present but in more recent studies, especially where the disinfectant is made by heating potassiun chlorate with oxalic acid (palin, ) its activity is better understood. fortuitously, chlorine dioxide does not readily react with nitrogenous compounds although it does experience a demand from phenolic chemicals. it is this property, resulting in the elimination of colour, taste and odour which has attracted much attention (ingols, ) . its assay in the presence of chlorine is complex but recently a specific spectrophotometric assay has been reported (knechtel, janzen and davis, ) which is useful. from the earliest times ozone has attracted a lot of attention as a water disinfectant. like chlorine dioxide, it has to be produced in situ but the equipment is simple depending on the convertion of oxygen in an electric arc. however, for accurate and reliable delivery considerable care has to be exercised, and its assay is frought with difficulty because its chemistry in aqueous solution is complex (peleg, ) . its disinfectant activity probably resides in the hydroxy and oxide radicals (hoigne and baden, ; kim, gentile and sproul, o) but its value also lies in its capacity to react with many organic carbon compounds incidently resulting in the removal of colour, odour and taste, a subject which has been critically reviewed (kinman, ) . it has been shown to be active against a range of viruses (evison, ) and its potential is greatly enhanced by good mixing particularly the application of ultrasound (dahi, , burleson, murray and pollard, ) which probably affects not only the bubble size (farouq, chian and engelbrecht, ) but may be useful in the break-up of viral aggregates and the release of virus from floe. its effectiveness has been compared to that of chlorine (bollyky and siegel, ; wyatt and wilson, ) , but its main disadvantage for effluent is the great demand which has to be met before the formation of the active residual, however there is a complete absence of harmful biproducts. there remains a small miscellany of chemicals which are known to have a disinfecting potential but which have not been adequately tested. prime amongst these is peracetic acid. its virucidal activity was first reported by klein and hull ( ) and later by sprossig and mucke ( ) and there is a recent laboratory study on viruses in effluent (hajenian and butler, i o) . this is complemented by a similar, although more extensive study of its bacteriocidal aativity in effluent (poffe et al., ) in aqueous solution nascent oxygen is produced which presumably imparts the disinfecting property:- the byproduct, acetic acid, is unlikely to be toxic although it is undoubtedly a microbial metabilite. it appears to suffer negligable demand in effluent since its activity is fully retained against added virus up to thirty minutes after its original application (hajenian and butler, i o) . another common peroxide,the peroxide of hydrogen is a very weak disinfectant (bayliss and waites, ) and is unlikely to be useful for wastewater treatment except for the control of hydrogen sulphide and bulking. one of the most interesting developments in the disinfection of effluents could be the application of two or more treatments sequentially or simultaneously to achieve a real or apparent synergistic effect. apparent synergism would occur, of course, when the action of one treamtent simply removed a substance capable of blocking or inactivating the other reactant. examples of this have already been noted, for instance the pretreatment of effluent to make it more suitable for chemical disinfection or the more special case of the value of ultra sound during ozonisation (dahi, ) .a good example of real synergism is demonstrated by the sequential addition of chlorine and chlorine dioxide where the nett disinfection is improved (tifft et al., ) . usually the advantages of such treatment or equivalent procedures with chlorine and ozone (ross, van leeuven and grabow, ; wyatt and wilson, ) is mainly in the production of a better quality water free from irritating or toxic residuals. true synergism has also been thought to be the result of the combination of monochl or amine and iodine (kerman and layton, ) and iodine with ozone (buddie, ) . a number of other combinations of disinfectants could be usefully explored, for instance chlorine with peracetic acid where the application of the latter causes a fall in ph to acid values (hajenian and butler, b) at which chlorine is at its greatest efficiency. a particular problem with such studies especially where the chemicals are applied simultaneously could be in the determination of specific disinfectant residual. in conclusion it should be stressed that few of the viruses present in effluent can be properly characterised. furthermore, the health hazard that any potentially pathogenic enteric virus represents in effluent can only be guessed at. however, although the disinfection of effluents, will improve the situation the variable quality of effluents will make it difficult to standardise any procedure. it should also be stressed that overdosing with disinfectant, especially chlorine, should be discouraged and critical studies of alternatives continued, especially, perhaps the development of the combination of disinfectants. proc. th water quality conf. univ. illinois, snoeyink introduction to ewnvironmental virology am. water wks. m water sew water sew. wks. ih i o) in: disinfection -water and waste water div, water air waste chem, pacs ϋ transmission of viruses byy the water route waste water eng inrdisinfection-water and wastewater in:discharge of sewage from sea outfalls water pollut. cont. fed. highlights (deeds & data) proc. biochem. j¿ chemistrv of water suddiv treatment and distribution water pollut viruses in water surveillance of drinking-water quality who monograph series no key: cord- - ycp m authors: elliot, elisa l; colwell, rita r title: indicator organisms for estuarine and marine waters date: - - journal: fems microbiology letters doi: . / - ( ) - sha: doc_id: cord_uid: ycp m abstract the use of indicator organisms for estuarine and coastal waters has been reviewed. the natural flora of the environment must be considered in selecting an indicator organism, but, more importantly, recent work which shows a viable but non-recoverable stage of pathogens entering the marine environment demonstrates that the conventional detection of indicator microorganisms is misleading, if not inaccurate. results suggest that the newly developed epifluorescent/immunofluorescent direct detection of pathogens in the environment may be the most reliable method for determining public health hazards in marine and estuarine waters. the use of indicator organisms for estuarine and coastal waters has been reviewed. the natural flora of the environment must be considered in selecting an indicator organism, but, more importantly, recent work which shows a viable but non-recoverable stage of pathogens entering the marine environment demonstrates that the conventional detection of indicator microorganisms is misleading, if not inaccurate. results suggest that the newly developed epifluorescent/immunofluorescent direct detection of pathogens in the environment may be the most reliable method for determining public health hazards in marine and estuarine waters. the microbiology of the world oceans and coastal waters has been under study since the late nineteenth century. among the earliest investigations concerning the number of bacteria and their * to whom correspondence should be addressed. distribution in seawater was that of russell [ ] [ ] [ ] who conducted a survey of the distribution of bacteria in the gulf of naples. the early workers established general distributional patterns for bacteria in the sea, using agar plate and dilution methods for the enumeration of bacteria. bacteria were found in small numbers, uniformly distributed in water several miles from shore and at all depths. early workers also found that the water of harbors and bays may contain large numbers of bacteria, but that the number of bacteria becomes very small at comparatively short distances from shore [ , ] . the work of fischer is, of course, classic, since he investigated sea water over a wide area of the atlantic ocean and found bacteria throughout, the largest numbers being found near land or in the presence of floating seaweed [ , ] . seasonal studies were done by lloyd [ ] in the clyde sea, where the waters are relatively shallow. the surface waters were richest in bacteria, the number decreasing with depth but usually increasing slightly at the bottom. a variety of studies was done in the early )'ears of marine microbiology, summarized by benecke [ ] and zobell [ ] . the observation that direct microscopic examination of seawater showed bacteria to be much more abundant in seawater than cultural methods would - / /$ . © federation of european microbiological societies suggest, was made as early as by cholodney [ ] . marine bottom deposits are known to contain much larger numbers of bacteria than the water column. russell [ ] showed that bacteria were distributed in sediments of the bay of naples at depths ranging from - m below the surface of the water. zobell and morita [ ] , in a study carried out about years ago, showed that bacteria are indeed present in sediment cores taken in the phillippines and marianas trenches. some years earlier, reuszer [ ] had demonstrated that bacteria are much more numerous in bottom deposits than in overlying water, and that the number of bacteria decreases with depth into the sediment. dale [ ] showed a strong relationship between bacterial numbers and sediment properties, i.e., the number of bacteria is highly correlated with grain size and other granulometric properties. sampling methods for microbiological analyses of water include the j-z sampler and the niskin sampler [ ] . deep ocean environmental samplers have also been developed in recent years [ , ] . sediment samples are unfortunately still obtained by core or grab [ ] . animals are collected by means of traps and trawls. the methods for the enumeration of microorganisms, estimating their biomass, and measuring their activities in the ocean, were the basis of a recent symposium [ ] , and include the classic methods of plate count and filtration. more recently developed methods include atp measurement, a biochemical technique for estimating total microbial biomass as a function of the luciferinluciferase reaction, which is quantitatively dependent on the amount of atp in the sample [ ] , and bacterial cell wall component analyses [ ] . several different approaches to microscopic bacterial counts are now available, all employing fluorochromes and epifluorescent microscopy. some of these methods enumerate total (i.e., living and non-living) bacteria [ ] [ ] [ ] , whereas others purport to count only living bacteria [ ] [ ] [ ] . recently, tabor and neihof [ ] compared of the direct viable count methods with amino acid uptake, and found that amino acid uptake consistently measured a larger number of cells. the epifluorescent-immunofluorescent method offers exciting new opportunities for .determining directly the presence of pathogens in water, even when these organisms are viable but non-recoverable [ ] [ ] . the results of the work of pioneers in marine microbiology at the turn of the century have been firmly established. more recently, sieburth [ ] surveyed seawater from the pacific, caribbean, and atlantic oceans, culturing bacteria in agar roll tubes indicated within . °c of the temperature of the water from which they were obtained. the isolates were keyed to genus, and a characteristic bacterial flora was reported in the ~m-thick surface film overlying 'low-count' waters off the west coast of central america in an area known for upwelling; in all the samples, pseudomonads were dominant. however, very recently, the same areas have been re-examined and the dominant organisms, especially at the ocean dumping sites, have been identified as vibrio spp. [ ] . murchelano and brown [ ] found that heterotrophic bacteria in the long island sound water column showed bacterial concentrations ranging from - per ml, with variations in seasonal concentration mirroring that of the phytoplankton. gram-negative rods constituted % of the isolates. genera were identified, but . % were a chromobacter, flaoobacteriurn, and pseudomonas, with pseudomonas dominant in the summer and flaoobacterium in the spring. again, more recent work suggests a far more prominent role for vibrio [ ] . there is a wide oceanic distribution of bacterial genera associated with geomicrobial cycling in nature. for example, nitrogen-fixing, nitrite-and nitrate-forming, nitrate-reducing, cellulose-decomposing, agar-decomposing, and chitin-decomposing bacteria have been isolated from seawater and sediment [ ] . denitrifying bacteria in shallowwater marine sediment were documented by patriquin and knowles [ ] . nitrogen-fixers have been isolated, including nitrogen-fixing vibrios ( [ ] ; m.l. guerinot and r.r. colwell, in press). thiobacillus-like bacteria, i.e., reduced sulfuroxidizing bacteria (thiosulfate or elemental sulfur), have been isolated from seawater and sediment in the atlantic ocean, black sea, and other areas [ ] [ ] [ ] . yeasts are readily isolated from coastal waters [ ] , especially in association with seaweeds [ ] . actinomycetes, mycelial bacteria, can also be isolated from bays, coastal and, offshore waters [ . however, more recent work [ ] suggests that the actinomycetes are perhaps more commonly of terrestrial origin, especially at ocean dumping sites. a very interesting group of bacteria is the 'filterable form' or 'mini-bacteria'. the occurrence of filterable bacteria in sea water was reported by oppenheimer [ ] , who found up to viable cells per ml of sea water filtrate after passage through a membrane with a pore diameter of approx. . /lm. brisou [ ] , using filtration procedures, showed that l-forms of bacteria can be isolated from marine molluscs. anderson and hefferman [ ] used a process of double filtration of sea water, filtering first through a . #m membrane and then through a . #m pore diameter filter. a group of microorganisms, not usually encountered by the conventional techniques of pour-plates or one-stage filtration, was obtained. many could not be identified, but the largest single group belonged to the genus spirillum. other isolates were placed in the genera leucothrix, flavobacteriura, cytophaga, and" vibrio. a group of microorganisms also often overlooked is the oligotrophic bacteria. these include organisms which require prolonged incubation periods or low nutrient conditions. such organisms have been studied in chesapeake bay and were found to include hyphomicrobium, nocardia, and sheathed bacteria [ ] . recent work of martin and macleod [ ] suggests that the oligotrophic bacteria might better be defined as organisms with narrowly defined patterns of nutrient utilization. for centuries man has used the sea as both a dumping ground, with wastes dumped into rivers and streams or directly into the ocean, and as a source of fish for food. when populations in the settlements along coastal regions of the world were small and sparse, and fishermen harvested offshore, the risk of contamination of fish by domestic and/or industrial wastes was low. nevertheless, even in the earliest recorded times, disease was widespread and life expectancy short. with the world population increase, and concomitantly more densely inhabited cities, domestic and industrial waste problems have magnified. the spread of disease via fecally contaminated water and foods in many countries is guarded against by hygienic treatment of sewage wastes. despite such efforts, outbreaks of disease still occur. as a result, standards have been proposed and employed to maintain water quality for specific use, i.e., drinking, agriculture, mariculture and recreation. standards are developed to ensure that water supplies are either free of disease-causing organisms, including pathogenic bacteria, viruses, and other microorganisms, or carry concentrations of pathogens at a level that does not pose an unacceptable risk to health. the list of potentially pathogenic organisms found in water is long, and even now, remains incomplete, since new pathogens are constantly being described, as for example, the legionella spp. the task of assaying fresh and salt waters for all such organisms on a regular basis would be monumental. for this reason, those concerned with water potability, as well as food safety, including microbiologists, sanitary engineers, and medical personnel, have devised schemes using indicator organisms as a warning of the unsafe condition of waters and foods, the protocol specifying the use to which the water is to be put. most indicator organisms were selected for fresh water systems, since river, lake, and ground waters are the most common sources of community water supplies. however, sewage wastes are dumped all too frequently into estuaries and oceans, as well as into rivers. although the oceans cover three-quarters of the earth's surface, the dilution of contaminated influent waters is rarely sufficient or rapid enough to rid estuarine, coastal and deep marine waters of microbial health hazards and the effect of pollutants carried in wastes. thus, indicator organisms appropriate for measuring the quality of estuarine and marine waters are needed. questions to be answered, with regard to estuarine and marine water standards include: (i) for which activities are water quality standards needed; (ii) are corresponding fresh water indicators adequate and appropriate indicators of health hazards or increased risk of disease arising from the presence of pathogenic organisms of fecal origin; and (iii) are such indicators appropriate and satisfactory, even for fresh water? of greatest importance is defining what the indicators will be used to indicate, i.e., fecal pollution or risk of disease. in addition to public health problems associated with infectious microorganisms transmitted by water, it has become evident that there are also risks of disease caused by organisms which occur naturally in the aquatic environment. gastroenteritis or wound infections are caused by vibrio parahaemolyticus and related vibrio spp., as well as by pseudomonas and aeronomas spp., none of which are necessarily associated with fecal pollution. it is increasingly evident that indicators of sanitary quality are not appropriate markers for the presence of pathogens that are autochthonous to the aquatic or marine environment. therefore, yet another indicator or set of indicators is required. historically, an appreciation of the spread of water-borne pathogens, e.g., salmonella spp. and vibrio cholerae, was developed in the late s. a dramatic example is the cholera epidemic in london. in the late s, klebsiella pneumoniae and klebsiella rhinoscleromatis were isolated from the feces of patients by von fritsch. escherich [ ] isolated bacillus coil, now classified as escherichia coil, and a erobacter aerogenes ( enterobacter aerogenes) from feces. these bacteria and other coliforms were used in the past as indicators of water-borne pathogens, that is, the presence of fecal contamination being correlated with the occurrence of pathogens, for which direct detection methods were not available. thus, 'indicator organisms' are markers of health hazards and/or water (or food) quality. mossel [ ] , when discussing markers in foods, differentiates indicators from index organisms, the latter suggesting the risk of occurrence of pathogens, indicators being more general and suggesting inadequate bacteriological quality. cabelli [ , ] also differentiates fecal indicators from water quality indicators. fecal indicators would be inadequate as markers of pollution by organisms from fecal wastes if these organisms could multiply in the environment. a water quality indicator would be necessary to indicate these organisms, in addition to those organisms which do not have a fecal source, but are health hazards and are present in the environment [ ] . indicators must fulfil basic requirements, including: (i) specificity to the source of pathogens or fecal contamination; (ii) sensitivity of detection; and (iii) resistance to disinfection and to the environment that either equals or slightly exceeds that of the pathogen [ , - ]. since an indicator of fecal pollution is usually considered to be an index of the risk of the presence of fecal pathogens, the indicator must be present when the pathogen is present, and its source should be feces. an indicator must be sufficiently abundant in feces that the probability of detection is high, and it should be present in higher concentration than the pathogens. also, an indicator should be as resistant, or more resistant, than the most resistant fecal pathogens, and it should not multiply in the aquatic environment, whether freshwater or marine. in addition to these basic requirements, simple techniques must be available for detection and quantification of the indicator. the ideal indicator, therefore, is the pathogen itself [ ]. in fact, salmonella spp. has been suggested as an index of pollution in fresh water [ ] . the major arguments against using pathogens directly as indicators have been that they are difficult to isolate and identify, which is in most cases no longer a problem, and that the absence of one pathogen does not rule out the presence of another, nor does it have predictive value for the concentration of the pathogen [ ] . indeed, testing for the presence of every possible pathogen would be very expensive in time, effort, and resources. indicators are needed for application in the marine environment to help protect the quality of shellfish beds and primary contact recreational waters, and to prevent the spread of enteric disease. sewage treatment and subsequent dilution obtained upon entry into rivers or by direct disposal into coastal waters via marine outfalls and sludge dumping is monitored by bacteriological methods. unfortunately, marine and estuarine waters present special problems, since the behavior of pathogens and indicators differs from that in fresh water. indicators used in freshwater environments are not always appropriate for salt water. estuaries are less saline than the ocean, and salinity, as one parameter, can range from % at the mouth of a river to ~ where the estuary meets the ocean. the waters .may be stratified into a cold saline bottom layer and a less saline top layer in the typical salt-water wedge type of estuary [ ] . most ocean waters have a salinity of - %~. the water temperature of estuaries is higher and more variable than that of the ocean. tidal flushing is controlled by influent fresh water and ocean water, as well as by the physical shape of the estuary itself. biologically, estuaries are more productive and densely populated than oceans, with the populations including microorganisms, plants, and animals. freshwater organisms and other material entering estuaries and oceans are subject to competition with, and predation by, the autochthonous flora and fauna, in addition to effects of changes in the chemical constituents and physical structure of the surrounding waters. clearly, any proposed indicator must be examined in the water in which it will be monitored and/or used as a regulatory aid. many groups of microorganisms have been proposed as indicators of water quality and fecal pollution. indicators discussed separately, and in detail in the symposium proceedings edited by hoadley and dutka [ ] , include: coliforms, fecal coliforms, clostridium perfringens, pseudomonas aeruginosa, vibrio spp., bifidobacteria, candida albicans, salmonella spp., fecal streptococci, and klebsiella spp. bacterial and biological indicators were also the subject of a symposium, the presentations of which were published by james and evison in [ ] . the coliform group, i.e., total coliforms, are aerobic and facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas formation within h at °c [ ] . total coliforms are the most universally used indicator group, but include bacteria, in addition to escherichia coil, that are not specifically associated with fecal pollution, i.e., klebsiella spp., citrobacter spp., and enterobacter spp. [ ] . fecal coliforms are differentiated from other coliforms by their ability to produce gas from lactose at . °c within h [ ] . several techniques are available for enumeration of total and fecal coliforms, including tube enrichment and membrane filter methods, which are continually being compared and modified. although a positive fecal coliform test has been equated with the presence of e. coli and the presence of fecal pollution, fecal coliform-positive klebsiella pneumoniae [ ] [ ] [ ] [ ] and enterobacter aerogenes [ , ] have also been isolated from the environment. hendricks [ ] pneumoniae, alone, as indicative of fecal pollution, and the concurrent isolation of fecal coliform positive k. pneumoniae and e. coil as indicative of recent fecal pollution. they concluded that the ability of bacteria to grow in ec broth at . °c was not sufficient proof of a fecal source, and that whether or not k. pneumoniae was correlated with fecal pollution, it was indicative of a potential health hazard. enterococci have been proposed as indicators of fecal pollution for coastal bathing waters [ ] . enterococci are gram-positive cocci which grow at temperatures between - °c, and survive exposure to °c for at least min, grow at ph . and also grow in . % sodium chloride [ , ] . the enterococci are part oft he genus streptococcus, and include certain group q streptococci (e.g., [ , ] . the enterococci group excludes streptococcus boois and streptococcus equinus, which are not usually found in human feces [ ] . recently, schleifer and kilpper-balz [ ] proposed that s. faecalis and s. faecium be transferred to the genus enterococcus nom. rev. as enterococcus faecalis comb. nov. and enterococcus faecium comb. nov. fecal streptococci comprise all these organisms, as well as streptococcus mitis, and streptococcus safiv-arius, and are, therefore, less indicative of human fecal pollution. in fact, moore and holdeman [ ] found that s. salivarius ranked th on a list of human fecal bacteria, making it the most frequently encountered fecal streptococcus in human feces, and placing just behind e. coli which ranked th (the most abundant species was bacteroides vulgatus ). fecal streptococci are characterized by their ability to grow in the presence of azide and ethyl violet at °c, in azide dextrose broth and ethyl violet azide broth, or to produce red or pink colonies on kf streptococcus agar, lack the cataiase enzyme, and grow in bile broth at °c [ . the enterococci have a fecal source, survive sewage treatment better than coliforms, and are present in concentrations of - times less than e. coli in treated sewage [ ] . the survival of enterococci in sea water is longer than that of e. coli and total coliforms, but growth does not usually occur in sea water, regardless of the total salt concentration [ ]. enterococci have not been considered to be ubiquitous in the natural environment or to multiply in water [ ], even though biotypes of s. faecalis have been isolated from insects and plant surfaces. the above cited qualities, in the view of some investigators, make enterococci useful indicators of fecal pollution. the use of fecal coliform-fecal streptococcus ratios was proposed by geldreich and kenner [ ] to differentiate between human and other animal fecal pollution. since fecal streptococci survive longer than fecal coliforms in fresh water, the changing ratio of fecal coliform-fecal streptococcus makes the use of this ratio of limited and questionable significance [ , ] . the most widely used indicators for primary contact waters and shellfish waters are total and fecal coliform estimates. the concentration of e. coil is also used in shellfish guidelines. although coliforms are used as indicators, their effectiveness is now being questioned for both fresh and salt water applications. one major problem with fecal coliforms is their low survival rate in sea water, compared to other indicators and pathogens. dilution alone does not account for the rapid decrease in coliform counts near sewage outfalls in marine waters [ ] . various mechanisms have been proposed for the 'die-off, including ultraviolet (uv) radiation [ ] , predation [ ] , competition [ ] , osmotic stress [ ] , antibiosis [ ] , and heavy metal toxicity [ , ] . solar radiation is considered by some investigators to be a significant factor responsible for the bactericidal action of seawater. in field studies, mortality rates of coliform bacteria exposed to seawater are much greater in daylight than in the dark [ , , ] . gameson and gould [ ] report that half of the lethal effect of light is due to wavelengths below nm, while the remainder results from light wavelengths of - nm. however, fujioka et al. [ ] stress the effect of visible light as more significant than that of uv light, with respect to the bactericidal action of light on both fecal coliforms and fecal streptococci in seawater. high turbidity, which blocks the penetration of sunlight, slows the rate of bacterial mortality [ ] . reduction in coliform counts has been shown to be correlated with intensity of sunlight and has been concluded to be related to diurnal and seasonal variations. these variations should be taken into consideration in designing ocean outfalls and sampling protocols for coastal waters [ . the mechanism of bacterial 'die-off caused by exposure to light may be related to the presence of sensitizers in cells, which absorb light and cause cell damage [ ] . another theory is that light energy reacts with oxides to form superoxides, which may damage cells [ ] . kapuscinski and mitchell [ ] demonstrated that, in e. coli, sunlight damages the catalase enzyme system, which is necessary for the degradation of peroxide. long uv and visible wavelengths affect membrane permeability and damage active transport [ ] . inhibition of amino acid uptake, traced to the effects of uv and visible light, has also been demonstrated for estuarine bacteria [ ] . interestingly, the effect of sunlight is a function of the salinity of water [ ] . fecal coliforms and fecal streptococci are considerably more resistant to sunlight when diluted in fresh streams than in marine waters [ . chojinowski et al. [ ] reported that the mortality rates of coliforms in fresh water exposed to light are approximately half those obtained in experiments carried out using estuarine water, and one third of those when ~ sea water was employed. osmotic stress alone adds to the 'die-off" of coliforms in sea water. sublethal stress has been recorded for e. coli after exposure in test media to sea water of various salinities and incubation for up to days in the dark [ ] . the time taken to reach ~ mortality for sewage coliforms in river water ( ~ salinity) was twice that for coliforms in sea water (> % salinity) incubated in the dark [ ] . studies of non-illuminated, highly saline water taken from great salt lake (ut), which has an ionic composition similar to that of seawater, showed a higher death rate for e. coii in water from the more concentrated north ann of the lake ( g/ chlorides) than in water from the southern portion ( g/l chlorides) [ ] . an inverse relationship was also found between salinity of estuarine water and survival of an e. coli strain isolated from the rhode river estuary in maryland, u.s.a. [ ] . temperature is directly related to coliform 'dieoff" rates in both fresh and sea water [ , ] . a °c increase in temperature doubles the mortality rate of e. coil [ ] and other coliform bacteria [ ] . in a multiple linear regression analysis of the effects of temperature, dissolved oxygen and salinity on e. coli survival in diffusion chambers in the rhode river estuary, temperature was associated with over ~ of the variation in numbers of viable e. coil [ ] . in highly saline water collected from great salt lake, low temperature had a sparing effect on mortality rates [ ] . cold temperatures allowed a lower death rate of e. coli in water collected from the more saline arm of the lake. however, hussong et al. [ ] suggest that numbers of false-positive total coliforms, i.e., presumptive total coliforms which are not confirmed at the completed step, are increased in sediment when the water temperature is < °c. parasitism by bacteriophage was suggested by mitchell [ ] as a factor responsible for the destruction of fecal microorganisms entering aquatic environments. interestingly, roper and marshall [ ] demonstrated that sorption of bacteria, and their specific phage, to saline sediment interfered with viral infections, thereby allowing for persistence of bacteria in sediment. destruction by bacteriophage is also thought to occur in sewage treatment plants, although predation may be more important in the removal of enteric pathogenic bacteria from the final clarified effluent [ ] . predation has been suggested as another mechanism contributing to the bactericidal effect of sea water on non-marine bacteria [ ] . ciliated protozoa were found to be responsible primarily in reducing the survival time of e. coli in activated sludge [ ] . in estuarine waters, protozoan predators, rather than lyric bacteria, were associated with the decrease in numbers of e. coil [ , ] . as an extension of this hypothesis, mccambridge and mcmeekin [ ] reported that sunlight and microbial predators acted synergistically to reduce the numbers of enteric bacteria. fresh and stored sea water yield higher mortalities than artificial seawater. thus, it appears that there are substances in seawater, in addition to the inorganic salts, which increase 'die-off" of e. coli [ ] . these include antibiotics and other antagonistic chemicals produced by marine bacteria and plankton. catalase-sensitive lipopolysaccharides, produced by some strains of pseudomonas and chromobacterium, are active against terrestrial bacteria [ ] . sunlight-induced injury to the catalase enzyme [ ] would render terrestrial bacteria more susceptible to these antibiotics. marine phytoplankton also secrete antagonistic substances during most of the year [ ] , which may affect the survival of bacterial pollutants. acrylic acid, produced from the breakdown of dimethyl-js-propiothetin of some marine algae, has an antibacterial effect both in sea water and in the intestinal tract of penguins [ ] [ ] [ ] [ ] . oyster extracts and sea water containing acrylic acid produced by phaeodactylum tricornutum were shown to have an antibacterial effect against e. coil [ ] . thus, the presence of acrylic acid in oysters would have the potentially dangerous effect of masking a possible health hazard posed by the presence of human pathogens. reduction in counts of bacteria in seawater can also be caused by sedimentation, in addition to 'die-off". in laboratory experiments, using natural seawater, the concentration of e. coil was shown to decrease because of starvation. interestingly, the addition of nutrients resulted in floc formation, followed by adsorption of the cells onto suspended particles, with concomitant precipitation [ ] . ogawa suggested that precipitation was a factor in the 'disappearance' of coliforms in coastal waters, and that, in sediment, these bacteria survive longer because of the higher concentrations of nutrients present in sediment. however, xu et al. [ ] reported that e. coli survived for at least weeks in salt water microcosms, but were not culturable (see below). sedimentation is considered to be the most important factor associated with reduction in numbers of bacteria in the water column in the deep ocean [ ] . the presence of indicator bacteria in sediments has been reported by investigators [ ] [ ] [ ] [ ] . bottom sediments were found to contain larger numbers of e. coli [ ] and enterobacter aerogenes [ ] than the overlying seawater, because of a higher concentration of organic matter in sediment. in laboratory studies using autoclaved estuarine sediments and seawater, e. coli has been shown to increase in number [ ] , whereas when non-autoclaved sediments and water were employed, persistence of e. coli was noted, with limited growth of the organism. lalibert~ and grimes [ ] made similar observations on the in situ growth and persistence of e. coli inoculated into dialysis bag-contained sediment. gerba and mcleod [ ] suggest that the type of nutrient and microflora present in sediment may differ from those present in sea water, allowing for greater competition with e. coil in sediment. the particle size of sediment was found to be inversely proportional to the concentration of nutrient, and to survival of enterobacter aerogenes [ ] . since the survival of fecal coliforms in sediment samples was prolonged at °c, the temperature of sediment in situ is also an important factor in their persistence [ ] . since the concentration of fecal indicators [ , , , ] , microbial pathogens including salmonella spp. [ , ] , and enteric viruses [ , ] are higher in sediment than in the overlying waters, samples of sediment, rather than water, should be taken for examination of the presence of indicator bacteria in the case of primary contact water and shellfishing areas. although de flora et al. [ ] found greater numbers of viruses and bacteria in surface water than in bottom water or sediment samples collected from moderately or grossly polluted coastal sites, they also suggested that sediment was a transient reservoir for viruses and that viruses survived longer in some sediments than in sea water. wave action, tides, currents, storms, run-off, and human activities, including dredging and pleasure-boating, can disturb sediments, stirring up particulates to which organisms which persist in these sediments are adherent. resuspension of sediment allows the sediment microorganisms to enter the water column which may previously have contained negligible numbers of indicator bacteria and pathogenic organisms. it has, in fact, been documented that dredging resulted in the release of sediment-bound fecal and total coliforms and fecal streptococci in the mississippi river [ ] [ ] [ ] . although the preceding section has focused on (i) die-off of e. coli and other indicator bacteria in seawater, and (ii) their persistence in sediment because of various factors associated with the particulate nature of the latter, it must be emphasized that recent evidence disputes claims that coliforms are rapidly killed by sea water. coliforms appear to be injured, stressed, or debilitated to such an extent that special techniques may be required to recover them [ ] [ ] [ ] . methods for recovery of stressed coliforms have been proffered for fresh water [ ] and for frozen or heated foods [ ] . for stressed coliforms in marine waters, the methods presently available which allow 'repair' include filtration through membrane filters which are subsequently placed on two-layered plates [ ] , modification of the most probable number (mpn) method requiring transfer of all presumptive tubes showing growth whether or not gas is produced [ ] , use of special membranes that 'nestle' the bacteria [ ] , and pour-plating followed by overlaying with a selective medium [ ] . the pour-plate method also has been used for enumeration of fecal coliforms and enterococci in seafood samples. dawe and penrose suspended dialysis tubes filled with bacterial suspensions or untreated samples of water in sea water ( - °c), and found that, although the rate of 'injury' for coliforms was high, the rate of survival was also high, based on atp content per cell [ ] . it was suggested that sea water, rather than being bactericidal, acts to protect 'injured' bacteria from death. recently, work in our laboratory has shown that e. coil cells in saltwaten microcosms remain viable, yet 'non-culturable' on laboratory media [ ] . viability of cells was determined using a direct viable microscopic technique [ ] . other microcosm experiments employing v. cholerae in salt water [ ] and salmonella enteritidis in fresh water showed the same result, i.e., all the cells remained viable, but were non-culturable on routine laboratory media, as had been observed for e. coli [ ] . this result was documented for at least days after inoculation. the apparent 'dormancy' of these enteric bacteria, possibly arising from depletion of available nutrients, compromises the use of e. coli and related coliforms as indicators of fecal pollution in fresh and salt waters, and renders highly questionable the phenomenon of 'dieoff' or genuine mortality of these bacteria. finding an appropriate indicator for the presence of enteric viruses, i.e., poliovirus, coxsackievirus a, coxsackievirus b, and echovirus [ , ] , in sea water, is a vexing problem, especially for those responsible for regulating the use of sewage-contamined sites, including fresh, estuarine, and marine water and sediment, and shellfish harvested from these waters. fecal coliforms and total coliforms have been used as indicators of contamination by domestic sewage, and therefore of the risk associated with exposure to bacterial and viral pathogens. however, in fresh water and waste water effluents [ ] [ ] [ ] [ ] [ ] [ ] [ ] , marine waters [ , , [ ] [ ] [ ] , and in shellfish meats [ , , ] , the total coliform and fecal coliform counts cannot be used to indicate the presence of enteroviruses. the lack of association of these indicators with enteric viruses is due to the fact that the latter survive longer in the 'recoverable' stage than fecal coliforms in fresh water [ , . ] and marine water [ ] , resulting in continuously changing ratios of viruses to the fecal indicators. however, goyal et al. [ ] observed a positive correlation between the concentration of virus in sediment and total coliform concentration. although de flora et al. [ ] found significantly larger numbers of viruses and bacteria in surface waters than in bottom water or sediment from polluted coastal waters, they also concluded that sediment was a transient reservoir for viruses and that survival of viruses was longer in some sediments than in sea water. in open ocean water, viruses persist longer when associated with, or attached to, solids, than when not associated with solids [ ] . viruses also survive longer in sediment than in overlying water [ ] , resulting in greater concentrations of enteric viruses recorded as being present in sediment than in water samples [ ] . since adsorbed viruses remain infectious [ ] , viruses in sediment and resuspended sediment are potential health hazards. most viruses also survive chlorination better than bacteria. grabow et al. [ ] reported that hepatitis a virus was more sensitive to chlorination than mycobacterium fortuitum, coliphage v , and poliovirus type , but more resistant than e. coil, streptococcus faecalis, coliphage ms , reovirus type , and rotavirus sa . total and fecal coliforms are also destroyed by chlorine more quickly than viruses [ ] . cold temperatures and factors associated with gross pollution appear to prolong virus survival in rivers [ ] . since fecal streptococci and enterococci survive longer than fecal cofiforms, in both fresh water and sea water, fecal streptococci and enterococci have been suggested as better indicators of enterovirus contamination in fiver water [ , ] , relatively less polluted waters [ , ] , and coastal recreation water [ ] . however, the viability in virus titers recorded for sediment and sea water samples is less than that of any of the bacterial indicators examined [ ] . isolation of enteric viruses when the number of indicator bacteria was extremely low has been reported for sediment samples [ ] , marine water [ ] [ ] [ ] , ] , and ground water [ ] . it is important to point out the disturbing finding of gerba et al. [ ] that enteroviruses can be present about % of the time in approved recreational and shellfish harvesting waters, approval being based on total or fecal coliform standards. although the number of total coliforms was reported to be correlated significantly with the number of viruses present in water samples, the amount of variation associated with the indicator was such that it could not be considered a good predictor. a positive correlation between coliforms and enteric viruses was found for ocean waters, but only for samples collected close to a sewage outfall. at further distances from the outfall, viruses could be isolated, even in the absence of fecal indicator bacteria [ ] . vaughn et al. [ ] found little difference in the viral quality of open and closed shellfish waters. furthermore, echovirus, the vaccine strain of poliovirus, and other, unidentified viruses, could be isolated from clams collected from both areas [ ] . contamination of shellfish with enteric viruses has been well documented, and the subject has been reviewed extensively by several investigators [ ] [ ] [ ] . enteric viruses have been isolated from shellfish collected from approved shellfishing waters. for example, fugate et al. [ ] reported the isolation of echovirus type , and poliovirus types and , from oysters collected in approved waters off the louisiana and texas coast. the coliform mpn of water and coliform mpn, e. coli mpn, and aerobic plate count of the oysters, thus, were not indicative of the presence of viruses [ ] . no correlation was found between fecal coliforms in water and the number of viruses isolated from oysters collected from approved and prohibited oyster harvesting sites in coastal mississippi [ , ] . rather than using fecal coliforms as indicators, it was proposed that a virus, such as poliovirus type , could be helpful, and that concentrations of a viral or bacterial indicator in the sediment might also be valuable as a predictor of health risk from viral contamination. similarly, goyal et al. [ ] found no significant correlation between virus concentration in oysters harvested from approved shellfish sites and the bacteriological quality of water and shellfish. the relationship between risk of viral disease and presence of viruses in water is not well known. the fact that a small number of viruses constitutes an infective dose has prompted the recommendation of less than one viral infectious unit per gallons ( . ) of water as acceptable [ ] [ ] . methods for the detection of viruses in water are still being improved, and recoveries are low. therefore, the detection of one virion indicates the presence of many more viruses which go undetected. sobsey and glass [ ] have developed filtration methods which have an average recovery efficiency for poliovirus from tap water of %, ranging between - %. in a comparison of positively charged microporous filters with conventional standard methods, i.e., negatively charged filters, for the recovery of enteric viruses from tap water, conventional methods averaged less than % recovery efficiency, whereas the new methods averaged % and . %, for -and -stage protocols [ ] . sobsey et al. [ ] have also devised methods for recovery of viruses from shellfish, with virus recovery efficiencies ave'raging %. but, as has been noted, epidemioiogical studies are needed for primary contact and shellfish harvesting waters [ , , , ] . to date, the evidence available suggests that the presence of viruses in shellfish can be linked to viral gastroenteritis and hepatitis outbreaks. enteric viral disease outbreaks, traced to swimming in polluted waters or eating contamined shellfish, have made it clear that coliform standards are not sufficient for monitoring the health hazards posed by the presence of viruses in sewage or the presence of viral pathogens in freshwater, marine waters, or in shellfish. viruses which may be transmitted by water, and are currently of interest, include enteroviruses, adenoviruses, reoviruses, hepatitis a virus, rotaviruses, coronaviruses, and the norwalkvirus [ , ] . total and fecal coliforms, e. coil, enterococci, fecal streptococci and s. faecalis do not survive as long as the viruses employed in studies accomplished to date, and therefore cannot be relied upon to be present and recoverable when enteric viruses are also present. coliforms can be a useful indicator for very recent contamination, but clearly not for contamination after a prolonged period of time after occurrence. fecal streptococci and enterococci, which survive longer in seawater than coliforms, but have not been studied as thoroughly as the coliforms, have been proposed as indicators for the presence of enteric viruses [ , ] , but, as stated above, cannot be relied upon to indicate the presence of viruses. viral indicators, suggested for waste-water, include sabin vaccine poliovirus [ , [ ] [ ] [ ] , cyanophage [ , ] , and coliphage [ , ] . the difficulties associated with the use of a virus as an indicator are many, the most important of which are: no single human virus is always present in every sewage-impacted area; viruses are often present only in relatively small numbers; and better methods for virus concentration are badly needed [ ] . total virus count was recommended as a method preferable to enumeration of poliovirus for those countries with high enterovirus infection rates [ ] . katzeneison and kedmi [ ] found that poliovirus was absent in about % of waste-water samples tested, although the total virus concentrations were high. they also recommended that a system be developed to isolate and identify total viruses in water. smedberg and cannon [ ] proposed the use of lpp-cyanophages, i.e., viruses of blue-green algae of the general lyngbya, phoridium and plectonema [ ] as indicators of the presence of human enteric viruses and reliability of disinfection for treated wastewater. cyanophages can be found throughout the year in wastewater. furthermore, they are more resistant to chlorine than coliforms, and can be correlated with the number of fecal coliforms present in wastewater [ , ] . coliphages have also been proposed as indicators [ , ] . correlations between the number of fecal bacterial pathogens and indicator bacteria and their respective bacteriophages in fresh and marine water have been reported [ ] . the concentration of coliphages was reported to be at least as high as the number of enteroviruses in waste-water and other waters [ ] . in estuarine waters, the survival time of coliphages is comparable to that of enteric viruses [ ] . coliphages are more resistant to chlorine than enteroviruses and survive longer in sand columns than poliovirus type [ ] . a rapid test for coliphages, as a water quality indicator for bacteria, was developed for fresh water [ ] . using this test, a linear relationship for the number of fecal or total coliforms with coliphages has been reported [ ] . the value of coliphages has also been investigated, with respect to indication of enteric viruses in shellfish and shellfish-harvesting waters [ ] . as a result of their studies, vaughn and metcalf [ ] recommended the use of coliphages as an indicator for raw sewage and treated effluents, but not for estuarine sheufishing waters, because coliphages were found to increase in number by replication in estuarine waters. furthermore, they were accumulated by oysters to a greater extent than enteric viruses, and showed shifts in dominant type over time [ ] . thus, the presence of coliphages was concluded not to be related to the presence of enteric viruses in estuarine water [ ] . in freshly shucked oysters contaminated with poliovirus and stored at °c, the virus persisted longer than days [ ] . in fact, viruses in contaminated oysters can survive processing, refrigeration, and freezing [ ] . in addition to bivalves, other marine invertebrates, including crabs, must be suspect agents in the transmission to man of enteric viruses present in polluted waters. even crabs caught in unpolluted waters may be contaminated with viruses accumulated when the crabs were exposed to polluted waters, either by swimming and/or feeding on contaminated shellfish in polluted waters [ ] . in laboratory studies, digirolamo et al. [ ] found that crabs concentrated high titers of poliovirus from contaminated mussels and filtered sea water that had been artificially seeded. in similar experiments, polioviruses were isolated from all parts of crabs, being present in highest concentrations in the hemolymph and digestive tract of the animal [ ] . poliovirus survived for up to days in crabs held at °c. recovery of poliovirus type , simian rotavirus sa , and echovirus type was reduced by . % in crabs after they had been boiled for rain [ ] . as the human populations located along urban coastal areas have increased, so too has the amount of material discarded into the ocean. it is now well known that larger concentrations of bacteria are found near the mouths of rivers and bays in highly populated areas and other coastal regions than in the open ocean. to protect bathers from waterborne enteric diseases acquired by swimming in coastal waters, some indication of the extent and amount of fecal pollution is needed. epidemiological studies are valuable for determining whether or not swimming in sewage-polluted waters poses a health risk, and, also, what organism or organisms are best suited to predict such health hazards. in addition to assessing the risk of enteric viral and bacterial diseases such as infectious hepatitis, typhoid fever, shigellosis, and cholera, the risk of contracting diseases affecting the eyes, ears, nose, throat and skin, as well as wound infections, is a matter of serious concern. organisms which cause the latter group of diseases are not necessarily sewage-associated, and may not be signalled by the traditional drinking water indicator bacteria [ ] . an important aspect of epidemiological studies is the determination of the infectious dose for a particular pathogen, and its virulence. in a review of water-borne pathogens, geldreich [ ] focused on enteric pathogens, including salmonella, shigella and leptospira (freshwater) species, enteropathogenic e. coli, francisella tularensis, vibrio cholerae, mycobacterium tuberculosis, hepatitis a virus, poliovirus, other human enteric viruses and parasitic protozoa and worms. not all of these organisms are necessarily of concern for the marine environment. in addition to the organisms listed by geldreich [ ] , moore [ ] cites as being communicable, otitis media, sinusitis, and gastroenteritis of unknown etiology, but dismisses certain organisms as irrelevant for coastal bathing waters, including staphylococcus aureus, clostridia, mycobacterium spp. and candida spp., based on their biology. however, staphylococci, especially staphylococcus hominis and staphylococcus epidermidis, and including s. aureus, have recently been isolated from coastal, offshore, and deep ocean sites [ , ] . also in conflict with moore [ ] are the frequent isolations of clostridium botulinum and clostridium perfringens from marine sediment [ ] [ ] [ ] . according to moore [ ] , typhoid fever is of concern at the present time only in areas of high enteric morbidity. wound and enteric infections caused by bacteria of the vibrionaceae and skin rashes caused by trematodes, acquired from swimming in estuarine and ocean waters, were also included in a review of recreational water use published by cabelli [ ] . most of the epidemiological studies of bathing water quality were done in the s in the u.s.a. and u.k. notable of these are the reports of stevenson [ ] in the united states and moore [ ] in britain. indicator organisms used to compare to the incidence of disease in both the american and british studies were total coliforms. however, the authors of these reports reached different conclusions. stevenson [ ] reviewed results of surveys of swimming pools and beaches on the ohio river, lake michigan, and long island sound, and concluded that a positive correlation existed for fresh waters between high total coliform concentrations and illness in swimmers, i.e., more swimmers than non-swimmers became ill, with more than one-half of the illnesses being ear, eye, nose, and throat diseases, one-fifth gastrointestinal, and the remainder skin or other types of infections. on the other hand, moore [ ] , who reported on an epidemiological study of bathing waters carried out on the coasts of england and wales, found no evidence to support the conclusion that sewage-contaminated water was a significant cause of illness. it should be pointed out, however, that isolation of salmonella spp. from seawater was interpreted as being evidence of the wide dissemination of this organism in the population at large, rather than a risk of infection for bathers. in a later publication, moore [ ] concluded that the environmental hazards of swimming are difficult to assess and require statistical proof of association, causation, and specificity of disease with cause of the disease. two other factors important in epidemiological studies were cited, these being the incubation time of the pathogen, and dose response of morbidity related to frequency of swimming [ ] . the most recent report of results of an epidemiological study of coastal bathing waters was of a study conducted over a -year span in the s at beaches in new york city, lake pontchartrain, la and boston, ma [ ] . a direct linear relationship was observed between swimming-associated gastrointestinal disease and water quality. other types of disease were not reported. bacteria enumerated in the waters included enterococci, total and fecal coliforms, e. coli, klebsiella sp., enterobacter sp., citrobacter sp., clostridium perfringens, pseudomonas aeruginosa, aeromonas hydrophila, and vibrio parahaemolyticus. the concentration of enterococci was strongly correlated with gastrointestinal symptoms, while fecal and total coliforms were found to be poorly correlated. as reported in earlier studies in the u.s.a. by stevenson [ ] , swimmers showed a higher rate of symptoms than non-swimmers [ ] , even in marginally polluted marine waters, the latter being indicated by the presence of enterococci. although total coliforms have been used as indicators of primary contact water quality, this approach has been discontinued in many areas in favor of the more fecal-specific fecal coliforms [ ] . enterococci have been suggested as better than either the fecal coliforms or e. coli as an indicator of fecal pollution for bathing waters since the correlation of enterococci with gastrointestinal symptoms was highest [ , ] . a wide array of indicator organisms has been proposed for brackish, estuarine, and coastal waters. it is clear that no single indicator, index or reference organism exists for titering public health safety of these waters. with the development of immuno-epifluorescent microscopy methods for direct detection of specific human pathogens, and considering the 'viable but non-eulturable' stage which allochthonous human pathogens undergo when released to natural bodies of water, it is concluded that the direct viable 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water-borne pathogens the present status of diseases connected with marine pollution a note on the isolation and numeration of gram-positive cocci from marine and estuarine waters numerical taxonomy of staphylococci isolated from the marine environment clostridium perfringens in the environment ciostridium perfringens as a water pollution indicator distribution of clostridium botulinum ) studies of bathing water quality and health sewage contamination of coastal bathing waters in england and wales: a bacteriological and epidemiological study a marine recreational water quality criterion consistent with indicator concepts and risk analysis key: cord- - l m yh authors: ishihara, masayuki; hata, yuuki; hiruma, sumiyo; takayama, tomohiro; nakamura, shingo; sato, yoko; ando, naoko; fukuda, koichi; murakami, kaoru; yokoe, hidetaka title: safety of concentrated bioshell calcium oxide water application for surface and skin disinfections against pathogenic microbes date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: l m yh immediately post-production, commercially available bioshell calcium oxide (biscao) water is colorless, transparent, and strongly alkaline (ph . ), and is known to possess deodorizing properties and broad microbicidal activity. however, biscao water may represent a serious safety risk to the living body, given the strong alkalinity. this study aimed to investigate the safety of biscao water for use as an antiseptic/disinfectant despite concerns regarding its high alkalinity. the change over time in ph of biscao water was measured during air contact (stirring biscao water in ambient air). when sprayed on metal, plastic, wood piece, paper, and skin surfaces, the ph of biscao water decreased rapidly, providing a white powder coating upon drying. scanning electron microscopy images, energy dispersive x-ray elemental mapping, and x-ray diffractograms showed that the dried powder residues of biscao water were composed primarily of calcium carbonate. these results suggested that biscao water is a potent reagent that may overcome the obstacles of being strongly alkaline, making this material appropriate for use in disinfection against pathogenic microbes. the recent epidemic of coronavirus disease , caused by a newly discovered coronavirus sars-cov- , is a worldwide crisis [ ] [ ] [ ] . some antiseptics/disinfectants, such as ethanol and naclo, show significant activity with broad microbicidal and antiviral activities, notably, activity against sars-cov- results from disruption of the viral envelope. however, such antiseptics/disinfectants are cytotoxic to cellular and organic components [ ] [ ] [ ] and require high concentrations for disinfection activity [ ] [ ] [ ] . furthermore, chlorine-derived disinfectants are ineffective in the presence of organic compounds and may generate compounds harmful to the environment and living tissue [ ] [ ] [ ] . therefore, novel disinfectants that can decrease the bacterial and viral bioburden without harmful side effects and environmental disruption are still desired for environmental hygiene and public health. biscao water was freeze-dried for sem observation and energy dispersive x-ray (edx) elemental mapping of particles contained. the images and edx spectra are shown in figure . the edx elemental mapping showed that the particles contained in biscao water were composed of oxygen, carbon, and calcium. the dried white powder was insoluble in water, and the ph of the supernatant of a suspension of this powder was below . these results suggested that the observed particles were generated by an interaction between ca + ions in biscao water and co in the air. furthermore, x-ray diffraction analysis revealed that the dried powder obtained from biscao water consisted primarily of calcium carbonate (caco ) (figure ). molecules , , x of biscao water was freeze-dried for sem observation and energy dispersive x-ray (edx) elemental mapping of particles contained. the images and edx spectra are shown in figure . the edx elemental mapping showed that the particles contained in biscao water were composed of oxygen, carbon, and calcium. the dried white powder was insoluble in water, and the ph of the supernatant of a suspension of this powder was below . these results suggested that the observed particles were generated by an interaction between ca + ions in biscao water and co in the air. furthermore, x-ray diffraction analysis revealed that the dried powder obtained from biscao water consisted primarily of calcium carbonate (caco ) (figure ). scanning electron microscopy (sem) and energy dispersive x-ray (edx) elemental mapping images and edx spectra of freeze-dried bioshell calcium oxide (biscao) water. two different regions of the sample were analyzed (left and right). yellow, red, and green pictures show the presence of carbon, oxygen, and calcium, respectively, by edx elemental mapping. when biscao water was exposed to air with stirring for h, the liquid became cloudy. furthermore, the ph decreased to . and . after and h, respectively, as shown in figure . following drying, the resulting white powder was insoluble; the supernatant of a suspension of this white powder had a ph of . . on the other hand, the ph of a biscao suspension ( . wt.% biscao) [ , , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , and colloidal dispersion ( . wt.% biscao + . wt.% pp) [ ] did not change during h of stirring in open air. these results suggest that the insoluble powder was caco generated by an interaction between ca + ions in biscao water and co in the air, and that the biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the alkaline ph. when biscao water was exposed to air with stirring for h, the liquid became cloudy. furthermore, the ph decreased to . and . after and h, respectively, as shown in figure . following drying, the resulting white powder was insoluble; the supernatant of a suspension of this white powder had a ph of . . on the other hand, the ph of a biscao suspension ( . wt.% biscao) [ , , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , and colloidal dispersion ( . wt.% biscao + . wt.% pp) [ ] did not change during h of stirring in open air. these results suggest that the insoluble powder was caco generated by an interaction between ca + ions in biscao water and co in the air, and that the biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the alkaline ph. when biscao water was exposed to air with stirring for h, the liquid became cloudy. furthermore, the ph decreased to . and . after and h, respectively, as shown in figure . following drying, the resulting white powder was insoluble; the supernatant of a suspension of this white powder had a ph of . . on the other hand, the ph of a biscao suspension ( . wt.% biscao) [ , , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , and colloidal dispersion ( . wt.% biscao + . wt.% pp) [ ] did not change during h of stirring in open air. these results suggest that the insoluble powder was caco generated by an interaction between ca + ions in biscao water and co in the air, and that the biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the alkaline ph. biscao water was sprayed onto the surfaces of a plastic plate, a steel plate, a piece of wood, and tissue paper (kimwipe), and the ph of each wet surface was measured over the course of the next min (figure ). for all tested surfaces, the ph immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the ph of the surfaces fell below after min, and continued falling to less than ph and after and min, respectively. molecules , , x of biscao water was sprayed onto the surfaces of a plastic plate, a steel plate, a piece of wood, and tissue paper (kimwipe), and the ph of each wet surface was measured over the course of the next min (figure ). for all tested surfaces, the ph immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the ph of the surfaces fell below after min, and continued falling to less than ph and after and min, respectively. in a parallel experiment, biscao water (~ ml) was sprayed onto the back skin of hairless rat, which then were rubbed lightly, and the ph of each wet skin was measured over the course of the following min ( figure ). the ph of the wet skin immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the phs on the backs subsequently fell to ph . after min, ph . after min, and ph . after min. we confirmed that none of the skin exhibited chapping or inflammation from use of biscao water. in a parallel experiment, biscao water (~ ml) was sprayed onto the back skin of hairless rat, which then were rubbed lightly, and the ph of each wet skin was measured over the course of the following min ( figure ). the ph of the wet skin immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the phs on the backs subsequently fell to ph . after min, ph . after min, and ph . after min. we confirmed that none of the skin exhibited chapping or inflammation from use of biscao water. molecules , , x of biscao water was sprayed onto the surfaces of a plastic plate, a steel plate, a piece of wood, and tissue paper (kimwipe), and the ph of each wet surface was measured over the course of the next min (figure ). for all tested surfaces, the ph immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the ph of the surfaces fell below after min, and continued falling to less than ph and after and min, respectively. in a parallel experiment, biscao water (~ ml) was sprayed onto the back skin of hairless rat, which then were rubbed lightly, and the ph of each wet skin was measured over the course of the following min ( figure ). the ph of the wet skin immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the phs on the backs subsequently fell to ph . after min, ph . after min, and ph . after min. we confirmed that none of the skin exhibited chapping or inflammation from use of biscao water. during incubation of bath tub water with % dulbecco's modified eagle's medium (dmem) and . wt.% bovine serum albumin (bsa) at • c for h the tc and cf values increased from ± colony-forming unit (cfu)/ml and ± cfu/ml (respectively) to . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. the cfu/ml for tc and cf following treatment for min with undiluted (final two-fold diluted) and two-fold diluted (final four-fold diluted) biscao water and . and . wt.% (final . and . wt.%, respectively) of biscao suspension, dispersion, and colloidal dispersion were below the detection limit (< cfu/ml), whereas - and - , cfu/ml of both tc and cf remained viable following treatment with . wt.% (final . wt.%) and . wt.% (final . wt.%) of biscao suspension, dispersion, and colloidal dispersion, respectively ( figure ). in contrast, around , cfu/ml of tc and cf remained viable, even following treatment with undiluted (final -fold diluted) biscao water, when the reagent had been subjected to h of stirring in ambient air. molecules , , x of during incubation of bath tub water with % dulbecco's modified eagle's medium (dmem) and . wt.% bovine serum albumin (bsa) at °c for h the tc and cf values increased from ± colony-forming unit (cfu)/ml and ± cfu/ml (respectively) to . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. the cfu/ml for tc and cf following treatment for min with undiluted (final two-fold diluted) and two-fold diluted (final four-fold diluted) biscao water and . and . wt.% (final . and . wt.%, respectively) of biscao suspension, dispersion, and colloidal dispersion were below the detection limit (< cfu/ml), whereas - and - , cfu/ml of both tc and cf remained viable following treatment with . wt.% (final . wt.%) and . wt.% (final . wt.%) of biscao suspension, dispersion, and colloidal dispersion, respectively ( figure ). in contrast, around , cfu/ml of tc and cf remained viable, even following treatment with undiluted (final -fold diluted) biscao water, when the reagent had been subjected to h of stirring in ambient air. the cfu/ml for tc and cf released from the contaminated wood pieces was . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. although the cfu/ml for tc and cf following treatment with undiluted (final two-fold diluted) biscao water and . (final . wt.%) biscao suspension, dispersion, and colloidal dispersion exhibited high disinfection activities (> log decreases in cfu/ml), a small part of tc (> cfu/ml) and cf (> cfu/ml) remained viable (figure ) . the cfu/ml for tc and cf following treatment with two-fold diluted (final four-fold diluted) biscao water . wt.% (final . wt.%,) biscao suspension, dispersion, and colloidal dispersion exhibited > log decreases, while - cfu/ml of tc and - cfu/ml of cf remained viable. in contrast, biscao water that had been subjected to -h stirring in ambient air exhibited weaker sterilization activity against both tc and cf, with tc (about , cfu/ml) and cf (about cfu/ml) remaining viable even following treatment even with undiluted (final two-fold diluted) biscao water with h stirring (figure ) . on the other hand, in biscao suspension, dispersion, and colloidal dispersion the ph did not decrease at all with stirring for h and their microbicidal activities against tc and cf also did not decrease (data not shown). the cfu/ml for tc and cf released from the contaminated wood pieces was . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. although the cfu/ml for tc and cf following treatment with undiluted (final two-fold diluted) biscao water and . (final . wt.%) biscao suspension, dispersion, and colloidal dispersion exhibited high disinfection activities (> log decreases in cfu/ml), a small part of tc (> cfu/ml) and cf (> cfu/ml) remained viable (figure ) . the cfu/ml for tc and cf following treatment with two-fold diluted (final four-fold diluted) biscao water . wt.% (final . wt.%,) biscao suspension, dispersion, and colloidal dispersion exhibited > log decreases, while - cfu/ml of tc and - cfu/ml of cf remained viable. in contrast, biscao water that had been subjected to -h stirring in ambient air exhibited weaker sterilization activity against both tc and cf, with tc (about , cfu/ml) and cf (about cfu/ml) remaining viable even following treatment even with undiluted (final twofold diluted) biscao water with h stirring (figure ) . on the other hand, in biscao suspension, dispersion, and colloidal dispersion the ph did not decrease at all with stirring for h and their microbicidal activities against tc and cf also did not decrease (data not shown). proper action plans for cleaning and disinfection play a critical role for minimizing the spread of infectious diseases such as covid- during an outbreak. an effective action plan should outline what should be cleaned, the frequency of cleaning, the proper materials and techniques for disinfection, and the training of healthcare workers. the action plans using proper materials and techniques will support a facility's pandemic response [ ] [ ] [ ] , ] . biscao water, which is a colorless and transparent disinfectant with a ph of . , can be sprayed and dried on smooth metal or plastic surfaces, providing a white powder coating. previously, sem observation for the dried powder obtained from biscao water showed the presence of agglomerated microparticles ( - µm) [ ] . in addition, cryo-sem observation has indicated that aggregates ( - nm in size) of nanoparticles ( - nm) were contained in biscao water [ ] . in the present work, elemental mapping of particles from biscao water revealed that the particles in the cloudy, air-exposed reagent, and the dried powder were composed of oxygen, carbon, and calcium ( figure ). the dried white powder obtained from biscao water was insoluble in water, and the ph of the resulting supernatant was less than . furthermore, x-ray diffraction analysis confirmed that the dried powder recovered from biscao water consisted primarily of caco (figure ). when biscao water was exposed to air with stirring, the liquid became cloudy after h. furthermore, the ph decreased to approximately . and . after and h of stirring, respectively ( figure ). in contrast, the ph of a biscao suspension, dispersion, and colloidal dispersion did not decrease when treated similarly. these results suggest that the insoluble powder from biscao water is caco , which presumably is generated by the interaction between ca + ions in biscao water and co in the air, and that a biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the elevated ph. biscao water after h stirring, which had decreased ph and contained caco , exhibited much less microbicidal activities than did fresh biscao water (figures and ) . we have previously reported that biscao water was a potent reagent with excellent deodorization and disinfection activities against pathogenic bacteria and viruses including both enveloped and nonenveloped viruses due to the strong alkaline [ ] . however, there has been serious concern about the safety of biscao water for use on living tissues, given that this reagent has a ph exceeding . . this study showed that biscao water, when biscao water was sprayed onto a variety of surfaces (plastic plate, steel plate, wood, and paper), gave an initial ph of . ± . , a value lower than the original value (ph . ) for biscao water, with the ph on the surfaces subsequently and rapidly decreasing to below , , and after , , and min, respectively ( figure ) . thus, the high ph of this fresh reagent rapidly decreases on surfaces and skin due to the generation of caco that lack obstacles following the interaction between ca + ions in biscao water and co in the air. in fact, caco powder is used as a gastric antacid [ ] , an anti-erosive agent [ ] , and a plant fertilizer [ ] . we infer that the ingestion of a small amount of biscao water would not cause problem if swallowed or inhaled, given that caco powder will be converted to a safe and soluble compound (calcium bicarbonate (ca(hco ) )) upon further interaction between caco and co . these results in this study suggest that spraying biscao water onto the surfaces of various materials is safe cleaning procedure in spite of its strong initial alkalinity in addition to the strong disinfectant activity. furthermore, we have already reported that cleansing p. aeruginosa-infected wounds with transparent supernatant liquids of . wt.% biscao suspension for days enhanced removal of the bioburden and wound repair without any complications such as acute inflammation, abscess formation, or seroma accumulation [ ] . similarly, the ph of biscao water sprayed onto hairless rat skin fell to ph . after min and ph . after min of application ( figure ) without apparent harmful effects such as rough skin. thus, biscao water appears to be friendly to skin. nevertheless, it is necessary to perform the safety study for biscao water to apply onto human such as hand sterilization and mouth cleaning with official ethics committee approval on use of human subjects. the world health organization (who) recommends "to ensure that environmental cleaning and disinfection procedures are followed consistently and correctly. thoroughly cleaning environmental surfaces with water and detergent and applying commonly used hospital-level disinfectants (such as sodium hypochlorite) are effective and sufficient procedures" [ ] . although some antiseptics/disinfectants, including ethanol and naclo, exhibit significant activity against sars-cov- by damaging the virus envelope, they are cytotoxic to cellular and organic components and require high concentrations for sufficient activity [ ] [ ] [ ] . furthermore, the presence of organic materials significantly diminishes the activity of chlorine-derived compounds [ , ] . therefore, novel antiseptics/disinfectants without harmful side effects or environmental disruption are still desired for environmental hygiene and public health. we anticipate that biscao water with attractive characteristics, including its apparent safety and virucidal activity against enveloped viruses, may be a trump card against respiratory viruses such as sars-cov- , even though further studies are required to validate the activity of biscao water against critical pathogens, including the coronavirus. scallop shell powder that had been heated at • c for h (average biscao particle diameter of µm) was purchased from plus lab corp., kanagawa, japan. according to the manufacturer, the cao concentration in all biscao preparations exceeded %. na hpo ( - ) and na-polypo (pp) ( - ) were purchased from fuji film wako pure chemical corp., osaka, japan. biscao was added to pure water followed by rotary mixing to generate biscao suspensions, to which a solution of % na hpo or % pp compared with biscao was added to prepare biscao dispersions or biscao colloidal dispersions, respectively [ , ] . biscao water was purchased from plus lab corp. according the manufacturer, biscao water was prepared by adding g of biscao to ml chilled (< • c) clean water; the combination then was gently mixed and allowed to stand for min. the resulting supernatant ( ml) was decanted to a l water tank. another chilled ml of clean water was gently added to the remaining biscao precipitate; again the combination was gently mixed and allowed to stand for min, and the supernatant was decanted into the collection tank. this process was repeated for a total of a hundred times. the resulting biscao water (total l) was obtained commercially from plus lab corp. sem observation and edx elemental mapping of freeze-dried powder of biscao water were performed by outsourcing to jeol, ltd., tokyo, japan. the observations were conducted using a jeol jsm- f microscope (jeol ltd., tokyo, japan) equipped with a jed- edx analysis system at an accelerating voltage of kv at room temperature. the content of dried powder derived from biscao water was determined using an x-ray diffractometer system (phillips x'pert-pro; phillips japan, ltd., japan). this analysis was performed at the kanagawa institute of industrial science and technology. each aliquot ( ml) of biscao water, biscao suspension ( . wt.% biscao) [ , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , or colloidal dispersion ( . wt.% biscao + . wt.% pp)) [ ] was dispensed into a -ml beaker and actively stirring using digital stirrer/hotplate (corning japan inc., tokyo, japan) with stirring bar ( mm in diameter × mm) (as one corp. osaka, japan) for h at room temperature in ambient air. the ph of each biscao formulation was measured (using a bench ph meter; f- , horiba, ltd., kyoto, japan) at , , , , , , and h during the process. when an aliquot (approximately ml) of biscao water was sprayed onto a defined surface (about cm × cm) of a culture plate and a steel plate, generations of large and small droplets (diameter: - mm, thickness: . - mm) were observed. the ph of large droplet was measured every min during min using diagonally placed micro tough electrode ( s- d; horiba, ltd.) attached to a bench ph meter (laqua ph meter, f- , horiba, ltd.). similarly, approximately ml of biscao water were sprayed onto the back skin of hairless rats (male, - g) from japan slc inc., shizuoka, japan and maintained under appropriate condition (i.e., • c and % humidity). in the experiment, the rats were placed under general anesthesia by intraperitoneal injection of pentobarbital sodium (dainippon sumitomo parma co., ltd., osaka, japan) [ , , ] . the ph of biscao water on the back skin of the rats was measured every min during min using diagonally placed micro tough electrode attached to the ph bench meter. all animal experiments were approved by the national defense medical college, tokorozawa, saitama, japan, and carried out following the relevant guidelines for animal experimentation (approval number, , / / ). on the other hand, the spraying approximately ml of biscao water onto surface (about cm × cm) of wooden pieces and paper sheets (kimwipe, nippon paper crecia, co., ltd., tokyo, japan) were permeated into them and did not generate droplets. however, the ph of their wet surface could be measured using diagonally placed micro tough electrode attached to the ph bench meter for min. we investigated the microbicidal efficacy of variously dilutions of biscao water and biscao water with -h stirring, as well as various concentrations of biscao suspension, dispersion, and colloidal dispersion. these reagents were tested against a contaminated suspension comprising normal bacterial flora (cf and tc), which was prepared by incubating the leftover bath water with % dmem and . wt% bsa at • c for h [ , , ] . equal volumes of each test sample and the contaminated suspension were mixed well and incubated at room temperature for min, and then the density of cfu/ml per sample was determined. for the disinfection assay of contaminated wood pieces, a wood piece was added to ml of each disinfectant, rinsed gently for min, and then tc and cf were released from the contaminated wood piece in ml of clean sterile water by vigorous vortexing for min. to evaluate cfu, ml of each mixture was gently added to individual petri dishes with pre-aliquoted portions of simple and easy dry medium for tc or cf (nissui pharmaceutical co., ltd., tokyo, japan) [ , , , ] , followed by incubation for h in a • c incubator (a ; ikuta sangyo co., ltd., ueda, nagano, japan). plating and counting experiments were conducted as a set of technical replicates (n = ). biscao water is colorless and transparent and has a ph of . . this reagent has higher deodorization and disinfection activities than do other microbicidal reagents such as ethanol, naclo, and povidone iodine [ , ] . however, concerns have been raised concerning the safety of biscao water when applied to the living body, given the strong alkalinity of this reagent. the present study showed that the high initial ph of biscao water following application to various surfaces and skin of hairless rat, rapidly decreases for areas that lack obstacles with the generation of caco as a result of the interaction between ca + in biscao water and co in the air. thus, the characteristics of biscao water, including both its safety and microbicidal activity, may be valuable for limiting the spread of various pathogenic microbes and viruses. persistence of coronaviruses on 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inactivation of avian influenza virus, newcastle disease virus and goose parvovirus using solution of nano-sized scallop shell powder sporicidal kinetics of bacillus subtilis spores by heated scallop shell powder comparison of antifungal activities of scallop shell, oyster shell and their pyrolyzed products disinfection treatment of heated scallop-shell powder on biofilm of escherichia coli atcc surrogated for e. coli o :h ability of heated scallop-shell powder to disinfect staphylococcus aureus biofilm heated scallop-shell powder treatment for deactivation and removal of listeria sp. biofilm formed at a low temperature comparison of various disinfectants on bactericidal activity under organic matter contaminated environments skin cleansing technique with disinfectant using improved high-velocity steam-air micromist jet spray development of antimicrobial biomaterials produced from chitin-nanofiber sheet/silver nanoparticle composites adsorption of silver nanoparticles onto different surface structures of chitin/chitosan and correlations with antimicrobial activities synthesis and application of silver nanoparticles (ag nps) for the prevention of infection in healthcare workers healing of pseudomonas aeruginosa-infected wounds in diabetic db/db mice by weakly acidic hypochlorous acid cleansing and silver nanoparticle/chitin-nanofiber sheet covering bioshell calcium oxide (biscao) for cleansing and healing of pseudomonas aeruginosa-infected wounds in hairless rats bioshell calcium oxide (biscao) ointment for the disinfection and healing of pseudomonas aeruginosa-infected wounds in hairless rats cleaning technique using high-velocity steam-air micromist jet spray preparation and application of bioshell calcium oxide (biscao) nanoparticles-dispersions with bactericidal activity application of colloidal dispersions of bioshell calcium oxide (biscao) for disinfection concentrated bioshell calcium oxide (biscao) water kills pathogenic microbes: characterization and activity evaluation of calcium hydrogen carbonate mesoscopic crystals as a disinfectant for influenza a viruses inactivation of scrapie prions by the electrically charged disinfectant cac- absorption characteristics of novel compound calcium carbonate granules: effect of gastric acid deficiency and exogenous weak acids anti-erosive effect of calcium carbonate suspensions influence of precipitated calcium carbonate (caco ) on shiitake (lentinula edodes) yield and mushroom size this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank forte, inc. (www.forte-science.co.jp), for their english language editing services. the authors declare no conflict of interest. key: cord- -xgw o gd authors: innes, elisabeth a.; chalmers, rachel m.; wells, beth; pawlowic, mattie c. title: a one health approach to tackle cryptosporidiosis date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: xgw o gd cryptosporidiosis is a significant diarrhoeal disease in both people and animals across the world and is caused by several species of the protozoan parasite cryptosporidium. recent research has highlighted the longer-term consequences of the disease for malnourished children, involving growth stunting and cognitive deficits, and significant growth and production losses for livestock. there are no vaccines currently available to prevent the disease and few treatment options in either humans or animals, which has been a significant limiting factor in disease control to date. a one health approach to tackle zoonotic cryptosporidiosis looking at new advances in veterinary, public, and environmental health research may offer several advantages and new options to help control the disease. infection occurs through oral ingestion of the oocyst stage of the parasite from contaminated faeces, food, drink, and pasture (for grazing animals), and following ingestion, the sporozoites are released from the oocysts and invade and undergo asexual development in the epithelial cells of the gastrointestinal tract of the host. this is followed by a sexual phase of development resulting in the production of potentially genetically diverse oocysts that are shed fully infective in the faeces. oocysts may also 'hatch' before they are shed from the host, causing reinfection and exponential increases in parasite burden, leading to chronic infection, particularly in immunocompromised hosts [ ] . cryptosporidium parasites have a remarkable capacity to reproduce in the host. the rapid multiplication of the parasite in the gut cells causes tissue damage and destruction of the intestinal epithelial cells with stunting of the villi, reducing the absorptive surface of the gut, leading to malnutrition, dehydration, and diarrhoea [ ] . during acute infection, and for a week or so following clinical recovery, the infected host will shed very large numbers of infectious oocysts in faeces [ ] . work with neonatal calves has shown that one calf can shed around million infectious oocysts in faeces, which is a massive source of environmental contamination and poses an infection risk for other vulnerable hosts [ ] . cryptosporidium oocysts are microscopic (most species are - μm in diameter) and have a tough waxy wall composed of lipids and glycoproteins, which enables the parasite to survive a wide range of conditions, including temperatures from - °c to °c. this wall protects the parasite against many common disinfectants, which makes cryptosporidium hard to control on the farm, in drinking water, swimming pools, and in fresh produce [ , ] . not all cryptosporidium spp. in livestock are of public health significance, but the species of most zoonotic importance for human infection is c. parvum [ ] , which will be the focus for this review. livestock, in particular young calves, are very vulnerable to cryptosporidiosis and a recent modelling study estimated the global load of cryptosporidium parasites in livestock manure to be in the region of . × oocysts per year, with cattle being the predominant source [ ] . therefore, improving our understanding of environmental transmission routes of zoonotic cryptosporidium and oocyst survival is important in assessing and mitigating against disease risk and is essential for a one health approach to tackle human and animal cryptosporidiosis. currently, there are no effective vaccines to prevent cryptosporidiosis in humans or in livestock and there are few safe and effective therapeutic options available. cryptosporidiosis is a disease that is well placed for a collaborative interdisciplinary approach to tackle it effectively and previous authors have advocated the benefits of adopting a one health approach [ ] . this review will discuss progress made in this area, with greater emphasis on the benefits and opportunities from tackling the disease in livestock, and look at recent advances in scientific technologies that may lead to much needed prevention and control strategies. human infection is usually of the small bowel via the faecal-oral route, although there is growing evidence for respiratory involvement in some populations [ ] . gastrointestinal symptoms, glossary calcium-dependant protein kinase (cdpk ): the calcium-dependant protein kinase family are expanded in apicomplexan parasites and are plantlike and do not occur in animal cells, which makes them interesting targets for drug development. crispr/cas : a technology that enables genome editing by removing, adding, or altering sections of the dna sequence. disability adjusted life years: a measure of overall disease burden, expressed as the number of years lost to ill health, disability, or early death. finished water: water that has passed through a water treatment plant. gnotobiotic: an animal derived by aseptic caesarean section and reared in isolator conditions. gp : a -kda glycoprotein gene of cryptosporidium used as a target for molecular typing studies. hyperimmune colostrum: produced by immunisation of cows during pregnancy, resulting in high levels of specific antibodies in the colostrum, the first milk produced after birth. mesophilic: an organism that grows best in moderate temperature ( °c- °c). multilocus fragment typing (mlft): multilocus fragment typing uses tandem repeat units within the genome, often called micro-or minisatellites, which are amplified by pcr and the amplicons visualised by gel electrophoresis. oocyst: environmentally resistant transmissive life cycle stage of cryptosporidium spp. organoids: self-organised threedimensional tissue culture organ models derived from stem cells that show realistic micro-anatomy. pk/pd: pharmacokinetics (pk) describes the drug concentration in body fluids and pharmacodynamics (pd) describes the observed effect of the drug. potable: water that is safe to drink or to use in food preparation. rapid gravity filtration system: type of filter used in water purification. thermophilic: an organism that grows best in high temperatures ( - °c) . zoonotic: pertaining to a zoonosis, a disease that can be transmitted from animals to people. cryptosporidiosis is a significant diarrhoeal disease in both people and animals worldwide. young children and people with compromised immune systems are very vulnerable to disease and the importance of cryptosporidium in human health was first recognised during the aids epidemic in the s [ ] . from to the global prevalence of cryptosporidium in hiv/aids patients was . % [ ] . a large-scale epidemiology study involving children in sub-saharan africa and south east asia found that cryptosporidium was a major cause of severe diarrhoea in very young children and was the only gastrointestinal pathogen presenting a significant risk of death [ ] . cryptosporidium is also recognised as an important food-borne pathogen [ ] , being responsible for more than million cases of food-borne illness annually [ ] . burden of disease studies, focussed on acute illness, estimate . million disability adjusted life years lost in children under years [ ] , with longer term sequelae, including growth faltering and cognitive defects. disease impact studies in high-income countries found longer term impacts following acute infection involving persistent abdominal pain, myalgia/arthralgia and fatigue [ ] , irritable bowel syndrome [ ] , and a recent study has shown a strong association between cryptosporidium infection and human colon cancer [ ] . cryptosporidiosis is a major diarrhoeal disease in neonatal calves and other animals. in the cattle industry, production losses associated with the disease include death of the calf, costs incurred in the diagnosis, treatment and supportive therapies employed, and the extra costs of feed and husbandry for the animals to reach market weight [ ] . cattle infected with cryptosporidium and monitored from birth to days showed a correlation with infection and a lower live weight gain along with poorer production performance [ ] . long-term production impacts on growth and carcase weights following acute cryptosporidiosis in lambs was also reported on australian sheep farms [ ] . outbreaks of cryptosporidiosis associated with contaminated water supplies can result in significant economic and health impacts. the large waterborne outbreak in wisconsin affected people and was estimated to cost usd . million [ ] . in sweden it was estimated that sick leave days were attributed following a waterborne cryptosporidiosis outbreak where the attack rate was % of residents [ ] . detection of cryptosporidium oocysts in public water supplies often results in condemnation of supplies, public notices to boil water, and provision of bottled water. in a recent waterborne outbreak in ireland a boil water notice was put on for days, affecting over people and costing around eur million [ ] . including profuse watery diarrhoea, abdominal pain, and nausea, typically start between and days after ingestion of oocysts and arise from malabsorption and inflammation [ ] . some populations are especially vulnerable to cryptosporidium, and the parasite was included in the world health organization's neglected diseases initiative in [ ] . this was in recognition of the links to poverty, the disproportionate impact on young, malnourished children with subsequent growth and development impairment [ , ] , and the severe impact on immunocompromised people. long-term health consequences are also now recognised in people (box ). globally, cryptosporidiosis affects a large number of animal species, including the main livestock animals such as: cattle, sheep, goats, pigs, rabbits, horses, donkeys, water buffalo, camels, and poultry [ ] and zoonotic cryptosporidium spp. have also been reported in wildlife species, including rabbits [ ] , deer [ ] , wild mammals [ ] , and fish [ ] . infection with c. parvum is one of the most important causes of diarrhoeal disease in neonatal (b weeks old) calves in many countries worldwide [ ] . the parasite is the most frequently diagnosed cause of neonatal calf diarrhoea in the uk i and c. parvum also causes significant disease in lambs and goats [ ] . sheep and goat production is an important agricultural industry in europe, particularly in less favoured areas for agriculture, where they are an important source of meat and dairy products. it is estimated that % of the goat population is in developing countries, mainly in africa and asia, where they are an important source of meat, milk, fibre, and fertilizer and help to improve the livelihoods of the world's poorest communities [ ] . children often play an important role in tending grazing livestock in many countries and, due to their close proximity to the animals, are very likely to come into contact with cryptosporidium parasites. recent data from china has also shown the presence of c. parvum in yaks, which may pose a public health risk [ ] . the parasite causes disease in both dairy and beef herds and the prevalence of infection can be high in young calves, particularly in intensively reared management systems, with various clinical outcomes being reported, ranging from death of the calf to growth retardation and some animals with no obvious clinical signs [ ] . the disease can also cause long-term production impacts in livestock (box ). the minimal infectious dose for neonatal calves is very low, with oocysts causing infection and a correlation of a dose-dependent relationship between oocyst intake and clinical signs [ ] . clinical signs in acutely infected calves include yellow watery faeces, reluctance to feed, dehydration, and in severe cases can result in death [ ] . the gut takes a few weeks to recover from infection and regain its ability to effectively absorb nutrients [ ] . oocyst shedding in the faeces of infected calves can be observed from day postinfection, lasting for a further days to weeks and shedding can occur after diarrhoea has ceased. therefore, asymptomatic animals may also pose a risk of infection to other animals and to people. this is particularly pertinent in managing risk of zoonotic transmission of cryptosporidium at open farms and petting zoos [ ] . transmission pathways for zoonotic cryptosporidium oocysts figure (key figure) illustrates some of the main sources and transmission pathways for zoonotic cryptosporidium parasites and further information about transmission sources to people are shown in box . environmental contamination with cryptosporidium oocysts is widespread globally and it has been suggested that cryptosporidium poses the biggest pathogen threat to the water industry [ ] . of documented outbreaks of human parasitic protozoal disease due to water contamination in the period - , cryptosporidium spp. accounted for the majority of outbreaks [ ] . how the oocysts reach water courses is not clear, as fate and transfer of oocysts from host faecal material through the environment to water courses is not well understood. most scientists and water industry experts consider this mainly to be due to overland flow, particularly in periods of high rainfall or around intense rainfall events [ ] . the effect of climate change (which is predicted to increase winter precipitation and summer extreme precipitation events) on pathogen loading to surface waters was recently explored, but predicted to have limited effect on the risk to public health, using models for catchment pathogen loads [ ] . as livestock pastures frequently surround reservoirs ultimately destined for human drinking water, this can cause problems for water providers. the true incidence of human cryptosporidiosis is not known, although the population at most risk is clearly children under the age of five and the immunocompromised. diagnosis requires laboratory confirmation, usually through testing a stool sample [ ] . stool samples will not necessarily be tested for cryptosporidium unless the patient falls into a selected group (e.g., young children; hiv-aids; recent foreign travel) or a request is made. traditionally, stained microscopy is used to observe oocysts, and infection rates among (presumably immunocompetent) diarrhoea patients of . % and . % in developing and developed areas, respectively, have been reported, contrasting with % and % among hiv positive diarrhoea patients [ ] . differences in the sensitivity, specificity, and the positive predictive value of the diagnostic tests can impact burden of disease estimates; in the global enteric multicentre study, an improved elisa was used and revealed the significant contribution of cryptosporidium to childhood morbidity and mortality [ ] . differences in access to health care, sample submission, and diagnostic practice contribute to the ascertainment of cryptosporidiosis and, along with variation in reporting requirements, make interpretation of surveillance data within and between countries difficult [ ] . in , there were . confirmed cases per population reported in the eu/eea by / countries [ ] . one study in the uk that measured under-ascertainment found that for every case reported to national surveillance there were an estimated . undiagnosed cases in the community [ ] . diagnosis of cryptosporidium infection in livestock is traditionally done by the direct examination of faecal samples using microscopy and modified ziehl-neelsen or auramine phenol staining. people may become infected directly by ingestion of infective oocysts through the faecal-oral route. this may be occupational [ ] , through contact with infected animals [ ] , or indirectly through the consumption of contaminated drinking water [ ] or food [ ] . proportional source attribution data are sparse for cryptosporidium. one study in canada attributed source to sporadic cryptosporidiosis cases from exposure data and found that water was the most commonly reported likely source of infection ( % of cases), followed by contact with livestock ( %), person-to-person contact ( %), food-borne transmission ( %), and contact with pets ( %) [ ] . more recent expert elicitation generally concurs with this order [ ] . general social and economic zoonotic risk factors have also been identified for human cryptosporidiosis. c. parvum was more common in areas with lower human population densities, where there are a higher ratio of the number of farms to human inhabitants, or a higher ratio of the number of private water supplies to human inhabitants and in areas with high ruminant livestock density [ ] . in england and wales, c. parvum was linked to living in an area with a high estimate of cryptosporidium applied to land from manure [ ] . additionally, through the identification of the infecting species and subtypes, the variable transmission and epidemiology of cryptosporidiosis becomes clearer and interventions can be directly applied, whether these are broadly hygiene and sanitation measures on farms, in homes, and institutions, or in potable or recreational water and the food chain. although most c. parvum subtypes are zoonotic, a subspecies that is human-adapted has been proposed as c. parvum anthroponosum [ ] . geographical analysis indicates that the human-adapted species (c. hominis, some c. parvum subtypes) are relatively more prevalent in resource-poor countries, whereas zoonotic c. parvum dominates in north america, europe, australia, and parts of the middle east [ ] . transmission varies seasonally; in most european countries cryptosporidiosis cases are mainly reported in late summer and autumn (august-october) [ ] , with some countries also having a smaller peak in spring. there is good evidence from outbreaks and molecular analytical epidemiology that the spring peak is mostly attributable to c. parvum and may be related to lambing and calving, while the summer/autumn peak is mainly c. hominis and is associated with swimming pools and foreign travel [ ] . immune chromatography lateral flow assays are used to give a rapid diagnostic and are widely used for point-of-care testing on farms. molecular assays are essential to give further information on parasite species and genotype [ ] . different cryptosporidium species can be detected using multiplex pcr assays [ ] or by direct sequencing but this is not done as a routine diagnostic test. molecular-based diagnostics are widely used in commercial labs, often using real time pcr (that is helpful to detect low concentrations of oocysts) and are increasingly used in human diagnostics. diagnostic epidemiology studies on a whole catchment scale are essential when considering riskbased water safety assessments [ ] . such studies are scarce but have shown links between livestock and wildlife c. parvum infection with water contamination. human infection through contamination of the public water supply has resulted in outbreaks and hospitalisation of consumers. in one study in scotland, where this had occurred, further investigation showed that livestock and wild red and roe deer were contributing to catchment parasite loading and water contamination [ ] . another catchment study in cumbria, uk, concluded that the distribution of cryptosporidium species in surface waters, livestock, and wildlife were also linked [ ] . gene sequencing and pcr-restriction fragment length polymorphism analysis showed the presence of the same c. parvum strain in water and faecal samples collected from neonatal calves on dairy farms in parana, brazil, highlighting the risk of zoonotic strains contaminating water supplies used for human and animal consumption [ ] . a recent study in australia showed the presence of zoonotic cryptosporidium spp. in livestock and wild mammals in water catchments [ ] . in addition, c. parvum has been detected in edible marine fish in european waters, indicating the extent of environmental contamination with this zoonotic pathogen [ ] . application of genotyping tools assignment to species level is useful in determining zoonotic potential of the parasite, but to determine transmission dynamics and source of infection, more discriminatory power is required [ ] . genotyping within c. parvum has mainly been based on a single locus through sequencing of a polymorphic region of the gp gene, which has a putative role in virulence. whilst this is a useful library typing tool, it does not provide adequate differentiation for local or regional epidemiological questions, such as outbreak investigations. a literature review of multilocus genotyping schemes for c. parvum showed that these are usually based on single nucleotide polymorphisms or on micro/minisatellite regions [ ] . in multilocus fragment typing (mlft), repeat units within the genome (micro/minisatellites) are amplified and either sequenced or analysed for size, reflecting the length polymorphisms due to variable numbers of repeat motifs, which forms the basis for genotyping. alleles at different loci, or markers, are combined to give a multilocus genotype. robinson and chalmers [ ] favour fragment sizing for surveillance and outbreak investigations, due to the potential to provide rapid, cost-effective results that are discriminatory enough to address source attribution. work in bovine-derived c. parvum assessed an mlft typing tool and demonstrated good discriminatory ability compared with gp sequencing alone [ ] . it would be desirable for a one health approach to develop an integrated genotyping approach that could be used by both veterinary and public health researchers, as advocated in a recent eu cost action ii . the markers that mlft is based on are single copy genes and as such this approach has limitations for environmental samples where there is likely to be a low quantity of c. parvum dna present. this presents a problem for whole-catchment approaches, where mlft could be a useful tool in determining c. parvum transmission dynamics and innovative application of amplification technologies could be used to overcome such barriers. there are currently few options to help prevent and treat cryptosporidiosis in humans with no vaccines and only one fda approved drug, nitazoxanide, available. nitazoxanide has efficacy in immunocompetent people but is not effective in severely immunocompromised individuals [ ] . education about routes of transmission and hand washing are practical preventative environmental public health measures to help reduce disease incidence [ ] . due to the lack of vaccines for cryptosporidiosis in livestock, prevention and control of disease currently involves supporting the resilience of calves in the first few weeks of life. this will include making sure they receive adequate quantities of good quality colostrum in the first few hours of life [ ] and housing in clean, dry, and warm pens with raised feeding and water troughs to minimise exposure to cryptosporidium parasites in the environment. deep and clean straw bedding will also help minimise contact with contaminated faeces and regular cleaning out of calf pens along with steam cleaning, as oocysts are inactivated at temperatures above °c [ ] . disinfection with recommended products can help to reduce build-up of contaminated faeces on the farm, where disinfectants containing hydrogen peroxide are the most effective [ ] . a recent study has also suggested that disinfection of calf pens with hydrated lime may help to reduce onset and severity of cryptosporidiosis in calves [ ] . it is advisable to house calves in similar age groups, as the younger calves are more susceptible to disease and may be vulnerable to infection if they are moved into an environment contaminated by older calves. calves affected by cryptosporidiosis should be housed separately until at least week after diarrhoea stops, as the animal may still be shedding oocysts. calves can be given supportive rehydration therapy and kept warm, clean, and dry. there are currently two licenced treatment products for use in calves; one is halofuginone lactate [ ] , which is used as a preventative treatment and is given within hours of the onset of clinical signs and continued for days. the drug acts to reduce shedding of the parasite and to reduce the severity and duration of diarrhoea, although it can be toxic in dehydrated animals so it needs to be used appropriately. paromomycin [ ] is recently available in uk under veterinary prescription and can be applied for days, following a diagnosis of cryptosporidium infection, and has been shown to reduce oocyst shedding and diarrhoea, although there is also a risk of toxicity with this drug. treatment of manure to reduce viability of cryptosporidium a recent study used spatially explicit process-based modelling to estimate the global cryptosporidium loads in livestock manure and estimated this to be in the region of . × oocysts per year, with cattle being the predominant source [ ] . as manure is applied to land, oocysts can be transported via run-off into surface waters and may be a source of infection to both animals and people. asia has the highest oocyst load from livestock manure, followed by africa, south america, and europe [ ] . the effective management of manure and slurry on farms to reduce viability of cryptosporidium oocysts will have an impact on disease risk in the wider environment and in particular for water catchments [ ] . such on-farm practices include the proper composting of manure, as heat (n °c) will inactivate the oocysts; slurry storage, as ammonia and low ph will help to inactivate oocysts; and treatment with mesophilic and thermophilic anaerobic digestion to significantly reduce oocyst viability [ ] . fencing of livestock away from streams and water courses and use of vegetated and riparian buffer strips can help to slow down the transfer of cryptosporidium oocysts from livestock faecal matter into water courses [ ] . applying measures to prevent and control cryptosporidiosis in livestock will have significant benefits for livestock health and welfare and will increase the efficiency of production, bringing economic benefits to livestock farmers in many regions of the world. in addition, applying methods on farm to minimise the environmental contamination with faeces containing infective cryptosporidium oocysts will also help to minimise risk to other animals and to people through protection of the environment and water catchments (figure ). there has been considerable recent investment in cryptosporidium drug discovery and there are now several classes of compounds of lead, late lead, and preclinical status. these include bumped kinase inhibitors that target cryptosporidium calcium-dependant protein kinase (cdpk ) and are effective in several in vivo models of cryptosporidiosis, including gnotobiotic piglets and calf models, which showed a reduction in oocyst shedding and clinical signs [ , ] . compounds that target trna synthetases, enzymes that charge trnas with their cognate amino acid for use in protein synthesis, have also proven effective against cryptosporidium [ ] . several groups carried out screens of compounds with activity on related parasites and identified new anti-cryptosporidial compounds [ ] . these compounds progressed from in vitro screens, to small animal models, and to neonatal calf studies. because c. parvum infects both humans and cattle, new medicines for treating human patients may also be effective for controlling livestock cryptosporidiosis. a major question in drug discovery is a requirement for gastrointestinal versus systemic exposure. because cryptosporidium are usually located in the brush border epithelium of the gastrointestinal tract, systemic exposure may not be required to cure the infection [ ] . current drug efforts are working to better understand the pk/pd distribution required to cure cryptosporidiosis. modelling this for both human and cattle will be important in developing effective treatments. vaccines for livestock? there are currently no vaccines available to prevent cryptosporidiosis in farm livestock and as the disease mainly occurs in very young calves it might be difficult to generate protective immunity to the parasite quickly enough through active vaccination. an alternative strategy such as that currently used to vaccinate dams against other pathogens causing diarrhoeal disease (e.g., rotavirus, coronavirus, and escherichia coli) may also work for cryptosporidium by generating specific immunity in the dam, which is passed to the calf through the colostrum, thus helping the calf to resist the disease during these crucial early weeks of life [ ] . promising results using a passive immunity approach have been achieved through immunisation of dams with recombinant p and cp proteins to generate hyperimmune colostrum containing specific antibodies and other immune factors that can help protect neonatal calves against disease caused by cryptosporidium infection [ ] . the protective immune mechanisms in calves against cryptosporidium parasites are not well understood and are likely to involve both humoral and cell-mediated immune responses [ , ] . antibody responses against several dominant cryptosporidium antigens, such as gp , cp , and cp that are thought to play a role in parasite attachment and invasion of host cells, may be important as vaccine candidates [ ] . both animals and humans develop protective immunity after exposure to and recovery from cryptosporidium infections. understanding what drives immunity will aid development of much needed vaccines. recent work by sateriale et al. [ ] developed a new mouse model to dissect immunological responses to cryptosporidium. they isolated a mouse-specific cryptosporidium tyzzeri strain that infects the small intestine of immunologically competent mice, closely mimicking human infection. this model is genetically tractable for both the host and parasite, and promises to provide mechanistic understanding of protective immunological responses that can drive rational vaccine design. an effective vaccine that would minimise disease in livestock and reduce shedding of oocysts in faeces would have a significant impact on livestock health and welfare and also would reduce the contamination of the farm environment and wider catchment areas with infective oocysts [ ] . a large outbreak of cryptosporidiosis that occurred in scotland in was associated with drinking water supplied from loch katrine and the catchment area surrounding the loch had a large sheep and cattle population. livestock were removed from the area by the water authorities as a precaution and a rapid gravity filtration system was introduced, which successfully reduced the cryptosporidium oocysts from the finished water [ ] . an important example of health authorities working together with farmers to help protect water catchments is one of the largest water supply systems in the usa, providing over million people in the new york city area with . billion litres daily from a single source of unfiltered water. farmers in the catskills area had intensified livestock production, leading to increased pollution from faeces and manure getting into the water shed [ ] . the new york city authorities worked with the farmers to set up a whole farm planning system to customise pollution control on each farm to maximise effectiveness and minimise cost iii . by continuing to work effectively with the farmers, the new york city authorities were able to protect the environment in the water catchment area and avoid the multi-billion dollar cost of filtering the water supply, illustrating very well how a managed environment will produce good quality water. adopting a one health approach offers several opportunities to help tackle cryptosporidiosis, in particular, disease caused by c. parvum and other zoonotic species that affect both animals and people. the impact of the disease is far reaching and recent studies have highlighted the longterm consequences of clinical disease in both humans and livestock animals (box ). the true burden of disease in both public [ ] and veterinary health is unknown, as many incidences of infection and disease go undiagnosed or unreported. with livestock farming moving towards increasingly efficient and sustainable production to minimise impact on the environment, there is much to be gained from tackling cryptosporidiosis. as livestock are also a major source of infectious and long-lived oocysts that can infect other animals, wildlife, and people [ ] , measures to reduce oocyst shedding and methods of storing and treating farm manure [ ] to prevent oocyst contamination of the wider environment are important mitigation strategies. new diagnostic targets rapid point-of-care detection is needed to enable quick and effective intervention and treatment. in addition, an integrated genotyping approach, which could be used by both veterinary and public health researchers [ ] , would help in disease surveillance identifying sources of infection and routes of transmission, examine evolving epidemiology patterns, and aid in the analysis of parasite virulence. sensitive detection and genotyping methods are required when looking at environmental samples that may contain very few oocysts and a test to confirm oocyst viability would be highly desirable for analysis of infection risk. a recent eu research project involving research organisations and industrial partners across europe looked at emerging technologies for early detection of waterborne pathogens, including cryptosporidium, to enable improved water safety and public health (aquavalens, iv) . collaboration and cooperation between countries on surveillance strategies and techniques would be of great benefit to enable sharing of information and disease epidemiology data. protective immunity and prospects for vaccination developing vaccines for use in livestock to prevent disease and reduce oocyst shedding would be a key priority for a one health approach. this intervention would not only benefit animal health and welfare, it would also have additional benefits to reduce parasite contamination of the environment through reduced oocyst shedding and would therefore also benefit public health [ ] . the most feasible approach would be to target cattle where the parasite is highly prevalent in the first instance, as they are the most significant contributors to contaminated manure globally [ ] . there are currently no human vaccines available and this would be a very desirable target, particularly for high-risk groups such as young children in developing countries and immunocomprised individuals, where infection with the parasite can have very severe clinical consequences. the feasibility of a vaccination approach has been supported by studies showing the development of a degree of resistance to the parasite following repeated exposure [ ] . in addition, sero-epidemiology studies in scotland looked at the prevalence of antibodies against cryptosporidium sporozoite antigens in populations before and after the introduction of enhanced filtration to remove cryptosporidium oocysts for the water supply and found a significant reduction in seroprevalence, indicating a potential reduction in immune resilience to the trends in parasitology parasite [ ] . several promising candidate antigens have been identified, along with knowledge of likely protective immune responses and different vaccine delivery strategies to target mucosal immune responses, recently reviewed [ ] . of interest is the level of cross-protective immunity between c. hominis and c. parvum, as this will be an important consideration for vaccine design. a serological study was conducted in children in bangladesh and found that although most children were infected with c. hominis, they also had cross-reactive antibodies to the c. parvum antigen cp [ ] . a further study looked at cross-protective immunity between the two species using an in vivo gnotobiotic pig model and showed than prior infection with c. hominis protected against a further c. hominis challenge and gave partial protection against a c. parvum challenge [ ] , showing that there is a degree of cross-immunity between the two cryptosporidium species. the gnotobiotic piglet model shows similar clinical signs of diarrhoea as seen in human infection following challenge with c. hominis and is therefore a relevant clinical model with which to test out new therapies and vaccines [ ] . neonatal cattle [ ] and sheep [ ] are also relevant clinical models for c. parvum in particular, to improve understanding of host-parasite interactions, to test out novel therapeutic agents, and to provide sources of cryptosporidium oocysts for research and diagnostic purposes. recently, there has been some exciting progress on the in vitro culture of cryptosporidium parasites using hollow-fibre technology [ ] and the demonstration of the complete life cycle of c. parvum when cultured in human and mouse intestinal organoids [ , ] . heo et al. microinjected cryptosporidium into the lumen of three-dimensional human-derived organoids [ ] , while wilke et al. infected monolayers of mouse-derived organoids cultured at an air-liquid interface in transwells [ ] . both culture systems support long-term growth (n days) and produce viable, infectious oocysts. additionally, wilke et al. generated transgenic parasites in vitro in organoid co-culture and used these parasites to perform a genetic cross [ ] . these advances will help to reduce the numbers of animals used in research and for production of oocysts. important advances have also been made in the genetic engineering of cryptosporidium parasites [ ] . use of crispr/cas in cryptosporidium has enabled the generation of reporter strains to advance drug discovery. manipulation of genes of interest allows us to interrogate the function of genes directly in the parasite for the first time. further tool development, including protein tagging and conditional knockdown, will improve our understanding of the parasite biology and host pathogen interactions [ ] . these new technologies are very exciting, as both host and parasite are accessible and tractable for fundamental research, and provide more relevant models for drug and vaccine discovery. similarly, other hosts and species of cryptosporidium may be amenable to culture and laboratory exploration in the future. this would be especially impactful for c. hominis, which lacks an in vitro system and relies on a resource intensive gnotobiotic piglet animal model. along with increasing awareness and understanding of the impact of cryptosporidiosis in people and animals, there is a renewed impetus to tackle cryptosporidiosis. understanding key transmission routes and main sources of infection can also help to develop mitigating strategies to prevent direct infection and to protect against indirect transmission through water, food, and the environment. cryptosporidium really lends itself to a one health approach and with the recent technological advances and increased awareness of the long-term impacts for people, animals, and our natural environment, the opportunity exists to work collaboratively across the different sectors to tackle this global disease (see outstanding questions). a key target would be the development of a outstanding questions would a vaccine relying on passive transfer of immunity be effective in neonatal calves to reduce incidence of cryptosporidiosis and shedding of oocysts into the environment? would an integrated genotyping approach in veterinary and public health help with source tracking, epidemiology, and surveillance? what treatments would be effective to reduce viability of cryptosporidium oocysts in manure? will some of the new anti-cryptosporidial drug compounds identified be effective in both people and livestock? will the application of crispr/cas in cryptosporidium enable the manipulation of genes of interest to facilitate the direct interrogation of the function of specific genes? trends in parasitology vaccine that could be used to help prevent disease and oocyst shedding in neonatal calves, as this would not only improve the health and welfare of the animals, it would also reduce environmental contamination as these animals are a main reservoir of c. parvum oocysts. due to the very young age of the calves when they first encounter the parasite, a vaccination approach involving passive transfer of immunity in colostrum is most likely to be successful. applying an integrated genotyping approach that may be used in both veterinary and public health would aid in source tracking and surveillance and inform epidemiology studies. treatment of livestock and human faecal waste to reduce viability of cryptosporidium oocysts would help to minimise contamination of the environment with infectious parasites and protect human and animal health. there have been some very exciting new advances in research into cryptosporidium parasites, involving the development and application of relevant clinical in vivo models of disease, new in vitro systems, and the ability to conduct genetic manipulation studies. these will greatly advance our knowledge of host-parasite interactions and help facilitate the testing of new therapeutic compounds and vaccine targets. by combining our knowledge and expertise from the veterinary, public, and environmental health areas, along with the new advances in biology, genetics, and host-pathogen interactions, we have an exciting opportunity to 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design. key: cord- -ewowkq o authors: ollivett, t.l.; mcguirk, s.m. title: salt poisoning as a cause of morbidity and mortality in neonatal dairy calves date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: ewowkq o nan cause of increased morbidity and mortality in calves < days of age on a large commercial dairy farm. historically, affected calves suffered from anorexia, diarrhea, abnormal gait, seizures, and death between and days of age. serum chemistry determinations and measurement of the sodium content of milk replacer and water used on the farm identified salt poisoning as the cause of the clinical signs. the use of high salinity water to mix milk replacer, mixing of oral electrolyte powder directly into liquid feed, adding additional milk replacer powder to the same volume of water for daily cold weather feedings, and limited access to water contributed to the salt poisoning. morbidity and mortality rates decreased after switching to an entirely whole-milk diet, mixing oral electrolyte powder in water according to label instructions, and providing adlibitum access to normal salinity water. sodium is the major cation in extracellular fluid, and under normal conditions serves as a marker of total body water. its osmotic effect is important in establishing concentration gradients that drive fluid shifts between the extracellular and intracellular fluid compartments. active and passive transport mechanisms in the body facilitate these processes. maintenance of sodium homeostasis is critical to maintaining cellular hydration and preventing rapid fluxes of water between compartments. , sodium imbalances may be caused by dysfunction in either sodium or water regulation. in dairy calves, milk replacer or milk sodium content and water consumption are the major dietary contributors to serum sodium concentration. less common contributors may be high salinity water, oral electrolyte powders, and sodium-containing feed additives or medications such as lasalocid that also might provide a substantial amount of sodium to the calf. inappropriately high concentration of the milk replacer solution, milk replacer mixing errors, lack of access to water, dehydration from disease, or a combination of these factors also can result in hypernatremia. [ ] [ ] [ ] regardless of the cause, hypernatremia causes inhibition of neuronal cell glycolysis and results in central nervous system (cns) depression. , hypernatremia also causes plasma osmolality to increase relative to tissue osmolality. this is particularly important with nervous tissue in which neurons effectively shrink as water is lost to the extracellular space in an attempt to equilibrate the osmotic gradient between the intracellular and extracellular space. a partially protective mechanism occurs with accumulation of amino acids, sugars, and alcohols in neuronal cells in an attempt to balance the intra-and extracellular osmolality. , correcting hypernatremia can be difficult because the animal is predisposed to additional neuronal damage as fluid shifts into the intracellular space creating cellular edema and possible brain herniation through the foramen magnum. , the current report describes acute and chronic salt toxicity in neonatal dairy calves on a wisconsin dairy from november to february in which abnormally high concentrations of sodium were detected in sera collected from these animals and in the water in which a commercial milk replacer powder was being mixed. the dairy that is the subject of this report milks approximately holstein, jersey, and holstein-jersey mixed breed cows. the calves are born at the dairy, separated from the dam, moved to an individual pen in the calf barn, and fed colostrum. calves are fed whatever volume of colostrum that they voluntarily suckle, up to a maximum volume of quarts for the jersey and quarts for the holstein calves. colostrum force-feeding occurs only when the calf does not suckle at all. water by itself is not provided until month of age. whole milk is fed for the first - days of life followed by milk replacer, which is fed until - weeks of age, when weaning occurs. the primary complaint by the farm owner that led to this herd investigation was high mortality in calves between and days of age, starting in november . the farm had lost % of its newborn calves in the interim period (approximately months) for a total of calves. calves that did not die had a tendency to develop diarrhea at - days of age with recovery approximately days later. abnormal gaits and seizures had occurred in several of the calves. since the onset of the problem, attempts at treatment included addition of electrolyte powder to the liquid feed, administration of sodium lasalocid to the liquid feed (which already contains lasalocid from the manufacturer) and with multiple antibiotics to no avail. upon further questioning, it was found that the owner began increasing the amount of milk replacer powder provided to the calves in late fall to provide additional energy during the winter months. additional history indicated that water quality had been a concern. the depth of the well had been increased and gallons of sodium hypochlorite had been added to the well water supplying the calf barn to improve water quality. the clinical signs reported by the owner included scours, ataxia, seizures, and lethargy before death. in the week before the farm visit, calf health seemed to have improved. the only management change at that time was switching the calves to an entirely wholemilk diet. upon investigation, of calves were showing signs of decreased mentation, base-wide stance, and mild ataxia. seventeen of ( %) calves had respiratory disease, seven of which had or more signs of respiratory disease (fever, cough, abnormal nasal discharge, ocular discharge, or otitis media) and were under no treatment. thirteen of ( %) calves had scours. based upon the history and clinical signs observed, hypernatremia was considered the primary differential diagnosis. other considerations were meningoencephalitis, lasalocid toxicity, bovine virus diarrhea (bvd) infection, enteric colibacillosis, salmonellosis or other causes of diarrhea, and associated metabolic derangements or toxemia. blood samples were collected for measurement of serum electrolyte concentrations from lethargic animals that stood with abnormal extension of the head and neck and were reluctant to move. fecal samples were obtained for salmonella enterica culture and acid fast stain for cryptosporidium spp. from each of untreated calves, five of which had diarrhea at the time of sampling. two milk replacer samples from the first and last calves fed for each of successive feedings after the farm visit were analyzed for total solids and sodium concentration. milk replacer total solids were determined by a brix refractometer a after construction of a standard curve, with known milk replacer concentrations. milk replacer sodium concentrations were determined by a vitros â , fs chemistry system b after the milk replacer solution was agitated, centrifuged, and an aliquot of the supernatant obtained for analysis. milk replacer powder c also was collected, to be mixed with university water for sodium analysis and to serve as quality control standards for total solids calculations by a brix refractometer. the farm protocol was to mix ounces ( g) of milk replacer in quarts ( . l) of water. results of analyses are presented in the tables as follows: milk replacer (table ) and well water ( table ) . postmortem reports from calves that died previously were reviewed. antemortem testing disclosed severe hypernatremia in the calves with neurologic signs that were sampled. serum sodium concentrations were , , and meq/l (reference range: - meq/l), respectively. chloride concentrations also were increased at , , and meq/l (reference range: - ), respectively. only of the calves had an increased anion gap of mmol/l (reference range: - mmol/l). extremely high salinity well water (farm water, , ppm; safe level, < , ppm ) used to mix the milk replacer on the farm resulted in extremely high milk replacer sodium concentrations, compared with considerably lower milk replacer sodium concentrations in milk replacer mixed according to farm protocol but by university water (table ) . fecal diagnostic testing showed no evidence of pathogenic or toxin-producing e. coli or salmonella enterica. one of ( %), of ( %), and of ( %) of the calves were shedding rotavirus, coronavirus and cryptosporidium spp., respectively. no postmortem testing was performed on the day of the farm visit but the reports from previously examined calves indicated no gross lesions and no evidence of meningoencephalitis, bvd infection, or salmonellosis. no lung or intestinal pathogens were identified, including toxinproducing e. coli. salt toxicity outbreaks have been reported in cattle on pasture [ ] [ ] [ ] [ ] [ ] and in a few calves, , but none have involved a herd problem associated with dairy calves. access to a salt lick without free-choice water created signs of salt toxicity, including diarrhea, abdominal pain, anorexia, convulsions, muscle twitching, paresis, and death in a group of -to -month-old holstein beef cattle. clinical signs seen in cattle grazing on coastal pastures with high salinity water ( , ppm) and marsh grass ( , ppm) included diarrhea and excitability. in another report, mature beef cows showed signs of recumbency, seizures, opisthotonus, caudal ataxia, and hypersensitivity after excessive consumption of a protein supplement containing % sodium chloride after breaking into a salt storage shed. severe neurologic signs including mentation changes (excitability or lethargy), head pressing, ataxia, blindness, recumbency, and death were noted in several adult steers that had been deprived of water after allowing access to salt after a period of salt restriction. the calves in this report exhibited many of these signs, including diarrhea, convulsions, anorexia, salt poisoning of calves ataxia, and death. lastly, the calves of this report were given approximately mg/kg lasalocid between the liquid medicated suspension and the medicated milk replacer, which is less than reported toxic dosage of mg/kg in neonatal calves. blood, liquid feed, and water analysis confirmed the primary differential diagnosis of severe hypernatremia secondary to high salinity water, lack of water availability, unusually high milk replacer total solids concentration (table ) , and history of adding additional sodium lasalocid to the milk replacer. the volume of the extracellular fluid (ecf) is determined by the total body sodium content, whereas the osmolality and sodium concentration of ecf are determined by sodium and water balance. sodium, glucose, and serum urea nitrogen (sun) are solutes that affect plasma osmolality. all conditions associated with hypernatremia reflect hyperosmolality and hypertonicity of the ecf. these conditions are caused by a deficit of pure water, such as lack of water access; loss of hypotonic fluid, such as occurs during osmotic diuresis; or gain of sodium, such as during salt poisoning which may result in either relative or absolute dehydration. hypernatremia caused by the gain of sodium results in expansion of the ecf at the expense of the intracellular fluid (icf), that is, relative dehydration in an attempt to return plasma osmolality to normal. hypernatremia because of deficient free-water intake or hypotonic fluid loss is associated with absolute dehydration and reduction in total body water content. characterization of dehydration is important when determining treatment options. it is likely that the calves tested in this report suffered predominately from relative dehydration because gain of salt was the primary factor predisposing to hypernatremia. specifically in cattle, the pathogenesis of hypernatremia involves excessive intake of sodium as a result of mixing errors, excessive water loss, or prolonged administration of iv isotonic crystalloids without access to free-choice water. water salinity of , ppm on this farm contributed . meq/l of sodium from water added to the milk replacer powder. additional sodium from the milk replacer powder (approximately meq/l), electrolyte powder ( % sodium), and times the recommended lasalocid concentration fed to calves with no access to free-choice water likely contributed to the early onset of clinical signs and death. no deaths occurred in calves fed colostrum and whole milk exclusively. multiple factors contributed to salt poisoning in the neonatal dairy calves on this farm. the importance of the availability of free-choice water, appropriate mixing of milk replacer and electrolyte solutions, as well as water salinity testing are evident. two of the calves with hypernatremia died. the surviving calf was given no access to water. instead, it was fed whole milk and offered a progressively more dilute oral electrolyte solution over a course of days. water was offered on day eight after the farm visit. treatment of sodium balance disorders. water intoxication and salt toxicity disorders of sodium and water balance in hospitalized patients a case of presumptive salt poisoning (water deprivation) in veal calves hypernatremia in calves hypernatremia in neonatal elk calves: cases ( - ) treatment of hypernatremia in an acidotic neonatal calf effects of hypernatremia on organic brain osmoles practical toxicologic diagnosis. mod vet prac water deprivation-sodium ion toxicosis in cattle acute salt poisoning in cattle salt poisoning in beef cattle sodium chloride poisoning in cattle salt poisoning in beef cattle on coastal pasture on prince edward island abutarbush sm, petrie l. treatment of hypernatremia in neonatal calves with diarrhea conflict of interest declaration: authors disclose no conflict of interest. key: cord- -de zhswl authors: ganesh, atheesha; lin, johnson; singh, moganavelli title: detecting virus‐like particles from the umgeni river, south africa date: - - journal: clean (weinh) doi: . /clen. sha: doc_id: cord_uid: de zhswl it is important to consider viruses in water quality because of their incidence as causal agents for diarrhoeal disease, and due to their characteristics, which allow them to survive in changing environmental conditions indefinitely. this study assessed the viral quality of the umgeni river in south africa seasonally. a two‐step tangential flow filtration process was setup to remove the bacteria and to concentrate the virus populations from large volume water samples. the concentrated water samples contained up to and pfu/ml of somatic and f‐rna coliphages, respectively. several virus families including adenoviridae, herpesviridae, orthomyxoviridae, picornaviridae, poxviridae and reoviridae were found in the river based on the morphologies examined under transmission electron microscopy. all concentrated water samples produced substantial cytopathic effects on the vero, hek , hela and a cell lines. these results indicate the potential of viruses in the water samples especially from the lower catchment areas of the umgeni river to infect human hosts throughout the year. the present study highlights the importance of routine environmental surveillance of human enteric viruses in water sources. this can contribute to a better understanding of the actual burden of disease on those who might be using the water directly without treatment. despite the advancement in water and wastewater management proficiency, faecal pollution of surface waters is still a cause of concern for the general public and can lead to waterborne illnesses which have community well being and socio-economic repercussions in both the industrialised and developing world [ , ] . waterborne diseases are frequently caused by enteric pathogens, which belong to the group of organisms transmitted by the faecal-oral route [ ] . the world health organisation (who), states that there are $ . million deaths related with hazardous water conditions, sanitation and hygiene, and millions more suffer numerous incidents of non-fatal diarrhoea every year [ ] . water quality concerns have increased in recent years, in part due to recurrent contamination of surface water resources by waterborne bacterial, viral and protozoan pathogens [ ] . human enteric viruses are imperative causative agents of waterborne illness, however, viral diseases are difficult to identify by current diagnostic techniques [ , ] . the centres for disease control and prevention [ ] projected that viral infection may be responsible for almost half of all acute gastrointestinal diseases, and therefore, viral contamination of recreational coastal water is of paramount importance and is a rising public health concern [ ] . enteric viruses have a relatively low infectious dose and can be responsible for a large range of human illnesses such as paralysis, meningitis, respiratory diseases, myocarditis, congenital abnormalities, epidemic vomiting, diarrhoea and hepatitis [ , ] . nevertheless, it is now accepted that viral contamination should be definitely taken into account in the microbiological analysis of water and food [ ] . currently microbiological quality standards for water include mostly bacterial faecal indicators, which are not correlated with the presence of waterborne viruses [ ] . routine viral monitoring is not required for drinking or recreational waters and neither is it required for wastewater that is discharged into the environment. this lack of a monitoring effort is due largely to the lack of methods that can rapidly and sensitively detect infectious viruses in environmental samples [ ] . phages share many properties with human viruses, particularly composition, morphology and structure [ , ] . phages, especially somatic coliphages and f-rna coliphages, have been recommended as alternate microbial indicators as they behave more like the human enteric viruses which pose a health risk to water consumers if water has been contaminated with human faeces [ , ] . somatic coliphages have been described as being a heterogeneous group of viruses, which could originate from faecal sources [ , ] . the presence of these viruses in faecally contaminated water means that they can serve as indicators of faecal pollution and may indicate the coexisting presence of pathogenic viruses. due to their resistance against environmental factors, somatic coliphages are more applicable than faecal bacteria for indicating faecal contamination of water [ , , ] . the occurrence of f-rna phages in surface waters generally indicates pollution by human or animal faeces [ ] [ ] [ ] [ ] . it was shown that for monitoring purposes, f-specific rna bacteriophages can indicate the possible presence of human pathogenic enteric viruses as they behave like waterborne viruses and their morphology and survival characteristics closely resemble that of some of the important human enteric viruses [ , , ] . the phage plaque assay has the advantages of technical simplicity and low cost but the choice of a suitable bacterial host is of paramount importance [ ] . one of the requirements for the virological analysis of water is an efficient and simple virus concentration method. it is well recognised that monitoring the presence of enteric viruses could be challenging due to the relatively low level of infectious viral particles towards the respective host species and small viral particle size existing in environmental waters, thus making it essential to start with a large water sample volume and concentrate it to several orders of magnitude [ ] [ ] [ ] [ ] [ ] . however, this limitation can be overcome by the use of improved methods for aquatic sample concentration, viral infectivity and more sensitive viral detection techniques [ ] . tangential-flow and hollow-fibre ultrafiltration (uf) have been used to investigate microbial contamination of drinking water [ , ] . the virus concentration method should be applicable to a broad spectrum of enteric viruses to facilitate concurrent downstream study [ ] . the present study was conducted to optimise procedures to extract and enumerate indigenous virus-like particles (vlps) and to determine the community structures and infectivity of these viruses from river water. a setup for the concentration of viruses from large ( l) volume water samples using a two-step tangential flow filtration (tff) process was established. the presence of vlps was determined using epifluorescence microscopy (em), transmission electron microscopy (tem) and their infectivity was determined on cell culture. focus in this study was on the umgeni river (five points along the river as shown in fig. ) due to it being the largest catchment ( km ) in the kwazulu-natal region of south africa. the sampling seasons and sites included five vast areas of the river starting from a dam water source, passing through: a nature reserve, waste treatment works, informal human settlement and finally ending of at the river mouth (were the river and ocean meet). sampling points were designated as follows: u (umgeni river mouth-estuarine/brackish water), u (reservoir hills-informal settlement/domestic waste), u (new germany wastewater works-treated water after chlorination that enters the adjoining umgeni river water), u (krantzkloof nature reserve-vegetation and conservation area) and u (inanda dam-restricted water containment). water samples from the river were collected during autumn (march-april ), winter (june-july ), spring (september-october ) and summer (december -january ) months, to cover the four seasons of the year. water samples were collected in l plastic drums with caps. prior to the sample collections, the alcohol-sterilised drums were rinsed with the water from the river source. the water samples were collected knee deep (ae . m) below the water surface facing away from the water current. all samples were protected from direct sunlight and transported to the laboratory within one hour of collection, and stored at °c until further analysis. escherichia coli atcc (american type culture collection) was used as the host for the somatic coliphage. the bacterial host was grown overnight at °c on a shaking incubator at rpm, in nutrient broth. salmonella typhimurium wg (provided by dr. maite muniesa, university of barcelona, spain) was used as the host for f-specific coliphage. the s. typhi host was grown in tryptone yeast extract broth and incubated at °c with shaking at rpm until the f-pili developed (iso ) . this growth suspension was monitored at min intervals from time against a blank reference at wavelength of nm until an absorbance of . was obtained indicating that the sex pili were produced [ ] . bacteriophages were enumerated by the double agar layer technique following the iso - standard (iso ) for enumeration of somatic coliphages and iso - (iso ) for enumeration of f-specific rna bacteriophages (f-rna). one hundred microliters of the concentrated samples were mixed with ml of overnight bacterial host and ml of soft agar and the mixture was poured over agar bottom plates [ ] . plaques were enumerated after - h of shown are the locations approximate sampling points of the umgeni river investigated in this study [ ] . incubation at °c and plaque forming units (pfu) were recorded per millilitre. viral community samples (virioplankton) were concentrated using a two-step tff process as given in fig. , according to the method of wommack et al. [ ] , with some modifications. concentration of virus particles from large volume water samples occurred in four steps: (i) twenty litres of river water were first pumped through a mm string-wound polypropylene sediment cartridge filter to remove large particles and cells at a flow rate of ml/min; (ii) a peristaltic pump (masterflex) was used at a flow rate of ml/min with pressure (p in ¼ psi and p out ¼ psi) to pump the pre-treated water samples through a . m , . mm pellicon tff cartridge filter (microporous filtration membrane) (millipore) to remove all bacterial cells; (iii) viruses in the cell-free permeate was then further concentrated to a final volume of ml using m -kda spiralwound tff filter (helicon, millipore); (iv) the retentate was allowed to re-circulate through the two step system until only ml to l of sample remained in the original vessel. retentates were then reconcentrated by ultracentrifugation. for ultracentrifugation, runs of h min each at  g (i.e. rpm), °c with a sw- ti rotor (optima l- xp, beckman coulter ultracentrifuge) were necessary to ultracentrifuge ml ( tubes of ml filled with ml of samples per run) of the ultrafiltered retentate [ ] . viral pellets were then resuspended in ml of  phosphate buffered saline (ph ). normal water permeability (nwp) testing was performed regularly on the tff filter membranes before and after running the water samples through the system to ensure filter quality. proper maintenance and preservation of filter quality was carried out according to manufactures instruction with some modifications. nwp was calculated using the following equation: where r is the permeate flow rate (l/h), p in the feed inlet pressure (psi), p out the retentate discharge pressure (psi), p p the permeate discharge pressure (psi), a the total filter area (m ) and f is the temperature correction factor (based on water fluidity relative to °c). sybr gold staining coupled with epifluorescent microscopy [ ] [ ] [ ] was used to enumerate vlps from the umgeni river virus filtrate. after uf one millilitre ( kda cut-off) of the concentrated water samples were fixed with ml of a % paraformaldehyde solution and then filtered through . mm anodisc filters (whatman, maidstone, kent, uk) with vacuum pressure kpa. the anodisc filters were allowed to air dry and were then stained with  sybr gold (invitrogen, carlsbad, ca, usa) for min in the dark. after staining the filters were wicked to remove any remaining solution and air dried. the filters were mounted onto glass slides, and counted digitally at  magnification under blue-green light excitation (fitch filter, excitation at - nm) with a nikon eclipse ( i) epifluorescent microscope in at least eight fields of view for each sample [ ] . images obtained were then analysed using the item software and nis-d elements software (d . ) to digitally count the fluorescent green vlp spots and compute the results. tem was used to examine the structures and morphology of vlps in the river water. these tem images were then compared to known viral images of human origin where possible [ ] . briefly one drop of freshly prepared vlps was spotted onto a formvar-carbon-coated mesh tem grid (electron microscopy sciences, fort washington, pa). the edge of the grid was gently blotted with a piece of whatman filter paper to drain away the excess fluid, and the grid was then stained with a % phospho-tungstic acid (pta) solution or a % uranyl acetate solution for s, washed with drop of deionised water for s, and air-dried before examination with a tem (jeol). photomicrographs of viruses were taken at magnifications of  to Â. morphological characteristics of vlps were compiled from multiple photomicrographs of phage particles in order to minimise size or shape anomalies. cell-culture (where cell lines were available) was used to determine infectivity of vlps. the total cultivable virus method as described in us epa [ ] was used as the infectivity protocol. the concentrated vlps from various water sources were fed into the various cell lines. three known viruses: human adenovirus, group a rotavirus and coxsackie b virus were used as positive controls. amongst the cell lines used for human virus investigations in water were, the a (adenocarcinomic human alveolar basal epithelial cell), hek (human embryonic kidney cell), hela (humancervical cancer cell), hepg (human hepatocellular carcinoma cell), plc/prf/ (human primary liver carcinoma cell) and vero (african green monkey kidney cell). cells were grown in % dulbecco's modified essential medium (gibco) supplemented with % foetal calf serum (gibco) containing a penicillin/streptomycin/fungizone mix ( : : ) by volume, to confluent monolayers in well tissue culture plates (corning, usa). approximately ml of viral concentrate was overlaid onto the monolayers of appropriate cell lines and incubated at °c for - days. the development of cytopathic effect (cpe) that is indicative of a viral infection in the cell cultures was monitored for up to days. presence or absence of cpe (loss of cell to cell contact and detachment) was confirmed as described in us epa [ ] . after three freeze-thaw cycles, cpe positive and negative samples were filtered through . mm syringe filters and were inoculated in new a , hek , hela, hepg and plc/prf/ cells for another seven days. the cpe of cell culture was inspected under an olympus microscope at  magnification. samples that showed cpe at the end of the confirmation step were reported as positive for infectivity. the mtt ( -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide) assay was performed using the cell lines mentioned above [ ] . when performing the mtt assay, the cells were plated into -well plates and infected by the viral samples (each sample in triplicate) approximately h later. after - days, the medium was removed and mtt solution was added to the wells, and further incubated at °c for h. thereafter the mtt solution was removed and dmso was added to the wells. the cell viability (%) was determined by the optical density of cells at nm using a mindray a microplate reader (vacutec, south africa) according to the method of heldt et al. [ ] . the cpe was calculated as the percentage of cell death due to the viral infection comparing to the control. pearson's correlation test was used to evaluate the correlations between the bacteriophages, vlps and environmental variables. data comparisons were performed using analysis of variance (post hoc tests) and the student's t-test. probability (significant level) was set at . . the spss program version (spss, illinois) was used for the statistical analyses [ ] . canonical correspondence analysis (cca) was used to reveal the association amongst the bacteriophages, vlps and the physical and chemical water quality variables, which were measured from the same sites and seasons in concurrent studies performed in this laboratory [ ] , with a view to defining the significant variables accountable for the observed spatial and temporal distribution of the communities. cca ordination allows assessment of the relationships between biological and environmental data, specifically the extent to which variation in biological data can be accounted for by the measured environmental variables. on cca ordinations, environmental variables are represented by arrows whose lengths reflect their relative importance in structuring the biological sample data [ ] . a monte carlo permutation test with random permutations was used to establish the environmental axis that significantly correlated with the biological variables. cca analysis was performed using the computer programme canoco for windows version . [ ] . somatic coliphage counts ranged from pfu/ml (u -autumn) to pfu/ml (u -summer) and f-rna coliphage counts from pfu/ml (u -autumn) to pfu/ml (u -summer), respectively (fig. ). vlps were detected using em (electrostatic force microscopy). point u during summer had the highest population of vlp/ml and point u and u had the lowest vlp counts of . vlp/ml (u winter) and . vlp/ml (u -autumn), respectively. mean concentrations of somatic coliphages and f-rna coliphages were comparable between the autumn and spring seasons and correlation analysis resulted in a strong pearson's correlation coefficient of . (p < . ). these coliphages had inverse correlations (r ¼ À . ) with the vlps detected during autumn but correlated (r ¼ . ) with the vlps found during spring along the umgeni river. in this study, cca was used to investigate the relationships between the water quality variables and the vlps, as well as the relationships between the bacteriophages and the physico-chemical parameters at all sites and seasons. cca ordination plot (fig. ) revealed strong relationships between the overall vlp and phage populations at different sites and seasons measured as well as with the physicochemical water quality variables. cca plot (blue circle), temperature, biochemical oxygen demand in five days, turbidity, ph, conductivity, orthophosphate and sulphate were the most important variables (fig. , long arrows) that impacted the community structures significantly at sites , and during the autumn, summer and spring seasons. site , , during autumn, winter and spring seasons correlated with the nitrate/nitrite profiles (green circle). site and in autumn and winter showed no correlations with the rest of the sites and variables measured. cca axis (fig. ) accounted for . % of total variance of the species data set and in total the speciesenvironment relation accounted for . % of the cumulative variance suggesting that there may be a correlation between the vlp and phage populations at the various sites and water quality variables during all seasons. positive significant (p < . ) pearson correlation of species and environmental scores for axis one and two were r ¼ . and . , respectively. the ordination plot revealed that the somatic phage, f-rna phage and vlps had little relationships with each other and existed independently (fig. ) . the sample scores were scattered in the ordination with a number of denser clusters (physico-chemical) spread out around the origin. cca axis (fig. ) accounted for . % of total variance of the species data set and in total, the speciesenvironment relation ordination accounted for . % of the cumulative variance. eigen values for axis point one and two were . and . , respectively. positive significant (p < . ) pearson correlation of species and environmental scores for axis one and two were r ¼ . and . , respectively. table classifies the different type of bacteriophage that could be detected in the umgeni river by tem. most of the detected phages ( %) had isometric heads and long non-contractile tails, belonging to morphotype b (siphoviridae) (tab. ). members of morphotypes a (myoviridae) were detected in % of the samples, and c (podoviridae) was present in substantial ( %) numbers. the phages tagged as a /b were not conclusive in morphological appearance and could not be classified. table illustrates the size range of the bacteriophages that could be observed during all four seasons. phage head diameters interesting to note was that the total length of phages varied extensively, ranging from nm (autumn), nm (winter), nm (spring) to nm (summer). figures and resemble bacteriophages detected in the umgeni river water samples. phage heads examined, appeared to be hexagonal in outline, with six sided profiles which were regular, with three symmetrical axes (e.g. fig. a, b, c, e and f) while others were irregular, with only one symmetrical axis (e.g. fig. i and k) . however, these data were not adequate to differentiate between icosahedra, octahedra and dodecahedra classification. figure c and h closely resemble members of the family myoviridae and podoviridae, respectively. figure l and p resemble members of the family b siphoviridae and b siphoviridae, respectively. figure represents phage particles detected in the umgeni river at sites u , u and u during spring and summer seasons that resemble known bacteriophages found in literature. these include known phages of: a- of vibrio vulnificus phage, phage t and t -like vibrio parahaemolyticus phage as shown by ackermann and heldal [ ] . figures - illustrate several presumptive virus types that were found in the umgeni river during all seasons. these presumptive viruses were compared to their known structures illustrated in literature. presumptive naked adenoviridae-like particles ranging in size from . to . nm are shown in fig. and these images are compared to known adenoviruses ( - nm) (fig. g) . figure represents tem images of presumptive naked picornaviridae (enterovirus)-like particles ranging in size from to nm, compared to a known coxsackievirus (fig. f) . poxviridae-like particles (fig. a-c) and herpesviridae-like particles ( fig. e-g) were also detected in the umgeni river water samples and these were found to be similar in literature to known poxvirus (fig. d ) and known herpesvirus (fig. h) . figure illustrates presumptive reoviridae-like particles ranging in size from to nm (fig. a-c) compared to a known rotavirus ( - nm) (fig. d) . presumptive caliciviridae-like particles are illustrated in fig. e -g and these had a size of nm, similar to that of a known norovirus (fig. h ) of - nm. presumptive enveloped coronaviridae-like particles (fig. a-c) were found to be similar to a known coronavirus (fig. d) , with presumptive orthomyxoviridae-like particles being similar to a known influenza virus (fig. h ). the cpe is an observable morphological (shape) change in tissue cells due to viral infection. figure illustrates the results obtained for the figure . tem images of phage morphotypes (a, c, e) present downstream of the umgeni river at the sampling sites u , u and u , respectively, during spring and summer, (b) known a- of vibrio vulnificus phage, (d) known phage t , (f) known t -like vibrio parahaemolyticus phage [ ] . images captured at -  magnification. scale bar: nm figure . tem images of presumptive naked adenoviridae-like particles and (g) a known adenovirus [ ] , present in the umgeni river at the sampling sites u , u , u , u and u during autumn, spring and summer tested. images captured at -  magnification. scale bar; nm. cpe of the vlps isolated from the umgeni river using six tissue cell lines. the identification of cpe in the cell cultures included the occurrence of elongated, granulated cells, loss of cell to cell contact and the appearance of vacuoles. all vlps isolated from all water samples during all four seasons were capable of inducing some cpe (cell death) on all tissue cell lines tested. the vlp samples from sites u , u and u produced no cpe on the hep-g cell line during autumn. vlps from site u produced cpe figure . tem images of presumptive naked picornaviridae (enterovirus)-like particles and (f) a known coxsackievirus [ ] , present in the umgeni river at sampling sites u , u , and u during spring and summer tested. images captured at -  magnification. scale bar: nm. figure . tem images of enveloped presumptive (a, b, c) poxviridae-like particles, (d) known poxvirus [ ] and (e, f, g) herpesviridae-like particles, (h) known herpesvirus [ ] , present in the umgeni river at site u during summer. images captured at -  magnification. scale bar: nm. detecting virus-like particles figure . tem images of presumptive (a, b, c) reoviridae vlps, (d) known rotavirus [ ] , (e, f, g) presumptive caliciviridae vlps, (h) known norovirus [ ] , present in the umgeni river at the different sampling sites during all seasons tested. images captured at -  magnification. scale bar: nm. figure . tem images of presumptive enveloped vlps (a, b,c) coronaviridae vlps, (d) known coronavirus [ ] , (e, f, g) presumptive orthomyxoviridae vlps, (h) known influenzavirus [ ] , present in the umgeni river at the different sampling sites during all seasons tested. images captured at -  magnification. (fig. ). microbiological contamination of water has long been a concern to the public and contamination of water resources, intended for use by general population, with enteric viruses is a great public health problem. although the occurrence of human enteric viruses has been demonstrated in surface water bodies as well as in drinking water supplies throughout the world and several outbreaks of enteric viral diseases (attributed to either contamination of drinking water or vegetables and seafood grown in such waters) have been recorded [ , ] . routine examination of water samples for the presence of enteric viruses is not largely performed in both developed and developing countries [ ] . in this study, the populations of somatic and f-rna coliphages as well as various enteric viruses were monitored using traditional techniques such as electron microscopy and tissue culture. phages have been proposed as microbial indicators of water quality, as they share many fundamental properties with human enteric viruses which pose a health risk, if present in water contaminated with human faeces [ ] . all sampling points throughout all seasons tested positive for the presence of somatic coliphages which ranged from pfu/ml (u -autumn) to pfu/ml (u -summer) especially in the lower reaches of the river and during summer. f-rna coliphage counts were significantly lower compared to those of somatic coliphage counts. f-rna coliphages, from pfu/ml (u -autumn) to pfu/ml (u -summer), were also detected more frequently in the lower reaches of the river. mean concentrations of somatic coliphages and f-rna coliphages were comparable between autumn and spring and correlation analysis resulted in a strong pearson's correlation coefficient of . (p < . ). bacteriophages are not a stable part of water micro-flora and their presence is usually associated with wastewater inflow rich in animal and human excrement [ ] . their survivability depends upon physico-chemical environmental conditions [ ] . somatic coliphages have been found to outnumber f-rna phages in waste water and raw water sources by a factor of about , and cytopathogenic human viruses by about [ , ] , thus making them valuable indicators for assessing the behaviour of and the possible presence of enteric viruses in water environments. male-specific (f-rna) coliphages are highly specific for sewage pollution and cannot be replicated in water environments, but detection methods are more complicated [ ] . vlps were detected using em at all sampling sites throughout all seasons, with point u during summer having the highest population of vlp/ml. umgeni river points u , u and u were more contaminated with phage and virus-like populations than points u and u . increasing in the phage and vlp populations from upstream to the river mouth were observed in all seasons. this was probably because the river flows through the more urbanised areas of durban and is subject to higher surface runoff [ ] . it can be speculated that increased human activity as well as informal settlements and trading activities in the storm water drain catchment areas contributed to the high levels of phage populations downstream the river at points u , u and u . the thicket and bushland that enclosed both the krantzkloof nature reserve (u ) and inanda dam (u ) can serve as vegetated pervious buffers as passive treatment systems allowing agricultural and storm water runoff to reach the river and dam in a less impaired state [ ] . mallin et al. [ ] found that human development along the land-seawater interface is considered to have significant environmental consequences resulting in a difference in abundance and distribution of the enteric pathogen indicator microbes and can often pose an increased human health risk. all variables tested had positive correlations with each other during all seasons. significant positive correlations were observed between the somatic phage and f-rna phage (r ¼ . , p ¼ . ) and somatic phage and vlp (r ¼ . , p ¼ . ). a correlation between phage quantity and degree of faecal pollution has been found elsewhere [ ] . similar results were also obtained by armon and knott [ ] who found positive correlations of phage number and bacterial pollution indicator factors. in this study, tff with the pellicon cassette system (millipore) for concentrating vlps from l river water samples was used. the twostep filtration procedure used in our laboratory involved fluid flowing parallel to the . mm tff filter surface. particles with molecular weights (m w ) smaller than the exclusion size ( . mm) of the filter passed through and collected (filtrate) in a reservoir which was further concentrated through a kda spiral wound tff unit. particles with larger molecular weights were retained and recycled to the original reservoir (retentate). thus, as the filtration continued, the retentate volume decreased until the desired volume was attained. the tem study demonstrated that the two-step tff procedure coupled with ultracentrifugation produced a viral concentrate devoid of bacteria, with most viruses being intact. the phage/vlps concentrations in every sample were high enough to view on formvar coated electron microscopic grids without enrichment. all water samples in this study contained a mixture of morphologically different tailed phage viruses, which were regarded as bacteriophages. it was possible to observe viral particles with long tails (b -siphoviridae), short tails (a -myoviridae and c -podoviridae), and without tails. the phages particles observed without tails may belong to a wide range of hosts, including eukaryotes [ ] . most of the phages from the umgeni river were intact, with discrete structures such as tail fibres, base plates, and other appendages which are pertinent for the recognition of and interaction with the host cell. these accessories may be an indication that a significant proportion of the phages are suspended in the water environment as potentially infective particles. the main purposes of phage classification are generalisation and simplification, which facilitates comparisons and understanding of viruses [ ] . the high morphological diversity of phage communities corresponds well with the great variability and dynamics of bacterial populations obtained in this current study. electron microscopy provides a direct insight into the morphological variability of phage present in the water environment, without being dependent on the isolation of suitable host strains. this is important, as bacteria which are not cultivable under laboratory conditions usually dominate aquatic environments and may be important phage hosts [ ] . great morphological variability was observed in the virus assemblage's from the umgeni river, which suggests that these viruses may also be diverse in terms of the hosts that they infect. most of the viruses were detected downstream of the river towards the river mouth area during the spring and summer seasons. the viral contamination of these widely used areas could be attributed to storm water discharge, surface runoff, sewage discharge and overflows, recreational exposure and other anthropogenic activities [ ] . the detection of viruses by tem in the sampling points upstream of the umgeni river was relatively low or nonexistent, probably due to the stagnant waters and limited recreational exposure. several presumptive virus types including adenoviridae, caliciviridae (norovirus), coronaviridae, herpesviridae, orthomyxoviridae (influenza virus), picornaviridae (enterovirus), poxviridae and reoviridae (rotavirus) were found in the umgeni river during all seasons based on the morphologies under tem. human viruses seen in negative stains fall into one of two major morphological categories: enveloped or naked [ ] . enveloped viruses have a nucleocapsid (the nucleic acid held together by some structural proteins) inside, whilst naked viruses are icosahedral, their protein coat or capsid is more rigid and withstands the drying process well to maintain their spherical structure in negative stains [ ] . naked human viruses are of three size ranges: (i) - nm (e.g. parvoviruses, enteroviruses and caliciviruses), (ii) - nm (polyomaviruses and papillomaviruses) and (iii)  nm (reoviruses, rotaviruses and adenoviruses) [ ] . as a general pattern, the virus population was dominated by small forms (< nm), and tails were rarely seen, which is in agreement with the results reported by several authors [ , [ ] [ ] [ ] . the negative staining technique for tem allowed for the examination of particulate material including determination of structure and size of particles and has proved important in virological studies [ ] . all water samples analysed were environmental in nature, thus natural degradation may have altered the morphological features of the viruses substantially [ ] . the application of pcr-based molecular technology and tem has advanced our knowledge of the occurrence and prevalence of human viruses in water. however, it has provided no information on viral viability and infectivity in a specific environment. the tissue culture assay is the only us epa approved method for virus infectivity monitoring in aquatic samples [ ] . virology laboratories have traditionally used tissue culture cell lines to amplify the amount of virus present in a water sample, express the viral antigens and in many cases monitor the cell death as a consequence of the viral infection producing characteristic cpes in the cell monolayer. the tissue culture technique has been used extensively for virus replication and infectivity studies and plaque assays [ ] . it has been established that for the recovery of a maximum number of infectious viruses, it is often necessary to inoculate each sample type into several different cell culture lines because no single cell culture is available with susceptibility to all enteric virus types [ ] . in the present study six cell lines (the a , hek , hela, hepg , plc/prf/ and vero) were used to determine cpe of the vlps from the umgeni river during four seasons. all vlp samples from all sites and seasons produced substantial cpes on the vero, hek , hela and a cell lines. this effect was tested against positive controls of three known viruses (voxsackievirus, rotaviruses and adenovirus) in the same cell lines. vero cells are sensitive to infection with many different viruses [ ] . human embryonic kidney cell line a and human lung carcinoma cell line a were the most penetrating to viral infection, especially to enteric adenovirus and [ ] . grabow et al. [ ] reported the propagation of both enteric adenoviruses and in the primary liver carcinoma (plc/prf/ ) cell line. hep-g cell lines are often used to study hepatitis b, c and other viruses [ ] . cpe of the vlps was examined by microscopy every day for the first week of growth to maximise the detection of viral progression. certain viruses take one day to three weeks to yield a cpe dependent on the initial concentration and virus type. cpe can be swelling, shrinking, rounding of cells to grouping or whole destruction of the monolayer [ ] . all the vlps isolated from water samples were capable of inducing the cpes on six tissue cell lines. the ability of these viruses to infect susceptible host cells and to replicate their dna/rna, confirms that they are viable and infectious and therefore constitute a health risk which is due to be investigated [ , ] . in this study, different approaches to determine viral pollution during four seasons and at five sampling points in the umgeni river were comparatively evaluated. a setup for the concentration of viruses from large ( l) volume water samples using a two-step tff process was successfully established. electron microscopy illustrated the presence of vlps in concentrated water samples at all sites sampled, and their infectivity was established based on their cpe on various cell lines. the results produced suggest the infectious potentials of the vlps in the umgeni river water samples, especially river water from the lower catchment areas, to infect the human hosts throughout the year. these observations have serious health care implications if raw untreated water is used for human consumption. however, it should also be considered that not all viral particles detected in this study correspond to infectious viral particles and a high percentage of non-infectious viral particles may be present in the environment [ ] . the present study thus highlights the importance of routine environmental surveillance of human enteric 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oligotrophic marine environments by tangential flow filtration bacteriophage electron microscopy direct electron microscopy study on the morphological diversity of bacteriophage populations in lake plubsee rapid diagnosis of plant virus diseases by transmission electron microscopy abundance of viruses in marine waters: assessment by epifluorescence and transmission electron microscopy frequency of morphological phage description distribution of viruses in the chesapeake bay human adenoviruses in water: occurrence and health implications: a critical review enteric virus isolation in different cell cultures the role of polyomaviruses in human disease propagation of adenovirus type and in the plc/prf/ primary liver carcinoma cell line role of cell culture for virus detection in the age of technology yield and berry composition of five vitis cultivars as affected by rupestris stem pitting virus detecting virus-like particles the authors would like to thank the water research commission of south africa and the national research foundation for funding.the authors have declared no conflict of interest. key: cord- -mso zfom authors: sunkari, emmanuel daanoba; korboe, harriet mateko; abu, mahamuda; kizildeniz, tefide title: sources and routes of sars-cov- transmission in water systems in africa: are there any sustainable remedies? date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: mso zfom governments across the globe are currently besieged with the novel coronavirus (covid- ) pandemic caused by sars-cov- . although some countries have been largely affected by this pandemic, others are only slightly affected. in this regard, every government is taking precautionary measures to mitigate the adverse effects of covid- . sars-cov- has been detected in wastewater raising an alarm for africa due to the poor water, sanitation, and hygiene (wash) facilities. also, most countries in africa do not have resilient policies governing sanitation and water management systems, which expose them to higher risk levels for the transmission of sars-cov- . therefore, this study unearthed the likely sources and routes of sars-cov- transmission in water systems (mainly wastewater) in africa through a holistic review of published works. this provided the opportunity to propose sustainable remedial measures, which can be extrapolated to most developing countries in the world. the principal sources and routes of potential transmission of sars-cov- in water systems are hospital sewage, waste from isolation and quarantine centres, faecal-oral transmission, contaminated surface and groundwater sources, and contaminated sewage. the envisioned overwhelming impact of these sources on the transmission of sars-cov- through water systems in africa suggests that governments need to put stringent and sustainable measures to curtail the scourge. hence, it is proposed that governments in africa must put measures like improved wash facilities and public awareness campaigns, suburbanization of wastewater treatment facilities, utilizing low-cost point-of-use water treatment systems, legally backed policy interventions, and community-led total sanitation (clts). sars-cov- in water systems can be inactivated and destroyed by integrating ozonation, chlorination, uv irradiation, and sodium hypochlorite in low-cost point-of-use treatment systems. these proposed sustainable remedial measures can help policymakers in africa to effectively monitor and manage the untoward impact of sars-cov- on water systems and consequently, on the health of the general public. countries, especially in africa. although some countries have been largely affected by this pandemic, others are only slightly affected. owing to this, every government is taking precautionary measures to mitigate the adverse effects of covid- . some of the symptoms associated with covid- include fever, cough, diarrhoea, and breathing difficulties (who, ) . the world health organization (who) has mentioned that the main routes of exposure of the virus to humans are inhalation of droplets generated when an infected person coughs, sneezes, or exhales (who, ) . however, recent studies have shown that sars-cov- can be found in the faeces of infected symptomatic and asymptomatic patients (foladori et al., ; pan et al., ; randazzo et al., ; tang et al., ; xiao et al., ; zhang et al., a) . clinical experiments and researches have also reported evidence of sars-cov- in urine samples of infected patients (lescure et al., ; ling et al., ) , and hospital and urban sewage (medema et al., ; wu et al., ) . it can be inferred from these studies that the water systems, especially municipal wastewater of areas hit hard by covid- might contain the virus. recently, sars-cov- has been detected in untreated wastewater in australia, the netherlands, usa, and france (ahmed et al., ; medema et al., ; nemudryi et al., ; wurtzer et al., ) corroborating the assumption that the virus could be detected in wastewater. this raises serious concerns for the water systems in developing economies like most african nations. most countries in africa do not have resilient policies governing sanitation and water management systems, which expose them to higher risk levels for the spread of policies regulating the siting of pit latrines in communities but these have been widely not adhered to in most countries. such acts expose people to cross-contamination of the pit latrines with the groundwater sources. consequently, if there are covid- infected patients living in such communities that have their pit latrines closer to their drinking water sources, the risk of getting infected with the virus through oral ingestion of the virus-contaminated water is very high. similar observations about water-borne diseases such as diarrhoea and gastroenteritis in africa are very common (oppong et al., ; samie et al., ) . indiscriminate open defection around surface and groundwater sources in africa have also been reported in the literature (elisante and muzuka, ; anornu et al., ; back et al., ; abanyie et al., ; houéménou et al., ; mutono et al., ; owamah, ) . through open defecation, the carriers of the virus may unknowingly transmit it through their stool to drinking water sources, which can have a debilitating effect on consumers of such virus-contaminated water. there is also a high risk of exposure to the virus by people that use the same toilets (a common practice in africa) because the virus may remain in the aerosol or droplet generated during the use of shared toilets built in small spaces without aeration. on july , senior government medical officers reported in ghana that the commonly known symptoms of cough, cold, fever, and breathlessness often exhibited by covid- patients are not lately shown by covid- patients. now, covid- patients show stomach upset, vomiting, and diarrhoea because the virus attacks the gastro-intestines rather than the previously known lungs attack. these are not well known symptoms of the virus and as such patients may not suspect these to be indicative of the fact they have come in contact with the virus. in addition, since these new symptoms of the virus are oblivious to most doctors, the virus could escalate at the community level. these symptoms are also akin to waterborne diseases and go to buttress the possibility of the virus being transmitted through the domestic usage of the virus contaminated water. also, untreated wastewater is mostly discharged into the environment, which may interact with surface and groundwater resources, thereby contaminating these dwindling resources (williams et al., ) . overall, since most of the people living in africa, especially those dwelling in rural and peri-urban settlements depend on surface and groundwater resources for their domestic water supply, the risk of contracting covid- through sars-cov- contaminated water is very high and thus, the sources and routes of community spread of the virus, which is currently being reported must be critically re-examined. therefore, this review examines the potential sources and routes of sars-cov- transmission in water systems in africa with emphasis on wastewater management systems and proposes sustainable remedial measures to deal with the potential risk of community spread of sars-cov- via wastewater in africa. hence, the review will be a framework for african policymakers in dealing with the covid- pandemic. it will also add to the pool of existing literature on the possible transmission of sars-cov- through wastewater. according to the who, as at , about billion people in the world are still living without basic sanitation facilities (who, ). out of this, about million people representing % of the total number are living in africa. in contrast to the developed world, there is overreliance on non-sewered wastewater and sanitation systems in africa (street et al., ) . however, this goal is yet far from being achieved in africa. for example, in ghana, which is one of the biggest economies in west africa, only % of the population has improved sanitation systems in place (appiah-effah et al., ) , slightly below the improved basic sanitation coverage of % in sub-saharan africa (who, ) . elsewhere in south africa, which happens to be one of the biggest economies in africa, about % of the general public does not have a water-borne sewage system and thus, relies on bucket toilets, pit latrines, and chemical toilets as an alternative (street et al., ) . the same situation is common in most of the african countries. it is well documented in the literature that sub-saharan africa has most of its population sharing toilets vis-à-vis other nations in the world (rheinländer et al., ) . the situation is much worrying in the densely populated urban regions of sub-saharan africa, where public or shared toilets are common (morella et al., ) . gudda et al. ( ) studied pit latrine faecal sludge accumulation in nakuru, kenya and pointed out that all the pit latrines in households were communal and the average number of shared users of a pit latrine was persons. there have not been any reported cases of sars-cov- transmission via faecal-oral or waterborne routes and due to quarantine or self-isolation of persons suspected to be infected with covid- , it is argued that infection through this route is unlikely (amirian, ). nevertheless, this might not hold for shared or communal spaces and the risk of faecal-oral transmission might be common in confined places of abode like hotels and places that are overcrowded . within the next decade (dos santos et al., ) . majority of the population growth is in informal and peri-urban areas, which usually do not have basic amenities like water and sanitation, as a result, urban access to water has been impeded overtime. current estimates reveal that approximately two-thirds of sub-saharan africa's urban residents live in informal settlements and only in nairobi, kenya, these areas contribute about % of urban growth (dos santos et al., ) . the access to water by residents of these informal areas is really appalling when compared to the core urban areas, thus, rapid population growth will only compound the situation (adams, ) . bain et al. ( ) revealed that during the period between and , the annual rate of change of total population with access to basic drinking water amenities in sub-saharan africa was . % and in north africa, it was . %. moreover, the annual rate of change of total population with access to basic sanitation in sub-saharan africa was reported as . % and . % in north africa (bain et al., ) . in terms of hygiene, sub-saharan africa recorded a positive change of . % whereas it was . % in north africa (bain et al., ) . even with positive changes in hygiene, the records suggest that access to basic water, sanitation and hygiene amenities still remains very low in sub-saharan africa. in addition, nhamo et al. ( ) , studied the chances of africa to achieve the sdg by using a composite index approach with three indicators of the sdg ; proportion of the population using safely managed drinking water amenities, proportion of the population using safely managed sanitation amenities, and level of water stress. the results indicate that most of the countries are at various stages of achieving the targets set out in the sdg ( table ). the fact that many of the african countries show declining trends of wash is a clue that it will be difficult for africa to reach the sdg targets by (nhamo et al., ) . j o u r n a l p r e -p r o o f there are several routes through which sars-cov- can enter the water systems, which can eventually lead to its transmission through the water medium ( figure ). some of these routes include wastewater discharged from hospitals, government-approved quarantine and isolation centres and houses used as quarantine and isolation centres. faecal contaminations can be transmitted through water supply network systems, which end up in contaminating the water (arslan et al., ; foladori et al., ) . in africa, domestic water supply is usually from surface and groundwater sources and these also serve as potential routes for transmission of sars-cov- . for example, wastewater is often discharged directly into surface water sources without pre-treatment or proper treatment. if the wastewater discharged into the surface water sources contains fragments of sars-cov- , which is the causative organism of covid- , then it is possible that the water will be contaminated and there is a potential risk of community transmission since most rural dwellers in africa rely on surface water sources for their domestic water supply. in the same way, groundwater sources can also serve as conduits for transmitting sars-cov- if the discharged wastewater that contains fragments of the virus is introduced into the aquifer. the virus contamination can as well occur during groundwater recharge. generally, groundwater contains lesser microbial contaminants known as pathogens than surface water, but the biological integrity of groundwater cannot be underestimated (alley and alley, ). governments to partner with the private sector to deal with the situation. there is the need to provide efficient but cheap sanitation services to the citizenry, especially for the poorest of the poor. the best solution to this menace is the application of financial solutions to support the access of households to improved wash amenities and to encourage wash-related entrepreneurial activities. if governments provide easy access to funds by households and private individuals through microcredit, loans, and micro enterprise financing, this will facilitate the provision of wash amenities for the poor. another important measure for dealing with the possible transmission of sars-cov- through wash-related activities is public awareness through regular behavioural change campaign to revolutionize the thinking of most africans on myths associated with wash activities. if all these measures are put in place in africa, the likelihood for the transmission of sars-cov- and other human enteric viruses that may originate from the stool of infected persons will be very low. adequate and centralized wastewater treatment facilities are a major problem in africa. as explained before, in most african countries, huge amount of the untreated wastewater generated is usually released directly into surface water sources. the adverse effects of this attitude on public health is unquestionable. it is even worrying to know that the quarantine and isolation centres of health care facilities used for testing and treating suspected and confirmed covid- carriers have a common sewerage system with the coterminous communities and cities (adelodun et al., ) . this situation exposes the people to the tendency of using water from surface water sources that might be contaminated with sars-cov- and thus, making them vulnerable to the virus. one of the ways of contracting sanitary-related diseases is through leakage of sewerage systems when conveying wastewater j o u r n a l p r e -p r o o f journal pre-proof contaminated with pathogens and excretes (gormley et al., ) . the best antidote to this threat is separating the sewerage systems and wastewater treatment facilities of the health care centres from that of the urban central systems to facilitate viral removal during treatment process. wastewater treatment facilities should be located in at least each city. this will ameliorate the situation and stop people from indiscriminately discharging wastewater into surface water bodies. wastewater generated in most industries in africa, unfortunately do not pass through the entire cycle or pathway of wastewater flow (figure ). mostly after generation, due to financial or infrastructural challenges, the wastewater does not get treated but is discharged with no benefit (figure ). however, with the needed finances and infrastructure in place, the wastewater generated must be properly treated and discharged or reused. some of the ultimate uses of treated wastewater are for portable water supply, irrigation, and hydro energy ( figure ). governments are overwhelmed with several equally important developmental works but there should be a willpower in dealing with the wastewater treatment issue in africa. to augment the situation, the private sector should be motivated to focus on investing in waste-to-value projects in parts of africa, where waste treatment or recycling facilities are absent. this will help improve the sanitary conditions around physical locations and also improve the health of the people. from water systems using cheaper low-cost point-of-use treatment devices such as zerovalent iron filter, iron-oxide bio-sand filter, nanocellulose-based filter, gravity-based ultrafilter, and many more (bradley et al., ; shi et al., ; chaidez et al., ; mautner, ) . these devices can be effectively used for inactivating and destroying the sars-cov- genetic material, especially in africa where there are no effective wastewater treatment systems. moreover, in view of the recent findings of researches that suggest that sars-cov- can be completely eliminated in water systems by the use of ozonation, chlorination, uv irradiation, and sodium hypochlorite (rosa et al., ; quevedo-león et al., ; wang et al., b) , there is the need for african countries to start integrating them into their low-cost point-of-use treatment systems. the model for such an integration is schematically presented in figure . in this schematic diagram, it can be observed that when the different disinfection techniques are integrated and control measures are put in place to prevent the water from being re-contaminated, the integrity and reliability of the reclaimed water can be enhanced. such an integrated approach of disinfecting contaminated water and wastewater can withstand influent flow and water quality fluxes. the current times require policy interventions that have legal support to direct the attitude of people living in africa. even with the advent of the covid- pandemic, some people living in africa believe covid- is a western disease and that the confirmed cases in their countries are only political gimmicks of their governments who they think largely depend on the western world. in these abnormal times, surface water sources such as streams and rivers, which hitherto were used as domestic water supply sources in some villages have to be j o u r n a l p r e -p r o o f protected from encroachment by the rural people. in this regard, governments have to find alternative ways of meeting the water supply needs of rural people. by doing so, sanctions can be meted out to violators. at this critical point in time, wastewater should be treated before discharge into the environment. hence, there must be stringent measures regulating wastewater treatment and culprits should be punishable by law. employees in wastewater treatment facilities must thus take maximum precaution to prevent themselves from possible infection. at least, it is encouraging that some african countries like ghana, nigeria, cameroon, south africa, angola, benin, burkina faso, equatorial guinea, ethiopia, gabon, guinea, kenya, liberia, rwanda, dr congo, sierra leone, zambia, and morocco, have already put law-supported punitive measures in place for people who do not wear masks in public places in the wake of the covid- pandemic. this sets the basis for others to follow suit if only they want to fight the covid- pandemic. however, the safety protocols provided by the who and ministries in charge of health in all countries should be strictly adhered to and people who violate them in public places should be sanctioned. this is an innovative way of allowing communities to initiate their own actions towards addressing their own problems. the aim is to help improve wash practices and it is currently being implemented in many developing countries through the guidance and support of non-governmental organizations. in africa, it is difficult for governments to be able to spearhead every developmental project. this calls for active participation of community members themselves and important stakeholders in society. one of the primary targets of the since previous approaches to curtail sanitation problems only set high standards with subsidies as incentives to rural people. overtime, this never became sustainable due to the uneven adoption and partial use of the toilet facilities. moreover, it has made rural dwellers over reliant on subsidies, which are not also regular. in this regard, faecal-oral transmission through open defecation persisted leading to the spread of waterborne diseases. so instead of merely providing the 'hardware' for wash practices, there is the need for behavioural change to ensure sustainable improvement and this can only be done through clts. investing in community mobilisation should be at the heart of the clts programs such that the focus on toilet construction for individual households will be shifted to making sure villages and communities are actually open defecation-free. through awareness creation with the message that the continuous practice of open defecation by even the minority in the community will expose everyone to the risk of diseases, clts will prompt the community's crave for collective change, will push people into taking actions and development of innovative ideas, will provide mutual support and proffer suitable local solutions to local problems, which in the end will lead to complete ownership and sustainability. the impact of the covid- pandemic is currently felt everywhere in the world and in all aspects of the economy. the impact is even envisioned to be more severe in the developing world like africa, which is still struggling to meet the basic necessities of life for the people living there. since most of the people living in africa, especially those dwelling in rural and peri-urban settlements depend on surface and groundwater resources for their domestic water supply, the risk of contracting covid- through sars-cov- contaminated water from wastewater systems is very high. hospital sewage, waste from isolation and quarantine centres, contaminated surface and groundwater sources, faecal-oral transmission, and j o u r n a l p r e -p r o o f assessment of the quality of water resources in the upper east region, ghana: a review thirsty slums in african cities: household water insecurity in urban informal settlements of lilongwe, malawi clinical and virological data of the first cases of covid- in europe: a case series persistence and clearance of viral rna nanocellulose water treatment membranes and filters: a review presence of sars coronavirus- in sewage and correlation with reported covid- prevalence in the early stage of the epidemic in the netherlands monitoring progress of the role of integration of environmental health education with water and sanitation services in changing community behaviours temporal detection and phylogenetic assessment of sars is too soon for africa to achieve the water and sanitation sustainable development goal? improper solid waste management increases potential for covid- spread in developing countries economic analysis of wastewater reuse in covenant university enteric pathogens associated with gastroenteritis among children under years in sub-saharan africa: a systematic review and metaanalysis a comprehensive assessment of groundwater quality for drinking purpose in a nigerian rural niger delta community. groundwater for sustainable development burden of disease from inadequate water, sanitation and hygiene for selected adverse health outcomes: an updated analysis with a focus on low-and middle-income countries inactivation of coronaviruses in food industry: the use of inorganic and organic disinfectants, ozone, and uv radiation sars-cov- rna in wastewater anticipated covid- occurrence in a low prevalence area redefining shared sanitation covid- coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist , and therapeutic drugs , and analysis of a proposed achilles' heel conserved region tominimize probability of escape mutations and drug resistance prevalence and genetic characterization of giardia lamblia in relation to diarrhea in limpopo and gauteng provinces water supply, sanitation and health risks in douala municipality removal of viruses and bacteriophages from drinking water using zero-valent iron covid- wastewater surveillance: an african perspective hydrochemistry with special reference to fluoride contamination in groundwater of the bongo district hydrogeochemical characterization and assessment of groundwater quality in the kwahu-bombouaka group of the voltaian supergroup surgo foundation, . africa covid- community vulnerability index (africa ccvi) detection of novel coronavirus by rt-pcr in stool specimen from asymptomatic child sarscov- rna detection of hospital isolation wards hygiene monitoring during the coronavirus disease outbreak in a chinese hospital disinfection technology of hospital wastes and wastewater: suggestions for disinfection strategy during coronavirus disease un-water global analysis and assessment of sanitation and drinking-water (glaas) report: investing in water and sanitation: increasing access, reducing inequalities who climate, sanitation and health modes of transmission of virus causing covid- : implications for ipc precaution recommendations billion people lack safe drinking water at home, more than twice as many emerging contaminants in a river receiving untreated wastewater from an indian urban centre prolonged presence of sars-cov- viral rna in faecal samples time course quantitative detection of sars-cov- in parisian wastewaters correlates evidence for gastrointestinal infection of sars-cov- hydrogeochemical controls and human health risk assessment of groundwater fluoride and boron in the semi-arid north east region of ghana isolation of -ncov from a stool specimen of a laboratory confirmed case of the coronavirus disease (covid- ) the first author thanks the scientific and technological research council of turkey (tÜbİtak) for the continuous support as a doctoral fellow of bideb graduate scholarship program for international students. the editors, especially prof. damià barceló and the anonymous reviewers, are also sincerely acknowledged for their useful reviews that improved the quality of this paper. key: cord- -nntcac k authors: retzlaff, kimberly j. title: water utility communications can build trust during the covid‐ pandemic date: - - journal: j am water works assoc doi: . /awwa. sha: doc_id: cord_uid: nntcac k in april , awwa hosted a webinar on how utilities can be a trusted source of information during the covid‐ pandemic. following a communications plan and using a spokesperson, water utilities should focus on simple messages that use basic terminology. utilities should express empathy, focus on facts, and be transparent to build and reinforce public trust during a crisis. the current public health emergency is different from anything the world has seen in recent human history. because the public is paying more attention to authoritative sources during a crisis, effective communication is even more important than during "normal" life. utilities have several tools available to craft their messaging and tailor it appropriately, both for internal and external audiences. honest, frequent, clear, and concise communication is essential in times of crisis. also important is demonstrating compassion, because "no one will care what you know until they know that you care," villegas said. to craft a caring message, one helpful tool is the cap rule ( figure ), which can assist utilities by focusing on three main elements of communication: • a caring message-demonstrate empathy and shared concern (e.g., i understand your concern; i'm concerned about this, too). • an action message-explain what actions have been or will be taken to address the problem (e.g., tap water is safe; disinfection kills viruses, including the coronavirus). • a perspective message-put the issue into context (e.g., the united states has some of the highest standards for tap water in the world, and we consistently meet those standards). even when a message shows caring, in times of stress, people are less able to absorb the cap rule for compassionate messaging figure courtesy of the center for risk communication; adapted with permission c caring message: provide a message indicating concern, empathy, or compassion. the message should communicate the seriousness of the situation. state actions you have taken, are taking, or will take to address the issue or problem. for example, the message might indicate you are cooperating with other organizations or investigating the situation. complicated messages, so it's important to use concise and simple language. to achieve this, follow the - - rule ( figure ): use no more than words that take seconds to say with pieces of information. this messaging format is particularly good for communicating face to face, on the phone, by social media, or on camera. additional information can be placed on the utility's website for those who want a longer read. during a crisis, communication efforts should build trust from the inside out, dearing smith said. start with employee communication and then expand that to communicate externally. depending on the utility's employee structure, any employee could be a point of contact for customers and the community, so sharing information with the entire team helps ensure consistent messaging. louisville water relied on its crisis team and existing communications plan, which was tailored to responding to the rapid coronavirus outbreak. for smaller utilities that may not have a communications team or robust response plans, communications planning can be arranged using a simple "key message" template ( figure ). message mapping is another useful tool-guidance and templates for this are available from the center for risk communication. at louisville water, internal communications consist of a crisis team meeting, officers' team meeting, and messages to staff-each of which take place daily. the crisis team meeting related to covid- response is led by the chief executive officer (ceo)/president and includes people who meet for minutes each morning to discuss safety and the local and state situation. for these types of meetings, it's important to include the utility's auditor, or in lieu of that, someone can be assigned to take notes so the utility's efforts are documented. the officers' meeting is a chance for utility leaders to discuss big-picture concerns, human resources (hr) and information technology needs, revenue, employee morale, and industry conversations. the daily email to staff is brief-two or three sentences, following the cap rule-to provide important information and keep everyone connected. additionally, a leadership video from the ceo/president, produced using a cell phone, goes out about once a week via the utility's intranet site or youtube channel as another way to keep employees and leaders connected. the covid- crisis provides an opportunity to raise public awareness of the critical service that water utilities provide every day. talking to customers about the safety of tap water offers positive news amid a lot of negativity, and it also offers reassurance to the public. the key to good crisis communication is to be clear, consistent, and concise. messaging can be crafted using the cap principle and - - rule. once the utility's message is developed, information should be shared through multiple channels, depending on the community's preference, including newspapers, local access television, radio, social media, utility and partner websites, and/or direct mail. the human brain can effectively process no more than words that can be spoken in seconds and that have (or fewer) pieces of information. seconds with information changing rapidly, as has happened during the coronavirus pandemic, transparency and consistency are more essential, so key stakeholders should meet regularly. if applicable, union leaders should be included in update meetings, too. external communications should be proactive, so think through potential questions and concerns and craft responses that support messaging. the utility's call center is a good place to start. public meetings are usually effective at engaging a community and disseminating important information; however, these need to be held via web conference during times of social distancing. there are many platforms that can be used for this type of virtual meeting (see the sidebar on page for examples), but before organizing a virtual meeting, leaders should agree on all communication goals and clarity of purpose. for example, is the meeting for information only, or is public input being sought for a decision? for in-person and virtual events, enlisting a professional moderator or facilitator can help keep the conversation productive and minimize disruptive or aggressive attendee behavior. while many employees may have points of contact with external stakeholders, it's a good idea to assign a spokesperson for the utility. this person could be part of the communications team if the utility has one, or it could be the ceo, the general manager, or another leader. regardless, the spokesperson must be a good public speaker and someone who is credible, affable, and trustworthy. it helps if the spokesperson can convey information calmly and communicate clearly. providing the spokesperson with training on presentations and media interviews can increase the speaker's comfort level. the - - rule can be used to compose messaging that audiences will understand and retain. any messaging should use simple terminology, not industry-specific or technical language. one tool to help determine an appropriate language level is the centers for disease control and prevention's clear communications index. social media is an excellent tool during crisis communications that can be used to reach employees and customers alike. however, it is important to consider that successful social media requires using the proper tone, including graphics and video, getting employees involved, and being responsive to engagement. besides including relevant information, messaging during a crisis often requires a serious or professional -traditional -neighborhoods -stakeholders tone, but it's acceptable to post lighthearted messages when they can inspire hope and positivity. the use of graphics and videos draws attention and boosts engagement. utilities can use content they already have on hand or have employees send in photos and videos from their daily work to post to their social media accounts. in the current environment, filming new videos should be avoided unless they demonstrate employee safety and good social distancing practices. monitoring a utility's social media accounts is always important, especially during a crisis. someone on staff should be responsible for responding to online questions and comments so customers and the broader community feel heard and community issues can be recognized. for louisville water and fort worth water department, engagement is up as a result of the crisis because customers are actively seeking information. one post on louisville water's social media about halting shutoffs reached approximately , people and was shared more than , times. fort worth water department's average engagement was up % in march across all platforms, and almost half a million people saw the utility's facebook content in march on both its english-and spanish-language pages. in cases where a utility shares broader city platforms for social media, it could be helpful to work with the city communications team to promote water utility activities, for example, showcasing how utility operations protect public health. one example from the city of denton, texas, is a youtube video showing how the city works for citizens through water and wastewater, police, fire, electric, and solid waste services. another option to help differentiate water content across a shared platform is to create a hashtag that is consistently used for all water utility posts. water utilities have a responsibility to protect their employees, and this includes providing transparent communication when employees are at risk. in the current pandemic, communication should include utility guidelines related to an employee who tests positive for covid- , an employee who feels ill with flu-like symptoms, an employee who is asymptomatic but has had direct or close contact with someone who tests positive for covid- , and an employee who has had direct or close contact with someone who had direct contact with someone who tested positive. the actions required on the part of the employee and the employer may vary in each scenario, and steps may be considered for communicating about each of them accordingly. when preparing to communicate with staff about these sensitive topics, it's important to consult with the utility's hr and legal teams and to ensure that employees feel supported. any communication activities must maintain employees' health data privacy as well. if an employee tests positive for the virus, the employer should communicate with staff that a colleague tested positive and-depending on hr and legal guidelinesspecify which department and locations were affected. cleaning staff should be directed to properly disinfect all affected areas, and utility staff should be told what actions are being taken to ensure their safety in the workplace. to minimize the time it takes to notify staff of an employee testing positive for covid- , template language can be prepared ahead of time for quick use. in the tragic event that an employee passes away from an illness related to covid- or under other tragic circumstances, a utility's leader should share the news with staff. delivering this kind of news face-to-face to everyone at once is best; if not in person, video conference is a good alternative. if the employee's family allows it, the utility can notify external stakeholders after employees have been notified as well. for external communications about employee health, it can be helpful to consult guidance from state regulatory agencies on required actions. messaging should focus on employee safety and privacy, the continued safety of tap water, and actions the utility is taking to protect employees and system operations, such as cleaning protocols or alternate work or housing arrangements. many sectors, including the water sector, have implemented alternate work arrangements to help ensure the given "safer at home" requirements throughout north america, online meeting tools are being improved and gaining in popularity. there are many platforms that can accommodate online discussions, presentations, comments, and question-and-answer sessions, such as the following: • clickmeeting: https://clickmeeting.com • gotomeeting: www.gotomeeting.com • microsoft teams: www.microsoft.com/ en-us/microsoft- /microsoft-teams/ group-chat-software • skype: www.skype.com/en • tele-town hall: https://teletownhall.com • webex: www.webex.com • zoom: https://zoom.us safety of staff and the community. in the case of water utilities, some staff can work remotely, and other arrangements can be made to help protect those who must be at the utility or in the community for work. operations staff may need special arrangements, including isolating critical members or crews, even sheltering onsite at the plant in extreme cases, as was done at california's carlsbad desalination plant. if employees are working from home, it's advisable to be transparent with the community about it. share what the utility is doing to protect employees and the community by allowing employees to work from home where feasible. in the case of louisville water, the message to the community has been that the utility is an essential employer, but that doesn't mean that all employees need to do their essential job functions in a traditional work setting. beyond allowing work from home, utilities should also tell their communities how they're keeping employees who must report to work safe and healthy. according to a new analysis prepared by raftelis for awwa and the association of metropolitan water agencies, it's estimated that drinking water utilities in the united states will see revenues from customer payments drop by nearly $ billion. in responding to financial hardship, many utilities have suspended water shutoffs during the crisis. it's also important to consider that many customers may be unemployed as a result of the pandemic and tailor messaging to be sensitive to this. the key is to get in front of the situation as much as possible and to proactively reach out to consumers to set up payment plans while being as understanding as possible. for example, fort worth water department is letting customers know when they are past due on bills but has softened the tone and changed the message to encourage customers to pay what they can. louisville water launched a campaign to "pay what you can" in mid-april and then worked on a larger strategy for messaging, bill assistance, and community partnerships to help customers who fall behind on payments as a result of the pandemic. water utilities still have financial responsibilities, too, and some may have planned for rate increases before the pandemic started. in the case that the increases can't be delayed, methods for communicating should be varied and include online tools, phone calls, and e-mail messages. not all customers will have access to online tools, and lower-income populations will be affected more dramatically. as with communicating about finances during a crisis, other topics also may need to be shared with the utility's community. for example, if a treatment process is changing or a contaminant is identified, the utility needs to share this information with the community in context. important regular communications should also continue to go out as planned. messaging about treatment changes or other safety concerns should be transparent, accurate, appropriate, and timely and should be shared in simple language that's easily understood. it should incorporate any language required by the applicable regulatory agency and be sensitive to the crisis. for example, the communication may start off by acknowledging that people are at home but that the water utility is continuing its normal operations to provide safe water, and then go on to explain any operational changes that have or may become necessary. as another example, customers may have questions about the effectiveness of a treatment technology related to a potable reuse project. the messaging that has been used for these projects, focusing on the efficacy of science and technology, is especially relevant to covid- discussions. in the case of a contamination issue, context is important. for example, in discussing per-and polyfluoroalkyl substances contamination, the utility can describe what actions have been taken over specific time periods to address the issue, reinforcing that water quality is a priority. regularly scheduled communications such as annual water quality reports should go out by mail and online as planned to help maintain consistency and build trust with customers. staying on track with regular communications reinforces the utility as a trusted resource. questions related to water quality and covid- should be expected, and customer service and field crews should be given up-to-date information that can be shared publicly. for any online covid- messaging, link directly to the utility's consumer confidence report, reinforcing the emphasis on water quality and showing where customers can go to find more information or speak with someone if they have questions. besides including relevant information, messaging during a crisis often requires a serious or professional tone, but it's appropriate to post lighthearted messages when they can inspire hope and positivity. beyond consistently providing safe and reliable service, water utilities can build and reinforce public trust through their communications efforts. during times of uncertainty, utilities should carefully craft all messages (for both internal and external audiences), convey empathy, and share facts. water treatment in north america is extremely reliable, and water professionals are dedicated to protecting public health. sharing these facts consistently and broadly bolsters communities' confidence in their utilities and reinforces system reliability, which in times like these can help reassure a stressed and anxious public. covid- and subsequent question-and-answer session, on which this article is based, and for the panelists' review retzlaff is senior managereditorial at awwa in denver. she can be reached at kretzlaff@awwa.org awwa resources • coronavirus (covid- ) • trending in an instant. awwa. . www.awwa.org/policy-advocacy/communications-outreach • reduction in u.s. water utility revenue may result in $ billion economic hit awwa, denver. www.awwa.org/awwa-articles/reduction-in-uswater-utility-revenue-may-result-in- -billion-economic-hit these resources have been supplied by journal awwa staff. for information on these and other awwa resources thank you to the panelists of the awwa webinar on key: cord- -uytnpn f authors: abu shmeis, reham m. title: chapter one water chemistry and microbiology date: - - journal: comprehensive analytical chemistry doi: . /bs.coac. . . sha: doc_id: cord_uid: uytnpn f abstract water is essential to life; therefore, providing access to safe and clean water is a major concern for health development at the local, national, and regional levels. enhancing water quality by improving sanitation systems needs a profound understanding of water chemistry and microbiology. this chapter will give an overview of water chemistry and microbiology. the chapter will be divided into two main parts: the first one will discuss water chemistry in terms of its physical properties and the main chemical processes that takes place within water bodies, the main parameters that govern the water quality, and water quality management. this section will finally mark out the basics of water treatment processes. the second part will be devoted to water microbiology where the main concepts that are encountered when discussing water microbiology will be outlined. an overview will be given of the different types of microorganisms and their classification as well as the main processes they carry out in living cell such as metabolism, respiration, and microbial growth. a brief synopsis will be depicted about the main diseases caused by microorganisms that could be present in water bodies, the main microbial indicator, as well as the main analytical tests for microbial indicators. this section will also give a short overview about microbial control and the terms related to it such as disinfection. the final part of this section will delineate the role of microorganisms in wastewater treatment plants. water is a simple molecule that has only three atoms, but its unique properties make it the most important molecule in life. this extraordinary importance of water molecule came basically from the arrangement of atoms in the molecule and the bonds involved. all life processes involve water. water is an excellent solvent for many ionic compounds, as well as for other substances capable of forming hydrogen bonds with water. water molecule with molecular formula h o has two hydrogen atoms and one oxygen atom; the oxygen atom is connected to the two hydrogen atoms by two covalent bonds (fig. ) . this bond is a polar covalent bond with concentration of the electron density around oxygen which is one of the most electronegative atoms. this arrangement gives water molecule a dipolar character where oxygen atom is the negative pole and hydrogen atoms are the positive poles. this dipole character enables water molecules to be connected among themselves by intermolecular forces called hydrogen bonds. hydrogen bonds are a relatively strong type of intermolecular interaction in which hydrogen atom from a certain molecule is connected to an electronegative atom (such as oxygen, nitrogen, or fluorine) of another molecule. although many compounds can form hydrogen bonds (such as nh and hf), the difference between h o and other polar molecules is that each oxygen atom can form two hydrogen bonds, the same as the number of lone electron pairs on the oxygen atom. thus, water molecules are joined together in an extensive three-dimensional network in which each oxygen atom is approximately tetrahedrally bonded to four hydrogen atoms, two by covalent bonds and two by hydrogen bonds. this confers on water its unique physical and chemical properties that will be discussed in the next sections (chang, ; ebbing and gammon, ; manahan, ) . table summarizes some of the basic physical properties of water. most of these properties are highly affected by temperature. therefore, some values are given at different temperatures. also the effect of these properties on boiling point °c at atm water behavior is highlighted briefly (chang, ; ebbing and gammon, ; manahan, ; venkateswariu, ) . before discussing the chemical properties of water we shall introduce some terms that will be encountered a lot in the coming sections. a solution is a homogeneous mixture of two or more substances, consisting of ions or molecules. solute is the component present in smaller amount and solvent is the component in greater amount. the general term concentration refers to the quantity of solute in a standard quantity of solution. concentration might have different expressions depending on the unit of the amount of solute and the unit the amount of solution or solvent. when the solvent is water, it is called aqueous solution. the molar concentration or molarity (abbreviated m) of a solution is the number of moles of the solute species that is contained in l of the solution. it has the dimensions of mol/l. percent concentration is another way of expressing concentration. percent composition can be expressed as mass percent (w/w %) which is the percentage of the mass of solute over the mass of solution. or simply concentration could be expressed in units of mass per volume (i.e., g/l, mg/l). we can divide the substances that dissolve in water into two broad classes, electrolytes and nonelectrolytes. an electrolyte is a substance that dissolves in water to give ions that make the solution an electrically conducting solution such as sodium chloride. a nonelectrolyte is a substance that dissolves in water to give a nonconducting solution because it does not form ions such as sucrose, table sugar (chang, ; ebbing and gammon, ). there are principally three types of chemical reactions that could take place in water: precipitation, acid/base, and oxidation-reduction reaction. in precipitation reaction, dissolved ions react with each other and form a solid compound or precipitate. a typical precipitation reaction that occurs in water is the formation of calcium carbonate solid when solution of calcium is mixed with solution of carbonate (davis and cornwell, many definitions are available for acids and bases. however, for the purpose of simplicity here, acids can be defined as proton donor and bases as proton acceptor. a neutralization reaction is a reaction of an acid and a base that results in an ionic compound and possibly water. when a base is added to an acid solution, the acid is said to be neutralized. the ionic compound that is produced in a neutralization reaction is called a salt (chang, ; ebbing and gammon, ). the net equation for most acid base reaction is: oxidation-reduction reaction is a reaction in which electrons are transferred between species or in which atoms change oxidation number. the substance that loses the electron is said to be oxidized and it is called a reducing agent. while the substance that accept the electron is said to be reduced and is called oxidizing agent. when one species release electron, another one should be available to accept the electron. iron pipe corrosion is an example of oxidation-reduction reaction in which iron metal oxidizes and loses two electrons, while hydrogen ions accepts those electrons and reduces to hydrogen gas (chang, ; davis and cornwell, ; ebbing and gammon, ) fe ! fe + + e À h + + e À ! h g ð Þ the ph of a solution is a measure of hydronium ion (h o + ) concentration, which is, in turn, a measure of acidity. in acidic solutions the ph is smaller than , while it is greater than in basic solutions. the ph range in water samples is rarely below or above . determining ph of water is essential as it affects many of the chemical and biological processes that take place in water (davis and cornwell, ; venkateswariu, ) . alkalinity refers to the capability of water to neutralize acid. it is defined as the sum total of all titratable bases down to about ph . . it is found experimentally by determining how much acid it takes to lower the ph of water to . in most waters the only significant contribution to alkalinity is the carbonate species and any free h + or oh À . the total h + that can be taken up by water containing primarily carbonate species is alkalinity ¼ hco À ½ + co +  à + oh À ½ À h + ½ in most natural water situation (ph - ) the oh À and h + are negligible; therefore, alkalinity ¼ hco À ½ + co +  à note that co À ½ is multiplied by because it can accept two protons h co ! h + + hco À ! pk a ¼ : hco À ! h + + co À ! pk a ¼ : from this we can see that: below ph . all carbonates are h co and the alkalinity is negative -ph . - . all carbonates are hco À and alkalinity equals hco À ½ above ph . all carbonates are co À and alkalinity equals co + ½ + oh À ½ by convention alkalinity is not expressed in molarity units as shown in the above equations, but rather as mg/l as caco (davis and cornwell, ; manahan, ) . hardness is a term usually used to characterize water. technically, it is the sum total of all polyvalent cations. practically it is the sum of calcium and magnesium ions which are predominant cations in natural waters. hard water requires more soap and synthetic detergent for laundry and contributes to scaling in boilers and industrial equipment. hardness is expressed in mg/l as caco , as in alkalinity. both calcium and magnesium have a valence of two when converting to caco . the sum of calcium and magnesium is the total hardness (th), which is subdivided to carbonate and noncarbonated hardness. carbonate hardness is often called temporary hardness because heating the water will remove it. when water is heated, the insoluble carbonates precipitate and tend to form bottom deposits in hot water heaters. carbonate hardness is equal to the total hardness or alkalinity, whichever is less. noncarbonated hardness is correspondingly called permanent hardness because it is not removed when water is heated. noncarbonated hardness is the total hardness in excess of the alkalinity. if the alkalinity is equal to or greater than the total hardness, there is no noncarbonated hardness (davis and cornwell, ; manahan, ) . the cyclic movement of water through the environment is called the hydrologic cycle. the global hydrological cycle is powered by solar energy. it is a closed system, in continual circulation. the hydrologic cycle, illustrated in fig. , begins as water moves from the ocean's surface into the air above through evaporation. water evaporates into the atmosphere and forms clouds above the ocean. additional water is drawn from the soil by plants, and is then evaporated into the atmosphere from leaves and stems in a process called transpiration. as the air rises and the temperature drops, the moisture-laden air condenses, forming clouds and eventually resulting in precipitation. precipitation is the term applied to all forms of moisture falling to the ground such as rain, snow, sleet, and hail. in some cases, the raindrops soak into the earth and move slowly into the groundwater. sometimes it runs off the land surface and moves quickly in a swift-flowing stream. other times, the raindrop rests in deep river pools or lakes, is taken up by plants and animals, or enters the atmosphere again through evaporation. other water seeps downward into the soil. this process is called infiltration. if the rock below the soil is permeable, then the water percolates the rock and is stored as groundwater. ultimately, all water makes its way back to the ocean, which is like a giant reservoir. water is stored in the ocean until it is delivered to the land as a result of evaporation and precipitation and the cycle continues (davis and cornwell, ; manahan, ; vigil, ) . according to the united states geological survey (winter et al., ) , most of the fresh water ( . %) is locked up as ice in glaciers. of the balance, . % constitutes groundwater, while that in lakes and reservoirs mounts to . %. another . % is in form of soil moisture and atmospheric water vapor. thus, only a very small fraction of fresh water, about . %, flows through rivers and streams. the volume of sea water is times greater than that of fresh water. natural waters can be classified into two categories: sea water (inclusive of estuarine water) and fresh water. surface waters might possess color, odor, taste, suspended solids, etc. groundwaters are expected to be free from organic odor and have a relatively less variable composition at the same source. the united states geological survey (venkateswariu, ; winter et al., ) has classified different waters on the basis of their total dissolved solids (tds) content as given in table . many diverse factors have to be taken into account before making comments on water quality. for this reason, the concentrations of inorganic (venkateswariu, ; winter et al., ) water quality tds (mg/l) less than , slightly saline , - , moderately saline , - , very saline , - , briny greater than , and organic substances dissolved in a body of water and their amount need to be monitored regularly. agents that alter water quality can be classified under four major categories. . . physical agents physical properties of water are related to the appearance of water, namely, the color, temperature, turbidity, taste, and odor. to be suitable for use, water must be free from all impurities that are offensive to the sense of sight, taste, or smell and one very important physical characteristic that should be encountered when discussing water quality is turbidity (davis and cornwell, ) . the presence of suspended materials such as clay, slit, finely divided organic material, plankton, and other inorganic materials in water is called turbidity. turbidity is a measure of the clarity of water. low-turbidity water is clear, while high turbidity water is cloudy or murky. the unit of measuring turbidity is turbidity unit (tu). turbidity larger than tu is easily detected in a glass of water and is objectionable for aesthetic reasons (davis, ; davis and cornwell, ) . those include all the major dissolved constituents in water such as humic substances, in addition to the minor constituents such as heavy metals, detergents, pesticides. the chemical analysis of a domestic water supply should ordinarily include the determination of water hardness, alkalinity, ph, conductivity, and the presence of chloride, sulfate, and nitrate. other chemicals of importance are: iron, manganese, fluoride, copper, sodium, and zinc, in addition to some toxic substances such as arsenic, barium, cadmium, chromium, lead, mercury, selenium, silver, and cyanides. the significant concentrations with respect to several chemicals that might be present in natural waters are given in table . above these levels, such chemicals can cause undesirable effects (davis, ; davis and cornwell, ; venkateswariu, ) . the american water works association (awwa) has issued its set of goals with respect to major physical and chemical agents. these goals are shown in table . biological agents are very important due to their relation to public health and may also be significant in modifying the physical and chemical characteristics of water. water for drinking and cooking should be free from disease-causing organisms (pathogens). these organisms include bacteria, protozoa, viruses, fungi, and helminthes (worms) (davis and cornwell, ) . (venkateswariu, ; winter et al., ) this factor must be considered in areas where there is a possibility that water comes in contact with radioactive substances. radioactive substances could find their way to drinking water from atomic energy power sources and the mining of radioactive materials, as well as naturally occurring radioactive materials (davis, ; davis and cornwell, ) . seasonal variations in the quality of some surface waters could be large enough to make the use of such waters more problematic. the selfpurification capacity and the water intake structure are also important factors that affect the quality of water. whatever might be the quality of water available to a user, it can certainly be upgraded by properly designed and executed treatment procedures. governments with the aid of organizations such as environmental protection agency (epa) and world health organization (who), as well as local water authorities have set specifications for water. these specifications vary according to the intended use of water (for drinking, agriculture, industry, or disposal wastewater into the aquatic system or landfills). water specifications lay down the maximum contamination levels or maximum permissible level with respect to several chemicals and pathogens that might affect water quality in order to meet minimum requirement for protection of public health (davis, ; davis and cornwell, ; weiner and matthews, ) . water pollutants are categorized as point source or nonpoint source. when water pollution arises from a single source, this is called point source pollution (an example would be chemicals from a single factory). conversely, when pollution affecting a body of water issues from multiple sources (multiple factories), it is called nonpoint source pollution. point source pollutants are all dry-weather pollutants that enter watercourses through pipes or channels. point source pollution comes mainly from industrial facilities and municipal wastewater treatment plants. storm drainage, agricultural runoff, construction sites, and other land disturbances are considered nonpoint source pollution (weiner and matthews, ) . the range of pollutants is vast. the major pollutants that could affect water quality are overviewed here. oxygen-demanding materials might be discharged from municipal wastewater treatment plants, food-processing plants, breweries, as well as paper mills, compose one of the most important types of pollutants because these materials decompose in the watercourse, and can deplete the water of dissolved oxygen. sediments and suspended solids (ss) may also be classified as a pollutant. sediments consist of mostly inorganic material washed into a stream as a result of land cultivation, construction, demolition, and mining operations. sediments interfere with fish spawning because they can cover gravel beds and block light penetration, making food harder to find. sediments can also damage gill structures directly, smothering aquatic insects and fishes. organic sediments can deplete the water of oxygen, creating anaerobic conditions, and may create unsightly conditions and cause unpleasant odors. nutrients, mainly nitrogen and phosphorus, can promote accelerated eutrophication, or the rapid biological aging of lakes, streams, and estuaries. phosphorus and nitrogen are common pollutants in residential and agricultural runoff, and are usually associated with plant debris, animal wastes, or fertilizer. heat may be classified as a water pollutant when it is caused by heated industrial effluents or from alterations of stream bank vegetation that increase the stream temperatures due to solar radiation. heated discharges may drastically change the ecology of a stream or lake. although localized heating can have beneficial effects like freeing harbors from ice, the ecological effects are generally harmful. heated effluents lower the solubility of oxygen in water because gas solubility in water is inversely proportional to temperature, thus reducing the amount of dissolved oxygen available to oxygen-dependent species. heat also increases the metabolic rate of aquatic organisms (unless the water temperature gets too high and kills the organism), which further reduces the amount of dissolved oxygen because respiration increases. synthetic chemicals and pesticides, herbicides, fertilizers, pharmaceutical substances such as antibiotics, cosmetics and personal care products, detergents, toxic chemicals, and heavy metals can adversely affect aquatic ecosystems as well as making the water unusable for human contact or consumption. these compounds may come from municipal wastewater, industrial effluents, or agricultural and urban runoff. pathogenic microorganisms are important pollutants that directly affect human health. water-borne pathogen contamination in ambient water bodies and related diseases are a major water quality concern throughout the world. water-borne diseases (i.e., diarrhea, gastrointestinal illness) are caused by various bacteria, viruses, protozoa, algae, and fungi. a major pathogen is fecal coliform bacteria (i.e., escherichia coli) that is the bacteria that normally live in the intestinal tract of warm-blooded animals and indicate contamination by animal wastes. other bacterial pathogens include vibrio cholera which cause cholera, and shigella and salmonella that cause dysentery. other types of microorganisms that could contribute to biological water pollution are: protozoa (such as cryptosporidium parvum, giardia lamblia, entamoeba histolytica that cause diseases such as cryptosporidiosis, giardiasis, and amoebiasis); viruses such as coronavirus, hepatitis a virus (hav) that cause hepatitis a, and poliovirus which cause poliomyelitis; algae such as desmodesmus armatus that cause desmodesmus infection; and several fungi such as aspergillus which most frequently affects the lungs. some higher organisms such as nematodes could be present in water and lead to water-borne disease (tortora et al., ) . such species can be introduced into water bodies as the result of municipal and industrial wastewater discharges, or as a result of aquaculture activities. in addition to causing diseases, the presence of these organisms in water could alter the original microbial floral community in those water bodies. oil pollution can result from leak out of oil from huge tanker loaded with crude oil and cause water pollution with petroleum compounds. acids and bases from industrial and mining activities can alter the water quality in a stream or lake to the extent that it kills the aquatic organisms living there, or prevent them from reproducing. sulfur-laden water leached from mines, including old and abandoned mines as well as active ones, contain compounds that oxidize to sulfuric acid on contact with air (davis and cornwell, ; manahan, ; weiner and matthews, ) . oxygen-demanding materials and nutrients are pollutants of significant importance; they deserve particular focus and will be the subject of discussion in the following sections. dissolved oxygen (do) refers to the level of free, noncompound oxygen (o ) dissolved in water or other liquids. the bonded oxygen in water (h o) is in a compound and does not count toward dissolved oxygen levels. do is an important parameter in assessing water quality because of its influence on the organisms living within a body of water. oxygen gets into water by diffusion from the surrounding air, by aeration, or as a waste product of photosynthesis. do is essential to the survival of organisms in a stream. the presence of oxygen is a positive sign and the absence of oxygen is a sign of severe pollution. waters with consistently high dissolved oxygen are considered to be stable aquatic systems capable of supporting many different kinds of aquatic life (davis and cornwell, ; weiner and matthews, ) . anything that can be oxidized in the receiving water with consumption of molecular oxygen is termed oxygen-demanding material. these materials are usually biodegradable organic compounds but also include some inorganic compounds. the consumption of do poses a threat to higher forms of aquatic life. the critical level of do varies greatly among species. for example, brook trout may require . mg/l of do, while crab may survive at mg/l. oxygen-demanding materials in domestic sewage come primarily from human waste and food residue. almost all naturally organic matters such as animal droppings, crop residue, or leaves, which get into water from nonpoint sources, contribute to the depletion of do (davis, ; davis and cornwell, ; weiner and matthews, ) . biochemical oxygen demand (bod) is the most commonly used method for measuring the quantity of organic oxygen-demanding materials. bod is a measure of the quantity of oxygen used by aerobic microorganisms (need molecular oxygen for living) in the oxidation of organic matter present in a given water sample at certain temperatures over a specific time period. the bod value is most commonly expressed in milligrams of oxygen consumed per liter of sample during a prescribed period of incubation at °c and is often used as an indicator of the degree of organic pollution of water. natural sources of organic matter include plant decay and leaf fall. however, plant growth and decay may be unnaturally accelerated when nutrients and sunlight are overly abundant due to human influence. urban runoff carries pet wastes from streets and sidewalks; nutrients from lawn fertilizers; and leaves, grass clippings, and paper from residential areas, which increase oxygen demand. oxygen consumed in the decomposition process robs other aquatic organisms of the oxygen they need to live. organisms that are more tolerant of lower dissolved oxygen levels may thrive in a diversity of natural water systems including aerobic bacteria. most of them feed on dead algae and other dead organisms and are part of the decomposition cycle. algae and other organism can produce oxygen through photosynthesis using the energy of sun. at night they use up oxygen in respiration. therefore, there is a continuous competition between the consumption of oxygen and its production through photosynthesis. the rate of oxygen consumption and therefore bod is affected by a number of variables: temperature, ph, the presence of certain kinds of microorganisms, and the type of organic and inorganic material in the water (davis and cornwell, ; weiner and matthews, ) . the most commonly used test for bod is called bod .this test provides a measure of the oxygen consumed in the biological oxidation of organic material in a sample at °c over a period of days. in the -day bod test (bod ), the sample of water is suitably diluted (in case of wastewater samples) with well-oxygenated water and an inoculum of microorganisms is introduced. the initial oxygen concentration is measured and the sample stored in darkness (to avoid photosynthetic oxygen generation) at °c for days. over the -day period, the oxygen concentration is measured at regular intervals and at the end of the -day period. the difference in oxygen concentration between the beginning and the end of the -day test period, taking due account of dilution, gives the bod (american public health association (apha), ). the concentration of oxygen could be measured using the traditional winkler titration method (apha, ) or is done automatically with modern bod apparatus equipped with manometer and do sensor. bod method is the most widely used method for assessment of water quality (weiner and matthews, ) . the amount of dissolved oxygen used by microorganisms in order to oxidize organic material in the absence of nitrification is called carbonaceous bod or cbod. however, additional amounts of dissolved oxygen could be depleted from the sample due to nitrification process, that is, the conversion of ammonia (nh ) to nitrate (no À ) by nitrogenous bacteria which is called nitrogenous bod or nbod. the forms of nitrogen in urine and proteins (urea, uric acids, ammonia, amino acids, and nitrates) are nutrients for algae and aquatic plant growth. the nitrogenous waste in municipal and industrial sewage is used by autotrophic bacteria, and they use a significant amount of oxygen as an energy source and convert ammonia to nitrates. the described bod test in section . . gives value for both carbonaceous and nitrogenous bod. thus, if we wish to determine the value of cbod only, nitrification suppressor or inhibitor such as atu (allylthiourea) should be added (davis and cornwell, ) . another closely related test to bod is the chemical oxygen demand (cod) test which is also considered as an important indicator of water quality. cod is a measurement of the oxygen required to oxidize soluble and particulate organic matter in water. this test gives the electron donating capacity of practically all the organic compounds in the sample, biodegradable or nonbiodegradable and soluble or particulate. the cod of a wastewater, in general, is greater than the bod because more compounds can be oxidized chemically than can be oxidized biologically. the method involves using a strong oxidizing chemical, potassium dichromate cr o À , to oxidize the organic matter in solution to carbon dioxide and water under acidic conditions. the sample is then digested for approximately h at °c. the amount of oxygen required is calculated from the quantity of chemical oxidant consumed (apha, ; davis, ). nitrogen and phosphorus are important elements that could be present in water and affect water quality fundamentally since they are essential nutrients for biological growth. nitrogen occurs in five major forms in aquatic environments: organic nitrogen, ammonia, nitrite, nitrate, and dissolved nitrogen gas. ammonia is one of the intermediate compounds formed during biological metabolism and, together with organic nitrogen, is considered an indicator of recent pollution. aerobic decomposition of organic nitrogen and ammonia eventually produces nitrite (no À ) and finally nitrate (no À ). therefore, high nitrate concentration may indicate that organic nitrogen pollution occurred far enough upstream that the organics have had time to oxidize completely. similarly, nitrate may be high in groundwater after land application of organic fertilizers if there is sufficient residence time (and available oxygen) in the soils to allow oxidation of the organic nitrogen in the fertilizer (andrews et al., ; manahan, ) . because ammonia and organic nitrogen are pollution indicators, these two forms of nitrogen are often combined in one measure, called kjeldahl nitrogen (apha, ). nitrate and nitrite could be determined separately by spectrophotometric methods (apha, ). the nitrogen cycle (fig. ) is the biogeochemical cycle that describes the transformations of nitrogen and nitrogen-containing compounds in nature. although there is an abundance of nitrogen in the atmosphere, most plants cannot use this form of nitrogen. instead, nitrogen must be in its "fixed" form, or in a compound, in order to be usable by plants. to get to this fixed form, nitrogen must first go through the cycle. the steps of the nitrogen cycle are the following: nitrogen fixation, nitrification, denitrification, ammonification, and assimilation. nitrogen fixation occurs when atmospheric nitrogen is converted to ammonia by a pair of bacterial enzymes called nitrogenase present in a few species of bacteria, including cyanobacteria. although ammonia (nh ) is the direct product of this reaction, it is quickly ionized to ammonium (nh + ). nitrification is the two-step process in which ammonia is converted to nitrites (no À ) and then to nitrates (no À ). two different genera of bacteria that are present in the soil oxidize the ammonia into inorganic forms of nitrogen: these are nitrosomonas and nitrobacter. denitrification is the process of reducing nitrate and nitrite, highly oxidized forms of nitrogen available for consumption by many groups of organisms, into gaseous nitrogen. it can be thought of as the opposite of nitrogen fixation. certain types of bacteria are responsible for this transformation. assimilation is the process by which living organisms incorporate no À and nh + (ammonium) formed through nitrogen fixation and nitrification. plants take in this form of nitrogen through their roots and incorporate them into nucleic acids and plant protein. animals are then able to receive and utilize the nitrogen from plant tissues through consumption. ammonification occurs when a plant or animal dies or excretes waste. decomposers, such as bacteria and fungi, first break down the proteins in the organic matter. this releases ammonia, which dissolves with water in the soil. ammonia then combines with a hydrogen ion to create ammonium. denitrification is the process in which microorganisms, such as bacteria, break down nitrates to metabolize oxygen. this releases nitrogen gas back into the atmosphere, completing the cycle (andrews et al., ; manahan, ) . phosphorus is found in the earth and rocks, and it is taken up by plants and animals. phosphorus is a very important chemical because it is essential in the formation of dna. in water bodies, phosphorus occurs almost entirely as organic phosphate and inorganic orthophosphate or polyphosphates (apha, ; davis and cornwell, ) . phosphorus is usually measured as total phosphorus (all forms combined) or dissolved phosphorus. dissolved orthophosphate (po À ) is an important indicator of water pollution because it is easily and rapidly taken up by bacteria, and therefore is almost never found in high concentrations in unpolluted waters. the various phosphorus forms can be measured analytically by spectrophotometric techniques (apha, ). phosphorous cycle could be defined as the biogeochemical cycle that describes the movement of phosphorus through the lithosphere (solid earth), hydrosphere (water), and biosphere (living organisms) (manahan, ; ruttenberg, ) . the global phosphorus cycle (fig. ) could be summarized in the following steps: . phosphorus starts out in rocks and then phosphates are washed off the rocks over a period of time by weathering and rain. after that, phosphate is distributed into soil and water. . plants then take up phosphate from the soil, and animals may consume these plants. when this happens, the phosphate becomes imbedded into organic molecules (dna). when plants and animals die and decompose, phosphate will return to the soil. . organic forms of phosphate can be made accessible to plants by bacteria which break down the organic matter. once this happens, the phosphate is changed to an inorganic form of phosphorus (this is known as mineralization). . once the phosphorus is in the soil, it can be transferred to waterways and then oceans. after this phosphorus can be integrated into sediments and rock and the cycle starts again. the cycle is largely affected by the production and use of fertilizers made from phosphorus and animal manure that contains phosphorus. soil cannot absorb all the fertilizers, so it eventually gets washed away with other surface runoff. this will cause an increased amount of phosphorus in the water bodies. consequently, eutrophication will occur. eutrophication occurs when extra nitrate and phosphate leach into a body of water and cause excessive growth of microorganisms. when phosphorus and nitrogen are introduced into the lake, either naturally from storm runoff, or from a pollution source, the nutrients promote rapid growth of algae on the surface layer of the lake. when the algae die, they drop to the lake bottom and become a source of carbon for decomposing bacteria. aerobic bacteria (that use oxygen for living) will use all available dissolved oxygen in the process of decomposing this material, and the dissolved oxygen may be depleted enough to cause the bottom of the lake to become anaerobic. as more and more algae die, and more and more dissolved oxygen is used in their decomposition, the middle layer of the lake may also become anaerobic (do not need molecular oxygen for living). when this happens, aerobic biological activity will be restricted on the surface layer only. the increasing frequency of this condition over the years is called eutrophication (davis and cornwell, ; manahan, ; weiner and matthews, ) . natural eutrophication may take thousands of years. however, eutrophication process may be shortened to as little as a decade as a result of excessive use of chemicals that provide nutrients such as fertilizers, detergents, and pesticides (davis and cornwell, ). water treatment is any process that makes water more suitable for a specific end use. the end use could be drinking or cooking, industrial supply, irrigation, fire fighting, river flow maintenance, water recreation, or just safe discharge of water to the environment. generally speaking, the characteristics of water and the final intended use of water determine the degree of treatment and the treatment method (davis, ; davis and cornwell, ; weiner and matthews, ) . mostly, the focus is on water treatment plants that produce safe drinking water and wastewater treatment plants that treat water for safe discharge or future reuse. drinking water treatment plant could be classified into: -disinfection plant which is used for high-quality water source to ensure that water does not contain pathogens -filtration plant: this is usually used to treat surface water -softening plant which is used to treat groundwater typical filtration plant is shown in fig. which is designed to remove odors, color, and turbidity as well as bacteria and other contaminants. filtration plant employs the following steps: a. rapid mixing: where chemicals are added and rapidly dispersed through the water b. flocculation: chemicals like alum (aluminum sulfate) are added to the water both to neutralize the particles electrically and to make them come close to each other and form large particles called flocs that could more readily be settled out c. sedimentation: during sedimentation, floc settles to the bottom of the water supply, due to its weight d. filtration: once the floc has settled to the bottom of the water supply, the clear water on top will pass through filters of varying compositions (sand, gravel, and charcoal) and pore sizes in order to remove fine particles that were not settled, such as dust, parasites, bacteria, viruses, and chemicals e. disinfection: involves the addition of chemicals in order to kill or reduce the number of pathogenic organisms softening plants utilize the same operational unit as filtration plants but use different chemicals in order to remove water hardness (davis, ; davis and cornwell, ; weiner and matthews, ) . municipal wastewater treatment may involve three major categories: primary treatment, secondary treatment, and advance treatment. fig. shows degrees of wastewater treatment. -primary treatment: the goal is to remove pollutants which will either settle or float in a clarifier tank. primary treatment will typically remove % of the raw sewage suspended solids and % of the bod . soluble pollutants are not removed in this process. -secondary treatment: the major goal is to remove bod that escapes the primary process and to provide additional removal of suspended solids. secondary treatment is typically achieved using biological processes. these processes could occur naturally in wastewater. nevertheless, secondary treatments are designed to speed up these natural processes. although secondary treatment may remove % of bod and suspended solids, it does not remove significant amount of nitrogen, phosphorous, or heavy metals, nor does it completely remove pathogenic bacteria and viruses. -advance treatments or tertiary wastewater treatment: these processes are normally applied when secondary treatment is not adequate. some of these processes may remove up to % of bod and phosphorous, all suspended solids and bacteria, and % of nitrogen. they can produce sparkling clean, colorless, and odorless effluent indistinguishable in appearance from high-quality drinking water. some of these processes involve: filtration or application of adsorbent such as activated carbon in order to remove persistent organic pollutants. tertiary treatment may involve addition of chemicals such as ferric chloride or alum to enhance the removal of phosphorous. nitrogen control step could be added to the tertiary treatment plant in order to facilitate the removal of nitrogen. this step could be accomplished biologically or chemically. the biological step is called nitrification/denitrification. specific types of bacteria must be present to cause these two reactions to occur. the chemical process is called ammonia stripping; in this process nitrogen is removed in the form of ammonia by raising the ph of the solution by addition of base such as lime to covert ammonium ion to ammonia (davis, ; davis and cornwell, ) . microbiology is the science devoted to the study of organisms that are too small to be seen by the naked eye. these microorganisms constitute a large and diverse group of free-living forms that exist as single cells or cell clusters. being free living, microbial cells are distinct from the cells of animals and plants as the latter are not able to live alone in nature but only in characteristic groups. a single microbial cell, generally, is able to carry out its life processes of growth, respiration, and reproduction independently of other cells, whether of the same kind or of different kinds. microorganisms could be bacteria, viruses, algae, fungi, or protozoa (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . in the following sections we will present an overview about microorganisms, their role in water, their classification, the main processes they perform, disease-causing microorganisms, and the role of microorganisms in wastewater treatment. most of the examples and discussion in the following sections will be assigned to bacteria as they outline the group of the most important microorganisms when encountering water and wastewater treatment; they can be pathogens or can help in wastewater treatment. despite their complexity and variety, all living cells can be arranged into two major groups, prokaryotes and eukaryotes, based on certain structural and functional characteristics. in general, prokaryotes are structurally simpler and smaller than eukaryotes. the dna (genetic material) of prokaryotes is usually in a single, circularly arranged chromosome and is not surrounded by a membrane; therefore, they do not have true nucleolus. they also lack membrane-enclosed organelles (specialized structures that carry on various activities). their cell walls almost always contain the complex polysaccharide peptidoglycan. they usually divide by binary fission. during this process, the dna is copied, and the cell splits into two. on the other hand, the dna of eukaryotes is found in multiple chromosomes in a membrane-enclosed nucleus. plants and animals are entirely composed of eukaryotic cells (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . taxonomy is the science of classifying living organisms. taxonomy is a basic and necessary tool for scientists, providing a universal language of communication. in a broader sense taxonomy consists of three separate but interrelated parts: classification, nomenclature, and identification. classification is the arrangement of organisms into groups (taxa, singular taxon), nomenclature involves assignment of names to taxa, and identification is the experimental determination of taxon to which an isolate belongs. in the currently accepted classification of life, there are three main domains which are eukarya, bacteria, and archaea. animals, plants, fungi, and protists are kingdoms in the domain eukarya. the domain bacteria include all of the pathogenic prokaryotes as well as many of the nonpathogenic prokaryotes found in soil and water. the organisms are classified as species. species is a group of closely related organisms that breed among themselves. a group of species that differ from each other in certain ways but are related genetically consist a genus. related genera make up a family. a group of similar families constitutes an order, and a group of similar orders makes up a class. related classes, in turn, make up a phylum or division. all phyla or divisions that are related to each other make up a kingdom, and related kingdoms are grouped into a domain. the hierarchal classification of organisms showing the eight major taxonomical ranks in addition to the phylogenetic tree of life are presented in fig. (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . according to this classification, the full taxonomical position of a bacterium (escherichia coli) can be described as follows (willey et al., ) nomenclature refers to the naming of microorganisms. two kinds of names are usually given to bacteria, common name and scientific name. the common or casual name for a microorganism varies from country to country and is usually known in the local language. for example, tubercle bacillus, typhoid bacillus, gonococcus are common names for communication at the local level. the scientific name is the international name that is accepted throughout the world. scientific nomenclature of microorganisms is governed by the international committee on systematic bacteriology and published as approved list of bacterial names in the international journal of systematic bacteriology (tortora et al., ) . by accepted taxonomic conventions, the order names end in ales (e.g., the order eubacteriales), family names end in aceae (e.g., the family enterobacteriaceae), and the tribe names end in eae (e.g., the tribe proteae). the order, family, and tribe names begin with capital letters. the genus name also begins with capital letter, but species name (e.g., coli) begins with running letter and not capital letter. both the genus (e.g., escherichia) and species names are either italicized or underlined when written in the text. the scientific name of the bacterium when written for the first time is written in full (e.g., escherichia coli), but later mentioned in an abbreviated form (e.g., e. coli). because this system gives two names to each organism, the system is called binomial nomenclature. when bacteria are referred to as a group, their names are neither capitalized nor italicized nor underlined (e.g., streptococci) (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . in the microbial world, bacteria and archaea are prokaryotes which constitute a vast group of very small unicellular organisms. other microorganisms such as fungi, protozoa, and algae are eukaryotes. viruses, as noncellular elements, do not fit into any organizational scheme of living cells. they are genetic particles that replicate but are unable to perform the usual chemical activities of living cells (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the majority of prokaryotes are bacteria. the thousands of species of bacteria are differentiated by many factors, including morphology (shape), chemical composition (often detected by staining reactions), nutritional requirements, biochemical activities, and sources of energy (sunlight or chemicals). bacteria comprise the highest population of microorganisms in wastewater treatment plants (gerardi, ; mara and horan, ) . therefore, special attention will be given to this group of microorganisms in the following sections. before that, a brief synopsis about the other types of microorganisms will be presented. bacteria and archaea may look similar since they are both prokaryotes. however, their chemical composition is different. archaea do not have peptidoglycan in their cell walls. they often live in extreme environments and carry out unusual metabolic processes (tortora et al., ; willey et al., ) . fungi are eukaryotes. their cells have a distinct nucleus containing the cell's genetic material and surrounded by a special envelope called the nuclear membrane. true fungi have cell walls composed primarily of a substance called chitin. fungi are nonphotosynthetic heterotrophic microorganisms. they are obligate aerobe. in general fungi are multicellular; however, yeast is a unicellular form of fungi. fungi can be reproduced by a variety of methods including fission, budding, and spore formation. the unicellular forms of fungi, yeasts, are oval microorganisms that are larger than bacteria. the most typical fungi are molds which form visible masses called mycelia. the cottony growths sometimes found on bread and fruit are mold mycelia. organisms called slime molds have characteristics of both fungi and amoebas. fungi form normal cell material using one half the nitrogen amount required by bacteria. in nitrogen-deficient wastewater they are found to be predominant over bacteria (davis and cornwell, ; gerardi, ; mara and horan, ; tortora et al., ) . algae are a group of eukaryotic microorganism. they might be unicellular or multicellular. the cell walls of many algae are composed of a carbohydrate called cellulose. algae have chlorophyll in their cells; therefore, they can perform photosynthesis. they reproduce sexually or asexually. algae are abundant in fresh and salt water, in soil, and in association with plants (davis and cornwell, ; gerardi, ; hogg, ; tortora et al., ) . protozoa are unicellular eukaryotic organisms. most protozoa are free living and solitary, but some do form colonies. most protozoa are aerobic chemotrophs (see section . . ), but some including amoebae and flagellates can survive anaerobic conditions. they reproduce by binary fission. they often consume bacteria. they are desirable in wastewater effluent because they act as polisher in consuming bacteria (gerardi, ; hogg, ; tortora et al., ) . to environmental factors microorganisms could be classified according to the energy and carbon source. carbon is the basic building block for cell synthesis. energy must be obtained from outside the cell to enable synthesis to proceed. if the microorganism uses organic material as a supply for carbon, it is called heterotrophic. autotrophs require only co to supply their carbon needs. organisms that rely only on the sun as source of energy are called phototrophs. chemotrophs extract energy from organic or inorganic oxidation/reduction reactions. organotrophs use organic material, while lithotrophs oxidize inorganic compounds (davis, ; davis and cornwell, ) . according to the ability to utilize oxygen as a terminal electron acceptor in oxidation-reduction reactions, microorganisms could be classified as obligate aerobes which are the microorganisms that must have oxygen as the terminal electron acceptor. obligate anaerobes are microorganisms that cannot survive in the presence of oxygen; they cannot use oxygen as a terminal electron acceptor. facultative aerobes are able to grow with or without oxygen being present. under anoxic conditions (depleted dissolved oxygen), facultative aerobes utilize nitrites (no À ) and nitrate (no À ) as the terminal electron acceptor. nitrate nitrogen is converted to nitrogen gas in the absence of oxygen. this process is called anoxic denitrification (davis, ; davis and cornwell, ; mara and horan, ) . according to the preferred temperature regime, four temperature ranges are used to classify microorganisms. those that best grow at temperatures below °c are called psychrophiles. mesophiles grow best at temperatures between °c and °c. between °c and °c, the thermophiles grow best. above °c stenothermophiles grow best. the growth range of facultative thermophiles extends from the thermophilic range into the mesophilic range (davis, ; davis and cornwell, ). bacteria come in a great many sizes and several shapes. most bacteria range from . to . μm in diameter and from to μm in length (tortora et al., ) . most bacteria of medical importance range from . to . μm in diameter and from to μm in length (parija, ) . the basic shapes of bacteria are spherical coccus (plural: cocci, meaning berries), rod-shaped bacillus (plural: bacilli, meaning little staffs), and spiral (see fig. for the different shapes of bacteria). cocci are usually round but can be oval, elongated, or flattened on one side. when cocci divide to reproduce, the cells can remain attached to one another. cocci that remain in pairs after dividing are called diplococci; those that divide and remain attached in chainlike patterns are called streptococci. those that divide in two planes and remain in groups of four are known as tetrads. those that divide in three planes and remain attached in cubelike groups of eight are called sarcinae. those that divide in multiple planes and form grapelike clusters or broad sheets are called staphylococci. bacilli divide only across their short axis. most bacilli appear as single rods. diplobacilli appear in pairs after division, and streptobacilli occur in chains. spiral bacteria have one or more twists; they are never straight. bacteria that look like curved rods are called vibrios. others, called spirilla, have a helical shape, like a corkscrew, and fairly rigid bodies. another group of spirals are helical and flexible; they are called spirochetes. spirochetes move by means of axial filaments, which resemble flagella but are contained within a flexible external sheath. in addition to the three basic shapes, there are starshaped cells, rectangular cells, flat cells, and triangular cells (hogg, ; madigan et al., ; parija, ; tortora et al., ; willey et al., ) . mostly water by composition. however, they have a semifluid nature due to a suspension of carbohydrates, enzymes, inorganic ions, lipids, and proteins. within this suspension we can find the following: -the nuclear region which consists mostly of genetic material in one large and circular chromosome. in addition to the chromosome, some bacteria have small molecules of genetic material called plasmids that may include genes encoding toxins or resistance to antibiotics. -ribosomes are small spherical bodies that serve as sites for protein synthesis. -inclusions which accumulate certain nutrients when they are plentiful and use them when the environment is deficient. -endospores which form in certain bacteria such as bacillus and clostridium. they are dormant forms of the cell that are highly resistant to extremes of temperature, ph, and other environmental factors, and germinate into new bacterial cells when conditions become more favorable. b. the cell membrane or plasma membrane is a flexible semipermeable membrane that surrounds the cytoplasm. the cell membrane contains two different layers that regulate the movement of substances in and out of the cell. the outer layer is hydrophilic (water loving), while the inner layer is hydrophobic (water fearing). together, these layers form a protective and regulating barrier between the cytoplasm and the environment. c. cell wall is an extremely porous and rigid structure that lies outside the cell membrane. the cell wall performs three functions. firstly, it provides protection. secondly, it maintains the characteristic shape of the cell. thirdly, it prevents the cell from bursting when fluids flow into the cytoplasm. d. a variety of external structures: bacterial cells might have several external structures such as the axial filament, capsule, flagella, glycocalyx, and pili. all external structures perform specific functions, but these structures are not necessarily found in all bacteria. based upon chemical composition of the cell walls, there are two types of bacteria, gram negative and gram positive. gram-positive bacteria retain the crystal violet stain after the gram-staining technique due to their thick cell wall layer of peptidoglycan. gram-negative bacteria on the other hand, cannot retain the crystal violet stain due to their thin cell wall that has a very thin layer of peptidoglycan, and this layer is reinforced with a second membrane on top. gram-negative bacteria are stained red with safranin at the end of the gram-staining technique (parija, ; tortora et al., ; willey et al., ) . in the following section we will take a look at the main microbial activities that are necessary for their life, namely; metabolism, respiration, and bacterial growth. metabolism is the term used to describe all the biochemical reactions that take place inside a cell; it includes those reactions that release energy into the cell, and those that make use of that energy. most microorganisms obtain their energy from the nutrients they take into the cell. these microorganisms break the nutrients into smaller molecules, and then use these molecules for synthesis of new cellular components. they release the chemical energy stored in the nutrients and use it later to perform other processes. on the other hand, some microorganisms obtain the energy needed for metabolism from the sun by means of photosynthesis (davis and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . catabolism is the term used to describe reactions that break down large molecules forming smaller molecules, usually associated with a release of energy. anabolism is the term used to describe reactions involved in biosynthesis of macromolecules, usually requiring an input of energy (davis and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the energy released in cabalism is used by the microorganisms in different aspects (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) : a. to synthesize new cellular components such as nucleic acids, polysaccharides, and enzymes b. to transport certain substances into the cell from its surroundings c. for the cell to grow and multiply d. for cellular movement e. to maintain the structural integrity of the cell by repairing any damage to its constituents the process of metabolism involves the use of some important molecules such as enzymes, and electron and proton carriers. biosynthesis and metabolic processes in the cell are generally catalyzed by molecules that are called enzymes which are large molecules usually protein, specific to a particular reaction or group of reactions. enzymes increase the rate of the reaction by lowering its activation energy through providing alternative pathway for the reaction. enzymes bring substrates together at spatial places on their surface called active sites. therefore, chances of effective interaction between substrates are enhanced and the rate increases (tortora et al., ; willey et al., ) . coenzymes may assist the enzyme by accepting atoms removed from the substrate or by donating atoms required by the substrate. some coenzymes act as electron carriers, removing electrons from the substrate and donating them to other molecules in subsequent reactions. many coenzymes are derived from vitamins. two important coenzymes are encountered while discussing metabolism are nicotinamide adenine dinucleotide (nad + ) and nicotinamide adenine dinucleotide phosphate (nadp + ); both are derivatives of the b vitamin niacin, and each can exist in an oxidized and a reduced form. nad + + h + + e À Ð À nadh nadp + + h + + e À Ð nadph nad + is primarily involved in catabolic reactions, while nadp + is primarily involved in anabolic reactions. the flavin coenzymes, such as flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad), contain derivatives of the b vitamin riboflavin and are also electron carriers. cytochromes are another type of electron carriers. cytochromes are proteins with an iron-containing group that could exist as fe + or fe + (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . energy taken up by the cell, either in the form of nutrients or sunlight, must be converted into a usable form. the most usable form of energy is a compound called adenosine triphosphate (atp). atp is by far the most important of a class of compounds known as high-energy transfer. atp stores energy derived from catabolic reactions (such as breaking down of glucose) and releases it later to drive anabolic processes (such as synthetic reactions) and perform other cellular works. atp molecule (fig. ) consists of an adenine, a ribose, and three phosphate groups. the terminal phosphate group requires a lot of energy for its formation and is often referred to as a high-energy phosphate bond. when this bond is broken, the same large amount of energy is released, so when atp is broken down, adenosine diphosphate (adp) is formed, and energy is released to drive anabolic reactions. the process of adding or removing a phosphate group is called phosphorylation or dephosphorylation, respectively (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the term cellular respiration refers to the biochemical pathway by which cells release energy from the chemical bonds of food molecules and provide that energy for the essential processes of life. all living cells must carry out cellular respiration. respiration could be aerobic or anaerobic according to the availability of oxygen. prokaryotic cells carry out cellular respiration within the cytoplasm or on the inner surfaces of the cells. in eukaryotic cells the mitochondria are the site of most of the reactions. the energy currency of these cells is atp, and one way to view the outcome of cellular respiration is as a production process for atp. prokaryotes take the carbohydrates into their cytoplasm, and through a complex series of metabolic processes, they break down the carbohydrate and release the energy. the energy is generally not needed immediately, so it is used to combine adp with phosphate ions to form atp molecules. during the process of cellular respiration, carbon dioxide is given off as a waste product. this carbon dioxide can be used by photosynthesizing cells to form new carbohydrates. also in the process of cellular respiration, oxygen gas is required to serve as an acceptor of electrons. this oxygen gas is identical to the oxygen gas given off in photosynthesis (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the overall mechanism of cellular respiration involves four subdivisions: a. glycolysis, in which glucose is oxidized to two molecules of pyruvate with the synthesis of two molecules of atp. glycolysis is summarized in fig. . after glycolysis, two options are present depending on the amount of available oxygen. in the presence of oxygen, the cell will undergo aerobic respiration. without a supply of oxygen, as in an anaerobic environment, the cell will undergo fermentation (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . b. krebs cycle, citric acid cycle (cac), or the tricarboxylic acid (tca) cycle: which results in formation of (per two molecules of pyruvic acid) two atp molecules, six nadh molecules, and two fadh molecules and six co molecules. the nadh and the fadh will be used in the electron transport system (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the main steps of krebs cycle could be summarized as in fig. . c. electron transport: in this step nadh and fadh produced in both glycolysis and the krebs cycle are now being used. electron transfer takes place in mitochondria in eukaryotic cells. the process is the same in prokaryotes, but it just happens at the main cell membrane. cytochromes and coenzymes act as carrier molecules and transfer molecules. they accept high-energy electrons and pass the electrons to the next molecule in the system. at key proton-pumping sites, the energy of the electrons transport protons across the cell membrane to the outside (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . d. chemiosmosis, in which the energy given off by electrons is used to pump protons across a membrane and provide the energy for atp synthesis. during cellular respiration (krebs cycle reactions, the electron transport system, and chemiosmosis), molecules of atp can be produced for each glucose molecule. additional two atp molecules are produced through glycolysis, so the net yield is molecules of atp. these atp molecules may then be used in the cell for its needs. however, the atp molecules cannot be stored for long periods of time, so cellular respiration must constantly continue in order to regenerate the atp molecules as they are used. each atp molecule is capable of releasing . kcal of energy per mole (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . when oxygen is not available as final electron acceptor, the process of respiration after glycolysis cannot continue as described above. therefore, an alternative pathway should be present such as fermentation or anaerobic respiration. fermentation is an anaerobic pathway for breaking down glucose in which organic carbon is the terminal electron acceptor, one that is performed by many types of organisms and cells. in fermentation, the only energy extraction pathway is glycolysis, with one or two extra reactions tacked on at the end. fermentative organisms use nadh and other cofactors to produce many different reduced metabolic by-products, often including hydrogen gas (h ). these reduced organic compounds are generally small organic acids and alcohols derived from pyruvate, the end product of glycolysis. examples include ethanol, acetate, lactate, and butyrate. fermentative organisms are very important industrially and are used to make many different types of food products. the different metabolic end products produced by each specific bacterial species are responsible for the different tastes and properties of each food. many microorganisms are facultative anaerobes, meaning they can switch between aerobic respiration and anaerobic pathways (fermentation or anaerobic respiration) depending on the availability of oxygen (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . in the process of anaerobic respiration, carbohydrate can be metabolized by a process that utilizes oxidative phosphorylation via an electron transport chain, but instead of oxygen serving as the terminal electron acceptor an inorganic molecule such as nitrate or sulfate is used. in addition, other organisms may turn to this form of respiration if oxygen is unavailable (facultative anaerobes). anaerobic respiration is not as productive as its aerobic counterpart in terms of atp production, because electron acceptors such as nitrate or sulfate have less positive redox potentials than oxygen. anaerobic respiration tends to occur in oxygen-depleted environments such as waterlogged soils. it must be underlined here that anaerobic respiration is not the same as fermentation. the latter process does not involve the components of the electron transport chain and generate much smaller amounts of energy (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . most organisms obtain energy by oxidizing complex organic compounds produced by other organisms, while other microorganisms are capable of producing the complex organic compounds from simple inorganic substances in a process called photosynthesis. photosynthesis involves two major processes: -the light reaction or energy fixing reaction in which light energy from the sun is converted to chemical energy stored in atp molecule. the organisms use the light energy, water molecules, nadp + , adp, and phosphate to produce atp, nadh, and oxygen molecule. -the dark reaction or carbon fixing reaction. in this reaction, organisms use atp and nadh produced in the light reaction plus carbon dioxide absorbed from the atmosphere to produce carbohydrates, mainly glucose (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . carbon fixation reaction is usually referred to as co fixation or calvin cycle. calvin cycle (fig. ) is the cycle in which carbon is fixed, reduced, and utilized. it involves enzymatically regulated chemical reaction used by autotrophic microorganisms for co fixation. the cycle produces phosphoglycerate and the glyceraldehyde- -phosphate molecules which are subsequently used to synthesize all the organic compounds needed by the cell. each turn of the cycle results in the fixation of one molecule of co . therefore, to synthesize one glucose molecule, the cycle must operate six times (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . fig. shows the net outcomes of six cycles. the energy generated in the catabolic pathways will be used by cells to manufacture the cellular components (biosynthesis) such as: nitrogenous substances, including proteins and nucleic acids, carbohydrates, including complex polysaccharides (such as capsules and the peptidoglycan of the cell wall), and phospholipids which are components of the cytoplasmic membrane. an autotrophic bacterium that can synthesize all its cellular constituents from co and simple inorganic compounds obviously is said to have great biosynthetic ability. in the same way, a heterotrophic bacterium that uses glucose as the only carbon and energy source, ammonium sulfate as the nitrogen and sulfur source, and a few additional inorganic compounds also has great biosynthetic capability. from these nutritional substances, the microorganisms can manufacture anything they need. bacteria are particularly adept at converting anything they can get hold of into any other item they need. glycolysis and the krebs cycle produce a number of intermediates that can be used in amino acids synthesis. amino acids can be converted to proteins or they can be incorporated in the production of nucleotides to form nucleic acids. these intermediates can be used also to form more complex carbohydrates. acetyl coa from glycolysis can be oxidized to form fatty acids. glucose can be converted into just about anything. fatty acids and amino acids can be catabolized to feed into glycolysis or the krebs cycle. these pathways are largely reversible and interconnected as shown in fig. . bacteria are extremely resilient, they just need something to work with, and they can manufacture the rest. for example, if they start with protein, they will break it down to amino acids, convert that into pyruvic acid, and be able to form carbohydrates (hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . bacterial growth refers to an increase in cell number, not in cell size. bacterial growth is a complex process involving numerous anabolic and catabolic reactions. ultimately, these biosynthetic reactions result in cell division which is done mostly by binary fission as shown in fig. . the requirements for microbial growth can be divided into two main categories: physical and chemical. physical requirements include temperature, ph, and osmotic pressure. chemical requirements include macronutrient and micronutrients (davies, ; davis and cornwell, ; gerardi, ; hogg, ; madigan et al., ; mara and horan, ; parija, ; tortora et al., ; weiner and matthews, ; willey et al., ) . macronutrients include the elements that are involved largely in the composition of bacterial cells, such as carbon, hydrogen, nitrogen, oxygen, phosphorous, and sulfur. these elements are required in large quantities. micronutrients, on the other hand, contain minor elements such as ca, fe, k, mg, and na; organic compounds such as vitamins; and trace elements such as co, mn, mo, ni, and zn. these micronutrients are required in relatively small quantities for most bacteria. some bacteria such as methane-forming bacteria require sulfur and some minor and trace elements such as co, fe, and ni in quantities, two to five times greater than other bacteria. calcium is required in large amounts by gram-positive bacteria for the synthesis of cell walls (davis and cornwell, ; tortora et al., ) . microbial culture is a method for increasing the number of organisms by letting them reproduce in controlled media. in a pure culture medium, cells are placed in a liquid medium in which the nutrients and environmental conditions are controlled to allow the reproduction of single species of microorganisms. if the medium supplies all nutrients required for growth and environmental parameters are optimal, the increase in numbers or bacterial mass can be measured as a function of time. the representation of the increase in numbers or bacterial mass as a function of time is called a growth curve. several distinct growth phases can be observed within a growth curve as can be seen in fig. . these includes the lag phase, the exponential or log phase, the stationary phase, and the death phase. each of these phases represents a distinct period of growth that is associated with typical physiological changes in the cell culture (davies, ; davis fig. growth curve. and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the first phase observed under batch conditions is the lag phase in which the growth rate is essentially zero. when an inoculum is placed into a fresh medium, growth begins after a period of time called the lag phase. the lag phase is thought to be due to the physiological adaptation of the cell to the culture conditions (davies, ; davis and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the second phase of growth observed in a batch system is the exponential phase. in this phase, the most rapid growth occurs. exponential increase in the number of bacterial cells is observed in the growth curve. during exponential growth, the rate of increase of cells in the culture is proportional to the number of cells present at any particular time (davies, ; davis and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the third phase of growth is the stationary phase. the stationary phase in a batch culture can be defined as a state of no net growth. carbon and energy source or an essential nutrient becomes completely used up; therefore, there is no net growth in stationary phase. however, cells still grow and divide. this is because dying cells can lyse and provide a source of nutrients (davies, ; davis and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . the final phase of the growth curve is the death phase, which is characterized by a net loss of culturable cells. even in the death phase there may be individual cells that are metabolizing and dividing, but more viable cells are lost than are gained so there is a net loss of viable cells. the death phase is often exponential, although the rate of cell death is usually slower than the rate of growth during the exponential phase (davies, ; davis and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . in nature, pure cultures do not exist, rather mixtures of species compete and survive within the limits set by the environment. population dynamic is the term used to describe the time-varying success of the various species in competition. it is expressed quantitatively in terms of relative mass of microorganisms. the prime factor determining the dynamic of the various microbial populations is the competition for food. the second most important factor is the predator-prey relationship. the relative success of a pair of species competing for the same substrate is a function of the ability of species to metabolize the substrate. the more successful species will be the one which metabolizes the substrate more completely. in so doing, it will obtain more energy for synthesis and consequently will achieve greater mass. because of their relatively smaller size and thus larger surface area per unit mass which allow a more rapid uptake of substrate, bacteria will predominate over fungi. for the same reason, fungi predominates over protozoa. when the supply of soluble organic substrate becomes exhausted, the bacterial population is less successful in reproduction and the predator population predominates. in a closed system with an initial inoculum of mixed microorganisms and substrate, the predominant populations will cycle as the bacteria give way to higher-level organisms that in turn die for lack of food and are then decomposed by different sets of bacteria. in an open system such as wastewater treatment plant or river, with a continuous inflow of new substrate, the predominant populations will change through the length of the plant (davis and cornwell, ; hogg, ; madigan et al., ; tortora et al., ; willey et al., ) . in nature, microorganisms seldom live in the isolated single-species colonies as we see on laboratory plates. they more typically live in communities called biofilms. biofilms are an assemblage of microbial cells; in this assemblage cells stick to each other and often these cells adhere irreversibly. these adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (eps). biofilms contain also dna and proteins that are often informally called slime. biofilms could be attached to a wide variety of surfaces, such as a rock in a pond, a human tooth, mucous membrane, indwelling medical devices, or water system piping. bacteria start to divide by binary fission. after their growth, those bacteria belonging to the same species will produce and secrete saccharide. this saccharide will form the extra polymer substance (eps). eps is very adhesive and is the protective membrane of a biofilm. living in biofilm community provides bacteria with resistance to antibiotic penetration. in addition, it allows bacteria to survive in low nutrient medium. if viewing an intact biofilm under microscope, one may immediately find that bacteria in biofilms do not randomly stick together, but rather form a well-organized community with numerous specialized configurations. cell-to-cell signaling or quorum sensing (qs) has been demonstrated to play a role in biofilm formation and cell attachment and detachment from biofilms. qs allows bacterial cells to communicate. it can occur within a single bacterial species as well as between diverse species. qs is highly associated with cell number. induction of qs gene system requires the accumulation of signaling molecules called autoinducers such as acyl homoserine lactones (ahls) or peptide pheromone. for example, gram-negative bacteria adapt luxi/luxr gene signaling system using ahls autoinducers. lux gene is initially expressed in low levels, leading to the production of small number of lux proteins including luxi and luxr. luxi code for synthesis of autoinducer ahl. autoinducer molecules rapidly diffuse out of the cell. the more the number of cells is in a given space, the faster the inducers build up and the more likely they reenter the cells, bind to receptor, and stimulate the expression of luxr. luxr protein will stimulate the expression of many genes including those for inducer synthesis. the produced gene could trigger many cellular activities such as the formation of biofilms. it has been suggested that biofilm formation results from a developmental program of gene expression. in addition, biofilm development and maintenance have been shown to require a wide range of genetic determinants and to involve bacterial subpopulations carrying out different functions. quorum sensing was found to play a significant role in coordinating biofilm formation for many species (donlan, ; irie and parsek, ; li and tian, ) . biofilms also might be useful in wastewater treatment plants. the floc that forms in certain types of sewage treatment is an example. many wastewater treatment plants include a secondary treatment stage in which wastewater passes over biofilms grown on filters, which extract and digest organic compounds. in such biofilms, bacteria are mainly responsible for removal of bod from water, while protozoa and rotifers are mainly responsible for removal of suspended solids (ss), including pathogens and other microorganisms. on the other hand, biofilms could have adverse effect on wastewater treatment process. for instance biofilm is a major drawback in the performance of membrane bioreactor (mbr) (davis, ; tortora et al., ; weiner and matthews, ) . the pathogenic microbiological agents that may be present in water intended for human consumption are essentially bacteria, viruses, or protozoa, most of them derived from human or animal waste. the consequences of exposure to pathogenic bacteria, viruses, and pathogens in the water may vary. the most common symptoms are nausea, vomiting, and diarrhea. infants, children, older people, as well as people with compromised immune systems may have more serious symptoms. in extreme cases, some pathogens can infect the lungs, skin, eyes, nervous system, kidneys, or the liver, and the effects may be more severe, chronic, or even fatal. the presence of pathogenic microorganisms in tap water is linked to a lack of protection of the resource, to a lack of water treatment, or to backflow into the drinking water system. worldwide, the presence of microorganisms in water kills millions of people every year. it is estimated that half of the world's population contract diseases that are the direct consequence of polluted water (typhoid fever, cholera, dysentery, toxoplasmosis, and cryptosporidiosis). thousands of children die every day from contaminated water. microbial waterborne diseases also affect developed countries. in the united states, it has been estimated that , people suffer from severe waterborne diseases yearly, and . million suffer from a mild to moderate infections, resulting in estimated , deaths a year (cabral, ; who, who, , . the most important bacterial diseases transmitted through water are listed in table (cabral, ) . according to the who in , % of the global population ( . billion people) use safe drinking water that is free from contamination. however, million people lack even a basic drinking water service, including million people who are dependent on surface water. safe and readily available water is important for public health, whether it is used for drinking, domestic use, food production, or recreational purposes. improved water supply and sanitation, and better management of water resources can boost countries' economic growth and can contribute greatly to poverty reduction. in some situations, it is necessary for us to destroy, or at least limit, microbial growth because of the undesirable consequences of the presence of microorganisms or their products. control of microorganisms can be achieved by a broad variety of chemical and physical methods. some common terms are associated with microbial control such as sterilization, disinfection, antisepsis, sanitation, decontamination, and degerming. sterilization is the removal or destruction of all forms of microbial life. sterilization is generally achieved by using physical means such as heat, radiation, and filtration. disinfection usually involves the destruction of vegetative pathogens which is not the same thing as complete sterility. antisepsis is the destruction of vegetative pathogens on living tissue and the chemical used for this purpose is called an antiseptic. sanitation is the treatment intended to lower microbial counts on eating and drinking utensils to safe public health levels. decontamination is the treatment of an object or inanimate surface to make it safe to handle. on the other hand, degerming involves removal of microbes from a limited area, such as the skin around an injection site. degerming is mostly a mechanical removal by an alcohol-soaked swab. some other terms are associated with treatments that cause the outright death of microbes. biocides or germicides are the agents that kill microorganisms; a fungicide kills fungi; a virucide inactivates viruses. antibiotic which is a metabolic product produced by one microorganism that inhibits or kills other microorganisms. static, on the other hand, is the agent that is static in action and will inhibit the growth of microorganisms (mcdonnell and russell, ; russell, ; tortora et al., ) . biocidal activity depends upon several factors, notably, concentration, period of contract, ph, temperature, the type, nature, and numbers of microorganisms to be inactivated, and the presence of interfering material. microorganisms vary considerably in their response to antiseptics and disinfectants. viruses with lipid envelopes are the least susceptible, followed by gram-positive bacteria, viruses without envelopes, fungi, including most fungal spores, then gram-negative bacteria, vegetative protozoa, cysts of protozoa, mycobacteria, endospores of bacteria, and finally prions which have the highest resistance (mcdonnell and russell, ; russell, ; tortora et al., ) . disinfection is a very crucial step in water treatment processes as well as many industries since it aims at destroying all harmful microorganisms. disinfection could be accomplished through the use of chemicals, ultraviolet radiation, boiling water, or steam. in practice, the term disinfection is most commonly applied to the use of a chemical (a disinfectant) to treat an inert surface or substance. several categories of disinfectants can be used for commercial and industrial purposes such as chlorine compounds, alcohols, aldehydes, phenols, and hydrogen peroxide. disinfection is the final component in most water treatment processes. disinfection serves two purposes: as a final stage of treatment, in order to remove any remaining microorganisms that have survived the preceding processes; it may also provide partial protection against recontamination within distribution. disinfection step in water treatment plants is commonly done with chlorination, ultraviolet (uv) radiation, or ozonation (davis, ; davis and cornwell, ; tortora et al., ; weiner and matthews, ) . chlorination which is the most common disinfectant in water and wastewater treatment plants is very powerful in eliminating coliforms or e. coli. however, it was found that some pathogens, especially viruses and protozoan (giardia lamblia and cryptosporidium), are more resistant than coliforms to chlorination. this emerges the need for using alternative disinfectant (i.e., chlorine dioxide) or coupling of more than one disinfectant in the disinfection steps. the occurrence of microbial pathogens is often difficult to determine due to a many factors. pathogens are usually present in low numbers so it is necessary to monitor waters using "indicator" organisms, e.g., e. coli, fecal, thermotolerant, or total coliforms. the assumption of this approach is that the presence of bacterial indicator organisms is associated with the presence of microbial pathogens. the traditional indicator group for all microbial pathogens has been the coliform bacteria. their ease of enumeration, crude relationship to pathogen and disease occurrence, and the lack of better indicators led to the adoption of coliform counts in most international standards documents. during the last years, numerous shortcomings of the use of coliforms as indicators of water-borne disease have been identified, and possible alternative indicators have been suggested; however, one all-purpose indicator has not emerged. for example, pseudomonas aeruginosa are alternative indicator for the traditional total coliforms, candida albicans are alternative indicator for e. coli, and bifidobacteria are alternative indicator for fecal streptococci (russell, ; tortora et al., ) . these indicators are distinguished using various methods based on enumeration procedures such as membrane filtration (mf) or multiple tube dilution or most probable number (mpn). these methods will be discussed briefly in the following section. the detection and enumeration of water quality indicators currently relies on the use of a few basic methods: membrane filtration (mf), multiple tube or most probable number (mpn) method, the standard plate count (spc) or total bacterial count, and presence-absence (pa) test. it should be noted that defined enzyme substrate technologies (dst) have been incorporated into each of these methods (pa, mpn/mtf, and mf) and are increasingly used in microbiological testing generally, both clinically as well as for food and water analysis and monitoring. each method has its advantages and disadvantages and has its applications according to the type of information needed and the type of water sample to be tested (mara and horan, ; russell, ; smith et al., ; tortora et al., ) . in the membrane filtration (mf) method, a measured volume of sample is passed through a membrane filter of known pore size that retains bacteria on or near the filter surface. the colonies that develop after incubation on a selective medium are presumed to have originated from individual bacteria, thus representing a direct count of the number of bacteria in the original sample. the mf method is often the method of choice in the analysis of most water types (russell, ; smith et al., ; tortora et al., ) . the multiple tube technique (mtf) or most probable number (mpn) technique involves three stages: the initial stage that is presumptive because the acid and gas, or growth/no growth, reactions may be caused by organisms other than those being selected; the confirmed test (the second stage which is usually the extent of testing), and the completed test (the third stage which is used for total coliform analyses only). although the completed test provides the greatest reliability, the workload of the testing laboratory and the time for all tests to be completed may restrict its use (russell, ; smith et al., ; tortora et al., ) . the mtf or mpn technique involves the inoculation of measured volumes of decimal dilutions of a sample into replicate tubes containing an inverted durham tube of a selective liquid medium. the tubes are incubated at °c or °c for h. positive tubes can be identified by the presence of gas in the durham tube. negative tubes are reincubated for a further h. at the end of this period, the tubes are checked again for gas production. gas production at the end of either or h incubation is presumed to be due to the presence of coliforms in the sample. confirmation test should be carried out after each incubation in order to confirm the presence of thermotolerant (fecal) coliform. the positive results of the confirmed test with the aid of probability tables are then used to obtain the most probable number (mpn) (russell, ; smith et al., ; tortora et al., ) . the viable aerobic and facultative anaerobic, heterotrophic bacterial count of water is generally enumerated by either pour plate or surface plate technique. the procedures provide data on the bacterial population capable of growth at the incubation temperature-time combinations used and do not estimate the total bacterial population present in the water. it is the most frequently used method of measuring bacterial populations. an important advantage of this method is that it measures the number of viable cells. a plate count is done by either the pour plate method or the spread plate method. in the pour plate method either . ml or . ml of dilutions of the bacterial suspension is introduced into a petri dish. the nutrient medium, in which the agar is kept liquid by holding it in a water bath at about °c , is poured over the sample, which is then mixed into the medium by gentle agitation of the plate. when the agar solidifies, the plate is incubated. with the pour plate technique, colonies will grow within the nutrient agar as well as on the surface of the agar plate. spread plate method is frequently used instead. a . ml inoculum is added to the surface of a prepoured, solidified agar medium. the inoculum is then spread uniformly over the surface of the medium with a specially shaped, sterilized glass or metal rod. this method positions all the colonies on the surface and avoids contact between the cells and melted agar. some recent methods include a fluorescent agent so that counting of the colonies can be automated. at the end of the incubation period, the colonies are counted by eye, a procedure that takes a few moments and does not require a microscope as the colonies are typically a few millimeters across (apha, ; russell, ; smith et al., ; tortora et al., ) . the presence or absence of pathogens, or other traditional indicator organisms, is frequently determined economically by presence-absence test (pa), which is a simplification of the multiple-tube procedure that permits qualitative information, as opposed to quantitative methods. the pa test involves analyses of one large test volume of water in a single culture bottle to obtain qualitative information. the utility of the procedure is its logistical simplicity relative to mf or mpn techniques. as regulatory limits for drinking waters are commonly < ml coliform or e. coli/ ml, pa format analysis allows such determinations by simply analyzing for the presence of the respective organism in that volume of sample with minimal processing (apha, ; russell, ; smith et al., ) . the basic objectives of wastewater treatment are to remove oxygendemanding organic wastes and to remove nutrients to levels where photosynthetic organisms in receiving waters are limited in their growth. biological, wastewater treatment plants are simply biological amplifiers. the plants permit organisms (biomass or sludge), primarily bacteria, to increase in number by using the pollutants (substrates and nutrients) in the wastewater and converting them to new organisms (biomass or sludge) and nonpolluting wastes and less polluting wastes (davis, ; davis and cornwell, ; gerardi, ; weiner and matthews, ) . there are three basic categories of biological treatment: aerobic, anaerobic, and anoxic. aerobic biological treatment involves contacting wastewater with microbes and oxygen in a reactor to optimize the growth and efficiency of the biomass. the microorganisms catalyze the oxidation of biodegradable organics and other contaminants such as ammonia, generating innocuous by-products such as carbon dioxide, water, and excess biomass (sludge). anaerobic (without oxygen) and anoxic (oxygen-deficient) treatments are similar to aerobic treatment, but use microorganisms that do not require the addition of oxygen. these microorganisms use compounds other than oxygen to catalyze the oxidation of biodegradable organics and other contaminants, resulting in safe by-products. the three individual types of biological treatment technologies (aerobic, anaerobic, or anoxic) can be run in combination or in sequence to offer greater levels of treatment. regardless of the type of system selected, accomplishing effective biological treatment is guaranteed by developing and maintaining an acclimated, healthy biomass, sufficient in quantity to handle maximum flows and the organic loads to be treated (davis, ; davis and cornwell, ; gerardi, ; weiner and matthews, ) . a typical wastewater treatment plant comprises three phases of treatment ( fig. ) : primary, secondary, and tertiary. secondary treatment is mainly biological and may contain several processes. the major goal of secondary treatment is to remove bod that escapes primary treatment and to provide further removal of suspended solids. however, due to the increasing recognition of the adverse effects of nutrients, secondary treatment often includes treatment of nitrogen and phosphorus. the main unit processes that are employed in secondary treatment are aerobic oxidation, nitrification, denitrification, phosphorous removal, and activated sludge selector. the objectives of these processes may be summarized as (davis, ; davis and cornwell, ; gerardi, ; weiner and matthews, ) : -aerobic oxidation which is concerned with the removal of bod. this process is accomplished by aerobic heterotrophic bacteria. -nitrification which involves the oxidation of ammonia to nitrate. aerobic autotrophic bacteria must be predominant to carry out nitrification. two genera are commonly recognized. ammonia is oxidized to nitrite by nitrosomonas. nitrite is oxidized to nitrate by nitrobacter. -denitrification involves the reduction of nitrate to nitric oxide, nitrous oxide, and nitrogen gas. a large number of genera are capable of denitrification, either heterotrophic or autotrophic. pseudomonas is the most common species. -phosphorous removal: in this process phosphorus is incorporated into cell biomass that is subsequently removed from the process. genera such as acinetobacter, arthrobacter, aeromonas, nocardia, and pseudomonas are responsible for this transformation. -selector: is a bioreactor design that favors the growth of floc-forming bacteria instead of filamentous bacteria so that the biomass has better settling and thickening properties. the unit processes for biological treatment may be either suspended growth, where microorganisms are suspended in the wastewater or attached growth, where the microorganisms grow on a solid surface. suspended growth methods include activated sludge processes, aerated lagoons, and aerobic digestion. attached growth methods include trickling filters, rotating biological contactors, and fixed-film reactors (davis, ) . one of the most commonly used methods of secondary treatment are activated sludge systems. the term activated sludge refers to particles produced in wastewater by the growth of organisms in aeration tanks. aerobic bacteria are the most common organisms in activated sludge. however, facultative bacteria along with higher organisms such as fungi and protozoa can be present. the exact composition of bacteria depends on the reactor design, environment, and wastewater characteristics. the sludge microorganisms oxidize the organic carbon in the wastewater to produce new cells, carbon dioxide, and water. as microorganisms grow, they form particles that clump together. these particles (floc) are allowed to settle to the bottom of the tank, leaving a relatively clear liquid free of organic material and suspended solids. the effluent can be discharged into a river or treated in a tertiary treatment facility if necessary for further use. there is a large variety of design; however, in principle all activated sludges consist of three main components: an aeration tank, which serves as a bioreactor; a settling tank or clarifier for separation of activated sludge solids and treated wastewater; and a return activated sludge equipment to transfer settled activated sludge from the clarifier to the influent of the aeration tank. the mixture of activated sludge and wastewater in the aeration tank is called mixed liquor. the average amount of time that microorganisms are kept in the system is called solids retention time (srt). the time that a fluid particle remains in the reactor is called hydraulic retention time (hrt). activated sludge processes show many advantages such as: resistant to organic and hydraulic shock loads, high reduction of bod and pathogens, and high effluent quality. on the other hand, these processes have some disadvantages such as: high energy consumption, high capital and operating costs, prone to complicated chemical and microbiological problems, and sludge and possibly effluent require further treatment and/or appropriate discharge. membrane bioreactor (mbr) technology has emerged as a wastewater treatment technology of choice over the activated sludge process. it overcomes the drawbacks of the conventional activated sludge process. mbrs have two fundamental process arrangements: ( ) integrated systems that have membranes immersed in an activated sludge reactor and ( ) separate systems that have a membrane module placed outside the reactor. immersed membranes are the most popular arrangement. the membranes (which could be flat sheet or tubular) have porosities ranging from . to . μm (which is considered between micro-and ultrafiltration). when immersed in wastewater, the membrane forms a barrier for impurities, while allowing pure water molecules to pass. process arrangements for implementation of mbr for nitrification, nitrogen removal, and complete biological nutrient removal could be also found. the mbr technology provides the following advantages over activated sludge process: it eliminates the requirement of large space for settling tank (or clarifier) which is replaced by the membrane, thereby reducing plant footprint which is usually %- % smaller than an equivalent conventional activated sludge facility; it produces high-quality effluent with higher volumetric loading rates; it needs shorter hydraulic retention time (hrt), and longer solid retention times (srt); it leads to less sludge production. a membrane bioreactor is able to process significantly higher sludge concentrations ( - g/l) and lower reactor volumes, compared to conventional activated sludge systems. however, the use of mbr technology has disadvantages, including higher energy costs, the need to control membrane fouling problems, and potential high costs of periodic membrane replacement. membrane fouling is believed to be a major drawback of mbr, as it significantly reduces membrane performances and membrane lifespan, leading to an increase in maintenance and operating costs and low treatment efficiency. membrane fouling in mbrs is attributable to suspended particulates in activated sludge that includes biomass of microorganisms and cell debris, colloids, solutes, and sludge flocs. these materials deposit onto the membrane surface and into the membrane pores, clogging the pores, and leading to a decline in the permeability of the membrane. when the clogging of membrane is due to accumulation of biomass of microorganisms, it is referred to as membrane biofouling. membrane biofouling is caused by several physicochemical and biological processes, which are highly dependent on the composition of feedwater, membrane characteristics, operation conditions, and microorganisms present. biofilms formation is thought to be essential in membrane biofouling. therefore, controlling biofilm formation in membranes can prevent mbr failures. in order to overcome the fouling problem, several operational strategies have been employed; however, membrane fouling remains an issue of investigation in mbr operation. understanding the biofouling phenomenon in mbr is crucial to be able to control this problem. hence, it is essential to characterize the microbial community involved in the development of mature biofilms in mbr. biofilm formation begins with the colonization of bacteria and subsequent surface attachment. the initial colonization starts with the detection of quorum sensing (qs) signal molecules (such as ahls) from the surrounding environment. the qs regulates the bacterial group behaviors in a cell density-dependent way and is closely associated with biofouling of the membrane in mbr treating wastewater. thus, characterization of activated sludge ahls will provide an additional insight into the bacteria involved in mbr. also it helps in developing more effective strategies to deal with the biofouling problem in mbrs, for example, by blocking of intercellular communication to prevent biofilm formation (lade et al., ; yeon et al., ) . apha), . standard methods for the examination of water and wastewater an introduction to environmental chemistry water microbiology. bacterial pathogens and water the biological basis of wastewater treatment water and wastewater engineering, first ed. the mcgraw-hill biofilms: microbial life on surfaces general chemistry essential microbiology quorum sensing and microbial biofilms isolation and molecular characterization of biofouling bacteria and profiling of quorum sensing signal molecules from membrane bioreactor activated sludge quorum sensing and bacterial social interactions in biofilms brock biology of microorganisms environmental science, technology, and chemistry; environmental chemistry handbook of water and wastewater microbiology antiseptics and disinfectants: activity, action, and resistance textbook of microbiology and immunology bacterial resistance to disinfectants treatise on geochemistry water microbiology microbiology: an introduction clean water: an introduction to water quality and water pollution control environmental engineering guidelines for drinking-water quality prescott's microbiology, th ed. mcgraw-hill education ground water and surface water; a single resource quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment guidelines for drinking-water quality, incorporating st and nd addenda key: cord- -egwo oc authors: aw, tiong gim; rose, joan b title: detection of pathogens in water: from phylochips to qpcr to pyrosequencing date: - - journal: curr opin biotechnol doi: . /j.copbio. . . sha: doc_id: cord_uid: egwo oc waterborne pathogens pose a significant threat to human health and a proper assessment of microbial water quality is important for decision making regarding water infrastructure and treatment investments and eventually to provide early warning of disease, particularly given increasing global disasters associated with severe public health risks. microbial water quality monitoring has undergone tremendous transition in recent years, with novel molecular tools beginning to offer rapid, high-throughput, sensitive and specific detection of a wide spectrum of microbial pathogens that challenge traditional culture-based techniques. high-density microarrays, quantitative real-time pcr (qpcr) and pyrosequencing which are considered to be breakthrough technologies borne out of the ‘molecular revolution’ are at present emerging rapidly as tools of pathogen detection and discovery. future challenges lie in integrating these molecular tools with concentration techniques and bioinformatics platforms for unbiased guide of pathogen surveillance in water and developing standardized protocols. water is essential to human health as a means to reduce diseases and also as the vehicle through which diseasecausing microorganisms or pathogens may be transmitted. a growing number of enteric microorganisms, including bacteria, viruses and protozoa in sewage have been identified as important waterborne pathogens. most waterborne pathogens are introduced into drinking water supplies and recreational waters by human and animal wastes and poor wastewater infrastructure as well as inadequate treatment. this includes leaking sewers, combined sewer overflows, urban storm water runoff, agricultural land runoff, faulty septic tanks/drain fields and poorly treated wastewater discharges. the vital role of public health in minimizing community wide-spread acquisition of these waterborne pathogens and infectious disease control of the hundreds of pathogens has drawn attention to the need for detecting and identifying bacteria, parasites and viruses directly in water. until recently, scientists have had no accurate and comprehensive way to detect and quantify the presence of microbial pathogens in various environmental samples. culture-based methods have long been extensively used to detect microbial pathogens in environmental matrices and are considered to be 'gold standard', but inherent problems exist, such as lengthy time of analysis, labor intensive and the inability to detect uncultivated species. the term ''the great plate count anomaly'' was coined by staley and konopka [ ] over years ago to describe great discrepancy between direct microscopic counting and numbers of bacteria that can be enumerated from environmental samples by plating procedures. the ability to access dna sequences of microbial genomes offers an exciting alternative for exploring and improving the characterization and rapid detection of a diverse group of pathogens in water. the advent of molecular tools has resulted in tremendous paradigm shifts in microbial water quality monitoring: (i) direct detection of pathogens, including uncultivated pathogens, is now possible and it takes hours instead of days to weeks, (ii) the true positivity rate of some pathogens in waters is known to be much higher than previously thought based on culture-based detection alone, and (iii) traditional bacterial indicators often fail to predict or correlate with the presence of waterborne pathogens. by increasing the capacity to detect a wide range of microbial pathogens in waters, novel and potential pathogens could be recognized and associated with waterborne diseases and better characterization of known pathogens can be undertaken for waterborne diseases of unknown etiology. according to web of science, articles with qpcr and water as key words have been published since with over articles in and . citations of this work grew to over /year by . this article reviews some of the most recent and significant progress in molecular tools for the detection of pathogens in water. eliminated many iconic waterborne enteric diseases such as typhoid and cholera in the north america and europe. however, microbial pathogens continue to be a major cause of water-related disease outbreaks globally and remain a key public health challenge in providing safe drinking water and recreational water in the st century. in addition to these traditional waterborne pathogens, a significant number of emerging and re-emerging pathogens have now been recognized and water transmission has caused significant outbreaks; examples include escherichia coli o :h , helicobacter pylori, legionella pneumophila, campylobacter jejuni, mycobacterium avium complex, enteric viruses such as noroviruses and hepatitis e virus, and the parasites cryptosporidium parvum and microsporidia. according to the latest world health organization (who)'s guidelines for drinking-water quality, zoonotic pathogens which make up % of the emerging pathogens, pose the greatest challenges to ensuring the safety of drinking water and ambient water [ ]. the list of potential waterborne pathogens is extensive [ ] and table provides examples of emerging waterborne pathogens which are also listed on the latest us epa's contaminant candidate list (ccl- ). because of the nature of waterborne pathogens, what is perceived as small concentrations are associated with high levels of community risks [ ] . the use of quantitative microbial risk assessment allows for an improved understanding of how concentrations, loading and transport influence probability of infection associated with microbial contamination of water. the limitations in detecting and identifying pathogens directly from environmental water samples by culture or microscopy can now be addressed by integrating concentration techniques with molecular tools to provide sensitive, specific and quantitative data on any pathogens of interest. the tools have a number of advantages. multiple pathogens can be characterized by microarray technology, however often sensitivity is not adequate. qpcr is the most rapidly growing technique for use in the water environment for both microbial source tracking and rapid pathogen specific quantification; and finally the power of pyrosequencing for exploring the water environment has offered new insights into the microbial world. developed in the early to mid- s, dna microarrays are arrays containing a high density immobilized nucleic acids (genomic dna, cdna or oligonucleotides) in an ordered two-dimensional matrix that enable the simultaneous interrogation of thousands of genes in a single assay via nucleic acid hybridization [ ] . unfortunately, current limitations of microarrays including the lack of sequence information for many pathogens, non-specific binding and inability to offer the low detection levels available by qpcr, make the use of microarrays for the direct detection of very dilute spectrum of pathogens in environmental matrices impractical. until recently, microarrays have not been used to quantitatively detect detection of pathogens in water aw and rose table potential waterborne pathogens, diseases caused and recent qpcr assays for their detection major disease(s) qpcr detection limit (per reaction) ref. pathogens in environmental water samples and a previous pcr enrichment with gene-specific primers was usually needed to increase the detection resolution [ ] . multiple displacement amplification, an isothermal dna amplification reaction using phi dna polymerase and random hexamer primers, offers a promising unbiased approach for increasing the amount of target dna in the environmental sample before microarray detection [ ] . the rapid growth of s rrna gene databases has helped pave the way for developing high-density phylogenetic microarrays to simultaneously analyze many taxa in complex environmental samples. phylogenetic microarrays such as the phylochip have been proven to be useful in determining microbial community structure [ ] . the phy-lochip that targets the known diversity within the s rrna gene is able to simultaneously identify any of thousands of taxa present in an environmental sample. the current version (g ) of the phylochip allows simultaneous detection of up to , bacterial, archaeal and microalgal taxa [ ] . besides monitoring bacterial population during environmental bioremediation, phylochip technology has been used for monitoring airborne bacteria for bioterrorism surveillance [ ] , microbial community analysis in biological wastewater treatment systems [ ] and microbial source tracking of pathogens in coastal urban watershed [ ] . more recently, two methods of pcr-independent microbial community analysis using the microarray phylochip: direct rrna hybridization and doublestrand cdna generation and hybridization have been developed, providing cost-effective and pcr bias-free alternatives to pcr-amplified microbial community analysis [ ] . the virochip, a pan-virus dna microarray containing thousands of unique -mer oligonucleotide probes to target all viral families known to infect humans has been described by wang et al. [ , ] . the authors demonstrated that this approach coupled with a random pcr amplification strategy, was able to detect multiple viruses as well as novel members of known viral families. recently, several high-density microarray prototypes such as the pan-microbial detection array (mda) [ ] and the greenechip system [ ] have been developed for the molecular surveillance and detection of a panel of pathogens including bacteria, viruses and parasites in clinical samples. these methods have yet to be applied to realworld water samples for the detection of waterborne pathogens. to make this practical, these high-density microarrays will need to be further coupled with water sample concentration and purification techniques, perhaps other amplification techniques and then could be used to assess the diversity of pathogens in water and potentially discover the presence of previously unknown pathogens. polymerase chain reaction (pcr) developed in the early s by kary mullis, is now a common and often indispensable molecular technique in both clinical and biological research laboratories. the concept of pcr remains unchanged since its inception. however, the agarose gel-based detection of pcr amplification products has undergone dramatically changes that have led to a revolutionary testing platform, namely quantitative real-time pcr (qpcr), which enables quantification of dna targets by monitoring amplified products during cycling as indicated by increased fluorescence [ ] . in the past few years, the development of qpcr has provided significant improvements in pcr-based assays for the detection of waterborne bacterial, viral and protozoan pathogens (table ) . currently, qpcr offers numerous advantages over conventional, end-point pcr techniques such as higher sensitivity and specificity, faster rate of detection, no post-pcr analysis and thereby minimizing the risk of carryover contamination, and the capability to provide quantitative results. the dual-labeled fluorescent probe such as taqman probe has been the most popular technique of choice for detecting pathogens in environmental samples owing to its higher specificity. the advantage of this assay is that the target amplicon is verified by the probe which recognizes internal amplicon sequences. inclusion of an internal control in each sample is essential for the accuracy of quantitative results, particularly for complex environmental matrices, by monitoring the efficiency of nucleic acid extraction and reverse transcription (for rna target) as well as the presence of pcr inhibitors. detection of human pathogenic viruses has undergone a significant transition from conventional cell culture to novel molecular techniques, particularly quantitative reverse-transcriptase (qrt)-pcr for rna viruses, with higher sensitivity and specificity. detection of pathogenic viruses by qrt-pcr does not necessarily indicate their infectivity, but it has been an excellent methodology for studying virus pollution in the water environment. moreover, the use of qrt-pcr for the analysis of non-cultivable noroviruses [ ] and slow growing hepatitis a virus [ ] has been fundamental to advancing the understanding of the occurrence of these human pathogens in water environments. surveys around the world have begun exploring the occurrence of both dna and rna viruses in water by qpcr (see [ ] for a review). however, there were no consistent trends found in regard to the viral types or levels. the molecular characterization of environmental viral strains has revealed a great genetic diversity of human pathogenic viruses in polluted surface waters [ ] . yet no global survey of viruses has been undertaken by evaluating the same virus targets, using the same protocols. a temporal assessment is needed to compare the diversity and level of viral infections and water pollution to contrast developing to developed regions of the world, from tropical environments to temperate conditions and to contrast rural versus urban populations. it may be possible to take on global virus characterization in the future with evolution in technology that allows higher throughput and hundreds of targets. advances in microfluidics and nanobiotechnology allow for the construction of high-density and low-volume qpcr platforms, such as biotrove openarray system (applied biosystems) that are capable of accommodating reactions per array. with a novel internal amplification control approach, this next-generation qpcr platform offers high-throughput testing of environmental samples against a wide range of pathogens [ , ] . since the invention of the dideoxy chain termination technique, often referred to as 'sanger sequencing', was first described more than three decades ago, technological advances have led to the increasing development of massively parallel dna sequencing platforms, including pyrosequencing. based on the sequencing-by-synthesis principle, pyrosequencing is a dna sequencing technology that utilizes enzyme-coupled reactions and bioluminescence to monitor the pyrophosphate release accompanying nucleotide incorporation, in real-time ( figure ) [ ] . a major advance for pyrosequencing technology came with the introduction of the roche sequencing platform. this next generation sequencing technology holds several advantages over the more traditional sanger sequencing include higher throughput, simplified sample preparation and the miniaturization of sequencing chemistries, enabling massively parallel sequencing reactions to be carried out at a scale and cost not previously possible [ ] . the latest sequencing platform, gx flx+ is able to produce one million reads (with a read length of up to bases) per -hour sequencing run. pyrosequencing technology is revolutionizing the study of microbial ecology as well as direct metagenomic detection detection of pathogens in water aw and rose high levels of several classes of resistance genes in bacterial communities exposed to antibiotic were identified. [ ] reclaimed and potable waters viral dna and rna tangential flow filtration, dnase treatment, mda, pyrosequencing ( gs-flx and gs sequencer) over % of the viral sequences with no significant similarity to proteins in genbank. bacteriophages dominated the dna viral community. the rna metagenomes contained sequences related to plant viruses and invertebrate picornaviruses. [ ] wastewater biosolids viral dna and rna dnase and rnase treatment, reverse transcription for rna, pyrosequencing ( gs-flx sequencer) optimal annotation approach specific for viral pathogen identification is described. parechovirus, coronavirus, adenovirus, aichi virus and herpesvirus were identified. [ ] lake water viral rna tangential flow filtration, dnase and rnase treatment, random amplification (klenow dna polymerase), pyrosequencing ( gs-flx sequencer) % of the sequences with no significant similarity to known sequences. presence of viral sequences ( viral families) with significant homology to insect, human and plant pathogens. of human pathogens in both clinical and environmental samples. unlike pcr and microarray methods where investigators are limited by known sequence information and must select the range of pathogens to be considered in a given assay, high-throughput sequencing approach is unbiased and makes it possible to detect novel pathogens. figure shows the basic steps that are needed for the detection of microbial pathogens in environmental waters by pyrosequencing. besides the direct detection of pathogens in stool specimens [ ] , several recent papers describe the application of pyrosequencing to investigate the diversity of bacterial and viral pathogens in environmental samples (table ) . a general finding from these studies is that the majority of the sequences in environmental metagenomes, particularly viral sequences, have no similarities to known genes in the database, indicating high abundance of uncharacterized viruses which may be potential human pathogens [ , ] . the extremely high-throughput of pyrosequencing has significantly expanded the repertoire of complete bacterial pathogen genomes, particularly strains associated with outbreaks [ , ] , since whole bacterial genomes can be sequenced in a fraction of the time and at a much lower cost. as the majority of waterborne disease outbreaks had undetermined etiology agents [ ] , pyrosequencing and other high-throughput sequencing technologies are likely to identify novel pathogens associated with waterborne illnesses in the future and could address multiple etiologies. moreover, sequencing of outbreak isolates is of public health interest, particularly if rapid data about virulence markers can be obtained. most recently, a new whole genome amplification and sequencing approach called 'single virus genomics', which enables the isolation and complete genome sequencing by pyrosequencing of the single virus particle (bacteriophages lambda and t ), has been described [ ] . this proof-of-concept study is likely to enhance uncultivated virus discovery as well as provide relevant viral reference genomes for the assembly of metagenomic data and the design of qpcr primers and probes targeting uncultivated viruses. further study is needed to optimize this approach for human pathogenic viruses in environmental waters. although high-throughput sequencing has made remarkable advances in the past few years, the most critical challenge for its application in pathogen detection is the development of improved user-friendly bioinformatics and visualization platforms to facilitate rapid and robust analysis and interpretation of high-throughput sequencing data. challenges for pathogen detection in water using molecular tools: upstream sample processing adapting novel molecular tools to meet the needs of waterborne pathogen monitoring is a significant technical challenge. rapid detection and identification of waterborne pathogens by novel molecular tools are often hampered by the relatively low abundance of target organisms in water samples. much work has been carried out in the development of rapid, sensitive and specific analytical methods. however, less emphasis has been placed on the pre-analytical or upstream sample processing which must be able to selectively separate and concentrate all target pathogens in each water sample. the preconcentration step is important in improving the detection sensitivity of microbial contaminants, particularly by molecular detection tools which use extremely small sample volumes (a few microliters). recently, tangential-flow, hollow fiber ultrafiltration, which uses size-exclusion as the mechanism of concentration, has been shown to be a promising technique to concentrate diverse pathogens in a single process [ , ] . moreover, ultrafiltration is currently the method of choice to concentrate viruses from large volumes of water for metagenomic detection by pyrosequencing [ , ] . the relationship between detection by molecular approaches and viability or infectivity of waterborne pathogens still remains a concern, particularly for risk assessment to public health. one of the most recent approaches to determine viability or infectivity using molecular tools is propidium monoazide (pma) treatment before qpcr to reduce pcr signals from dna originating from dead cells [ , ] . although pma treatment has also shown promise in excluding signals from dead cells with microarrays [ ] and pyrosequencing [ ] , further validation is needed in environmental water samples, particularly after disinfection. disinfection mainly chlorination of water serves as the major process for inactivating pathogens. viable qpcr methods using pma for distinguishing live cells and dead cells (heat killed) has not been verified for water after disinfection. varma et al. [ ] and srinivasan et al. [ ] found that there was no observed reduction in the qpcr signals after disinfection of wastewater effluent samples. thus, currently, qpcr can be used as a tool to monitor pathogen loading and physical removal or dilution but cannot be used to address viability. this review shows that considerable progress has been made to achieve sensitive, specific and high-throughput detection of pathogens in water. overall, qpcr is among the most reliable and cost effective of the molecular tools owing to the shorten time to result as well as its potential to increase sensitive and specific, and it is therefore not surprising that qpcr is increasingly being used for the detection of waterborne pathogens. despite the potential for development of high-throughput analytical systems for the detection and differentiation of pathogens in water, further assay optimization is needed for high-density microarrays to demonstrate reliability and match the sensitivity of qpcr. roche pyrosequencing and other commercially available high-throughput sequencing platforms such as solexa/illumina genome analyzer, applied biosystem solid sequencing as well as the most recently released ion torrent system [ ] have revolutionized the study of microbial diversity. nevertheless, these high-throughput sequencing platforms are currently in their infancy for the direct detection of pathogens in water and are exploratory. whereas many technological and methodological challenges remain, high-throughput sequencing technology has the potential to provide an unbiased detection approach for waterborne pathogens with a single common protocol. by integrating with qpcr, concentration of key pathogen groups can be subsequently determined. a recent study by svraka et al. [ ] where metagenomic sequencing was used to identify viruses in samples that exhibited consistent cytopathogenic effects in cell culture but remained unexplained by established pcr, demonstrates the need for unbiased approach for pathogen detection in a public health setting. however, several questions arise when molecular tools are being introduced to microbial water quality monitoring. which systems offer the greatest promise for more routine water pollution diagnoses? can current laboratory personnel be trained to perform some of these complex assays on a routine basis? what changes are necessary in water sampling, transport and storage? are the improved sensitivity and decreased turnaround time worth the additional cost for water quality monitoring? currently, success is being achieved more in the downstream part of the detection process and strategic sampling as well as concentration techniques of pathogens in environmental waters should be exploited further. it is clear that the field of environmental pathogen detection is and will remain highly dynamic with tremendous potential for development of new tools and continuous improvement of existing concepts. however, the major challenge facing environmental microbiologists is to apply these tools for detecting pathogens in real environmental matrices on a routine basis. multi-laboratory round robin test should be performed to evaluate novel molecular tools and refine the prototype protocols into standardized methods with appropriate technology transfer. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats microbial health risks and water quality challenges of multiplexed detection: detection of pathogens in water and wastewater using microarrays a comprehensive review describing recent advances and challenges of microarrays for the detection of waterborne pathogens something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology high-density universal s rrna microarray analysis reveals broader diversity than typical clone library when sampling the environment deepsea oil plume enriches indigenous oil-degrading bacteria urban aerosols harbor diverse and dynamic bacterial populations bacterial community structure in geographically distributed biological wastewater treatment reactors characterization of coastal urban watershed bacterial communities leads to alternative community-based indicators pcr amplification-independent methods for detection of microbial communities by the high-density microarray phylochip microarray-based detection and genotyping of viral pathogens viral discovery and sequence recovery using dna microarrays a microbial detection array (mda) for viral and bacterial detection panmicrobial oligonucleotide array for diagnosis of infectious diseases why the need for qpcr publication guidelines?-the case for miqe the author provides guidelines to improve entire qpcr workflow, from experimental design to statistical analysis and reporting. general implementation of these guidelines is important for producing consistent and high quality data from qpcr assays. transcription-pcr assay for quantification of hepatitis a virus in clinical and shellfish samples molecular detection of pathogens in water -the pros and cons of molecular techniques prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments accurate quantification of microorganisms in pcr-inhibiting environmental dna extracts by a novel internal amplification control approach using biotrove openarrays development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume pcr on the openarray platform pyrosequencing: history, biochemistry and future an informative introduction to the principle of pyrosequencing the development and impact of sequencing this article provides an overview of the sequencing technology direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach metagenomic analysis of viruses in reclaimed water metagenomic analysis of rna viruses in a fresh water lake these papers describe the metagenomic detection of viruses in environmental water samples by massively parallel pyrosequencing technology high-throughput genome sequencing of two listeria monocytogenes clinical isolates during a large foodborne outbreak variation in virulence among clades of escherichia coli o :h associated with disease outbreaks causes of outbreaks associated with drinking water in the united states from to single virus genomics: a new tool for virus discovery the authors describe a new approach that combines flow cytometry, whole genome amplification and pyrosequencing for the isolation and complete genome sequencing of a single virus particle. this approach has the potential to discover novel viruses and facilitate comparative genomic analyses by providing relevant reference genomes dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water tangential-flow ultrafiltration with integrated inhibition detection for recovery of surrogates and human pathogens from large-volume source water and finished drinking water quantification of viable legionella pneumophila cells using propidium monoazide combined with quantitative pcr use of propidium monoazide in reverse transcriptase pcr to distinguish between infectious and noninfectious enteric viruses in water samples selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by pyrosequencing quantitative real-time pcr analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater escherichia coli, enterococci, and bacteroides thetaiotaomicron qpcr signals through wastewater and septage treatment landscape of next-generation sequencing technologies the authors provide the current state of the art in next-generation sequencing technologies the authors demonstrate that metagenomic sequencing can provide an improved detection of unidentified pathogens in a public health setting rapid identification and quantification of campylobacter coli and campylobacter jejuni by real-time pcr in pure cultures and in complex samples quantification of viable but nonculturable escherichia coli o :h by targeting the rpos mrna failure to detect helicobacter pylori dna in drinking and environmental water in dhaka, bangladesh, using highly sensitive real-time pcr assays specific real-time pcr for simultaneous detection and identification of legionella pneumophila serogroup in water and clinical samples influence of environmental gradients on the abundance and distribution of mycobacterium spp. in a coastal lagoon estuary detection of live salmonella sp. cells in produce by a taqman-based quantitative reverse transcriptase real-time pcr targeting inva mrna development of a multiplex real-time pcr assay with internal amplification control for the detection of shigella species and enteroinvasive escherichia coli real-time pcr with an internal control for detection of all known human adenovirus serotypes a broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection of hepatitis e virus development and evaluation of a real-time pcr assay for quantification of giardia and cryptosporidium in sewage samples a duplex real-time pcr assay for the quantitative detection of naegleria fowleri in water samples pyrosequencing of the s rrna gene to reveal bacterial pathogen diversity in biosolids pathogenic bacteria in sewage treatment plants as revealed by pyrosequencing examples of how massively parallel pyrosequencing can be used to determine the diversity of bacterial pathogens in wastewater based on s rrna pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements using pyrosequencing, the authors demonstrate high levels of resistance genes in bacterial communities in river sediments exposed to antibiotics viral metagenome analysis to guide human pathogen monitoring in environmental samples another example of how pyrosequencing can be used to study the diversity of human pathogenic viruses in environmental samples. the authors also describe an improved bioinformatic approach for identifying pathogens in a virome data set key: cord- -t n dv authors: Żaczek, maciej; weber-dąbrowska, beata; górski, andrzej title: phages as a cohesive prophylactic and therapeutic approach in aquaculture systems date: - - journal: antibiotics (basel) doi: . /antibiotics sha: doc_id: cord_uid: t n dv facing antibiotic resistance has provoked a continuously growing focus on phage therapy. although the greatest emphasis has always been placed on phage treatment in humans, behind phage application lies a complex approach that can be usefully adopted by the food industry, from hatcheries and croplands to ready-to-eat products. such diverse businesses require an efficient method for combating highly pathogenic bacteria since antibiotic resistance concerns every aspect of human life. despite the vast abundance of phages on earth, the aquatic environment has been considered their most natural habitat. water favors multidirectional brownian motion and increases the possibility of contact between phage particles and their bacterial hosts. as the global production of aquatic organisms has rapidly grown over the past decades, phage treatment of bacterial infections seems to be an obvious and promising solution in this market sector. pathogenic bacteria, such as aeromonas and vibrio, have already proved to be responsible for mass mortalities in aquatic systems, resulting in economic losses. the main objective of this work is to summarize, from a scientific and industry perspective, the recent data regarding phage application in the form of targeted probiotics and therapeutic agents in aquaculture niches. fisheries and the aquaculture sector are key elements in the food industry that supply food to every part of the world. according to the food and agriculture organization of the united nations (fao), global fish production reached million tons in , nearly % of which has been consumed by humans [ ] . the numbers speak for themselves. in the past years, global aquaculture production increased by over % and fish consumption by % [ ]. in the united states alone, the estimated freshwater and marine aquaculture production reached a value of usd . billion in , an increase of over usd million from [ ] . on a global scale, china is the largest producer of seafood products in the world, followed by india, indonesia, vietnam, and bangladesh. the most commonly farmed species in the fish industry are carps, tilapias, and salmon. in addition to fish species, aquaculture production consists of, among others, clams, oysters, and shrimp [ ] . fish constitute crucial nutrition in many areas. it is not only one of the healthiest foods on earth but also one of the least impactful on the natural environment. seafood is rich in protein, vitamins, and minerals. the most recognized nutritional benefits come from the omega- fatty acids control and treatment options for a vast range of fish pathogens using vaccines and drugs are either inadequate, inefficient, or impracticable [ ] . furthermore, seafood is more susceptible to microbial spoilage than meat and has a relatively short shelf-life. there are several reasons for the differences between red meat and fish. first of all, red meat has a lower ph and is less moist. postmortem ph in fish flesh increases due to the utilization of large amounts of amino acids and other low molecular weight compounds by fish spoilage bacteria [ ] . in addition, contamination of seafood may originate not only from water but also from soil [ ] . microorganisms can be found on all the outer surfaces (skin and gills) and in the intestines of live and newly caught fish. it has been noted that the warmer the natural waters are, the higher the bacterial count associated with fish [ ] . global warming, which is on the rise, will certainly challenge the entire industry in terms of eliminating bacterial pathogens both in the water as well as during the food processing steps in the upcoming decades. moreover, global warming may shift the diseases traditionally confined to warm subtropical geographical areas, such as shellfish-related gastroenteritis caused by vibrio parahaemolyticus [ ] , northwards. the three most common bacterial genera identified as fish pathogens are aeromonas, flavobacterium, and vibrio, followed by edwardsiella, yersinia, renibacterium, streptococcus, and mycobacteria [ ] . examples of pathogenic bacterial flora associated with live fish, as well as fish muscle (meat) intended for consumption, are presented in table . mycobacteria * alcaligenes enterobacter pseudomonas moraxella acinetobacter shewanella putrefaciens flavobacterium raoultella edwarsiella yersinia typical of marine waters [ ] . typical of fresh waters [ ] . found on modified atmosphere-packed salmon [ ] . found in spoiled sardine [ ] . predominant during the ice storage of fish and shrimp [ ] . pathogen of japanese flounder, rainbow trout, tilapia, and coho salmon [ ] . causative agent of bacterial kidney disease affecting salmonid fish [ ] . * described by some authors as neither gram-positive nor -negative (a gram stain hardly penetrates the waxy cell wall). notably, fish pathogens can freely invade the flesh of dead fish by moving between the muscle fibers [ ] . fish and seafood spoilage is inevitably associated with the formation of chemical compounds that can be toxic for humans, such as trimethylamine, followed by ammonia, h s, and indole [ ] . there are numerous different bacteria capable of producing biogenic amines that have been isolated from fish muscle [ ] . even fish products with high salt content may spoil due to the growth of halophilic bacteria or anaerobic bacteria [ ] . although fish spoilage is usually easy to detect by observing changes in color, smell, or tenderness of fish flesh, many water-associated pathogens are opportunistic and may remain undetected in living organisms [ ] . in such a way, fish and seafood pathogens transferred to the human body can cause septicemia. for instance, marine vibrio bacteria (in particular, v. vulnificus and v. parahaemolyticus) can be found on oysters, a popular seafood that is consumed raw. this model of consumption puts people at great risk. it is estimated that in the usa, approximately , people contract a foodborne infection from vibrio spp. every year, with symptoms such as diarrhea, along with abdominal cramps, nausea, vomiting, headache, chills, and low-grade fever; fatal cases have been reported as well [ ] . v. vulnificus can also be responsible for chronic liver disease accompanied by high mortality [ ] . analysis of samples of blue mussels, seawater, or sediment revealed the presence of v. parahaemolyticus in over % of the samples collected at all time-points throughout the year [ ] . recently, the expansion of fish pathogens into new geographic areas and widening of their host range has been noted, leading to an emergence of new pathogens [ , ] . contrary to human or animal medicine, fish treatment concerns the entire local population and, therefore, is more difficult to control [ ] . prior to an investigation of therapeutic phage connotations in the fish and seafood industry, it is important to understand that environmental waters are their natural habitats. hence, the idea of phage application in aquaculture does not seem to be innovative or groundbreaking. suffice to say that phage discovery was inseparably associated with waters, and even over years later, this phenomenon is still being investigated in the ganges [ ] . these days we know that in aquatic ecosystems, there is typically a -to -times greater number of phage particles when compared to their bacterial hosts [ ] . viruses in aquatic habitats are responsible for the mortality of nearly - % prokaryotes every day, affecting community composition and impacting global biogeochemical cycles [ , ] . despite the small size of phages (approx. nm), they constitute the second-largest biomass in the oceans, just after the prokaryotic biomass [ ] . they tend to accumulate in coastal waters rather than in offshore waters, although phages can be found under any latitude, including arctic sea ice [ ] . analyses of phage genomes revealed not only seasonal phage distribution in waters but also different phage allocations in upper versus deeper water layers [ ] . their existence is shorter in upper layers, possibly due to environmental factors such as sunlight or relatively higher temperatures. experiments conducted to date have revealed that phages were found in % of the water samples collected from environmental habitats and industrial systems, including water treatment plants and drinking water [ ] . there is clear evidence that waters have been the most promising source of therapeutic phages since their discovery over years ago [ ] . one might assume that the actual known number of phages associated with aquatic environments is significantly larger, as the plethora of their bacterial hosts is difficult to culture in laboratory settings [ ] . the situation has changed, thanks to newly developed virome analyses. without the need to culture both bacteria and phages, scientists can finally become acquainted with a growing number of phage genome sequences. these days, the importance of phages in aquatic environments is of ongoing relevance. when lysing their hosts, viruses cause the release of dissolved organic matter (dom) in the process called "viral shunt" [ ] . bacteria have a high protein-to-dna ratio and, therefore, a high nitrogen-to-phosphorus (n:p) ratio. as a result of phage lysis, this larger amount of organic n than organic p is released and made available to other bacteria and phytoplankton for growth. scientists hypothesize that the effect of lysis on uninfected bacteria production and abundance, as well as on ecosystem functions, including the carbon cycle, might depend on which nutrient (n or p) is limiting. it is estimated that dissolved organic carbon constitutes the largest pool of organic matter in the oceans, and its regulation is a major biogeochemical process. recently performed studies showed that mutual phage interactions with bacterial hosts are responsible for conversion from organic to inorganic carbon in water environments [ ] . in tested samples, the increase in inorganic carbon was - % larger in samples with bacteria and phages when compared to samples with bacteria alone. notably, the accumulation of inorganic carbon in the form of co may have a physiological effect on feed intake, appetite, digestive function, and slowed growth in fish hatcheries. further, high dissolved co levels have been associated with the formation of mineralized deposits in the kidneys of salmonids (nephrocalcinosis), eye cataracts, and increased susceptibility to pathogens [ ] . last but not least, a plethora of phages in water make them ideal candidates to be utilized in other industry sectors. thanks to their abundance, sometimes restricted to narrow water sources, they can be useful as human-associated fecal indicators [ , ] or in pathogen reduction in wastewater treatment plants [ ] . a recent report suggests that marine phages may act as a microbial tracer for the transport of colloidal particles and water flow and contribute to better risk analysis [ ] . it is worth noting that the role of phages in spreading antibiotic resistance genes has been raised in a growing number of articles as well, which clearly reflects an increased interest in phage-based therapeutics in general [ ] [ ] [ ] [ ] . metagenomic studies have confirmed the presence of such genes in phages isolated from a vast range of environments, which is in line with their ability to transfer genetic material between hosts. the above referenced vast phage abundance in waters likely contributes to the intensification of the transduction of genes conferring resistance in aquatic niches. in fact, a recent study detected that environmental waters contain a large reservoir of resistance genes, conferring resistance to tetracyclines and β-lactamases. those genes, detected in phage dna, were isolated from freshwater, water treatment plants, and oceans [ , ] . aquatic environments may promote dissemination and acquisition of antibiotic resistance genes [ ] , which should be taken into consideration in aquacultures. fish farms, exposed to antibiotic stress, along with high densities of microorganisms and closed water circulation, can provoke horizontal transfer of unwanted genes and the danger of their leakage to the environment [ ] . the latest data indicate that antibiotic-resistant bacteria isolated from both aquaculture and agriculture share the same resistance mechanisms, indicating that aquaculture is contributing to the same resistance issues established by agriculture [ ] . the presence of remnants of antibiotics and antibiotic resistance genes in major aquacultures in southeast china has also been pointed out by chen et al. [ ] . such a phenomenon could be intensified in cases of prolonged, continuous, preventive phage application. in fact, among analyzed genomes of aeromonas veronii, a common human and animal pathogen abundant in aquatic environments, only two strains were free of phage elements [ ] . in contrast, there are sources questioning the active role of phages in spreading antibiotic resistance genes in waters. petrovich et al. [ ] , in their recent study, could not find strong evidence for phages as vectors for those genes in a hospital wastewater treatment system, suggesting that false-positive samples are possible. one must be aware that the use of antibiotics in fish hatcheries will result in accumulated and unabsorbed antibiotics that are likely to affect phage-host coevolution outside the host [ ] . certainly, the issue of spreading antibiotic resistance is not to be disregarded, and exaggerated optimism should not be a driving force for the extensive use of phages in aquacultures. antibiotic resistance genes isolated from wastewater samples have been reported to persist longer in phages than in bacteria [ ] . notably, there is a growing number of fish pathogens isolated from aquacultures carrying crispr-cas systems. this pattern was observed mostly in flavobacterium and vibrio species [ ] . the more contaminated the waters, the more likely it is that a higher count of microorganisms (including phages and their bacterial hosts) will be found in such samples. our team has been utilizing this phenomenon for several years during phage isolation. the most valuable samples come from water treatment plants and hospital sewage systems [ ] . one could assume that the cumulation of organic matter, chemotherapeutics, and other contaminants in aquacultures could result in a blooming plethora of utterly different phages, with an unexpected impact on environmental balance. notably, phages have been shown to be resistant, at least to some extent, to chemical agents used in water disinfection and can survive under unfavorable conditions for a long time. we explore this phenomenon further in section . . interestingly, lytic phages may be responsible for biofilm formation [ ] . such an occurrence was noted in marine tunicate ciona intestinalis, whose gut is colonized by shewanella. a study showed that mutual interactions between shewanella and its lytic phages enhanced biofilm formation. although shewanella is an unusual human pathogen, infections caused by these bacteria have been increasingly accompanied by symptoms such as cellulitis, abscesses, bacteremia, and wound infection [ ] . moreover, prophage induction can also stimulate biofilm formation in shewanella through the release of extracellular dna, which can be found in the biofilm matrix [ ] . temperate phages were found to act as disrupting agents in the nitrification process, which is one of the most important processes in waters [ ] . the authors revealed that the induction of the lytic cycle led to a decrease of the total nitrosospira multiformis count, a bacteria that is responsible for converting ammonia to nitrite. ammonia is severely toxic and represents an environmental threat for aquatic life, particularly in small reservoirs and at lower ph values. freshwater fish excrete ammonia directly and immediately into the water and, therefore, its concentrations may rapidly increase in aquacultures of high fish density. furthermore, dissolved ammonium is colorless and difficult to observe at an early stage of intoxication. as nitrifying bacteria occur together, a rapidly decreasing count of one group could disrupt the entire cycle. however, there is a greater chance for disruption of the nitrogen cycle in water through the use of aquaculture drugs, and phage lytic activity towards nitrifying bacteria should be studied more precisely in terms of possibility, not a real threat. a simplified scheme of the nitrification process is shown in figure . temperate phages were found to act as disrupting agents in the nitrification process, which is one of the most important processes in waters [ ] . the authors revealed that the induction of the lytic cycle led to a decrease of the total nitrosospira multiformis count, a bacteria that is responsible for converting ammonia to nitrite. ammonia is severely toxic and represents an environmental threat for aquatic life, particularly in small reservoirs and at lower ph values. freshwater fish excrete ammonia directly and immediately into the water and, therefore, its concentrations may rapidly increase in aquacultures of high fish density. furthermore, dissolved ammonium is colorless and difficult to observe at an early stage of intoxication. as nitrifying bacteria occur together, a rapidly decreasing count of one group could disrupt the entire cycle. however, there is a greater chance for disruption of the nitrogen cycle in water through the use of aquaculture drugs, and phage lytic activity towards nitrifying bacteria should be studied more precisely in terms of possibility, not a real threat. a simplified scheme of the nitrification process is shown in figure . ( ) decomposition of organic matter (e.g., fish excrement and uneaten fish food) leads to an increase in ammonium levels in fish tanks. in addition, fish excrete ammonium directly into the water. ( ) nitrifying bacteria (nitrosomonas, nitrosospira) oxidize ammonium to nitrite. ( ) nitrifying bacteria (nitrobacter, nitrospira) oxidize nitrite to nitrate. the latter is utilized as a plant fertilizer. indisputable differences between natural aquatic niches and aquacultures need to be carefully studied in order to properly conduct phage application. water parameters such as temperature, salinity, content of dissolved organic matter, ph, and oxygen saturation are subjected to rapid change in small water tanks loaded with different varieties of fish. obviously, such events should never occur, but small tanks are always at greater risk, especially in systems with high densities of fish, where biomass growth and substrate utilization can be difficult to control. consequently, spreading disease among fish would be much faster and more lethal in densely-stocked fish tanks. the need to maintain continuously available sources of food to prevent starvation and to promote rapid growth challenges fish workers on a daily basis [ ] . a large amount of easy-digestible food increases levels of organic pollutants, such as dioxins and pcbs (polychlorinated biphenyl). a study from reported on contaminated farmed atlantic salmon carrying such high levels of pbcs that it could be harmful to humans [ ] . all these factors pose a great danger to phage-bacteria interactions through changes in water parameters and may affect the outcome of phage application. below, we characterize the most crucial aspects that should be considered in aquacultures. ( ) decomposition of organic matter (e.g., fish excrement and uneaten fish food) leads to an increase in ammonium levels in fish tanks. in addition, fish excrete ammonium directly into the water. ( ) nitrifying bacteria (nitrosomonas, nitrosospira) oxidize ammonium to nitrite. ( ) nitrifying bacteria (nitrobacter, nitrospira) oxidize nitrite to nitrate. the latter is utilized as a plant fertilizer. indisputable differences between natural aquatic niches and aquacultures need to be carefully studied in order to properly conduct phage application. water parameters such as temperature, salinity, content of dissolved organic matter, ph, and oxygen saturation are subjected to rapid change in small water tanks loaded with different varieties of fish. obviously, such events should never occur, but small tanks are always at greater risk, especially in systems with high densities of fish, where biomass growth and substrate utilization can be difficult to control. consequently, spreading disease among fish would be much faster and more lethal in densely-stocked fish tanks. the need to maintain continuously available sources of food to prevent starvation and to promote rapid growth challenges fish workers on a daily basis [ ] . a large amount of easy-digestible food increases levels of organic pollutants, such as dioxins and pcbs (polychlorinated biphenyl). a study from reported on contaminated farmed atlantic salmon carrying such high levels of pbcs that it could be harmful to humans [ ] . all these factors pose a great danger to phage-bacteria interactions through changes in water parameters and may affect the outcome of phage application. below, we characterize the most crucial aspects that should be considered in aquacultures. studies performed with the use of water samples collected throughout the territory of poland revealed an unexpected presence of phages in municipal tap water systems [ ] . this evidence demonstrates that water disinfection, both chemical and physical, does not significantly affect the lytic activity of at least some phages. studies performed by yates et al. [ ] revealed that the prevalence and activity of coliphages in groundwaters may be affected by uv irradiation, the presence of organic compounds (particularly, acids from soil humus), and metabolic activity of other aquatic organisms. interestingly, data collected from numerous water tanks in different parts of the usa showed that ph fluctuations, water clarity, and hardness had no major impact on phage survival. in fact, the only factor inhibiting phage activity was temperature [ ] . the importance of temperature for phage lytic activity has been pointed out countless times by several authors over the years [ ] [ ] [ ] [ ] , along with in-depth molecular analyses of this occurrence. leon-velarde et al. [ ] revealed that yersinia enterocolitica outer membrane protein ompf, which acts as a receptor for phages, is subject to strong repression at • c. thus, the tested phage lysed its host when grown at • c but not at • c. intriguingly, recent articles imply that temperature may define the outcome of phage-bacteria interactions through the determination of the phage life cycle [ , ] . switching between cycles possibly regulates the population of the bacterial host throughout the seasons. investigations performed by madsen et al. [ ] on flavobacterium psychrophilum phages in salmonid aquacultures have shed more light on the ph-dependent activity of phages. in a laboratory setting, ph had only minor effects on long-term ( months) phage infectivity within a ph range of . to . but inhibited lytic activity below ph . particularly, low ph turned out to be the greatest obstacle in phage attachment to bacterial receptors [ ] . notably, marginal ph values, which proved to be lethal for phages [ , ] , are rarely found in environmental waters or fish tanks. notwithstanding, even in those extreme habitats, phages show unusual adaptation and are profoundly represented in niches of extreme ph, temperatures, and/or salinity. for phages isolated in from a haloalkaline lake in kenya, the optimal ph ranges from as high as to , values that are not tolerated by most of the lab-cultured phages [ ] . another important factor in terms of phage viability in waters is salinity. although phages are generally considered more resistant to salinity than bacterial species, osmotic shock has been shown to inactivate phages by even % [ ] . very interesting data on phage lytic activity in waters, with possible connotations for fish and seafood industry, come from silva-valenzuela et al. [ ] . three vibrio cholerae phages, well-known for their lytic activity in cholera patients in bangladesh, were not able to infect v. cholerae in fresh water. a significant decrease in osmolarity, inorganic nutrients, and carbon sources in the aquatic model caused a loss of bacterial viability and resulted in a lack of phage infection. the authors noted that high salinity was a crucial factor for some vibrio phages' predation. salinity in waters is subject to constant change, mostly due to water evaporation at high temperatures. this phenomenon may occur on a much larger scale in fish tanks characterized by limited water capacity, with a possible further impact on the therapeutic outcome of phage treatment. fluctuations in both viral and prokaryotic heterotrophic production were demonstrated in experimental cross-infections between viruses and prokaryotes from three tropical sites of west africa, with distinct salinities [ ] . the addition of native viruses consistently stimulated viral production. however, the lowest rates of this phenomenon were observed in hypersaline ( % ) water. choudhury et al. [ ] conclude that salinity and ph are two crucial abiotic factors affecting the growth and survival of aquatic organisms. the authors found that the v. harveyi phage was active against its host in all three tested salinities, but the highest rate was observed at a salinity of ppt, which was also optimal for the host. a similar analogy was found for the ph. a neutral ph turned out to be the most favorable for both the phage and its host. the ratio of infecting phage particles to bacterial prey is crucial in terms of the effective annihilation of pathogens. conditions in fish and seafood hatcheries may significantly differ from what we observe in wild waters. thus, quite a different approach is needed in the context of the therapeutic evaluation of phages in aquatic systems. typical phage and bacteria densities in waters are often too low for successful phage replication and more favorable conditions are rarely expected to be found in natural water niches [ ] . nilsson et al. [ ] observed phage-bacteria dynamics in the baltic sea. the number of rheinheimera-infecting phages was strictly correlated to host abundances from june to august between and . furthermore, alonso-saez et al. [ ] found that a high abundance of some marine phages does not imply they are highly active in waters. our group came to similar conclusions following the testing of aeromonas phages and their bacterial hosts in a small-scale water treatment plant [ ] . there was no increase in phage titer after a -h incubation period, despite the presence of a specific bacterial host. after several days, we were still able to recover phages introduced to the tank at the beginning of the experiment, which clearly confirms the theory that phages can be resistant to environmental factors, including filtration processes, but they are unable to amplify due to low values of cfu. in conclusion, proper dosing should always be carefully considered before phage application into fish hatcheries. the necessity of high moi values was pointed out by kalatzis et al. [ ] . phages against v. alginolyticus, an agent responsible for vibriosis in marine aquaculture hatcheries, were effective in vitro only at moi = and moi = (a -and -times higher phage ratio to bacteria, respectively), whereas at moi = , the lytic effect was barely noticeable. kim et al. [ ] used an moi as high as , to achieve a protective effect of aeromonas salmonicida phage pas- on rainbow trout. interestingly, another group revealed quite different results. an in vitro investigation performed on phages against waterborne fish pathogen aeromonas salmonicida showed limited lytic activity at moi = and almost complete lysis at moi = . [ ] . notably, in the first case, the bacterial count started to rise h following phage inoculation but remained at the same low level over the h (end of experiment) at low initial moi. the authors conclude that heavy phage loads could accelerate the occurrence of bacterial resistance to phages in a relatively short period of time. such discrepancies require further attention to disentangle complex phage biology in vitro, as well as in vivo, prior to designing phage-based therapeutics. the plethora of interactions was pointed out by bettarel et al. [ ] , who observed that viral enrichment in waters, and hence the rise of moi, elevates chances for contact between phages and bacteria in an environment-dependent manner. daniels and wais [ ] proposed the theory that a natural environment characterized by a low bacterial load may promote slow phage adsorption to reduce the frequency of release of dna from phage particles in response to encounters with nonhost material. irrespective of moi values, phages applied in low doses could act as preventive agents; this has been utilized several times for crops [ ] . it must be emphasized that our aforementioned experiment was performed without the involvement of fish or other aquatic organisms and, therefore, we were unable to evaluate possible therapeutic connotations in the described model [ ] . studies carried out by almeida et al. [ ] have shed more light on the prophylactic value of phages tested in comparable conditions. the authors used similar phage doses ( ml of flavobacterium fcl- lysate with the titer of × pfu/ml), achieving a final phage density of × pfu/ml in each tested tank with no addition of a bacterial host. although the authors confirmed that phages recovered during the experiment were derived from the original lysate and not from the environment or natural phage infection, applied doses had a positive impact on the overall health status of the fish. phages were reisolated from fish mucus and gills, providing a protective effect against f. columnare infections. furthermore, phage addition did not cause negative alterations in water quality or fish health. prolonged phage persistence in both the described experiments indicates that the protective, rather than therapeutic, effect of phage preparations on fish may be relatively easy to obtain. the absence of lytic activity in f. psychrophilum phages challenged with a low bacteria count was presented by madsen et al. [ ] . in the absence of host cells, phage infectivity in pond water decreased by , times over days. additionally, the infectivity of the same tested phage decreased over time in a buffer kept at • c, which indicates the necessity of constant supplementation of phage-based preparations in water. the use of phages in the form of probiotics has already been proposed by soliman et al. [ ] as a natural remedy in intensive fish farming. successful phage therapy can be greatly affected by immune deficiencies of organisms undergoing treatment, which has already been shown on the human model [ ] [ ] [ ] [ ] . furthermore, phages are capable of influencing the immune system of eukaryotes in a variety of ways [ , ] . in aquatic hatcheries, fish are subjected to constant stress due to the extensive use of antibiotics and other drugs and overstocking. hence, their ability to fight infections may be greatly weakened in different epidemic scenarios. one could predict that phage addition in aquacultures could modulate the immune response and act as an immunostimulant for the induction of antibodies. silva et al. [ ] noted that phage application against aeromonas salmonicida in juvenile senegalese sole did not impact natural bacterial communities but moderately affected the bacterial community associated with the fish intestinal tract. evidence for a large amount of naturally occurring phages in the digestive tract of fish was provided by studies performed by he and yang [ ] . in the gut of cultivated freshwater fish, researchers found phages, including vast diversity of aeromonas phages ( ), followed by vibrio ( ), citrobacter ( ), serratia ( ), enterobacter ( ), proteus ( ), buttiauxella ( ), plesiomonas ( ), kluyvera ( ), morganella ( ), and providencia ( ). the phages contribute to the microbiota balance in the gut ecosystem of fish and modulate their immunity. the innate immune system consists of barriers limiting the pathogens' ability to spread throughout the body. the inherent part of the innate immune system is mucus. this is a site of attachment for most pathogens, which causes mucosal infections responsible for high mortality and morbidity among fish [ ] . however, phage attachment to mucosal surfaces has been observed as well. almeida et al. theorize that such binding could create a ubiquitous nonhost-derived immunity against bacterial invaders [ ] . as fish are naturally covered by mucus, the authors investigated flavobacterium columnare fcl- myophage phage adherence to mucus layers in rainbow trout. although phage titer in water tanks decreased rapidly below the level of detection, phage particles remained in the mucus of rainbow trout for one week. it is possible that the persistence of fcl- phage in the mucus could be the result of the subdiffusive motion created by phage ig-like folds interacting with mucins, which appears to be stronger than mucus shedding and water flow. furthermore, the described pretreatment with phages resulted in a delay in disease onset and increased fish survival. the authors also observed increased bacterial susceptibility to phage infection in the mucosal environment. some authors suggest that phage-bacteria interactions in mucus play a role in lysis-lysogeny switches [ , ] . whereas the bam (bacteriophage adherence to mucus) model may promote phages by enabling them to have contact with the host and eliminating potential pathogens from deeper mucus layers, the ptw (piggyback the winner) model may favor bacteria through lysogeny. interestingly, barr et al. observed that only binding between phage ig-like domains and mucin resulted in the reduction of bacterial load, which suggests targeted action rather than accidental movement and attachment [ ] . reports on phage immunomodulatory activity in rainbow trout subjected to phage treatment also came from schulz et al. [ ] . the authors used a commercially available phage cocktail called bafador, intended for use in aquaculture niches. besides obvious antibacterial action against aeromonas hydrophila and pseudomonas fluorescens, which decreased the mortality of rainbow trout, elevated levels of immunoglobulin, protein, and lysozyme were noted, along with the increased activity of spleen phagocytes and proliferation of pronephros lymphocytes. the same outcome, after the application of bafador, was observed on european eels after experimental challenge with the aforementioned a. hydrophila and p. fluorescens [ ] . these results are consistent with reports of a chinese group who treated common carp with a phage lysate antigen as a vaccine active against the fish pathogen a. hydrophila [ ] . the survival rate of fish immunized with the phage vaccine was higher when compared to immunization with formalin-killed bacterial cells six weeks postvaccination. such a favorable result was accompanied by a robust immune response in the form of higher il- β and lysozyme c gene expression, along with higher tnf-α gene expression. in another experiment, rainbow trout, after intramuscular administration of the aeromonas salmonicida phage, showed significant neutralizing properties of its sera at and days postadministration, which declined by days [ ] . this sera neutralization did not correspond with phage particles accumulating in kidneys and occurred after the phage was cleared from fish kidneys below the level of detection. one must be aware that these preliminary reports require follow-up studies on the nonantibacterial action of phages. nevertheless, reports to date leave hope for the efficient and long-lasting prophylactic effect of phage preparations in fish hatcheries. interestingly, laanto et al. [ ] proved that phage resistance, developed over time in emerging fish pathogen f. columnare, declined bacterial virulence outside the fish host. thus, prophylactic use of phages could work in three different ways: (a) through direct lytic activity, (b) as immunomodulators in fish tanks through interactions in mucus, and (c) due to phenotypic changes in bacteria that lead to lower bacterial virulence. a countless number of sources have hypothesized that phages are a promising alternative to antibiotics in industry and medicine. the fish and seafood industry is certainly no exception here. this well-deserved rebirth is a result of numerous more or less analyzed factors, like relatively low cost, safety for the environment and treated organisms, as well as the vast abundance of natural therapeutic agents in the form of phage particles. the latter may be easily investigated and amplified in laboratory settings using well-known and simple techniques. table presents a summary of recent attempts to control fish and seafood pathogenic bacteria with the use of phage-based preparations. articles focusing on phage application in aquaculture facilities have skyrocketed in the past decade, which we wanted to emphasize by adding the year of publication to the table. as in-vitro results may differ from what can be achieved in the field, we focused solely on in-vivo experiments conducted over the past decade. undeniably, one of the major challenges in phage treatment is the use of a single phage versus a phage cocktail [ ] . customizing phage cocktails may be time-consuming, but universal phage therapeutics may not target all desired bacteria. studies performed by holmfeldt et al. [ ] found a large variation in host range diversity among cellulophaga baltica phages isolated from swedish and danish coastal waters. the authors suggest that the well-known narrow host range of marine phages may be the result of inadequate methods for phage isolation (e.g., single-host enrichment). summarized reports from recent years show the proportional use of both single-phage preparations as well as phage cocktails, along with the use of different moi ratios. a lack of information regarding applied cfu or pfu in the third column (method of application) means we were unable to obtain such information in some cases. vibrio parahaemolyticus blue mussels (mytilus edulus) . -l glass beakers positioned at • c with - infected ( cfu/ml) mussels and approx. . × pfu of phage cocktail ( phages) added prior to experiment phage cocktail was effective in significantly reducing v. parahaemolyticus to undetectable numbers in mussels [ ] diet supplemented with three phages alone or as a cocktail was implemented for days before immersion in seawater with × cfu/ml of bacterial pathogen survival rate during the next ten days was % for the control group, % for the phage cocktail, and %, %, % for the three phages applied alone [ the phage approach has ignited hope among scientists and fish workers for an imminent breakthrough in the fish and seafood industry, challenged by the abuse of antibiotics and growing demand for high-quality fish products [ ] . notably, the development of other methods in fighting antibiotic resistance, such as bacterial vaccines, has not diminished the importance of a phage approach in any way. in fact, the phage approach is becoming increasingly popular [ ] . at first glance, phage treatment appears acceptable and easy to conduct. however, the deeper we investigate this issue, the more complex it appears. phage administration in aquacultures imposes the most obvious route in the form of water additives or phage-impregnated feed (the latter seems to be more appropriate in prophylactic efforts as infected fish may not take up their food). in every application scenario, preventive or therapeutic, it is important to enable contact between phage particles and bacterial hosts in water, as well as on the surface or inside macroorganisms. those obstacles should be easily overcome in fish tanks where water circulation is usually maintained by installed pumps or even extensive fish motility in high-stocking tanks. other obstacles seem to be more challenging. first of all, phage lytic activity in vitro and in vivo may greatly differ as outdoor facilities are exposed to natural fluctuations in physical and chemical water parameters [ ] . such disparity is crucial when biocontrol strategies in the field are taken into account. comprehensive phage biology, including latency period, burst size, moi, adsorption rate, lytic spectrum, stability, and host range, should always be evaluated in models mirroring different environmental scenarios. selecting phages with a broad host range imposes hurdles in finding a proper one. the majority of all marine phages are highly host-specific. although there are reports suggesting that selected vibrio phages were able to infect % of tested strains, possibly due to the conservative structure of lps phage receptors [ ] , the majority of reports indicate vast species diversity in marine niches [ ] . further, zhang et al. [ ] showed that polar flagella in v. parahaemolyticus, bacteria inseparably associated with contaminated seafood, can reduce phage infectivity. notably, such a reduction was not caused by the physical presence of flagella but its rotary movement. although phages can decrease bacterial virulence outside fish hosts, some sources imply the opposite. lysogenic phages have been shown to have the ability to transform nonvirulent bacterial strains into virulent ones [ ] . in a review focusing on phage treatment in aquaculture from , the authors mentioned the narrow specificity of phages, which would not harm normal fish intestinal flora, and their self-replicating nature in the presence of susceptible bacteria [ ] . these days, scientists are aware that phages can interact with intestinal flora in a way that goes beyond antibacterial action, and their self-replicating nature is noticeable only at a high bacterial count. one must be aware that it is hard to predict how all the abovementioned aspects of phage activity in waters would affect hatcheries when used on a large scale globally. nevertheless, the pros arising from the use of phages in aquaculture facilities seem to outweigh the cons. rapid growth and an increasing number of bacterial outbreaks have forced the fish industry to take novel appropriate actions. this was acknowledged by the european commission, which created and sponsored a special network within the seventh framework programme entitled "network for the development of phage therapy in aquaculture-aquaphage" [ ] . we can easily assume that the number of such projects will increase in the upcoming years. funding: this research received no external funding. the authors declare no conflict of interest. the state of world fisheries and aquaculture overview of the seafood industry omega- fatty acids epa and dha: health benefits throughout life seafood industry struggling to stay afloat amid outbreak. ap news antimicrobial use in aquaculture re-examined: its 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water disease in salmonids eating oysters without risk of vibriosis: application of a bacteriophage against vibrio parahaemolyticus in oysters clinical application of bacteriophages in polar flagella rotation in vibrio parahaemolyticus confers resistance to bacteriophage infection the use of probiotics in aquaculture bacteriophage therapy of infectious diseases in aquaculture the seventh framework programme-aquaphage. available online this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - rrq o n authors: sivakumar, bellie title: covid- and water date: - - journal: stoch environ res risk assess doi: . /s - - - sha: doc_id: cord_uid: rrq o n the coronavirus disease, called covid- , is caused by severe acute respiratory syndrome coronavirus (sars-cov- ). since it was first identified in china in december , covid- has spread to almost all countries and territories and caused over , deaths, as on may , . the impacts of the covid- pandemic are now seen in almost every sector of our society. in this article, i discuss the impacts of covid- on the water sector. i point out that our efforts to control the spread of covid- will increase the water demand and worsen the water quality, leading to additional challenges in water planning and management. in view of the impacts of covid- and other global-scale phenomena influencing water resources (e.g., global climate change), i highlight the urgent need for interdisciplinary collaborations among researchers studying water and new strategies to address water issues. droplets can also land on objects and surfaces around the person, such as tables, doorknobs, and handrails. people can become infected by touching these objects or surfaces, then touching their eyes, nose or mouth. this is why it is important to wash one's hands regularly with soap and water or clean with alcohol-based hand rub. the spread of covid- and resulting mortality in a region is influenced by many factors, including the general health of the people, the personal and environmental opportunities for the spread, and the type of treatment and welfare schemes available. for instance, people with good health (generally young people) are less affected by covid- , while people with pre-existing health conditions (e.g., diabetes, kidney diseases, high blood pressure, cancer) are affected more; indeed, far greater percentages of deaths have been reported among elder people and those with pre-existing conditions when compared to younger people and those with good health. high population density (which influences the distance between people), low temperature (which facilitates the spread of the virus), and less water/disinfectants generally lead to more spread. numerous studies have examined the spread, impact, and control of covid- . such studies have addressed a wide variety of aspects. these include: how population flow drives the distribution of covid- (e.g., jia et al. ) ; effects of travel restrictions on the spread of covid- outbreak (e.g., chinazzi et al. ) ; links between climate factors and covid- spread (e.g., bashir et al. ; wu et al. ) ; drug development against covid- (e.g., abd el-aziz and stockland ); and plight of migrants and refugees during covid- and calls for their inclusion in response to covid- (e.g., orcutt et al. ) ; and impact of lockdown on the environment, including surface water quality and air quality (e.g., yunus et al. ; mahato et al. ; muhammad et al. ) . in the context of covid- spread and control, water plays a particularly important role. this is because, according to who (and governments around the world), one of the most effective ways to reduce one's chances of being infected or spreading covid- is to regularly and thoroughly wash one's hands with soap and water or clean them with an alcohol-based hand rub. washing one's hands with soap and water or using an alcohol-based hand rub kill viruses that may be on one's hands. therefore, the availability and accessibility of water can significantly influence the extent of spread of covid- in a region. in this regard, developing and under-developed regions have a particular disadvantage, since the accessibility to water in such regions, even for the most basic needs (e.g., drinking, sanitation), is generally limited (e.g., sivakumar a). it is also important to note that these are also the regions generally with high population and population density. therefore, mitigating or controlling the spread of covid- is highly challenging in these regions, as it necessitates identifying, accessing, and supplying significant quantity of additional water resources all around (not to mention the social-distancing). despite this, i believe, the issues associated with water demand and supply to tackle covid- has not gained serious attention until now. a key reason for this may be the so-called ''lockdown'' or ''stay-at-home'' restriction that has been and continues to be imposed in many countries (at national level or local levels), including in some of the most populous countries, such as china, india, and the united states. the lockdown has forced almost the entire population in many countries to stay at home. this has greatly reduced the need for washing or cleaning one's hands and, hence, reduced the need for additional water use. this situation will certainly change, however, once the lockdown is partially relaxed or completely removed. once people start going out of their homes to their workplaces and other public places, as was before, they will realize the need to wash their hands more often and, thus, will need more water. this will be not only in their workplaces and other public places but also at their own homes, since they will have to wash their hands after returning to their homes from outside, to make sure they are not spreading the virus at home. it is important, therefore, to ask whether there are enough water facilities in workplaces and other public places (and also at their homes) for everyone for the foreseeable future. this question assumes particular significance, since covid- will likely last for years (not just months), according to many health professionals, epidemiologists, and others in fields related to infectious diseases and their effects. indeed, there are concerns that covid- may never go away and that we will have to get used to living a ''new normal'' way of life, i.e. ''living with this means that our demands for water will significantly increase in the future, especially in the domestic, industrial, and recreational sectors. for instance, considering a country like india, with a population of over . billion, even a small amount of additional water requirement for washing one's hands will increase the water demand by at least a few billions of liters. with the already significant demand for water for the agricultural sector, which currently uses more than % of water globally (and even more than % in some regions), there will be enormous pressure on our water resources. this raises several important questions: where can we find the additional water resources? how to bring the water to where it will be needed (both urban and rural areas, and throughout the countries)? what will be the additional water storage and treatment facilities required for water supply? how much will it cost? will water pricing need to be introduced (or increased) to cover the costs, where water pricing currently does not exist (or where there is already water pricing)? how will the society respond to water pricing, especially when it is introduced for the first time in a region, as in many developing and under-developing regions? in addition to the increase in the demands for water quantity it imposes, covid- also presents challenges from the perspective of water quality. for instance, use of more water (and also soap and disinfectants) to wash one's hands to control the spread of covid- will increase the quantity of wastewater, with perhaps even poorer quality, coming from homes, workplaces, and other public places. this will, in turn, increase the required capacities of sewer/ drainage structures and wastewater treatment facilities, before such wastewater can be discharged safely to appropriate places, including to nearby water courses. this means, there will be additional costs for such potentially new drainage and treatment facilities required. it is relevant to note, at this point, that in many developing and underdeveloped regions, it is common for the untreated wastewater (particularly from many industries, but also from homes) to be discharged into the nearby water courses. assuming that this trend will continue, at least for the foreseeable future, the additional quantity and poorer quality of wastewater will have serious effects on water quality in nearby water bodies, including rivers and lakes. it is indeed encouraging to note that the quality of water in rivers, lakes, and other water bodies around the world, especially in developing and under-developed regions, have significantly improved during the lockdown period [e.g., yunus et al. ( ) ]. this, however, is only temporary, and there is no doubt that the situation will become ''normal'' again once the lockdown is over! furthermore, there are serious concerns about the handling and disposal of products and materials used to control the spread of covid- and to treat the covid- patients (e.g., face masks, hand gloves, and personal protective equipment) after their use. this is because their collection, handling, and disposal are dangerous and require undertaking extreme precautions. it is also important to note that disposing the bodies of covid- patients after their death has been an enormously challenging and excruciating experience, as has been witnessed in many places around the world. handling and disposal of wastes related to covid- often require a large quantity of water, both during and after. the above observations suggest that covid- will have serious impacts on the water sector, in terms of both water quantity and water quality. this will bring new and enormous challenges to water planning, management, and governance. such challenges include aspects related to water sources, infrastructure for storage and distribution, sewer/drainage structures, wastewater treatment and disposal, and water pricing, among others. it is also appropriate to note, at this point, that there are already significant challenges in water planning and management, due to population growth and improving living standards. global climate change is anticipated to bring its own challenges to the water sector (e.g., sivakumar b), in terms of both water quantity and water quality. there are also concerns about our ability to produce food and generate energy to meet the future demands, which depend, to a great extent, on water (for irrigation and hydroelectricity) (chen et al. ) . how will all these affect our future water planning and management activities, especially if covid- and its impact last for a long time? will totally new planning and management strategies be needed, and in what shape and form? what can the governments around the world do to address these? what can the water managers do? what can the people do? these are some of the basic questions policy makers, water managers, environmentalists, non-governmental organizations, and also common people should be starting to ask. needless to say, water researchers have a particularly important role in asking these questions and in finding solutions. there is no question that this will require interdisciplinary collaborations among water researchers from diverse fields, including earth sciences, engineering, economics, law, social sciences, and psychology. while there have already been some efforts towards such interdisciplinary collaborations to address water issues, there is still a long way to go, especially in view of any new globalscale disasters, such as the covid- pandemic. looking at the exponential developments we have recently made in diverse fields, especially in technology and medicine, not many people would have imagined that an infectious disease would almost literally ''lockdown'' the entire world and force us to ''stay at home.'' however, that is exactly what is happening now. what is even more, we cannot be sure that this kind of thing will never happen again. indeed, something may just be lurking around to cause havoc! whether infectious disease or other, water seems to offer solutions to almost all the problems on the earth. however, where to find solutions when the problem is water itself? we indeed need to have a long vision to look after and take care of our precious water. i hope we will steer our efforts in this direction much more than we have until now. if we do not have water to wash our hands, then we will be washing our hands of our problems! recent progress and challenges in drug development against covid- coronavirus (sars-cov- )-an update on the status correlation between climate indicators and covid- pandemic population, water, food, energy and dams the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak population flow drives spatio-temporal distribution of covid- in china effect of lockdown amid covid- pandemic on air quality of the megacity delhi covid- pandemic and environmental pollution: a blessing in disguise? global call to action for inclusion of migrants and refugees in the covid- response water crisis: from conflict to cooperation-an overview global climate change and its impacts on water resources planning and management: assessment and challenges world health organization (who) ( ) novel coronavirus-china effects of temperature and humidity on the new daily cases and stochastic environmental research and risk assessment deaths of covid- in countries covid- and surface water quality: improved lake water quality during the lockdown publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -h ulh y authors: eliades, theodore; koletsi, despina title: minimizing the aerosol-generating procedures in orthodontics in the era of a pandemic: current evidence on the reduction of hazardous effects for the treatment team and patients date: - - journal: am j orthod dentofacial orthop doi: . /j.ajodo. . . sha: doc_id: cord_uid: h ulh y the purpose of this critical review is to list the sources of aerosol production during orthodontic standard procedure, analyze the constituent components of aerosol and their dependency on modes of grinding, the presence of water and type of bur, and suggest a method to minimize the quantity and detrimental characteristics of the particles comprising the solid matter of aerosol. minimization of water-spray syringe utilization for rinsing is suggested on bonding related procedures, while temporal conditions as represented by seasonal epidemics should be considered for the decision of intervention scheme provided as a preprocedural mouth rinse, in an attempt to reduce the load of aerosolized pathogens. in normal conditions, chlorhexidine . %, preferably under elevated temperature state should be prioritized for reducing bacterial counts. in the presence of oxidation vulnerable viruses within the community, substitute strategies might be represented by the use of povidone iodine . %- %, or hydrogen peroxide %. after debonding, extensive material grinding, as well as aligner related attachment clean-up, should involve the use of carbide tungsten burs under water cooling conditions for cutting efficiency enhancement, duration restriction of the procedure, as well as reduction of aerosolized nanoparticles. in this respect, selection strategies of malocclusions eligible for aligner treatment should be reconsidered and future perspectives may entail careful and more restricted utilization of attachment grips. for more limited clean-up procedures, such as grinding of minimal amounts of adhesive remnants, or individualized bracket debonding in the course of treatment, hand-instruments for remnant removal might well represent an effective strategy. efforts to minimize the use of rotary instrumentation in orthodontic settings might also lead the way for future solutions. measures of self-protection for the treatment team should never be neglected. dressing gowns and facemasks with filter protection layers, appropriate ventilation and fresh air flow within the operating room comprise significant links to the overall picture of practice management. risk management considerations should be constant, but also updated as new material applications come into play, while being grounded on the best available evidence. minimizing the aerosol-generating procedures in orthodontics in the era of a pandemic: current evidence on the reduction of hazardous effects for the treatment team and patients the purpose of this critical review is to list the sources of aerosol production during orthodontic standard procedure, analyze the constituent components of aerosol and their dependency on modes of grinding, the presence of water and type of bur, and suggest a method to minimize the quantity and detrimental characteristics of the particles comprising the solid matter of aerosol. minimization of water-spray syringe utilization for rinsing is suggested on bonding related procedures, while temporal conditions as represented by seasonal epidemics should be considered for the decision of intervention scheme provided as a preprocedural mouth rinse, in an attempt to reduce the load of aerosolized pathogens. in normal conditions, chlorhexidine . %, preferably under elevated temperature state should be prioritized for reducing bacterial counts. in the presence of oxidation vulnerable viruses within the community, substitute strategies might be represented by the use of povidone iodine . %- %, or hydrogen peroxide %. after debonding, extensive material grinding, as well as aligner related attachment clean-up, should involve the use of carbide tungsten burs under water cooling conditions for cutting efficiency enhancement, duration restriction of the procedure, as well as reduction of aerosolized nanoparticles. in this respect, selection strategies of malocclusions eligible for aligner treatment should be reconsidered and future perspectives may entail careful and more restricted utilization of attachment grips. for more limited clean-up procedures, such as grinding of minimal amounts of adhesive remnants, or individualized bracket debonding in the course of treatment, handinstruments for remnant removal might well represent an effective strategy. efforts to minimize the use of rotary instrumentation in orthodontic settings might also lead the way for future solutions. measures of self-protection for the treatment team should never be neglected. dressing gowns and facemasks with filter protection layers, appropriate ventilation and fresh air flow within the operating room comprise significant links to the overall picture of practice management. risk management considerations should be constant, but also updated as new material applications come into play, while being grounded on the best available evidence. (am j orthod dentofacial orthop ;-:---) t he pandemic outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ) has had a large impact on the frontline of health care workers, and among those, on dentists and orthodontists. besides the public health and economic burdens of the coronavirus disease , it is now evident that its massive spread around the world has imposed great occupational challenges, with the implementation of routine dental services being at stake. the nature of the virus' infectious route, with direct implication of airborne droplets in the form of aerosol, has revealed certain potential hazards underlying conventional and standard oral health care procedures. orthodontic practices are not to be left aside. an aerosol is defined as a suspension system of solid or liquid particles in a gas. the term was introduced by frederick g. donnan to describe an aero-solution-clouds of microscopic particles in air. the various types of aerosol, classified according to physical form and how they were generated, include dust, fume, mist, smoke, and fog. aerosol should be differentiated from solid particles staying airborne for some time in the air and the splatter of relatively large sized droplets of water generated by splashes in a dental setting, such as those produced by using the water syringe. aerosol-producing dental procedures, along with upcoming concerns, are not new to the dental discipline and at most, these concerns should not be selectively twisted, hampered, or emphasized under the light of the present pandemic or potential future endemics. they are effectively there since more than years, and protective measures for dentists and clinic personnel should be prioritized in practice, irrespective of the presence of a pandemic, epidemic, normalized conditions, or otherwise. furthermore, these concerns and protective measures should effectively be carried forward through advancements in technologies as well as evidence directed by new knowledge over the years. the current pandemic situation has boosted our thinking and endorsements on how to efficiently manage and minimize aerosol production in contemporary practice. evidently, common categories and burdens of orthodontics-related applications producing aerosol and/or airborne particulates are focusing on bonding and debonding strategies. the former involve application of water-spray practices in connection to enamel etching, before conditioning with bonding agents and bracket bonding; the latter pertain to enamel clean-up practices after removal of fixed appliances on completion of orthodontic treatment. of late, in the line of debonding strategies, an additional procedure liable to aerosol generation has emerged in the clinical field; composite attachment removal after aligner therapy or possible attachment replacement and/or removal cycles during treatment with aligners should not be neglected. , this is particularly striking if one considers that most orthodontists and/or other clinicians utilizing aligner methods to straighten teeth and treat malocclusions have adopted wide application of these adjuncts in everyday practice. , with regard to bonding strategies, the conventional acid-etching stage may be employed with the use of a gel etchant of very thick consistency, a gel of lower viscosity, or a liquid etchant (fig ) . implications for the first alternative are rather straightforward, as it might require a considerably higher pressure of water flow to be rinsed off, as well as a longer rinsing period; but practically, there is more. very thick consistencies of gel essentially negate the action of acid for the amount of material not in contact with the enamel surface owing to limited wetting. thus, the other alternatives are often selected. however, high water pressure used generates splatter, which does not belong to the aerosol classification, but may too contribute to the contamination of the operatory. water pressure is normally set at psi in the dental units, with existing air pressure at psi. the american dental association (ada) has suggested testing of water squirt of more than . m ( ft), as a practical measure of raised water pressure. regarding debonding strategies of fixed appliances, implication of rotary instruments used to remove remnants of composite compounds after fixed appliance removal, as well as utilization of water as cooling agent during handpiece usage form priority factors that should be considered. cutting efficiency and aerosolized dust formation are also discussed. this narrative article aims to discuss the hazards arising from routine orthodontic practices implicated to aerosol generation, sometimes on par with and following examples from standard dental procedures, and also to elucidate potential interventions or alterations of conventional orthodontic applications as an attempt to minimize substantial hazards or adverse effects. the narrative is built on basic pillars regarding aerosol generation; the microbiologic on one side, and particulate production and toxicity related implications on the other. etching agents with variable viscosity. note the considerably lower viscosity of the green-colored agent, resembling a liquid etchant state. the green, blue (right side) and red agents should be preferred over the first , because they would require less water pressure to be rinsed away. the pathogenic pervasiveness of dental aerosol rests in its dependence on the concentration of bacterium or virus load in compressed air, or water-spray spatter mixed-up with tooth material, plaque, blood, calculus, and saliva debris that are theoretically and practically produced during routine dental practice, which makes use of an intraoral service handpiece. as such, orthodontic practices fall within the range of these procedures, seemingly within a more limited extent, but it is important that they are not neglected. the presence of dental unit waterlines (duwls) microbiota has also been considered an additional intriguing factor, especially because pathogens get carried forward through the water supply system directly to the handpiece in use. when coolants are used during service, the interaction of the cooling agent with fluids and debris produced within the oral environment as a result of composite or tooth grinding practices or use of ultrasonic scaling is present, and inductively, it may be detected in air-suspended particles and aerosol. the centers for disease control and prevention has established a safety maximum level of colony forming units (cfus) emitted and detected in the air at the threshold of cfus per ml as a result of dental handpiece and water and/or air supply instrumentation usage, excluding coliform bacteria for nonsurgical procedures. these levels are liable to reduction when immunocompromised patients are in chair, and are lowered to cfus per ml. evaluation of pathogen levels may be done through simple commercially available test strips or kits. in addition, air and/or water related dental instrumentation (handpiece, spray syringe, and/or ultrasonic scaler) in direct usage to patients' oral cavity should be flushed and pseudotested for minutes at the start of each day, as well as for seconds between patients. a recent systematic review on bioaerosols in dental environment has pinpointed the presence of types of micro-organisms, including bacteria and fungal genera, indicating a high variety of a range of species, whereas it was interesting that none of the included articles reported on the presence of viruses or parasites; seemingly, this is not linked to their absence from airsuspended droplets, but rather to the line of focus of the primary studies, partially in favor of the abundance and commonness of the former pathogens and their easier and nonspecific detection through wide air sampling techniques. a mean bacterial load range of to . cfus in logarithmic scale has been reported after procedural produced aerosol, while the most eminent load has been reported in the range of . meters from the oral cavity, even higher compared with closer distance measures such as that of meter from the patient. fusobacterium family pathogens have been identified in aerosols produced after ultrasonic scaling in practice through checkerboard dna-dna hybridization techniques. , of the family, fusobacterium nucleatum has been identified as a bacterium related to pathologic, ophthalmic, and respiratory implications, while also inductive of cellular apoptosis in vivo. , in addition, it has been reported as related to the launch and progression of periodontitis, or as attenuating attribute of gingival fibroblast mesenchymal cell proliferation. however, the results of checkerboard hybridization techniques should be interpreted with caution as per the exact bacteria species eligible for identification, because such practices are close ended, checked in preselected dna-probe panels and other pathogens not prespecified might be present within droplet spatters as well. nonetheless, studies assessing mostly periodontal pathogens have identified an increased prevalence of species belonging to the socalled orange complex in aerosols generated during usage of ultrasonic scaler. , these mostly pertained to campylobacter rectus, prevotella intermedia, and others, including f. periodonticum in addition to f. nucleatum. apart from directly exposed aerosolized bacteria, another potential contamination source within dental offices or in hospital based dental units has been identified and special attention has been placed to the presence of legionella pneumophilla as well as pseudomonas spp in duwls. , these might well serve as routes of infection for patients and/or dental personnel indirectly and via droplet suspension after aerosol-generating handpiece or water and/or spray syringe usage. other sources of l. pneumophilla constitute air-conditioning systems or cooling towers within dental settings. , interestingly, the novel sars-cov- has also been lately reported to demonstrate capacity of emanation via the airflow of air-conditioning units in business environments. an array of clinical studies, since more than years and until recently, have attempted to identify effective methods of reducing pathogen load stemming from aerosol forming procedures in dental settings (fig ) . the vast majority have studied in-service utilization of ultrasonic scaling, whereas some have reported on orthodontic related strategies of debonding procedures, or other dental prophylaxis or restorative procedures. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] largescale efforts have been lately endorsed to collectively appraise all available evidence and provide justifiable ranking of the efficiency of these methods. , the most prevalent recorded approaches were preprocedural mouth rinse using a wide variety of potentially antimicrobial agents, such as, chlorhexidine (chx) . %, chx . % or tempered chx . %, cetylpiridinium chloride . %, povidone iodine (pi) %, chlorine dioxide, herbal-based agents, or others pertaining to ozone irrigation, use of high volume evacuators and/or dental isolation systems, or agents added to duwls to reduce the load. , , , evidence from a study on bacterial load during orthodontic procedures comparing bracket debonding followed by enamel clean-up with high-speed handpiece and water cooling versus standard orthodontic care involving archwire and/or ligature change, and replacing procedures, highlighted the increased pathogenic state of aerosols produced by the former, with a mean difference of . ( % ci, . - . ) in total cfus. this highlights the exposure hazards of orthodontists related to certain orthodontic procedures in practice and draws attention to additional prophylactic measures to be selectively taken within the dental operating office. effectively, bacterial load in aerosol in the dental and/or orthodontic cabinet has shown to be significantly raised immediately within min of service for an aerosolgenerating procedure, including enamel clean-up. further evidence on microbiologic assessment of aerosol produced after debonding of fixed orthodontic appliances and during composite clean-up has elucidated the increased potential of aerosolized particles, particularly those with aerodynamic diameters of mm or less, to surpass the respiratory barriers and invade deep into the lungs, along with pathogen contaminants. , bioaerosol infiltration has been detected in simulation studies all the way to the respiratory tree from the pharynx to the bronchial alveoli of the lungs. although decreased particulate size seems to exhibit increased potential to penetrate deep into the lungs, the viability of pathogens has been shown to simultaneously decrease, also impacting biodiversity at the deep respiratory levels. , use of preprocedural mouthrinse with chx of either . % or . % concentration has been identified by individual studies as an important decontaminating agent contributing to identification of decreased bacterial amounts of infected aerosol; latest data coming from an endorsement to compare all direct and indirect evidence from examined interventions (mouth rinses, evacuators, decontamination of duwls, and others) across studies and within dental settings, has revealed this supremacy of preprocedural chlorhexidine mouthrinse over other measures for s to min, but also with documented prevailing of tempered ( c) chx . %. , , , , , , tempered chx solution at c, has been reported to offer increased anti-microbiologic action against bacteria of the human dental plaque, while also preserving adverse effects on tooth and pulp vitality to the minimum. the increase in bacterial kill rate has been determined to reach as high as % surplus, while to avoid storage contamination with toxic compounds such as p-chloroaniline, freshly made chx solutions should undergo heating. as this measure might be potentially considered impractical for the routine management of clinical practice, it might still be the treatment of choice for highly prone to aerosol induction procedures, with water cooling involvement; other solutions could also be considered for more conservative procedures. among the priority treatments of choice and apart from chx solutions (either tempered or nontempered), pi % has also been considered a viable alternative. , aforementioned documented evidence originates, as discussed, primarily from ultrasonic scaling clinical studies, randomized in most cases, while total bacterial count in generated aerosol has been the outcome of interest, leaving virus load aside. extrapolation to other potentially producing aerosolized compounds procedures, however, seems reasonable within a dental cabinet setting and certain orthodontic procedures, such as fixed appliance debonding, may benefit from such measures. at present and in the middle of sars-cov- pandemic mid- , there is no evidence from clinical trials on the effectiveness of interventions taken preprocedurally in dental offices against viral load in airsuspended droplets or aerosols. however, it would be reasonable to assume that mouth rinses or irrigates with proven capacity to interact with viral molecules and its cellular membranes might prove beneficial. on the basis of the oxidative action of such agents against the lipid membrane of coronaviruses, latest reports as well as primary guidelines of the national health commission by the people's republic of china on measures against sars-cov- , have indicated a decreased effectiveness of chlorhexidine as a measure of choice, mostly because of the lack of oxidative action, while use of hydrogen peroxide %, or pi . % to % appear more realistic as effective alternatives. [ ] [ ] [ ] oxidative agents act directly on the lipid shell membrane of the virus and destroy cellular components. in particular, pi action is enhanced by the slow and gradual release of iodine as carried by the povidone vehicle, while any adverse effects of iodine are reduced, allowing for a toxicity-free simultaneous interaction. based on the absence of clinical trials in the field of virus load of aerosols, latest calls have emerged and suggest the use of flavonoids or cyclodextrine agents to fight or attenuate sars-cov- infection through saliva expectorations or spatters secretions. however, their effectiveness remains to be tested. composite grinding and particulate production during handpiece instrumentation usage in routine dental practice has been considered an additional source of potentially hazardous concern for dentists and orthodontists in general, but also in particular in the middle of a pandemic of a novel sars-cov- , with unprecedented impact worldwide. an initial notion before any consideration of produced aerosolized dust is cutting efficiency and types of dental rotary instruments that might effectively reduce grinding duration. knowledge on the topic may largely be attributed to the extensive research and work on this field by a.j. von fraunhofer et al. [ ] [ ] [ ] [ ] [ ] type of cutting bur and mode of action first, discrimination between commonly used burs in terms of cutting mechanism is discussed, roughly between of the most prevalent cutting instruments in use, tungsten carbide and diamond burs. the tungsten carbide burs differ from diamond burs, as they are considered to achieve material removal through a flow-dependent fracture process (plastic flow), occurring as a result of elevated shear forces between the carbide blades and the material surface; this makes them rotary instruments of choice for cutting ductile substrates including composites, dentin, or metals. dissimilarly, diamond cutting burs induce brittle fracture of substrates, functioning by creating grooves and making use of dislocation motion and subsequent radial flow of the material, ultimately leading to propagation of cracks by the generated tensile stresses produced and chip formation. evidently, diamond burs are mostly efficient for ceramics or enamel surface. latest innovations for adhesive removal after completion of orthodontic treatment, entail the use of fiber-glass or fiber-reinforced composite burs, which have been reported to exhibit a potential for reduced enamel surface roughness on enamel clean-up, compared with standard carbides. , however, no data is currently available with respect to the effect of these cutting burs on particulate composite dust dynamic. moreover, water supplementation and spray patterns of the handpiece during tooth or material grinding, apart from the straightforward effect on preservation of temperature within tooth and pulpal tolerable standards, have also been implicated as a medium for achieving efficiency during the cutting procedure. water spray during tooth preparation within a proximal value of ml/min room temperature has been considered reasonable for avoiding pulp interactions. in reality, water or other lubrication medium has been considered to play a significant role in cutting efficiency following reynold's hydrodynamic lubrication theory. in particular, across dental setting environments where standard and known length and material cutting instruments are used for commonly used , rpm bur rotation speed, it appears unlikely that effects of dynamic viscosity of coolant media may be significant. testing across water coolant, alcohol ( %) as well as glycerol ( %) solution has revealed comparable effects. further, water application as coolant usage during material grinding in practice, including enamel cleanup from bonding remnants after orthodontic treatment, offer a thin line layer of interproximal matter between the carbide and material interface. this is considered to induce surface adsorption alterations in the substrate material after reduction of the surface-free energy, produced by changes in the strength of association of the interatomic bounds between interactive entities, thus resulting to surface hardness changes. to this respect, and as discussed above, cutting with carbide burs in ductile substrates such as resin remnants after debonding of fixed appliances or bonded attachment removal after or during aligner therapy, shall be advantaged, in terms of cutting efficiency, by water supplementation targeted directly to the carbide-composite interface, in the following manner: initial groove formation after bur application is generated, followed by lateral displacement of the substrate, pilling-up material dislocation, and crack propagation, resulting in chip formation. the described procedure broadly follows the original work of rehbinder et al back in s, who suggested that chemically-induced surface hardness changes bear the potential to increase drilling efficiency of the cutting tool in mining settings with aqueous surfactant solutions, within a range of %- %. gain is fold, with subsequent extrapolation to orthodontic and dental practice: faster advancement of the bur into the substrate and decreased demand for heavy load application in practice, thus reduction in operating time and total amount of aerosol production. resin composites are known to possess a wide range of applications in dentistry, with orthodontics usage in bonding procedures of both fixed appliances as well as treatment with aligners and attachment adjuncts being in the spotlight. normal composite composition comprises of the resin matrix (usually represented by bisphenol-a [bpa] diglycidyl dimethacrylate, triethylene glycol dimethacrylate, and ethoxylated bpa glycol dimethacrylate), the inorganic filler compounds as well as a coupling agent to guarantee bonding between the two. - filler compounds usually fall below . mm and may serve in a wide range of particulate sizes and even fall within the nano-range. , orthodontic adhesives have also been considered to acquire quartz-type filler particles as well. heavy metal oxides are preferred, namely barium, strontium, zinc, aluminum, or zirconium, while their primary service remains to offer enhanced physical and mechanical properties to the material, including polymerization shrinkage water sorption and solubility, radiopacity, and reduction of biodegradation in-service. [ ] [ ] [ ] [ ] during debonding strategies, but also lately increasingly during attachment removal in the course of and/or after the end of aligner treatment with thermoplastictype devices, breakdown of the bulk of composites takes place, with material micro-and/or nano-fragments being aerosolized. these particulates bear the aerodynamic potential to surpass the respiratory fraction barriers and natural defense mechanisms of the clinician, patient and office personnel and find their way deep into the lungs. , a foremost effort to provide evidence in the field of aerosolized composite compounds in dental settings, has been mainly initiated and driven by separately working groups in leuven, belgium, and bristol in the united kingdom, in essence after simulation in clinical conditions. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] evidently, aerosols comprising of particles lower than mm or . mm (pm or pm . , particulate matter) are gaining attention due to their potential to enter the respiratory tract; interestingly, even smaller particulates within the range of dozens of nanometers (\ nm) have been associated with an increased dynamic to surpass the primary boundaries of the respiratory system and reach deepest levels of the terminal epithelial bronchioles of the lungs because of their increased surface to volume ratio, offering an amplified reactive potential when in interaction with cellular interfaces. [ ] [ ] [ ] [ ] [ ] [ ] [ ] several studies have investigated the content compounds of composite dust produced in aerosols in dental and orthodontic setting, and it has been claimed that percentage and concentration of nano-sized identified filler particles in the aerosols might be related to the original filler content of the composites. however, this is far from the case, because all types of composites, irrespective of filler size, have been reported to exhibit significant amounts of nanoparticles within the range of - nm during grinding and clean-up. , , , in particular, surface friction and heating shock during composite grinding results to matrix decomposition of the substrate, aging, c c conversion of bonds on surface, and ultimately production of respirable composite dust. [ ] [ ] [ ] wet or dry conditions apart from water supplementation contribution to the cutting efficiency of grinding tools on the composite substrate during debonding, thus offering minimization of (bio)-aerosol production duration, the effect of water as per emanation, and generation of airborne dust has been disputed, however, with scarce evidence from few research efforts, across variable settings. in essence, a recent study inspected the effect of water cooling in slow-handpiece usage on bulk composite sticks containing an array of filler sizes under simulated conditions of dry and wet grinding. their work suggested consistent findings for all types of composites, which demonstrated a significant reduction in the number of detected nanoparticles being released when water spray was in-service ( . - . numbers per cubic centimeter), denoting a half-pace reduction, compared with dry settings. interestingly though, both dry and wet grinding alternatives produced high numbers of nano-sized particulates being aerosolized overall. the highest amounts have been detected during the last minute of grinding, reaching levels of approximately numbers per cubic centimeter. particulate agglomeration has been considered to occur across time, thus contributing in increasing average particulate diameter, overall. to this respect, under water usage conditions, airborne generated nanoparticles have been considered particularly prone to being trapped within water droplets, resulting in increased matter sizes, which are less likely to achieve penetration of the epithelial bronchial barriers and find their way to the lungs. the aforementioned conditions and settings could be considered as vastly resembling to the bulk attachment material removal during orthodontic treatment with aligners. as previously discussed, aligner usage for treatment of malocclusions currently involves increasingly frequent adoption of composite grips bonded to tooth enamel, sometimes more than per tooth, as attachments of various sizes and shapes, with nonnegligible dimensions, varying within the range of - mm and also width or thickness that may exceed mm. , these adjuncts target to the achievement of modes of tooth movement, either rotational or translational, within all planes of space, which would otherwise be non-manageable with the early phase plain thermoplastic aligner usage, that do not necessitate enamel involvement. this compares to the thin layer of composites used as a layer of "sandwich-type" pattern between the bracket base and the enamel surface in a conventional case fixed appliance treatment, with an average estimated thickness of to mm; one may evidently cognize that the bulk and thickness of the attachment grips in aligner therapy is implicated in conditions: first, the occurrence of an excessive amount of composite polymerized material within the oral cavity, allowing for the potential risk of bpa release or monomer leaching, depending on the number and shape or size; second, grinding procedures for attachment removal may prove extremely exhaustive and timely, bearing an increasing risk of excessive production of aerosolized composite dust. , , , handpiece role furthermore, an earlier report on human extracted teeth and subsequent simulated bracket removal and enamel clean-up, has examined the effect of handpiece, water coolant, and high volume evacuator as well as surgical facemask, on the amount of particulate production and particle concentration during composite grinding after debonding; however, the baseline effect of handpiece was variable, because slow-speed handpiece was used in absence of water coolant, whereas high-speed handpiece only under water-spray emission. findings structured on nonparametric data revealed a significantly higher concentration of airborne particulates under wet conditions and the use of high-speed instrumentation. in addition, use of facemask appeared considerably effective, contributing to the reduction of the detected concentration, while high volume evacuator was not identified as a critical parameter in this respect. to date, there is no further evidence on the direct crude effect of handpiece variation and rotary instrumentation speed with regard to airborne particulate generation, under otherwise comparable conditions. following research about cytotoxicity and xenoestrogenic effects of bpa and/or monomer release of adhesive compounds within the oral cavity, airborne particulates produced during grinding of composites after fixed appliance removal or aligner's attachment elimination, are seemingly a potential source of similar concerns. , a mild but gradual reduction of human bronchial epithelial cell viability in laboratory conditions has been documented, giving rise to speculations on the reactive dynamic of such particulates. [ ] [ ] [ ] composite filler particles and matrix composition of restorative adhesives did not appear to play a role. interestingly, the latest report encompassing orthodontic adhesive material evaluation at grinding stages after simulated conventional orthodontic treatment, pinpointed the aptitude of aerosolized particles of adhesives comprising of quartz-type fillers to demonstrate disrupting effects on interacting cell membrane integrity and cellular viability, while also to intervene with cellular growth potential of epithelial bronchial populations at an early stage. these effects are probably related to the size and shape of such fillers' configuration, following the increased surface to volume ratio they present. related evidence on orthodontic adhesives comes also from the assessment of in vitro estrogenicity of orthodontic composited ground under simulated bonding-debonding settings. estrogenic effects appear as a result of residual monomer release (bpa), which follows action as an endocrine disruptor because of the very similar structure with beta-estradiol. , under the use of highspeed handpiece without water-spray, eluents containing airborne particulates, after grinding different types of adhesives (ie, chemically or light-cured), have shown an increased proliferating capacity on mcf- breast cancer cells in vitro. such findings are of particular interest and raise considerable awareness when it comes to the largescale removal of attachment grips implicated in aligner therapy. the bulkiness and volume of these adjuncts evidently requires a great amount of grinding efforts and intraoral cutting instrumentation service. it is therefore likely that a significant amount of heat influx occurs first at the surface of the composite substrate if not substantially cooled, resulting in heat shock of the matrix. resultant effects on chemical decomposition of the produced aerosolized dust with further implications on monomer release and bpa diglycidyl dimethacrylate compounds might be alarming. , thus, broad and time-consuming composite removal, as required in extensive removal of attachments, with no water cooling in-service, should largely be avoided, while further research in the field is critical to detect specific effects of water supplementation to the emanation of monomer and other potentially estrogenic compounds. direction of measures taken to minimize effects of aerosol production in orthodontic practice should target in basic routes: bonding and debonding procedures, in essence those are interconnected (table) . the former basically comprises procedures that take place before bracket placement on tooth surface and involve rinsing actions for enamel preparation agents and use of certain types of bonding materials. as previously stated, very thick consistencies and substantial amounts of etchant acid gels applied on tooth surface, apart from presenting compromised action per se, evidently require higher water and/or spray pressure to be rinsed off, thus increasing the likelihood for spatter emanation and droplet formation, but also resulting in prolonged working times. conventional acid-etching agents entailing low viscosity or even liquid gels should be prioritized. self-etching primer alternatives have also been proposed, although these may require careful pumicing to ensure a precipitations-free enamel substrate. , in the same line and to avoid rinsing application and aerosol production, glass-ionomer cements as compared with conventional light-cured counterparts may be preferred. these material alternatives present a chemical interaction and adherence with enamel surface, do not involve prior conventional enamel conditioning, or involve a thin layer of polyacrylic acid agent in contact with enamel, with an induced shallow depth of penetration of approximately to mm. they are also less susceptible to moisturized oral cavity conditions, thus offering a viable alternative to classic adhesives bringing the aforementioned advantages, but also bearing a reduced risk for iatrogenic damage to the enamel surface. , however, all currently and widely adopted bonding alternatives do not target on the desirable minimization of adhesive remnants covering the enamel surface after debonding. starting from the necessity of an enamel-friendly bonding agent, there has been an endorsement and inspiration, following nature and wildlife environment, to design new material structures on par with living creatures' observations. these form the so-called biomimetic materials. for example, gekkonidae lizards (geckos) acquire a unique adhesion ability attributed to their foot pad, the "contact splitting." in particular, geckos' foot pad contains densely packed ultrafine hair, split in the endings, thus offering increased number of contact points per unit area, contributing to greater adhesion forces generated. as such, geckos are capable of sustaining their weight upside-down, with a gravity defying ability, without mediation of any chemical agent, relying only to physical forces, otherwise being impossible to achieve. this type of strong gecko-feet grip has inspired the design of medical adhesives and might attain applicability in orthodontic bonding agents for dry environments. moreover, to overcome failures of geckos' inspired materials, in wet conditions, scientists have studied the use of mussel adhesion as a combination approach, with a resulting new material named "geckel," which might exhibit enhanced adhesion potential both in dry and wet conditions. mussel biomimetic polymers are based on l- , -dihydroxyphenylalanine (dopa), offering "sticky" and "glue" resembling properties in the materials. in essence, biomimetic based bonding primers such as l-dopa might offer clinicians a significant tool against oral environment conditions. in combination with geckos' related properties and applicability to bracket bases, sufficient bond strength to enamel surface might be achieved, without necessitation for prior enamel conditioning, also making debonding practices and enamel clean-up at the end of treatment, effortless. pertaining to debonding procedures, calls and endorsements for aerosol containment, in general, should be focused first on preventive measures to minimize composite remnants after bracket removal in conventional orthodontics, and second on effective grinding patterns to reduce dust, particulate generation, and operating time, with further speculations on bio-aerosol formation and microbiologic perspectives, as well as xenoestrogenic action of the produced particulate matter. the compositebracket base interface may play a significant role in achieving a desirable limited amount of adhesive remnant for grinding. alterations in the adhesive-base interlocking characteristics may take place by induced modifications in the resin filler content and also in the adhesive retention patterns within the bracket base. targeting an efficient combination of bracket base mesh, size, and shape with adhesive composition that may result in a cohesive composite fracture on debonding, would allow for minimal enamel clean-up (fig ) . in this respect, applications from high technology and automotive industries might offer reformative solutions in orthodontic procedures in the near future. lately, adhesives that debond on command have been used in interlocking joint positions in technology adjuncts to allow for a temperature-controlled initiation of the debonding process. , this is achieved mostly through the embedding of thermally expandable particles (tems) into the adhesive matrix. the idea about tems dates many decades back and resides in the transformation of the particles through heat shock, occurring by softening of the cell particulate matter jointly with gasification of the inner liquid phase hydrocarbon. , in the same line, ferrous microparticles within the micron range, have been introduced as fillers and act by being preferentially distributed after external magnet polarity reversal, thus inducing destabilization of the polymer structure and initiating crack states within the resin matrix that may easily be diffused. other initiatives might also entail application of irradiation to reverse polymerization and produce a highly viscous adhesive state easily to be removed. wide adoption of bpa-free adhesives has been suggested for a range of dentistry applications including orthodontic bracket or fixed retainer bonding. to this line, advantages of such alternatives which miss bpa monomer derivatives, have been directed towards the elimination of the reactive oxygen species produced after bpa leaching in the oral cavity, after incomplete polymerization of the adhesives and being able to incite an estrogenic potential. the majority of such alternatives make use of aliphatic co-monomers based on triethyleneglycol dimethacrylate, urethane dimethacrylate, and cycloaliphatic dimethacrylates or are effectively represented by a single aromatic dimethacrylate derivative. these efforts might prove beneficial also with regard to elimination of bpa release in aerosolized dust at the debonding stage. , conclusion in all, wide and consistent adoption of occupational measures to control generation of aerosol in orthodontic practice should be universal, with microbiologic considerations, particulate matter production as well as toxicity related perspectives being on the spot, even more within the course of a pandemic. realistic management in practice, should focus on bonding and debonding strategies, while careful selection of procedures and application of safety measures depending on individualized patient needs is fundamental. in particular, minimization of water-spray syringe utilization for rinsing is anticipated on bonding related procedures, while temporal conditions as represented by seasonal epidemics should be considered for the decision of intervention scheme provided as a procedural mouthrinse, in an attempt to reduce the load of aerosolized pathogens. in normal conditions, chx . %, preferably under elevated temperature state should be selected for minimization of bacterial load. in the presence and spread of oxidation vulnerable viruses within the community, substitute strategies should be opted, effectively represented by the use of pi . %- %, or hydrogen peroxide %. after debonding, largescale enamel clean-up strategies should entail the use of carbide tungsten burs under water cooling conditions, to augment cutting efficiency, timely fulfillment of the procedure, as well as reduction of aerosolized nanoparticles. attachment clean-up at the end of aligner therapy falls into this category; however, selection strategies of malocclusions eligible for aligner treatment should be reconsidered, and a more confined use of attachment grips might also be a viable future perspective. for more limited clean-up procedures, with traces of adhesive remnants left on enamel substrate or individual "re-bracketings" or grinding after bracket breakage in the course of treatment, water cooling rotary instrumentation might not be the treatment of choice, whereas hand-instruments for remnant removal might represent better an effective strategy. furthermore, in-office measures of self-protection should never be neglected. dressing gowns and facemasks with filter protection layers and face shields for all clinic personnel, appropriate ventilation, and fresh air flow within the operating room are of paramount importance. risk management considerations should be constant but also updated as new material applications come into practice and/or epidemiologic equilibrium of the community is disrupted. coronavirus disease (covid- ): emerging and future challenges for dental and oral medicine the hallmarks of covid- disease aerosol technology: properties, behavior, and measurement of airborne particles infection control in dentistry the use of attachments in aligner treatment: analyzing the 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restaurant efficacy of preprocedural mouth rinsing in reducing aerosol contamination produced by ultrasonic scaler: a pilot study comparison of suction device with saliva ejector for aerosol and spatter reduction during ultrasonic scaling comparative evaluation of two different ultrasonic liquid coolants on dental aerosols efficacy of two pre-procedural rinses at two different temperatures in reducing aerosol contamination produced during ultrasonic scaling in a dental set-up -a microbiological study effect of aloe vera as a preprocedural rinse in reducing aerosol contamination during ultrasonic scaling comparative evaluation of colony forming unit count on aerobic culture of aerosol collected following pre-procedural rinses of either . % chlorhexidine gluconate or % stabilized chlorine dioxide during ultrasonic scaling: a clinical and microbiological study the efficacy of preprocedural mouth rinse of . % chlorhexidine and commercially available herbal mouth containing salvadora persica in reducing the bacterial load in saliva and aerosol produced during scaling evaluation of aerosol contamination during debonding procedures microbiological assessment of aerosol generated during debond of fixed orthodontic appliances reducing bacterial aerosol contamination with a chlorhexidine gluconate pre-rinse efficacy of preprocedural rinsing in reducing aerosol contamination during dental procedures interventions to reduce aerosolized pathogens in dental practice. a protocol for a systematic review and meta-analysis interventions to reduce aerosolized pathogens in dental practice. a systematic review with network meta-analysis of randomized controlled trials periodontal disease and rheumatoid arthritis: a systematic review comparative evaluation of chlorhexidine and cinnamon extract used in dental unit waterlines to reduce bacterial load in aerosols during ultrasonic scaling studies on dental aerobiology. i. bacterial aerosols generated during dental procedures size relationship between airborne viable bacteria and particles in a controlled indoor environment study compare the effcacy of two commercially available mouthrinses in reducing viable bacterial count in dental aerosol produced during ultrasonic scaling when used as a preprocedural rinse efficacy of . % tempered chlorhexidine as a pre-procedural mouth rinse: a clinical study antiplaque effect of tempered . % chlorhexidine rinse: an in vivo study transmission routes of -ncov and controls in dental practice national health commission prc. guidance for corona virus disease . prevention, control, diagnosis and management review of disinfection and sterilization -back to the basics covid- : a recommendation to examine the effect of mouthrinses with b-cyclodextrin combined with citrox in preventing infection and progression enhanced dental cutting through chemomechanical effects handpiece coolant flow rates and dental cutting the effect of handpiece spray patterns on cutting efficiency comparison of sectioning rates among carbide and diamond burs using three casting alloys dental burs-what bur for which application? a survey of dental schools comparative evaluation of enamel surface roughness after debonding using four finishing and polishing systems for residual resin removalan in vitro study enamel surface roughness after debonding: a comparative study using three different burs in vitro comparison of the cutting efficiency and temperature production of different rotary cutting instruments. part i: turbine the effect of medium and adsorption layers on plastic flow of metals materials science for dentistry bisphenol a and orthodontics: an update of evidencebased measures to minimize exposure for the orthodontic team and patients update on dental nanocomposites direct esthetic restorations based on translucency and opacity of composite resins particulate production and composite dust during routine dental procedures. a systematic review particulate production and composite dust during routine dental procedures. a systematic review with meta-analyses cytotoxic and genotoxic potential of respirable fraction of composite dust on human bronchial cells investigations on mechanical behaviour of dental composites determination of polymerization stress of conventional and new "clustered" microfill-composites in comparison with hybrid composites phillips' science of dental materials dental materials in orthodontics airborne particles produced during enamel cleanup after removal of orthodontic appliances should we be concerned about composite (nano-)dust? nanoparticle release from dental composites cytotoxic effects of composite dust on human bronchial epithelial cells release of monomers from composite dust the effect of water spray on the release of composite nano-dust quantitative and qualitative analysis of particulate production during simulated clinical orthodontic debonds the health effects of pm . (including ultrafine particles) mucociliary and long-term particle clearance in the airways of healthy nonsmoker subjects pulmonary effects of inhaled ultrafine particles the potential risks of nanomaterials: a review carried out for ecetoc the nanosilica hazard: another variable entity casarett and doull's toxicology: the basic science of poisons scientific update on nanoparticles in dentistry unreacted methacrylate groups on the surfaces of composite resins characterization and in vitro estrogenicity of orthodontic adhesive particulates produced by simulated debonding detection of nanoparticles released at finishing of dental composite materials effects of variable attachment shapes and aligner material on aligner retention the effect of orthodontic adhesive and bracket-base design in adhesive remnant index on enamel release of bisphenol-a from a light-cured adhesive bonded to lingual fixed retainers bpa qualtitative and quantitative assessment associated with orthodontic bonding in vivo assessment of bisphenol-a release from orthodontic adhesives biodegradation of acrylic based resins: a review the effect of temperature on bisphenol: an elution from dental resins failure rate of selfligating and edgewise brackets bonded with conventional acid etching and a self-etching primer: a prospective in vivo study self-etch primers and conventional acid-etch technique for orthodontic bonding: a systematic review and meta-analysis effect of intraoral aging on the setting status of resin composite and glass ionomer orthodontic adhesives future of bonding advances in glass-ionomer cements. carol stream. quintessence publishing orthodontic materials research and applications: part . current status and projected future developments in bonding and adhesives structural properties of a scaled gecko foot-hair gecko lizard toe hairs inspired the design of medical adhesives a reversible wet/dry adhesive inspired by mussels and geckos debonding on command of adhesive joints for the automotive industry an overview of the technologies for adhesive debonding on command. the annals of expansible thermoplastic polymer particles containing volatile fluid foaming agent and method of foaming the same development and testing of novel bisphenol a-free adhesives for lingual fixed retainer bonding key: cord- -p ld tun authors: bonadonna, lucia; la rosa, giuseppina title: a review and update on waterborne viral diseases associated with swimming pools date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: p ld tun infectious agents, including bacteria, viruses, protozoa, and molds, may threaten the health of swimming pool bathers. viruses are a major cause of recreationally-associated waterborne diseases linked to pools, lakes, ponds, thermal pools/spas, rivers, and hot springs. they can make their way into waters through the accidental release of fecal matter, body fluids (saliva, mucus), or skin flakes by symptomatic or asymptomatic carriers. we present an updated overview of epidemiological data on viral outbreaks, a project motivated, among other things, by the availability of improved viral detection methodologies. special attention is paid to outbreak investigations (source of the outbreak, pathways of transmission, chlorination/disinfection). epidemiological studies on incidents of viral contamination of swimming pools under non-epidemic conditions are also reviewed. swimming pools have been implicated in the transmission of infections. the risk of infection has mainly been linked to fecal contamination of the water, generally due to feces released by bathers or to contaminated source water. failure in disinfection has been recorded as the main cause of many of the outbreaks associated with swimming pools. the majority of reported swimming pool-related outbreaks have been caused by enteric viruses [ , ] . sinclair and collaborators reported that % of viral outbreaks occur in swimming pools, % in lakes or ponds, and the remaining % in fountains, hot springs, and rivers ( % each) [ ] . viruses cannot replicate outside their host's tissues and cannot multiply in the environment. therefore, the presence of viruses in a swimming pool is the result of direct contamination by bathers, who may shed viruses through unintentional fecal release, or through the release of body fluids such as saliva, mucus, or vomitus [ ] . evidence suggests that skin may also be a potential source of pathogenic viruses. we carried out a comprehensive literature review aimed at investigating waterborne viral outbreaks linked to swimming pools, to explore the etiological agents implicated, pathways of transmission, associations between indicator organisms and disease, and key issues related to chlorination/disinfection procedures. viral outbreaks are summarized in table . the presence of enteric viruses in swimming pools under non-epidemic conditions was also reviewed. different databases (scopus, pubmed, and google scholar) were accessed using the terms norovirus, norwalk virus, adenovirus, enterovirus, echovirus, coxsackievirus, and hepatitis a, in combination with terms recreation, swimming, pool, and water. switched off, to avoid eye irritation with chlorine. within one week, of the exposed swimmers became ill. children swimming in the afternoon, when chlorination was still working, did not get sick. the infection was shown to spread in families having index cases ( infected contacts). analyses of water were done approximately days after the presumed exposure. for this reason, attempts to isolate the virus from the water failed. an outbreak of acute conjunctivitis due to hadv type occurred in kansas, usa in : cases and one hospitalization were documented [ ] . eye symptoms predominated (red or pink eyes, swollen eyes), but a variety of other signs were also noted (mainly fever, headache, and nausea). a school swimming pool was identified as the source of infection. chlorine concentration was low due to an equipment failure. the epidemic was easily controlled by raising the pool's chlorine level. unfortunately, tests for viruses and bacterial indicators were carried out after super-chlorination and consequently results were negative. in , two outbreaks associated with swimming pools occurred in georgia, usa. the first, due to hadv type , involved at least cases [ ] , with patients showing different symptoms, including sore throat, fever, headache, anorexia and conjunctivitis. in this case, a private swimming pool was the source of infection. a temporary malfunction in the water filtration system of the pool associated with inadequate chlorine levels was recorded. both waterborne and person-to-person transmission occurred. in the second outbreak, hadv type was recognized as the etiological agent of pharyngoconjunctival fever in persons [ ] . an insufficient amount of chlorine was found in the water of the pool. to stop the spread of infection, the pool was closed during the summer and adequately chlorinated. adenovirus was recovered from the water sampled from the pool. in oklahoma, usa, an outbreak of pharyngitis caused by hadv type a was recorded in among children attending a swimming pool [ ] . symptoms included conjunctivitis, fever, sore throat, headache, and abdominal pain. two cases were hospitalized with dehydration from persistent vomiting. a malfunction of the automatic pool chlorinator was identified as the cause of the outbreak. in fact, during the two weeks preceding the epidemic, its failure forced the pool operator to manually add chlorine to the pool. another outbreak was recorded in , in greece, where athletes under years of age presented with fever, conjunctivitis, sore throat, weakness, and abdominal pain, after swimming in a pool [ ] . seven athletes were hospitalized. virological analyses on clinical samples were not performed and the illness was attributed to hadv on the basis of clinical symptoms alone. water samples from both the pool and the distribution system were tested for hadv, enterovirus, and hepatitis a virus by molecular methods. the water of the pool tested positive for hadv and negative for the other viruses, demonstrating its role as the source of infection. samples from the water system were negative for all viruses tested. chlorine levels were found to be low, probably due to a malfunctioning of the pool chlorination system. five hadv outbreaks associated with swimming were recorded in the s. in the year , an outbreak of pharyngoconjunctival fever occurred in north queensland, australia, where, after a school camp, children aged - , got sick [ ] . in addition to primary cases acquired at the camp (n • = ), nine other cases were acquired within the households. the school camp had a large saltwater swimming pool. adenovirus was isolated from eye and throat swabs. a pcr analysis of water samples for hadv did not yield positive results. it was, however, demonstrated that the pool was not properly maintained, and that the level of residual chlorine was inadequate. an outbreak of pharyngoconjunctival fever affecting children under was recorded in a municipality of northern spain in july [ ] . forty-three cases were recognized as primary cases, all of whom attended a municipal swimming pool. the remaining children were secondary cases, which had been in close contact with a primary case. adenovirus type was detected in pharyngeal swabs. electrical system failures causing the intermittent breakdown of the pool's bromine dosing pumps and the slowing down of water circulation were assumed to have been the cause of the outbreak. swimming was only allowed after the disinfection system was restored and appropriate concentrations of bromine were reached. due to logistic problems, no water samples were taken from the swimming pool for virological analysis. in , children ( - years old) who had attended a swimming training center in eastern china showed symptoms of pharyngoconjunctival fever. adenovirus type was recognized as the etiological agent [ ] . a total of cases were confirmed from among amateur swimmers, with an incidence of . %. fourteen hospital admissions were documented. fever, tonsillitis, sore throat, headache, sneezing, cough, conjunctivitis, fatigue, and diarrhea occurred among the bathers. the low level of residual chlorine in the water, along with excessive crowding in the pool were suggested as having caused the epidemic. in the same year, in a primary school in taiwan, an outbreak of hadv infection occurred among students, with four hospitalizations [ ] . most of the students attended a swimming course in two swimming facilities outside the school and presented with fever and symptoms of upper respiratory tract infection. other symptoms included diarrhea, vomiting, skin eruptions and conjunctivitis. throat swabs of affected students were tested for influenza virus, adenovirus, respiratory syncytial virus, coronavirus, metapneumovirus, parainfluenza types - , and herpes simplex virus. samples were found positive only for hadv type . water samples were not obtained from any of the facilities for virological analysis. in , an outbreak of pharyngoconjunctival fever involved people ( students and six staff) at a university in beijing, china [ ] . fifty patients ( %) attending the same swimming pool two weeks before the onset of symptoms were considered primary cases. the other five subjects ( %) who had not swum in the pool were defined as secondary cases (person-to-person transmission). human adv type was identified from both eye and throat swabs of the patients and from concentrated swimming pool water samples. gene sequences obtained from the water samples exhibited a % match with the sequences obtained from swab samples. control measures included the emptying and closing of the pool, and the disinfection with a high dose of sodium hypochlorite ( mg/l). enterovirus is a genus in the family picornaviridae, consisting of four human enterovirus species. enteroviruses can cause many illnesses, including paralysis, meningitis, and cardiomyopathy, although most infections are asymptomatic or cause less severe conditions, such as colds and fever. a number of reports have described enterovirus infections linked to swimming pools. the first enterovirus swimming pool-related outbreak occurred in , at a municipal pool in colorado, usa. twenty-six children presented with fever along with at least one additional symptom such as malaise, headache, stomachache, nausea, or diarrhea [ ] . it was found that the pool chlorination system was operating improperly, with chlorine levels close to zero. stool specimens collected from the children affected were tested for common enteric bacterial pathogens (salmonella, shigella, aeromonas, and campylobacter), but not for viruses. enterovirus was suggested as a likely etiological agent based on clinical manifestations, course of disease, incubation time, and the exclusion of likely bacterial pathogens. an enterovirus outbreak occurred in ireland in , with cases experiencing vomiting, diarrhea, and headache after attending an outdoor swimming pool in a small seaside village. one subject had vomited into the pool, and echovirus was isolated from this case and from six other cases. chlorine levels were found to comply with health standards, but were inadequate to contain the risk of infection from vomitus [ ] . another echovirus outbreak occurred in rome, italy, in late [ ] . children from two schools showed clinical manifestations after swimming in a pool. twenty children had meningitis-like symptoms (fever, headache, and vomiting), and six of them were hospitalized. other children had respiratory symptoms consistent with enterovirus infection. echovirus was isolated from the cerebrospinal fluid and stools of the hospitalized children. based on the epidemiological characteristics, it was hypothesized that person-to-person transmission occurred both at the swimming pool and in a number of classrooms. data on chlorination at the time of the outbreak were not available. virological analysis of pool water was performed one month after the outbreak, but yielded no positive results. in south africa, an outbreak involving children occurred following a summer camp in [ ] . camp activities included swimming and other aquatic sports. symptoms included mainly headaches, sore eyes, and/or abdominal discomfort, with one case of vomiting. four children were hospitalized for meningitis. echovirus was detected in cerebrospinal fluid and stool samples from symptomatic and asymptomatic children. the presence of viruses in the pool was not investigated. water contamination was confirmed through a total coliform count. in germany, cases of aseptic meningitis were recorded from july to october [ ] . swimming in a public, nature-like pond was identified as a risk factor for disease. up to people visited the pond each day during the summer holidays. echovirus and were detected in cerebrospinal fluid samples taken from some of the patients. an echovirus sequence obtained from one water sample collected from the pond showed a high level of genetic similarity ( % nucleotide homology) with sequences obtained from patient isolates. in august , an outbreak of meningitis occurred among campers staying at a campground in connecticut, usa [ ] . a total of cases of aseptic meningitis, four hospitalized patients and cases of enterovirus-like illness with symptoms such as headache, neck stiffness, photophobia, sore throat, chills, or exanthema were identified. echovirus serotype was detected in cerebrospinal fluid samples from three of the patients. the spread of the virus was associated with swimming in a crowded pool, which had low chlorine levels. as a result, the pool water was intermittently contaminated with enterovirus. hepatitis a is a virus causing mild to severe liver disease. globally, there are an estimated . million cases of hepatitis a every year. the virus is transmitted mainly via the fecal/oral route through the ingestion of contaminated food and water, or through direct contact with an infected subject. there is evidence to suggest that hepatitis a can be acquired by swimming in contaminated water. in september , an outbreak of hepatitis a affecting children ( - years old) and causing hospitalizations was recorded in hungary [ ] . all of the children swam in a pool at a summer camp. the pool was a non-chlorinated thermal pool/spa, which was overcrowded during the month of august. it was concluded that crowding and poor hygienic conditions, with a suspected accidental fecal release, contributed to the outbreak. another outbreak of hepatitis a was described in the usa during . it involved cases, probably associated with a public swimming pool [ ] . it was hypothesized that a cross-connection between a sewage line and the pool water intake line may have been the cause of the outbreak. according to another hypothesis, it was a swimmer who contaminated the water in the pool. however, disinfectant levels in the pools met local standards. seven hepatitis a cases among children from six families were documented in australia in [ ] . the children had attended an outdoor spa pool treated with hydrogen peroxide solution. it was hypothesized that hepatitis a virus was shed by the index case in the spa pool, and subsequently ingested by the others, who became secondary cases. virological analyses of water samples were not performed. noroviruses (novs), formerly known as norwalk-like viruses, are small viruses within the family caliciviridae, subdivided into at least seven genogroups (gi-vii), with gi, gii, and giv infecting humans. they are recognized as a major cause of sporadic and epidemic gastroenteritis in both industrialized and non-industrialized countries. outbreaks have been associated with a variety of settings including childcare centers, hospitals, nursing homes, cruise ships and restaurants. noroviruses are mainly transmitted via the fecal-oral route through contaminated food or water. norovirus-contaminated water-both recreational and drinking water-can thus lead to waterborne infections. a number of nov swimming-pool related outbreaks have been described. in , an outbreak of acute gastroenteritis with the typical symptoms of vomiting, cramping, nausea and diarrhea was documented among students and teachers at a primary school in ohio, usa [ ] . serologic studies suggested infection by norwalk virus to be the cause of the outbreak. the first cases recorded were caused by swimming in a contaminated pool, and a person-to-person transmission followed. the water of the pool tested negative for both bacterial and viral pathogens. water contamination was linked to both the pool chlorinator, which was accidentally turned off at the time of the school visits, and a leak in the water supply pipes. a large outbreak of gastroenteritis due to novs was recorded in july in helsinki. it involved people (children and adults) after bathing in an outdoor wading pool [ ] . norovirus and astrovirus were detected in both patient stool samples and pool water, with identical nucleotide sequences. the pool was found to be heavily contaminated with human fecal material carried from public toilets. the pool water had been manually chlorinated three times per week, thus not continuously. to control the outbreak, the pool was emptied, refilled, and the water was heavily chlorinated (up to mg/l of free chlorine). for the subsequent swimming season, the pool was equipped with both a continuous chlorination system and a water filtration system. in , a nov outbreak associated with a swimming pool was reported in minnesota, usa. thirty-six persons of three different youth sports teams became ill after swimming in a hotel pool and spa [ ] . unfortunately, there is no other information available on this epidemic. in , an acute gastroenteritis epidemic affected people who had swum in a pool in vermont, usa. vomiting and/or diarrhea occurred within h of attending a private indoor pool. specimens tested positive for nov. at the time of the inspection, no equipment failures or irregularities were identified. nevertheless, deficiencies in pool operation and maintenance, including poorly trained operators, inadequate maintenance checks, failure to alert management, and insufficient record keeping were reported [ ] . finally, yoder and coworkers documented a nov outbreak linked to a hotel pool in wisconsin, usa in , with persons exhibiting symptoms of gastroenteritis, related to inadequate disinfection and continued use by ill swimmers [ ] . hadv spain not tested [ ] hadv china not tested [ ] hadv taiwan not tested [ ] yes. gene sequences obtained from the water samples were % identical to the sequences obtained from the swab samples. [ ] to date, a limited number of studies examined the extent of viral contamination in swimming pools under non-epidemic conditions. the first isolation of viruses from urban wading pools was documented in albany, ny, usa in [ ] . two enteroviruses (echovirus and echovirus ) were identified from two chlorinated pools, both filled with water from the municipal supply. the same echovirus strains were found to be present in raw sewage sampled at the albany treatment plant, reflecting widespread infection in the community. in toronto, canada, coxsackievirus b was isolated from children with pleurodynia, myalgia, and primary peritonitis during . examination for the virus content of a gauze swab, which was placed daily in a wading pool with high bather load located in a congested city area, revealed the presence of the same coxsackievirus type [ ] . in in israel, swimming pool samples were found positive for enterovirus: three for echovirus , two for coxsackievirus b , and one for echovirus [ ] . viruses were isolated from water samples with no detectable fecal or total coliform bacteria. different enteroviruses were detected in swimming pools and wading pools equipped with gas chlorine and sand/gravel filters in texas, usa, in [ ] . after virus concentration from water, samples were assayed on cell culture and plaque assays. enteroviruses were found in / ( %) of the examined samples. coxsackieviruses b and b , poliovirus , and echovirus were isolated in pool waters. no correlation was found with total coliform bacteria, as six among the positive virus samples were negative for coliforms. in three samples, viruses were detected in the presence of free chlorine exceeding . ppm and in the absence of coliforms, indicating that viruses can survive low levels of biocides in actively used pools. cell cultures used in the study were suited for the isolation of enteroviruses, but it is likely that other viruses, not capable of growing on those cell lines, could also have been present in the water. three years later, in , enteroviruses were detected in . % of water samples [ ] collected from three outdoor swimming pools. a direct correlation was established between viral and microbial contamination, and the low exchange of water in the pools. in , van heerden and coworkers detected hadv in of samples ( . %) from an indoor swimming pool and in three of samples ( . %) from an outdoor swimming pool [ ] . quantitative data were also obtained by real-time pcr. application of these results in an exponential risk assessment model, assuming a daily ingestion of ml of water during swimming, indicated a daily risk of infection ranging from . × − to . × − . no acceptable microbial risk has thus far been established for swimming pool water. however, pool water quality is generally considered comparable to drinking water quality (absence of fecal indicators and pathogens). for this reason, a maximum of one infection per , consumers per year has been recommended as an acceptable level of microbial risk for swimming pools. the risk of hadv infections calculated for the swimming pool water in the study exceeded this acceptable risk. more recently, in in cyprus, hadvs and enteroviruses were detected in public swimming pools complying with bacteriological standards (such as fecal coliforms and enterococci) [ ] . the investigation was performed over a period of months, from april to december . a total of samples were obtained from swimming pools located in five major cities. bacteriological marker analysis showed that % of pools complied with the national regulations. enteroviruses were identified in four swimming pools, one containing echovirus , two containing echovirus and one containing poliovirus sabin . in four swimming pools, hadvs were detected, all characterized as type . in - , a study investigated the presence of human enteric viruses (adenovirus, norovirus, and enterovirus) in indoor and outdoor swimming pool waters in rome. bacteriological parameters (fecal indicator bacteria, heterotrophic plate count, pseudomonas aeruginosa, and staphylococcus aureus) were also investigated [ ] . moreover, the study was the first to examine the occurrence of non-enteric viruses in swimming pool waters: human papillomavirus (hpv) and human polyomavirus (hpyv). interestingly, enteric viruses were not detected, while both hpvs and hpyvs were identified in / swimming pool water samples, by means of molecular methods. neither of these viruses had previously been recognized as potential recreational waterborne pathogens, although the who guidelines for safe recreational water environments do include hpvs among non-fecally-derived viruses as viruses associated with plantar warts [ ] . a variety of hpvs and hpyvs were found in another study investigating spa/pool waters in rome [ ] . recently, disinfected water from sixteen pools and spa collected in rome between and were examined for the presence of human enteric viruses (adenovirus, norovirus and enterovirus. viruses were detected in % of the analyzed samples by molecular methods: two samples were positive for adenovirus (type ) and three samples for norovirus gii (type gii. ) (bonadonna et al., unpublished data). starting with the first hadv outbreak recorded in , we reviewed all of the reports concerning swimming-pool related viral illness. the data collected here confirm the involvement of viruses in cases and outbreaks associated with swimming pool attendance. a number of considerations emerge: • the paper reviews viral outbreaks due to adenovirus, enterovirus, hepatitis a virus, and norovirus, accounting for more than cases. nevertheless, there are likely to have been many other undetected cases and outbreaks. in fact, waterborne diseases are difficult to record because of their wide variety, the difficulty associating symptoms with water use/contact, and the limitations of pathogen detection methods. in the studies described, viruses responsible for reported cases were detected in pool waters only in % of the outbreaks, and were found to match with viruses of clinical origin. currently, better and more rapid methods for the detection of viruses in water samples are available than in the past, resulting in better studies and improved reporting of viral recreational outbreaks worldwide. this allows researchers to identify the causes of outbreaks and possible contributing factors for them. an excellent model to follow is the us-waterborne disease and outbreak surveillance system (wbdoss) that has been collecting and reporting data related to occurrences and causes of waterborne disease outbreaks associated with drinking and recreational waters since . • some of the studies found that waters meeting state or local water quality requirements contained enteric viruses and were the source of disease outbreaks, confirming that bacterial indicators are unreliable indicators of the presence of viruses and that enteric viruses are important hazardous waterborne pathogens. indeed, despite the relatively low concentration of viruses in water, they may nevertheless pose health risks due to their low infectious doses ( - virions). the human illnesses associated with enteric viruses in the reviewed studies were diverse: the most commonly reported symptoms were gastroenteritis, respiratory symptoms, and conjunctivitis. more severe symptoms were also documented, however, including hepatitis and central nervous system infections (aseptic meningitis). the majority of the outbreaks described involved mainly children and young people less than years of age. this may be attributable to differences in behaviors, susceptibility and/or immune defenses between children and adults. children are known to experience more severe symptoms than adults. • low concentrations of disinfectant/disinfection malfunction in swimming pools were reported in the vast majority of the outbreaks. only in one case the concentration of biocide was considered high. in 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associated with recreational water-united states hollos, i. an outbreak of hepatitis a due to a thermal spa an outbreak of hepatitis a associated with swimming in a public pool an outbreak of hepatitis a associated with a spa pool an outbreak of norwalk gastroenteritis associated with swimming in a pool and secondary person-to-person transmission wading pool water contaminated with both noroviruses and astroviruses as the source of a gastroenteritis outbreak outbreak of norovirus illness associated with a swimming pool surveillance for waterborne disease and outbreaks associated with recreational water use and other aquatic facility-associated health events-united states enteric viruses in wading pools enteroviral syndromes in toronto relationship of viruses and indicator bacteria in water and wastewater of israel occurrence of enteroviruses in community swimming pools enterovirus contamination of swimming pool water; correlation with bacteriological indicators analysis of enterovirus and adenovirus presence in swimming pools in cyprus from first detection of papillomaviruses and polyomaviruses in swimming pool waters: unrecognized recreational water-related pathogens? detection of oncogenic viruses in water environments by a luminex-based multiplex platform for high throughput screening of infectious agents author contributions: l.b. and g.l.r. conceived and wrote the paper, and approved the submitted version.funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -n le ix authors: saxena, abhishek; tiwari, archana; kaushik, rinku; iqbal, hafiz m.n.; parra-saldívar, roberto title: diatoms recovery from wastewater: overview from an ecological and economic perspective date: - - journal: nan doi: . /j.jwpe. . sha: doc_id: cord_uid: n le ix alarming water pollution is toxic to the aquatic ecosystem leading to a sharp decline in species diversity. diatoms have great potency to survive in contaminated water bodies, hence they can be compelling bioindicators to monitor the change in the environmental matrices effectively. around the globe, researchers are intended to evaluate the impact of pollution on the diatoms recovery and techniques used for the assessment. the diatoms are precious for futuristic need viz. value-added products, energy generation, pharmaceuticals, and aquaculture feedstocks. all these applications led to a significant rise in diatoms research among the scientific community. this review presents different isolation practices, cultivation, and other challenges associated with the diatoms. a precise focus is given to diatoms isolation techniques from highly polluted water bodies with the main thrust towards obtaining an axenic culture to elucidate the significance of pure diatom cultures. recovery of “jewels of the sea” from polluted water signifies the prospective ecological and economic aspects. diatoms are the most successful contemporary group of photosynthetic eukaryotic microbes that inhabit almost every kind of aquatic ecosystem. they can occur as endosymbionts in dinoflagellates [ , ] . diatoms have arisen because of an endosymbiotic event between red algae and heterotrophic flagellate approximately million years ago [ ] . although there are several types of microalgae present in water, diatoms are the most important and unique as they are the only species with the presence of silica in their cell [ , ] . diatoms are surrounded by a silica cell wall that allowing them to have varied shapes and sizes. their silica cell wall is popularly known as frustules that offer protection from photoinhibition; supply appropriate nutrients uptake; manage sinking rate and playing a significant role as a mechanical barrier against grazers [ ] . their silica content varies from one species to another depending upon their size, growth pattern, and environmental variables such as light, temperature, salinity, nutrients, and growth phase [ ] . there are more than , extant species of diatoms distributed to a different ecosystem [ ] . they are widely present in all aquatic habitat constituting lentic, lotic habitat and sand. it is estimated that % of species occur in freshwater, and % are benthic [ ] . aquatic ecosystems such as rivers, oceans, lakes, etc. support the lives of a large variety of organisms but excessive urbanization and industrialization transform them into the heavily polluted zone [ , ] . the rising pollution level deteriorates the water quality index leading to a huge decline in species diversity due to untreated discharge of metals, pesticides, aromatic polycyclic hydrocarbons, etc., which slows down the survival of an aquatic species, thus posing a big threat [ ] . several organisms have been used as bioindicators such as protozoa, bacteria, fishes, zooplankton, algae, etc. however, diatom-based monitoring for the assessment of ecosystem remedy is in great demand [ ] . because diatoms produce organic matter to a large extent that permits natural inbuilt capacity to withstand toxicity levels in water bodies, extended survival rate, short regeneration time than microalgae, fishes, and other micro invertebrates thus making them one of the best candidate for water quality monitoring, and excellent bioindicators of aquatic biological integrity [ , ] . water quality analysis is essential as it allowed to detect the habitat linked information of the related organisms [ ] . as diatoms also produce eicosapentaenoic acid (epa), docosahexaenoic acid (dha), and other nutritional compounds, they can be used as a food source to every aquatic organism such as zooplankton and fish. they constitute % of oxygen in water bodies, fix excessive chemical compounds and inorganic nutrients in the water, such as carbon, phosphorous, silica etc., [ ] . as fossil fuels are diminishing quickly, therefore, the demand for biofuel production has increased many folds since the last decades. the crop and food-producing plants will never be the top priority for biofuel production. in the future, diatoms will remain as the best candidate for biofuel production. some strain of diatoms produces lipids as % to % of the dry weight that can be used in the production of biofuel thereby focusing on the role of diatoms as an ideal substitute for fossil fuels [ ] . despite having all these important characteristics, they are the least explored aquatic species because isolation and maintenance of axenic culture is a tedious job [ ] . in this review, an effort has been put forward on the details related to the isolation of diatoms from polluted water bodies, the significance of diatoms, and their future potential. rapid industrialization and urbanization releasing a toxic substance into the aquatic environment. thousands of chemicals without any proper risk assessment and legislation are being introduced, which are endangered to aquatic species [ ] . algae, bacteria, protozoa, fishes, micro invertebrates, and zooplankton have been extensively used in biomonitoring purposes, but recently, diatoms have gained much more attention because they are primary producers and plays a crucial role in biogeochemical cycles of the aquatic food web [ , , ] . diatoms survive well in a variety of habitat, such as oceans, lakes, estuaries, wetlands, etc. the use of diatoms in ecotoxicological studies is advantageous to examine any possible effects of toxic elements. this makes diatom a potential tool in bioassay than any other aquatic species because of its easy sampling and frustules identification [ , ] . diatoms are highly sensitive to metal pollution than macroinvertebrates, which could very well explain gradation in metal composition, thus allows early identification of the presence of any heavy metal pollution in an aquatic ecosystem [ ] . diatom responds quickly to any organic matter or nutrient contamination, for example, metolachlor, atrazine, simazine, phenols, nitrate, phosphate, heavy metals, and pahs [ , ] . even though diatoms are ecologically diverse, their potential in biomonitoring is underutilized in evaluating any risk assessment because of traditionally available metrices such as cell density, biovolume, and relative abundance. new and advanced metrices are more advantageous in recognizing the dynamics of diatoms communities by demonstrating the sub-lethal effect that is not evident in diatom counts [ , ] . nuclear abnormalities, cell membrane, cytoplasmic content, and photosynthetic apparatus alteration change in lipid content and diatoms classification using ecological status are the main parameters that were infrequently reported [ ] [ ] [ ] . this behavior demonstrates sensitivity and efficiency in quantifying the toxic elements, stress detection after ultraviolet (uv) exposure, which remains unnoticed by the conventional method [ ] . as reported, calculating live diatom species in the biofilms seems to be a lucrative bioassessment tool. similarly, for assessing weak cytoplasmic content of freshwater species, the ecological association of diatoms is examined for evaluating the environmental health of water bodies because these newer metrices are easy to acquire, rapid detection, low labor-intensive, good reproducibility, standardized and worldwide acceptance [ , ] . diatoms as bioindicators check and observe the integrity of the precious environment by enumerating the change in water quality over large geological areas, provide variability, and any possible backdrop precondition [ ] . investigator worldwide have examined various water bodies using pollution tolerant diatom species to measure the pollution level by employing algal indices such as palmer's algal index; chlorophycean index; nygaard's phytoplankton indices to better realize the water quality. for example, some investigator utilized nygaard's algal index to evaluate the trophic status of hatia dam revealing the oligotrophic condition of water bodies [ ] . mishra and co-workers studied the bhoj wetland, employing nygaard's algal index to illustrate the wetland's eutrophic nature [ ] . palmer index was utilized in determining the seasonal variation in phytoplankton diversity of pichhola lake of udaipur, rajasthan [ ] . sharan and rekha used nygaard's algal index in their evaluation of the trophic status of hatia dam, finding the waterbody to exhibit an oligotrophic status [ ] . but still, diatoms potential is hardly exploited as bioindicators in determining the economic conditions as compared to other groups of microalgae [ ] . the environmental conditions of the target species must be taken into account as understanding their habitat and mimicking them in the laboratory play a crucial role in obtaining pure strains. each aquatic ecosystem has different attributes. river ecosystems may have less alkalinity than a marine ecosystem [ ] . these attributes directly or indirectly affect the occurrence of diatoms in a diverse aquatic ecosystem. thus, several factors, such as seasonal variation, ph, temperature, salinity, etc. should be considered as important factors [ ] . the representatives of the genus gomphonemoid can be examined from both marine and freshwater. gomphonemoid is widely present often in freshwater epiphytic and epilithic communities [ ] . gomphonema, one of the most common diatom strains, its valve and girdle view, the heteropolar cuneate shape can be recognized easily. gomphonema is attached to the substratum by a mucilage stalk, which assists in recognize strain from the rest of the other diatoms found in the freshwater ecosystem. the valves are wide at the head than foot pole with both uniseriate and biseriate nearby amid individual areolae cover-up externally by crescent-shaped volae [ ] [ ] [ ] . the raphe is straight or sinuous with terminal fissures expanding both apices to the valve margin, and inner central endings turn aside to one or the same side, respectively. the raphe is always shorter from the central nodule to the head pole, thus extending towards the foot pole. consequently, a perforated distinct pole field of different structures is also present at the foot pole. in many species, isolated punctatum near the central nodule occurs. the cingulum is arranged into many open bands; every second band is shorter than the next one to form lingual at the foot pole. each band is punctured by more than one row of pores. many other diatom species with the same cuneate shape, growing in the marine ecosystem can be compared to gomphonema by various workers [ ] . benthic diatoms are the significant controller in freshwater owing to their excellent adsorption, absorption, oxidation, decomposition, precipitation capability to store nutrients. they are very well utilized in biomonitoring purposes since the last decade because these diatoms provide crucial linkage in the aquatic ecosystem stipulating the flow of energy, cycling of materials, and reveal the water environment [ ] . diatom based indices were first produced by kolkwitz & marsson [ ] . since more than indices have been created from to such as humic degree, trophic level indicators, eutrophication, biological diatom index to name a few. however, the impact of indices has become less focal owing to the differences between floral geographical area and environment fluctuations guiding species to adapt according to the water quality criteria of the particular region in a particular instance of time [ ] . isolation and identification of benthic diatoms are problematic in comparison with planktonic species due to difficulties in sample treatment, sampling, and microscopic observation though benthic diatoms play the main role as bioindicators in the aquatic ecosystem because they attached to the substratum with secreted mucilage from their cell wall [ , ] . the morphological features help in the classification of particular species. morphological analysis by light microscope (lm) and electron microscope, for instance, transmission electron microscope (tem), scanning electron microscopy (sem) provides the better-detailed result of diatom biodiversity [ ] . the detection of diatom communities on various substrata can be observed as the slimy layer of biofilm appears brown or yellow-brown. although there are several types of substrata for isolating diatoms, the most preferred is from cobbles (epilithon). epiphytes also have diatoms attached to them [ ] . the taxonomy of the epiphyte should be recorded as specific strains attach to specific macrophytes. a typical substratum can be introduced to the water manually if the preferred substratum is absent. the isolated biofilm can be preserved or can be used for the preparation of axenic culture. a well-concentrated sample collected from a highly polluted habitat may have a more significant number of microalgae rather than diatoms. sometimes an association between diatoms and other microalgae can be seen. it may also contain empty or broken frustules. highly polluted water may have a high concentration of biogenic compounds, which may provide less nutrition or over nutrition. this may profit in the growth of those strain, which requires fewer nutrients or high nutrients, respectively. salinity, ph, light, etc. factors also play a role in the growth of such diatoms [ ] . the productivity of diatoms is higher than the other class of microalgae. they have undergone - cell divisions per day. they can survive in adverse climatic conditions since their silica cell wall is a nanoporous structure which allows them to act as a potential buffer system by improving carbonic anhydrase activity and helps in the transformation of bicarbonate to carbon-di-oxide (co ). they actively participate in wastewater remediation and quenching of heavy metal, leading to a degradation of environmental pollution [ ] . though bacteria, fungus, yeast, and algal biomass are commonly used in the treatment of heavy metal, diatoms are most preferred due to their large surface area, mucilage, nutritional requirement, and faster growth rate. bioremediation of heavy metal involves biosorption to metal-binding ligands on the cell surface, followed by bioaccumulation consists of inorganic molecules and related enzymes. in biosorption, the uptake of heavy metals is either carried out by the physiochemical pathway devoid of atp or during metabolism engaging atp energy. this mode of heavy metal remediation is a cost-effective approach through filtration. it is an extracellular passive route of adsorption of heavy metal, which is an energy-independent process occurring at the cell surface in equilibrium [ ] . biosorption is dependent upon temperature, ionic strength, ph, contact time, biomass and metal concentration, the composition of the cell wall, and metallic ions complexation. the principle behind biosorption is that diatoms are equipped with polymers, their outer wall has a negative surface charge due to functional groups like carboxyl, phosphoryl, and amine. the heavy metal with a positive surface charge attracts a negative charge through electrostatic interaction, which finally supports biosorption. bioaccumulation is an active intracellular process driven by energy. the heavy metals grow along with nutrients that assist in bioremediation. when heavy metals are accumulated, the microalgae produce reactive oxygen species (ros) to control cellular metabolism. there is an underlying passive uptake followed by an active process that is brought about by the detoxification of heavy metal at the cell surface by dehalogenation and denitrification process. this triggers the exclusion of heavy metals to maintain equilibrium conditions, thus eliminate toxic effects. despite this, a lot of work needs to be done in the treatment of wastewater by testing more species and study their capability of heavy metals removal. the biological degradation of heavy metal is quite challenging but not impossible [ , ] . previously, diatoms have been differentiating according to shape, size, pattern, and ultrastructure of their exoskeleton, which consists of silica, known as frustules. microscopic identification is based on the shape of their frustules, but still, they are challenging to identify, and improper examination reflects misleading observation upon identification beyond the genus level [ ] [ ] [ ] . to combat such a drawback, deoxyribonucleic acid (dna) sequence analysis has become a more potent approach in diatom research, thus opens a new leaf into their systematic and evolution. the main aim of the taxonomic examination is to make a hierarchical classification accurately reflecting evolutionary relationship, which then converted into a hierarchy of names related to different levels in a hierarchy of taxonomic ranks. recently, diatoms phylogenetic analysis at the generic and infrageneric levels require internal transcribed spacer (its) region of the s- . s- s nuclear ribosomal dna and among all, s ribosomal ribonucleic acid (rrna) sequencing resulting in the largest reference sequence dataset among the different markers used for diatoms [ ] [ ] [ ] . microarray technology enables parallel analysis of many genes in a single reaction. here, ordered matrices of dna sequence marked on the glass slide used for hybridization experiment with fluorescently labeled target genes and have been extensively used in both basic and applied aspects of the biological sciences [ ] . though several papers related to taxonomy and most of them provide line drawings or light microscopic images that are unable to give enough ultrastructure details of the diatom cell [ ] . a complete taxonomic survey can give the entire morphology of lesser-known microalgae or diatoms. as there can be a minor difference in the morphological structure of two or more organisms, such organisms are difficult to identify even under the lm. organisms like cyclotella brebisson may comprise more than complex species, which includes small specimens with a diameter of ≤ μm [ ] . based on frustules morphology, observation of these small specimens under a sem provides notable interspecific similarities that help in taxonomic studies. as some species may have similar morphological characteristics which may be ecologically distinct, their identification should be fundamentally accurate as it can provide quite an information about biodiversity which can be represented as an essential tool for different kind of studies such as ecological and applied studies. proschkinia is a rare diatom of the family proschkiniaceae within the naviculales. based on light microscopy, it was classified as a relative of nitzschia. the phylogenetic position of proschkinia was resolved with sequencing the complete mitochondrial genome of proschkinia complanatoides and compared the data set to other published diatom mitochondrial genes [ ] . to differentiate between diatoms strains that have similar morphology, it is vital to know the taxonomy of the target species. the nutritional requirements of various species also vary. some species require less nutrition composition, light temperature variables, while some require more. the taxonomy also helps in understanding the basic requirements of the target species [ ] . once the culture has been collected and identified, they will be preserved in the world federation of culture collection centers, which then made available to researchers and industry for intending applications and further experiments. table shows the list of available algae, microalgae, and diatom culture collection centers worldwide. the field procedures play a crucial role in the isolation of diatoms, microalgae, or other species. in a riverine environment, small boulders (rocks) and cobbles are the most favored substratum for diatoms screening. almost all diatom indexed to the eplithion community found here where epilithion`s ecology is recognized better than any other group [ ] . bricks, concrete, bridge supports, canal walls, etc. may be used as an alternative substratum, whereas simulated substrate can be set up into the stream if pebbles, cobbles, boulders, or macrophytes do not exist from the site though sampling should be done if they have been plunged for several weeks [ ] . sampling is an important step towards the collection of diatoms as the whole isolation process depends on it. the collected water sample may contain dead cells or damaged cells, which is not considered good, so it becomes very crucial to ideally collect the sample and isolate the most viable cells at the earliest. keeping the water sample for longer times is not recommended because environmental conditions change very frequently, which leads to cell death. some species multiply quickly and suddenly die, in that case, isolation should be done rapidly. sometimes, a planktonic net can be used to concentrate the sample as it removes other unwanted algal species, microorganisms, or debris but should be avoided as more concentrated sample leads to more damaged cells [ , ] . the collected sample should be kept according to the target species` environmental conditions. for example, several marine species are susceptible to sample concentration. for a sampling of these strains of species, special water bottles can be used. as several factors like water depth, pressure, temperature, or light influence the sample conditions, so instead of using a concentrated sample, a normal water sample taken in sterile containers can provide a better result. the collected sample should always be kept in sterile containers at a stable temperature [ ] . often sample collected from different water bodies contains zooplankton or other species which feed on algae or specifically on diatoms. so, it is essential to remove the unwanted organisms gently by filtering the sample at the sample site. however, sometimes tiny organisms may pass through the filter, which may pose threats to target species, in that case, dilution methods can be used to isolate the target species. the health of species in nature also plays a crucial role as an isolated cell will have successful growth only if they were in a good state at the sample collection time. it is advised to collect the sample carefully. the use of sterile equipment is mandatory, as dirty equipment may lead to contamination. all the isolation techniques should be performed under sterile conditions [ ] . a general idea about field procedure, laboratory procedure, culture preparation, and finally aim towards obtaining axenic culture is presented in fig. . the identification of a suitable substrate is a pivotal step to recognize the diatom communities in the natural environment. diatom communities form a slimy layer or thin golden-brown layer on the substratum. it can be more noticeable by feel or touch and can be identified at a specific time depending on species. the substratum can be differentiated into a preferred substrate and an alternative substrate. diatom attaches to different substrata or facilitates locomotion by releasing mucilage from various structures of the cell wall [ ] . diatom community composition is mainly influenced by chemicals present in water, water turbulence, temperature and light present in water, being eaten by large microorganisms, etc. the most preferred substratum is the solid substratum. it mainly includes cobbles/pebbles and small rocks (epilithon). fig. depicts various substrates intended for diatoms sampling. they are widely available in almost every aquatic habitat and throughout the year. in the absence of a solid substrate, submerged or emerging macrophytic plants (epiphyte) should be sampled as it also provides a suitable habitat for diatom communities. it is ideal to note the microhabitat of diatoms species [ ] . the most effective method to eliminate contaminants is achieved by various techniques such as filtration, sedimentation, and centrifugation as gravity separation. the most successful technique is filtration as it removes all unwanted microorganisms or other zooplankton, which feed on diatoms from the sample. in this, a planktonic net of a specific size is used, which does not allow any other organism to pass through except the target species. in gravity separation, diatoms settle down as sediments while leaving other microalgae in the suspension, which can be discarded further [ ] . field operators should always wear thigh waders for the protection of feet. they should wear life jackets while sampling. they should never go in-depth for sampling. they should wear globes when sampling heavily polluted water bodies. they should be very careful in habitats dominated by dangerous animals posing a threat to people and blood- fig. . flow-chart depicting isolation of diatoms from sample collection procedure to obtaining axenic culture of target species. sucking worms like leeches [ ] . anthropogenic pressure from the urbanized area causes changes in the water quality of a river and leads to the eutrophication of reservoirs and the proliferation of algae. the informal settlements without sanitary infrastructure aggravate the deterioration of water quality in urban water sources [ ] . the water quality index, conducted by using the planktonic diatom index (pdi) for monitoring the lake erie's nearshore pelagic zone, provides a robust assessment of water quality [ ] . the collected water sample should be adequately analyzed as it gives full information about the diatom habitat and its environment. several factors should be measured. hydrological characteristics of the stream, which mainly examined with stream velocity and channel depth and channel breadth. physical variables of water mainly involve water temperature and turbidity. physico-chemical variables are examined with ph, conductivity/total dissolved solids. water contains nutrients such as orthophosphate-phosphorus (po [ ] . for monitoring the pollution of water bodies, several indices have been developed, such as the index of saprobity-eutrophication (idse/ ), which utilizes the diatom population in terms of organic pollution [ ] . water can be divided into two types such as groundwater and surface water and both are at the risk of pollutants ranges from heavy metals, pesticides, fertilizers, hazardous chemicals, etc. an accepted water quality criterion according to american public health association (apha), the world health organization (who), indian standard institution (isi), indian council of medical research (icmr), and central pollution control board (cpcb) consisting of various parameters are presented in table [ ]. examination of the collected sample from different substrata should be done as quickly as possible to make sure that diatom assemblage contains live cells and not dead ones. sample with more dead cells should be discarded, and sampling should be performed again. the environmental conditions of samples collected from various aquatic habitats should be mimicked in a laboratory. it should be noted that all equipment like glass-slide, coverslip, pipette, forceps, inoculating loop, centrifuge tube, glass test tube, watch glass, etc. must be sterilized before use. uncleaned equipment may lead to contaminations that further result in the death of cells. after examination of diatoms in the collected sample, standard isolation methods should be performed [ ] . as the collected sample contains various microorganisms and other microalgae, these standard isolation methods play a crucial role in the isolation of target species of diatom. the isolator should focus on finding the target species in the collected sample. viable cells should be cultured as soon as possible while avoiding contaminants. all the following methods should be performed carefully [ ] . the simplest method of single-cell isolation is performed with the help of micropipette, first attempted by zumstein ( ) later improved by pringshiem ( ) [ , , ] . a capillary is attached to the pasteur pipette to make it ideal for single-cell isolation. this method requires a lot of practice. as diatoms have a minimal size, one single cell can also be picked separately with the help of a micropipette. single-cell isolation with micropipette is to pick a single cell of target species without getting it damage and avoiding contaminants and deposit it into a sterile tube or watch glass and then transfer it to a culture tube. the whole process is done with the help of a microscope, especially an inverted microscope, as it allows the isolator to work conveniently [ , ] . this method is very useful while isolating cells from very contaminated samples with a tiny microorganism, which is harder to separate from the sample. the aim of this method to separate one single cell of target species and deposit it in a culture test tube. usually, dilution is repeated serially up to : [ ]. it is probably the oldest and standard method available to isolate microorganisms. most of the diatom species usually grow well in agar. isolation using agar can be used with both "streaking" and "spreading" methods. later single cells or colonies can be picked from the culture plate and observed under the microscope. to study the colony/diatom community characteristics, agar plate method is advantageous. the agar concentration should be between . % to . % and . %. while observing glass slide under a microscope, if it contains a single diatom cell, it can be replicated on the agar plate for culture. agar pour plate method can also be used to isolate cells that do not grow on the surface of the agar plate [ ] . this is a physical technique for the pre-separation of single cells of diatoms from polluted water. the centrifugal technique applies gravity settling to isolate more prominent species from the microalgae. low dense cells, for example, microscopic organisms and others present in the supernatant, are emptied while diatoms species stay at the base as a pellet. the speed and time of centrifugation vary depending upon the target microalgal species. even though reasonably successful, however, it might harm delicate cells through sheer pressure [ ] . this method involves the use of enrichment media. enrichment media is rich in one constitute which provides nutrition to only one/two types of cells (species) while inhibiting the growth of other cells (microorganisms). for example, it has been noted that the presence of silica in a culture media boost the growth of diatoms but inhibit the growth of other microalgae. sometimes even a trace element makes an enormous difference in the growth of species. so, it is necessary to know the nutritional requirement of the target species, and it should be reached as it will improve the growth of the target species [ , ] . this is a pre-isolation step towards the separation of diatoms. samples can break into two portions depend upon particle size difference. sometimes enrichment media can also be utilized for better outcomes while separating fungal cells from diatoms. bigger species can be held at the membrane however, diatom cells pass through the filter quickly. this strategy is helpful, advantageous, and very adaptable [ ] . another technique where alginate beads were utilized to isolate the algal species from the mixed algal culture [ ] . in this method, the contaminated water is placed in nutrient media and incubate for one week or more to affirm the development of different species. a known concentration of sodium alginate solution containing a mixed culture of diatoms is added drop by drop to a calcium chloride solution to form alginate beads and kept for - h to make beads stronger, stiff, and firm. the beads transferred to a well enzyme-linked immunosorbent assay (elisa) microplate (one bead per well), smashed partially containing standard culture media and incubated under controlled conditions in the presence of light for one week to confirm the growth of isolated diatom species. the trapped diatom species in the beads viewed microscopically for the confirmation of isolated species. this methodology is simple, easy, cost-effective compared with existing methods and can be easily applied for the mass cultivation of specific species [ ] . there are other different strategies for the isolation of diatom species like anti-microbials, uv radiation. anti-microbial treatment hinders growth & development by eliminating microorganisms, thus help with getting the confined unadulterated culture of diatoms. uv radiation act as a disinfectant that prevents bacteria and other contaminants since diatoms are better resistant to uv radiation over bacterial cells [ ] . photoinhibition is an immediate strategy that harms biomolecules by engrossing uv light, which prompts the loss of natural capacity of microscopic organisms [ ] . diatoms are valuable for the aquatic food chain and give significant bioactive compounds to human prosperity. segregating unadulterated species or getting axenic culture from a polluted water source is a complicated and tedious procedure with the current strategies. hence, it becomes necessary to search for other advanced isolation techniques. micromanipulation is one such method permitting refined, confining single species in a more cleansed manner. prior fine capillary tubes were used focusing on target cells under microscopic observation, which is a laborious task and threat to contamination [ ] . with the arrival of a modern, sophisticated micromanipulator exploiting micromanipulator and stereomicroscope, a high level of precision can be achieved for screening and isolation of species of interest [ ] . a cell of interest can be captured utilizing a focused laser and transferred to a sterile media of interest. this system is still in its early stages because of the high state of expertise and time required [ ] . the requirement for cutting edge new methods push researcher to redesign the flow cytometer coupling facs (fluorescence-activated cell sorting). the premise of this procedure is light scattering and fluorescence [ ] . cells absorb the laser beam transmit fluorescence and give information on cell size, pigments, and reliability of species, which is identified with the morphology and other characteristics of the species accordingly, thus allowing characterization of up to , cells in a fraction of second [ ] . this technique can be employed to build up an axenic culture and get rid of any bacterial contamination. in some cases, mixed culture or aggregation of cells may make issues in the precise identification of cells, which can be overcome by cell disruption through appropriate sonication under controlled conditions. this technique is generally excellent for quick screening of organisms overproduces metabolites of interest in conjunction with fluorescent dyes. for example, bodipy or nile red facilitates the selection of desired mutants from a mixed population [ ] . an overview of the main techniques used to establish axenic diatom cultures is presented in table . diatoms, the most productive phytoplankton found all over the planet from antarctic glaciers to brick walls, have drawn a tremendous awareness in the research field, i.e., for the quantitative reconstructions of ocean surface conditions to establish palaeoceanographic records [ ] . they are one of the most promising candidates for various applications such as pharmaceuticals, bioenergy, industrial chemicals, nutraceuticals, and aquaculture [ , ] . to make most out of it, an axenic culture of diatom must be a prerequisite bearing in mind that a pure culture of diatoms is undoubtedly required in genome sequencing [ ] , to identify the producer of any novel bioactive compound for large scale manufacturing of nutraceutical [ ] , building a consortium for bioremediation [ ] and to elucidate the relationship between other microalgae using omics tool [ ] . maintaining an axenic culture for a longer duration is very difficult because bacteria are vulnerable, which frequently attacks the diatom. the primary focus is to isolate pure species of diatoms and their maintenance [ ] . nevertheless, an axenic strategy is dependent upon contamination and desirable organisms disclosing the possible relationship between them leading to the understanding, selecting, and developing an axenic culture, which is a critical step, thus alleviate the cultivation method. mimicking the natural environment for optimum growth under laboratory conditions facilitates the development of an axenic culture, but this step requires the correct isolation plan and approach [ ] . the fundamental question is, are axenic culture genuine.? what is the acceptability of their purity level.? with the direct symbiotic association between diatoms and other aquatic organisms, the development of a new advanced technique to assess the purity level cannot be ruled out. therefore, emphasis should be given towards improvement and innovation in isolation methods, to standardize and establish the correct isolation practices to solve future energy crises, nutritional requirement, nutraceuticals, pharmaceuticals by choosing the right technique for right diatom species for the benefit to the mankind, society and last but not least the ecological balance [ ] . therefore, constant effort to develop a new scientific method will surely pave the way towards the isolation of diatoms from mixed culture and maintaining their purity level to a greater extent. diatoms-virus interactions are rather difficult in obtaining pure culture. the virus kills diatoms, thus benefitting other algae. the limitation in silicon levels in the oceans facilitates infection by the virus. accelerating diatoms mortality due to viral infection is a major concern affecting the carbon cycle leading to global warming and ultimately changes climatic conditions drastically. also, ocean study is a difficult task. the main emphasis is given towards the marine environment rather than terrestrial causing disparity about diatoms-virus interactions. as a result, more isolations techniques and characterization is necessary for controlling the action of the virus in the regulation of host populations [ ] . several species of diatoms are found in a highly acidic environment, and they continue to grow consistently near or in the acidic conditions both in marine and freshwater ecosystems. they exhibited a significant response to the alterations in the growth conditions. they are capable of integrating numerous physiological as well as morphological adaptations, which favors their persistence. diatoms growth inhibited majorly in silica limitations than any other nutrients because cell division cannot sustain for a more extended period under silica deprived conditions [ ] . highly acidic conditions resulted in decreased si, indicating that close to the end of this century ocean acidification might persuade the c and si cycle and alter the composition of diatoms. [ ] . considerable human interference is turning aquatic bodies towards the acidic zone, and many countries have seen ocean acidification even in extreme low-temperature conditions such as north america, canada, and italy. due to the high concentration of hydrogen ions, diatoms flora like nitzschia, pinnularia, eunotia, and frustularia are exceptionally rich in habitats within the ph range of . - [ ] . in an acidic environment, a higher cell volume, chlorophyll, and productivity were observed due to a change in water chemistry because in acidic conditions number of grazing macroinvertebrates and microheterotrophs were less in number [ ] . however, it is unattainable to understand the natural influx of pre-acidification, natural fluctuations in conditions, and the level of nutrients that tend to vary from habitat to habitat. abundance and fall in diatom species can be explained based on physiological ph, availability of the essential nutrients, and biological interactions, which could be the factor for the productivity of diatoms in extreme conditions. very little is known about the influence of acidification on diatoms. hence the effect of acidic environment on diatoms changes from the open sea, near to the sea and deep-sea, and indeed not a decrease in diatoms productivity and growth [ ] . since diatoms nurture associated aquatic species to a greater extent within the marine environment so achieving utmost purity and break communication with unwanted organisms is a significant challenge. therefore, emphasis should be given to improve and channelize knowledge towards proper isolation techniques that will separate diatoms from contamination and any possible intervention to promote appropriate growth and development of diatoms [ , ] . an outline of isolation of pure diatom species getting affected by the surrounding contaminants is challenging since they get heavily occupied with different interfering organisms, which pose a significant threat in obtaining axenic culture, as presented in fig. . although there are several culture media are available, but the most recommended culture media for isolation and growth of diatoms are f/ -si, pm, and wc media. the composition of each culture media is prescribed according to the selection of diatoms for culturing for the desired application. f/ -si culture media is widely used as the most effective media, whereas pm media is utilized for culturing diatoms in an acidic environment. wc media is generally used for culturing diatoms existing in an alkaline environment [ ] . all these culture media differ in their composition of major and minor nutrients and provide proper nourishment to diatom culture. f/ -si media is required for marine species while pm and wc media supports the cultivation of freshwater species. for marine, it is advised to prepare both media with seawater and for freshwater prepare media with distilled water [ ] . the chemical composition of different culture media for diatoms cultivation are presented in table . elimination of contaminants is important to maintain the pure culture of isolated species. as stated, the above contamination can be eliminated with gravity separation, dilution techniques, etc. in the case of bacterial contamination, antibiotics should be used in a specific amount in culture. every equipment, glassware used in isolation or culture should be sterilized before performing experiments. culture should be maintained in aseptic and optimum conditions [ ] . after preparing the axenic culture, it should be subcultured after over a specific period. subculturing should be done before the decline phase of the primary culture. each subculture should be prepared in aseptic conditions. after each subculture, it should be observed for contaminations, and in case of occurrence of contamination that specific subculture should be discarded, and the process is repeated [ ] . an axenic culture contains only one target species and is free of all contaminations. after isolation of target species of diatoms, it is necessary to maintain the culture free from contaminants so that a pure culture can be established. pure isolated cultures can be obtained from a combination of techniques such as flow sorting, pasteur pipette, and agar plate methods. the polluted water collected from various habitat will be immediately examined by monitoring ph and temperature at the site and will be carried to the laboratory in - liters plastic bottles. the raw wastewater was filtered twice with a . μm pore size whatman filter paper to remove the large suspended solids particles and debris. the wastewater will be autoclaved for sterilization and further used for the cultivation of the microalgae. the physico-chemical parameters of wastewater will be characterized as per the standard procedure of apha guidelines [ , ] . initially, the water sample will be serially diluted in a microwell plate and test tube to get a pure culture and observed under the microscope to confirm the presence of microalgae species. isolation will also be carried out by spreading or streaking wastewater in a solid agar medium supplemented with silica. once confirmed, the microalgae species were transferred slowly and gradually in different volumes of erlenmeyer flasks containing artificial seawater enriched with f/ -si media at ph . and maintained in the culture room at h dark/ light diurnal cycle with a desired luminous intensity at - • c. finally, strains will be identified, and their taxonomic classification will be established. fig. shows the overview of obtaining pure culture diatoms from polluted water bodies. diatoms are very young as compared to other phytoplanktons but have evolved rapidly over some time. a decline return on investment in research and development (r&d) and slow-growing companies accelerating the demand for diatoms cultivation serving as a superfood. the term nutraceutical refers to nutrition that provides physiological benefits coupled with the protection and prevention of disease. these functional foods promote health by adding novel ingredients that are similar to conventional foods but with rich nutrition or bioactive compounds that may target the physiological mechanism of our body as characterized by the us department of agriculture, agricultural research service [ ] . on account of large content of polyunsaturated fatty acids (e.g., pufas n and n ), essential amino acids (e.g., leucine, isoleucine, and valine) and pigments (e.g., lutein and β-carotene) and vitamins (e.g., b ), the diatoms biomass have gained much popularity across the globe [ ] . nutraceutical industries manufacturing products on a large scale in collaboration with the food and pharmaceutical industry, thus benefitting consumers. diatoms are emerging as leading nutraceutical ingredients from a biotechnological point of view since they are equipped with polyunsaturated fatty acids, essential amino acids, pigments, and vitamins. according to the world health organization who, the most severe challenge of the st century is a lifestyle disease, i.e., noncommunicable disease. the current focus is to develop functional food and their products, which combat the disease development [ ] . diatoms are underutilized among the microalgae, and only a few species have been characterized thoroughly. diatom based products are an fig. . shows the main interfering agents or treats in obtaining axenic culture of diatoms. . × - . × - . × - excellent source for multifaceted use covering the health and nutraceutical sector. therefore, the cultivation of diatoms under different cultural conditions helps to understand their biochemistry to a large extent [ ] . diatoms cells, after degradation, settled down in the form of silica, which is known as diatomaceous earth. these death remains have tremendous applications for industrial and agriculture purposes. ongoing covid- impacted the world economy. the diatomite industry has also suffered significantly but successful in maintaining an optimistic growth for four years. the average annual growth rate of the diatomite industry will reach millions usd in . probably by , the market potential will see a significant expansion [ ] . diatomaceous earth is one of the major causes of their successful existence on the globe and also act as carbon dioxide sequester on the ocean floor. however, more information is needed about nutrient upwellings and the causes of their bloom degradation in their natural habitats. further, a more in-depth study on their self-defense mechanisms will reveal the unfolded truth about the interaction of diatoms and their feeders in the food web. to overcome the constraints of fossil fuels, the generation of bioenergy makes renewable energy particularly interesting. nowadays, renewable resources are contributing % of the total energy of the world. diatoms synthesize oil for human consumption, which is rich in nutrition showing a promising alternative to meet the demand of future growing populations. we need to emphasize the latest, more productive technologies to overcome the cost of their cultivation and harvesting for optimum utilization of diatoms as biofuels, valuable products, wastewater remediation, and aquaculture [ ] . the generation of bioenergy from novel sources like diatoms is incredibly significant as they contribute towards carbon dioxide mitigation generation of renewable energy concomitant with a plethora of value-added products. due to their unique evolutionary history and their adaptations to varied environmental conditions, diatoms have spread all around the world. undoubtedly, diatoms are playing a significant role in the reduction of global warming gases such as carbon dioxide and provide possibilities to change the present climatic conditions. but still more researches are needed on their biogeographic distribution and to learn more about factors which are responsible for their more successful existence. diatoms cells, after degradation, settled down fig. . protocol for adopting isolation strategies employed for diatom axenic culture. a. saxena et al. in the form of silica, which is known as diatomaceous earth. these death remains have tremendous applications for industrial and agriculture purposes. diatomaceous earth is one of the major causes of their successful existence on the globe and also act as carbon dioxide sequester on the ocean floor [ ] . however, more information is needed about nutrient upwellings and the causes of their bloom degradation in their natural habitats. further, a more in-depth study on their self-defense mechanisms will reveal the unfolded truth about the interaction of diatoms and their feeders in the food web. to overcome the constraints of fossil fuels, the generation of bioenergy makes renewable energy particularly interesting. nowadays, renewable resources are contributing % of the total energy of the world. diatoms synthesize oil for human consumption, which is rich in nutrition showing a promising alternative to meet the demand of future growing populations. we need to emphasize the latest more productive technologies to overcome the cost of their cultivation and harvesting for optimum utilization of diatoms as biofuels, valuable products, wastewater remediation, and aquaculture. they are the primary food of most of the herbivores in freshwater, as well as in marine water. furthermore, being the rich source of poly unsaturated fatty acid (pufa), they have a great scope in aquaculture. therefore, diatoms are the cost-effective approach which sequesters co , excess amount of heat, reduce the extra amount of nutrients, and remove metal contaminations from their habitats. they could be a possible solution to energy savers and to reduce the threat of global warming. conversely, they may be a viable source of food and feeder for living beings. diatoms and wastewater are an advanced integrated system that, on the one hand, utilize the potential of diatoms in purifying the wastewater thus, it helps in restoring the original quality of habitat. on the other hand, their biomass can be utilized in the production of antiobesity, antibacterial, antioxidant, biofuels, anticancerous, antiviral compounds. diatoms have received great attention because they are the significant producer of lipids and biomass for various commercial applications such as biofuels comprising of biodiesel and aviation fuel. they participate in cleaning the environment by fixing the atmospheric co and contributes to the reduction of greenhouse gas (ghg) [ ] . diatoms based biofuels are projected to be economically beneficial in the future, but still, some ambiguity exists whether they equate with fossil fuels or not is remain a question. obtaining a biofuel require several steps like harvesting, extraction, conversion of biomass to target biofuel is economically not feasible. one of the key bottlenecks is cell density, which is similar to the water and negative surface charge that put off settling owing to gravity, thus makes harvesting a most crucial and challenging step. there is an urgent need for an efficient harvesting technique to improve the economics and efficiency of the whole downstream process [ , ] . at present, the technologies of the water purification industry have been used for harvest and recovery of microalgae, but still, some technical glitch needs to address that are sole to microalgae harvest. firstly, harvesting techniques must be species-specific. secondly, a blend of different harvesting techniques must result in synergistic outcomes. thirdly, a complete cell separation from a diluted suspension to lower the cost of the downstream process. fourthly, steps such as lipid extraction and biofuel conversion should be minimized. finally, to come up with the idea of more advanced harvesting techniques in the future [ ] . the ultimate goal of economic feasibility is the conversion method by utilizing inexpensive chemicals and the release of the least toxic waste. microalgae biomass is the best option for fossil fuels for transportation, but the commercialization of microalgae-based biofuel is a significant task. new strategies should encourage innovative elements to the existing downstream process that can appreciably reduce the cost towards the realization of biofuel commercially and dispose of harmful co production. to sum up, harvest, extraction, and conversion steps must be environment friendly, while making biofuels as future transportation fuels [ , ] . all the above-mentioned factors enhancing the popularity of diatoms globally strengthen their market potential and growth in the coming years. their medicinal characteristics are likely to provide more interest in diatoms cultivation with a rising population. considering health benefits, the global diatoms market is expected to witness as one of the most emerging economies across the world. a general question raised by everyone about diatom`s attractive potential and advantages but still exploring their true value is a long journey because high rich diversity limits their studies making it obscure and inaccurate. it is high time to investigate new class and species of diatoms and understand their mechanism, research models, and the potential role for future perspectives. the past few decades have seen a tremendous rise in diatoms research, as reflected in the publication. an undercurrent excitement is moving around, but challenges are roaring high. practically, a good isolation practice is the only solution that will fill this void. smart isolation practices are prerequisites that must help quick isolations and speedy recovery of diatoms from polluted water. diatoms`axenic culture is the first step towards a complete study of an organism and maintaining their pure culture can be further used in different applications such as bioindicators, nutraceuticals, cosmetics, phycoremediation, aquaculture, etc. thus, growing 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challenges and current status of microalgae biomass applications in industrial wastewater treatment preparation, characterization and catalytic performance of mesoporous silicates derived from natural diatomite: comparative studies ocean acidification interacts with variable light to decrease growth but increase particulate organic nitrogen production in a diatom methods of downstream processing for the production of biodiesel from microalgae innovative microalgae biomass harvesting methods: technical feasibility and life cycle analysis dr. archana tiwari is thankful to the department of biotechnology (dbt), new delhi, india for providing financial support under project grant no: bt/pr/ /aaq/ / / . all listed authors are grateful to their representative universities for providing the literature access. key: cord- - mbn b x authors: harrington, gregory w.; xagoraraki, irene; assavasilavasukul, prapakorn; standridge, jon h. title: effect of filtration conditions on removal of emerging waterborne pathogens date: - - journal: j am water works assoc doi: . /j. - . .tb .x sha: doc_id: cord_uid: mbn b x this study evaluated removal of live emerging waterborne pathogens by pilot‐scale conventional treatment with alum coagulation, flocculation, sedimentation, and filtration. the microbes tested were cryptosporidium parvum oocysts, encephalitozoon intestinalis spores, enteropathogenic escherichia coli o :h , aeromonas hydrophila, and bacteriophage ms . the study showed the effects of filter run time, alternative loading rates, alternative filter media, and ph on pathogen removal. results indicated that turbidity breakthrough was accompanied by breakthrough of all pathogens tested in this study. results also suggest that the breakthrough of a. hydrophila and e. coli o :h occurred more rapidly than that of turbidity. in general, filtration rate or alternative filter media configurations had no apparent effect on pathogen removal. merging infectious diseases are defined as those whose documented incidence in humans has increased during the past years or threatens to rise in the near future (institute of medicine, ) . a variety of viral, bacterial, and protozoan agents have been implicated as potentially important emerging waterborne agents. although little is known about the fate, ecology, and distribution of these microorganisms in the aquatic environment, some of these microorganisms have already been identified as the cause of waterborne disease outbreaks (mullens, ; cdc, ; cdc, a; cdc, b; kaplan et al, a; kaplan et al, b) . beyond these documented cases, other emerging pathogens have been identified in natural water sources and therefore have the potential to cause waterborne disease outbreaks (moe, ; avery & undeen, ; clark et al, ) . emerging viral pathogens include the caliciviruses, such as norwalk and norwalk-like viruses, and other small round structured viruses such as astroviruses and coronaviruses. enteric adenoviruses are also considered to be emerging viral e filtration pathogens. several bacteria, including escherichia coli o :h , aeromonas hydrophila, mycobacterium avium, and helicobacter pylori, have also been identified as emerging waterborne pathogens. emerging protozoan parasites include five genera of microsporidian protozoa that are known to cause disease in humans: nosema, pleistophora, enterocytozoon, encephalitozoon, and microsporidium (garcia & bruckner, ) . other recent waterborne outbreaks have been attributed to the protozoa cyclospora cayetanensis and toxoplasma gondii (mullens, ; cdc, b) . regulatory response to pathogen emergence. giardia and cryptosporidium were the primary emerging protozoan pathogens of concern in the s and s. the emergence of giardia as a waterborne pathogen in the late s and early s helped reinforce the need for a multiple-barrier approach to water treatment. to address this need, the us environmental protection agency (usepa) issued the surface water treatment rule (swtr) in (usepa, ) . this rule required filtration for most utilities that treat surface water and established minimum levels of disinfection for all utilities that treat surface water. in addition, the emergence of cryptosporidium in the late s and s forced the usepa to issue and propose a series of enhanced swtrs (usepa, ; usepa, ; usepa, ; usepa, ) . these regulatory actions trace how a pathogen can emerge to substantially alter regulatory policies and treatment practices. given the significant effect these new rules have had on the drinking water industry, it is appropriate to ask whether some other organism could emerge to trigger more changes in both policy and practice. the observed or suspected presence of pathogens in water systems implies that they entered the systems by direct contamination of the distribution system or by surviving the conventional, multiple-barrier approach to drinking water treatment. the potential for the latter of these two possible routes has not yet been addressed for the emerging pathogens noted previously. this article provides data that begin to fill this information gap. objective. the objective of this research was to evaluate the removal of several emerging waterborne pathogens by using pilot-scale conventional treatment with alum coagulation, flocculation, sedimentation, and filtration. the study focused on the effect of filter run time, alternative filter loading rates, alternative filter media, and coagulation ph on pathogen removal. selected microorganisms. six pilot-scale challenge experiments were performed to investigate the effects of various filtration conditions on the removal of live waterborne pathogens by alum coagulation, flocculation, sedimentation, and rapid-rate granular media filtration. the microbes tested were cryptosporidium parvum oocysts, encephalitozoon intestinalis spores, enteropathogenic e. coli o :h , a. hydrophila, and bacteriophage ms . test microorganisms were pooled to produce a microbial "cocktail" that was used to seed raw waters for all pilot-scale experiments. table summarizes the morphological characteristics of these pathogens; however, the characteristics presented here may have only an indirect influence on pathogen removal. once coagulation and flocculation have been completed, these pathogens would likely be associated with floc particles having significantly different sizes and shapes from those shown in the table. it is the characteristics of these floc particles that have a direct influence on pathogen removal through sedimentation and granular media filtration. nevertheless, the pathogen characteristics listed in table play a role in determining the physical and chemical characteristics of the floc par- although many studies have focused on the effects of particle size on particle removal in clean bed filters, little research has been conducted to determine the breakthrough rates of different-sized particles. f fl lo ow w c ch ha ar rt t f fo or r e ev va al lu ua at ti in ng g e ef ff fe ec ct t o of f f fi il lt te er r r ru un n t ti im me e o on n r re em mo ov va al l o of f e em me er rg gi in ng g w wa at te er rb bo or rn ne e p pa at th ho og ge en ns s ticles. floc characteristics will also depend on other factors including the type and dosage of coagulant being used, the ph of coagulation, and the characteristics of other particles such as suspended silts and clays. methods common to all pilot experiments. continuous-flow pilot-plant experiments were run at the university of wisconsin's pilot-scale water treatment facility in madison. the pilot plant received raw water from lake mendota in madison and included two parallel treatment trains ( figures - ) operated at a flow rate of gpm ( . l/s) per treatment train. raw water was pumped to a constant-head reservoir and flowed by gravity through two rapid-mix tanks, three flocculation chambers, and one sedimentation basin on each train. for the duration of each run, pathogen cocktail and alum were fed at a constant dosage to the first and second rapid-mix chambers, respectively. for each treatment train, settled water was pumped to a constant-head tank and then flowed by gravity to a rack of four granular media filter columns. pumping of settled water was provided because of space limitations in the laboratory, but similar designs have been used in previous studies on cryptosporidium oocyst and giardia cyst removal (patania et al, ) . because pumping breaks up floc particles that are not removed by sedimentation, these experiments may represent a worst-case analysis of pathogen removal in conventional treatment of lake mendota water. a detailed description of the treatment plant operating conditions is provided elsewhere (harrington et al, ) . experiments were conducted to evaluate hydraulic residence time distributions and the influence of settled water pumping on turbidity removal (harrington et al, ) . the hydraulic residence time distribution showed that filter effluent samples could be collected no earlier than h after initiation of pathogen feed to the raw water supply. the other tests indicated that settled water pumping had no effect on turbidity removal through filtration provided that the sedimentation basin was cleaned out one day prior to a challenge experiment. the day before a pathogen challenge run, the rapid-mix tanks, flocculation chambers, and sedimentation basins were drained, and solids were cleaned out and flushed to the sanitary sewer. the alum dosage for each pilotscale test was determined by a series of jar-test experiments using pathogen-seeded water approximately h before the pilot-scale experiment. the selected alum dosage was based on optimal turbidity removal. after the cleaning, the pilot-scale system was brought back on line without pathogen feed and allowed to run overnight with the selected alum dosage. on the following day, feeding of the pathogen cocktail was initiated once the pilot plant was determined to be © american water works association f fl lo ow w c ch ha ar rt t f fo or r e ev va al lu ua at ti in ng g e ef ff fe ec ct t o of f f fi il lt tr ra at ti io on n r ra at te e o on n r re em mo ov va al l o of f e em me er rg gi in ng g w wa at te er rb bo or rn ne e p pa at th ho og ge en ns s t ty yp pi ic ca al l m mo or rp ph ho ol lo og gi ic ca al l c ch ha ar ra ac ct te er ri is st ti ic cs s o of f s se el le ec ct te ed d m mi ic cr ro oo or rg ga an ni is sm ms s operating in a stable fashion. table shows average pathogen concentrations in the seeded lake mendota water. these concentrations were measured at a location between the two rapid-mix basins on each train (see yellow circles on figures - ) . the cocktail was continuously fed to each pilot train at a flow rate of ml/min from a l ( . gal) high-density polyethylene container. the container was placed in a cooler with ice packs to help maintain the stability of the pathogen cocktail. a mixing system was designed to help maintain a uniform suspension of pathogens within the container. details of the pathogen feed system and the preparation methods for the stock pathogen suspension are provided elsewhere (harrington et al, ) . five hours after the pathogen feed was initiated, the filter effluent was presumed to be at steady state with respect to the pathogen feed. during this h period, pathogen samples were collected at a location between the two rapid-mix tanks (yellow circles on figures - ) to evaluate the stability of the pathogen feed during this part of the run. these samples were collected min, h, and h after initiation of the pathogen feed. after the h period, all filters were backwashed and simultaneously placed back on line after backwashing was completed. to track turbidity during the ripening phase, filter effluent turbidities were monitored every min for h after the filters were placed back on line. head loss across each filter was recorded every min during the ripening phase. once ripening was completed, turbidity and head loss were monitored every - h until the experiment was terminated. methods specific to evaluating filter run time. the pilot plant was used on two separate occasions to evaluate the effect of filter run time on the performance of dual-media filters operated at gpm/sq ft ( . mm/s). a flow chart for these tests is shown in figure . duplicate filters were run on a single train during each test to characterize experimental repeatability. the dual-media filters consisted of in. ( cm) of crushed anthracite over in. ( cm) of sand. table lists the effective size and uniformity coefficient of each filter medium. in these two experiments, feeding of the pathogen cocktail was initiated and the pilot plant was operated for h, at which point steady-state conditions were assumed. both filters were then backwashed and placed back into service. forty-five minutes after backwashing, pathogen samples were collected from the spiked raw water, the settled water, and both filter effluents to evaluate removal during the ripening stage (see yellow circles on figure ). additional sets of pathogen samples were collected from the same locations at , , and h after backwashing. the h time was selected because routine operation of the pilot plant showed that terminal head loss was typically achieved at ± h. f fl lo ow w c ch ha ar rt t f fo or r e ev va al lu ua at ti in ng g e ef ff fe ec ct t o of f a al lt te er rn na at ti iv ve e f fi il lt te er r m me ed di ia a c co on nf fi ig gu ur ra at ti io on n o on n r re em mo ov va al l o of f e em me er rg gi in ng g w wa at te er rb bo or rn ne e p pa at th ho og ge en ns s a av ve er ra ag ge e p pa at th ho og ge en n c co on nc ce en nt tr ra at ti io on ns s i in n p pa at th ho og ge en n-s se ee ed de ed d l la ak ke e m me en nd do ot ta a w wa at te er r methods specific to evaluating alternative filter loading rates. on two separate occasions, the pilot plant was used to evaluate the effect of alternative loading rates on the performance of ripened granular media filters ( figure ). the loading rates studied were , , , and gpm/sq ft ( . , . , . , and . mm/s). all eight filters were dual-media with in. ( cm) of crushed anthracite over in. ( cm) of sand; see table for effective size and uniformity coefficient. duplicate treatment trains were operated during each test to assess experimental repeatability. once filter effluent turbidities indicated that ripening was complete, samples were collected for pathogen analysis. these samples were collected at all of the locations designated by yellow circles on figure . to demonstrate the stability of the pathogen feed, additional samples were collected from the location between the two rapidmix tanks on each train. these samples were collected at h intervals throughout the experiment. methods specific to evaluating alternative filter media. pilot-scale tests were also performed to evaluate alternative filter media configurations. as shown in figure , these tests compared the performance of monomedium filters, dual-media filters, and trimedia filters. the monomedium filter contained in. ( cm table lists the effective size and uniformity coefficients for all of the filter media. all filters were operated at a loading rate of gpm/sq ft ( . mm/s). once filter effluent turbidities indicated that ripening was complete, samples were collected for pathogen analysis. these samples were collected at all of the locations designated by yellow circles on figure . to demonstrate the stability of the pathogen feed, additional samples were collected from the location between the two rapidmix tanks on each train. these samples were collected at h intervals throughout the experiment. methods specific to evaluating coagulation ph. this evaluation was combined with the evaluation of alternative filter media (figure ). for this experiment, alum coagulation was performed at ambient ph in one treatment train and at a lower ph in the other treatment train. the lower ph was achieved with the addition of sulfuric acid. all other procedures for this test were as previously described. control reactions. for each trial, a separate batch reactor containing the microbial cocktail diluted into untreated source water was maintained to monitor any changes in the stability of test microorganisms over the course of each experiment. for each set of samples collected (pretreatment, postsedimentation, and postfiltration), a sample was collected from the control reactor to monitor changes in microbial densities associated with growth or decay over the course of the experiment. turbidity. turbidity was measured with a turbidimeter in accordance with method b (standard methods, ) . the turbidimeter was calibrated several times throughout the project with primary standard formazin sus- bacteriophage ms cryptosporidium parvum oocysts encephalitozoon intestinalis spores escherichia coli o :h e ef ff fe ec ct t o of f f fi il lt te er r r ru un n t ti im me e o on n p pa at th ho og ge en n r re em mo ov va al l pensions (hydrazine sulfate and hexamethylene tetramine) and before every experiment with secondary standards. ph. sample ph was measured with a glass-bulb, liquidjunction electrode with manual temperature compensation. the ph meter was calibrated before every experiment with ph , , and buffers. absorbance. absorbance of ultraviolet light was measured at nm (uv ) with a spectrophotometer in accordance with method b (standard methods, ) . enumeration of microorganisms. this section provides a general overview of the methods used for microbial enumeration; methods used for each microbe are detailed elsewhere (harrington et al, ) . relative removals of c. parvum oocysts and e. intestinalis spores were determined by immunolabeling samples with specific antibodies conjugated to fluorescent antibodies and subjecting samples to enumerative analysis by flow cytometry. samples were supplemented with precise numbers of an internal standard (fluorescent beads), and the absolute numbers of oocysts or spores were then corrected for original sample volume and expressed per millilitre. in cases in which high turbidity or interfering materials rendered flow cytometry analysis ineffective or inadequately sensitive, oocysts and spores were enumerated manually by examining immunolabeled samples under epifluorescence conditions in a fluorescence microscope. reductions of oocysts and spores were then computed as the log-transformed ratio of pretreatment and posttreatment microbial densities. bacterial concentrations were quantified using a spreadplate technique with selective agar specific for each test microorganism. serially diluted samples in volumes of µl were inoculated onto selective agar and were distributed uniformly over the agar surface with sterile stainless-steel rods. for e. coli o :h and a. hydrophila, plastic petri dishes were prepared with ml of mac-conkey sorbitol and ampicillin dextrin agar, respectively. samples were incubated for to h at o c and enumerative results were expressed as colony-forming units per millilitre. to increase the precision of the results and to determine analytical error, plates were prepared in triplicate for each dilution. bacteriophage ms concentrations were determined according to infectivity assay using the double-agar-layer method in mm ( . in.) plastic petri dishes. samples ( µl) of each sample dilution were added to ml molten top agar, supplemented with µl log-phase e. coli f-amp, and plated over nutrient bottom agar containing streptomycin and ampicillin. samples were incubated at o c for to h, with sample dilutions plated in triplicate to characterize analytical error. virus concentrations were expressed as mean plaque-forming units per millilitre. effect of filter run time on pathogen removal. the alum dose for the experiment described here was set to mg/l; raw water temperatures during the experiment © american water works association e ef ff fe ec ct t o of f f fi il lt te er r l lo oa ad di in ng g r ra at te e o on n p pa at th ho og ge en n r re em mo ov va al l were . ± . o c. raw water ph averaged . ± . during the experiment, and alum addition lowered the ph to an average of . ± . in settled waters. treatment reduced uv by an average of %, from . ± . cm - to . ± . cm - . these standard deviations showed that both ph and uv were stable throughout the experiment, suggesting that raw water quality and alum dosage varied to an insignificant degree during the experiment. the variation in ph and uv between the two filters was < . ph units and . cm - , respectively. therefore, the chemical quality of the water through filtration was reproducible. filter effluent turbidities for the entire experiment are shown in figure , which indicates that turbidity breakthrough was evident after approximately h of filter run time. although breakthrough appears to have begun at around h of run time, the turbidity values at h of run time were not significantly different from values during the mature phase. from h of filter run time to h of filter run time, filter effluent turbidity averaged . ± . ntu. after this period, filter effluent turbidities increased steadily from an average of . ntu at h to an average of . ntu at h of filter run time. figure also shows that the two filters produced similar turbidity results, with filter producing an average turbidity that was . ntu lower than the average turbidity produced by filter . a statistical analysis of the data from the two filters showed that the experimental uncertainty was . ntu (see harrington et al, , for details of uncertainty calculations). therefore, turbidity differ-ences between the two filters were within experimental error and not significant. pathogen samples were collected from both filters at all four of the run times shown in figure . figure shows average pathogen removals for all four of these sampling times. the removals shown in figure reflect the performance of the entire treatment train from seeded raw water to filtered water. the experimental uncertainty for pathogen removal was ± . log for c. parvum oocysts, adenoviruses, and polioviruses. this uncertainty was ± . log for the remaining microorganisms. with the exception of bacteriophage ms , differences in removal between the min and h sampling times did not exceed the experimental uncertainty for any of the microorganisms. therefore, pathogen removal during the ripening phase was similar to pathogen removal in the mature filters. between and h, removals of e. intestinalis spores and c. parvum oocysts remained stable, and this stability was consistent with that observed for filter effluent turbidity. however, removals of a. hydrophila and bacteriophage ms declined significantly between these two sample times, suggesting that breakthrough of these microbes occurred earlier than that of turbidity. beyond h, significant breakthrough was observed for all microorganisms and was consistent with the extensive breakthrough of turbidity. results showed that e. coli o :h was poorly removed throughout the run, implying that breakthrough of this pathogen took place prior to the min sampling time. dual media trimedia figure e ef ff fe ec ct t o of f a al lt te er rn na at ti iv ve e f fi il lt te er r m me ed di ia a o on n p pa at th ho og ge en n r re em mo ov va al l a at t p ph h . . a second experiment with all of the test pathogens also showed early breakthrough of a. hydrophila and e. coli o :h . two additional tests run with only a. hydrophila and e. coli o :h showed that the latter microorganism achieved breakthrough in less than h of filter run time (results for one of these tests are shown in figure ). the reasons for the rapid breakthrough of a. hydrophila and e. coli o :h are unclear, but it is possible that this was associated with the size of the bacteria. filtration theory suggests that -µm particles are the most difficult ones to remove through clean bed, rapid-rate granular media filters (yao et al, ). this size is comparable to the two bacteria that were studied. however, a. hydrophila was more readily removed than any of the test microbes during the initial h of the filter run. this size also corresponds to that of e. intestinalis spores, which were more poorly removed than most of the other microbes but did not break through as quickly as the two bacteria. in addition, it is not known whether the bacteria were associated with floc particles when entering the filter. although many studies have focused on the effects of particle size on particle removal in clean bed filters, little research has been conducted to determine the breakthrough rates of different-sized particles. previous research has concluded that particles in the size range of to µm were associated with early breakthrough, but that research did not evaluate particles smaller than µm in size (moran et al, ) . it is also possible that surface charge played a role, because measurements showed that a. hydrophila was less negatively charged than either e. coli o :h or e. intestinalis (harrington et al, ) . however, the surface charge of particles entering the filter was not measured. therefore, additional research is needed to evaluate the reasons for the rapid breakthrough of a. hydrophila and e. coli o :h . effect of filter-loading rate on pathogen removal. figure shows the setup for this experiment. as noted previously, pathogen samples were collected after h of filter run time, when the ripening phase was complete. the alum dose was set to mg/l, and raw water temperature during the experiment was . o c. raw water ph averaged . ± . during the experiment, and alum addition lowered the ph to an average of . ± . in the settled water. treatment reduced uv by % from . to . cm - . the variation in ph and uv between the two treatment trains was < . ph units and . cm - , respectively. throughout the experiment, raw and settled water turbidities averaged . ± . and . ± . ntu, respectively. figures and show the filter effluent turbidities and the log removals achieved for the various microorganisms, respectively. these figures indicate that filtration rate had no observable effect on effluent turbidity or pathogen removal across the range from to gpm/sq ft ( . to . mm/s). although loading rate did not appear to affect effluent quality, higher loading rates were associated with greater head loss. the results were similar to those observed for cryptosporidium oocysts and giardia cysts in pilot studies at contra costa, calif., and seattle, wash. (patania et al, ) . however, the results observed in this study apply to filters that recently completed the ripening phase. further studies are needed to determine the effects of loading rate on time to pathogen breakthrough. effect of alternative filter media on pathogen removal. the experiment described in this section compared monomedium sand filtration, dual-media anthracite/sand filtration, and trimedia anthracite/sand/garnet filtration (see figure ). raw water temperature and ph were ± o c and . ± . , respectively. the alum dose for this experiment was set to mg/l. in one treatment train, alum was fed with sulfuric acid to produce ph . ± . in the settled water. in the train having no sulfuric acid addition, alum addition lowered the ph to an average of . ± . in the settled water. the treatment train achieved % removal of uv -absorbing substances, lowering uv from . to . cm - . all filters were loaded at gpm/sq ft ( . mm/s). raw water turbidity averaged . ± . ntu through the pilot run. there was no statistically significant difference among the filters for turbidity control. at the time of pathogen sample collection on the ph . train, filter effluent turbidities varied from . ntu for the trimedia filter to . ntu for the dual-media filter. turbidities varied from . ntu for the dual-media filter to . ntu for both the sand and trimedia filters on the ph . train. figures and show the pathogen removals achieved at ph . and ph . , respectively. with the exception of bacteriophage ms , there was no statistically significant difference among the three filters for microbe removal on the conventional coagulation train. the sand filter was observed to achieve the highest removal of bacteriophage ms . similarly, there was no difference among the three filters on the enhanced coagulation train except for removal of c. parvum oocysts. for both exceptions, the sand filter was observed to achieve the highest performance. effect of coagulation ph on pathogen removal. this experiment was conducted as part of the evaluation of trimedia, dual-media, and sand filtration. experimental conditions were described in the previous section. to summarize, an alum dose of mg/l was used for this study, and coagulation was carried out at ph . in one treatment train and at ph . in the other treatment train. figure shows the flow schematic for this experiment. a small but statistically significant difference was observed between the two ph values for turbidity control. the filters at ph . achieved effluent turbidities of . to . ntu, whereas the filters at ph . had slightly higher effluent turbidities ranging from . to . ntu. pathogen removals through sedimentation and dualmedia filtration are shown in figure . as shown in this figure, higher removals of e. coli o :h and bacteriophage ms were achieved with a coagulation ph of . . in both cases, the difference exceeded . log and was statistically significant. there was no statistically significant effect of coagulation ph on the other microorganisms. the same conclusions were reached for removals through sedimentation and trimedia filtration. with monomedium sand filtration, the ph . treatment train achieved a significantly greater removal of c. parvum oocysts than did the ph . treatment train ( figure ). in this case, oocyst removals through sedimentation and sand filtration were . log for ph . and . log for ph . . there was no statistically significant effect of coagulation ph on the removal of the other microorganisms. turbidity breakthrough at the end of a filter run was accompanied by the breakthrough of all pathogens tested in this study. however, the results suggest a more rapid breakthrough of a. hydrophila and e. coli o :h . although the reasons for this are unclear, this finding signals a public health concern because protection of water consumers from these pathogens may depend solely on disinfection. even if these pathogens are not resistant to disinfection, there may be little protection from these microorganisms in the event of a disinfectant feed system failure. filtration rate and alternative filter media configurations had no apparent effect on pathogen removal for the pilot-scale experiments. it is important to stress that pathogen samples were collected from ripened dual-media filters. there is a strong likelihood that the onset of pathogen breakthrough would occur at different times for each filtration rate. additional studies are needed to quantify the effects of filtration rate and filter type on pathogen breakthrough. in a limited number of cases, a pretreatment and filtration ph of . achieved better pathogen removal than a pretreatment and filtration ph of . . this enhanced removal was observed for c. parvum oocysts through sand filtration and for e. coli o :h and bacteriophage ms through both dual-media and trimedia filtration. there was no observable effect of ph on any of the other pathogens tested. garnet sand, silica sand, and crushed anthracite for trimedia filtration studies. installation and startup of the pilotplant facilities were achieved with the substantial involvement of jim buchholtz, chris bone, and andrew harris. andrew harris and kristine hahn provided assistance with the pilot-scale experiments. david a. battigelli provided laboratory support and consultation throughout the project; brad argue, rebecca hoffman, elizabeth weisshaar, and david polchert provided additional laboratory support. secretarial support was provided by nanette kelsey in the university of wisconsin's department of civil and environmental engineering. the authors also thank kathryn martin, awwa research foundation project manager, and the project advisory committee-gene koontz, rick sakaji, and stanley states-for their support and suggestions throughout the project. gregory w. harrington suggest a more rapid breakthrough of a. hydrophila and e. coli o :h , a finding that signals a public health concern because protection of water consumers from these pathogens may depend solely on disinfection isolation of microsporidia and other pathogens from concentrated ditch water preventing foodborne illness: escherichia coli o :h waterborne-disease outbreaks outbreaks of diarrheal illness associated with cyanobacteria (blue-green algae)-like bodies-chicago and nepal characterization of indicator bacteria in municipal raw water, drinking water, and new main water samples diagnostic medical parasitology institute of medicine, . emerging infections: microbial threats to health in the united states frequency of a norwalk-like pattern of illness in outbreaks of acute gastroenteritis epidemiology of norwalk gastroenteritis and the role of norwalk virus in outbreaks of acute nonbacterial gastroenteritis waterborne transmission of infectious agents. manual of environmental microbiology particle behavior in deep-bed filtration: part -ripening and breakthrough i think we have a problem in victoria": mds respond quickly to toxoplasmosis outbreak in bc optimization of filtration for cyst removal cfr parts , , and ; national primary drinking water regulations. long-term enhanced surface water treatment; final rule stage microbial and disinfection by-products federal advisory committee agreement in principle cfr parts , , and ; national primary drinking water regulations. interim enhanced surface water treatment; final rule cfr parts and ; national primary drinking water regulations. enhanced surface water treatment requirements; proposed rule cfr parts and ; national primary drinking water regulations. filtration, disinfection, turbidity, giardia lamblia, viruses, legionella, and heterotrophic bacteria; final rule water and waste water filtration: concepts and applications university of wisconsin grants were used to purchase pilot-plant equipment for the pilot-scale phase of the project. the f.b. leopold company (zelionople, pa.) provided crushed anthracite filter medium; best sand (chardon, ohio) and the northern gravel company (muscatine, iowa) donated filter sand and support gravel, respectively. the oak creek (wis.) water and sewer utility contributed key: cord- -r a vxl authors: liu, haizhou; yu, xuejun title: hexavalent chromium in drinking water: chemistry, challenges and future outlook on sn(ii)- and photocatalyst-based treatment date: - - journal: front environ sci eng doi: . /s - - - sha: doc_id: cord_uid: r a vxl chromium (cr) typically exists in either trivalent and hexavalent oxidation states in drinking water, i.e., cr(iii) and cr(vi), with cr(vi) of particular concern in recent years due to its high toxicity and new regulatory standards. this account presented a critical analysis of the sources and occurrence of cr(vi) in drinking water in the united states, analyzed the equilibrium chemistry of cr(vi) species, summarized important redox reaction relevant to the fate of cr(vi) in drinking water, and critically reviewed emerging cr(vi) treatment technologies. there is a wide occurrence of cr(vi) in us source drinking water, with a strong dependence on groundwater sources, mainly due to naturally weathering of chromium-containing aquifers. challenges regarding traditional cr(vi) treatment include chemical cost, generation of secondary waste and inadvertent re-generation of cr(vi) after treatment. to overcome these challenges, reductive cr(vi) treatment technologies based on the application of stannous tin or electron-releasing titanium dioxide photocatalyst hold extreme promise in the future. to moving forward in the right direction, three key questions need further exploration for the technology implementation, including effective management of residual waste, minimizing the risks of cr(vi) re-occurrence downstream of drinking water treatment plant, and promote the socioeconomic drivers for cr(vi) control in the future. • wide occurrence of cr(vi) in us source drinking water. • a strong dependence of occurrence on groundwater sources. • elucidate redox and equilibrium chemistry of cr (vi). • sn(ii)-based and tio -based reductive treatments hold extreme promise. • key challenges include residual waste, cr(vi) regeneration and socioeconomic drivers. the future. exposure to cr(vi) through respiratory, oral and dermal pathways induces cancers and other terminal illnesses. chromium mainly existing as hexavalent cr (vi) and trivalent cr(iii) are the two stable forms in the aquatic environment. at circumneutral phs, cr(vi) exists as an oxyanion, i.e., chromate cro -, whereas cr(iii) exists as different low-soluble solids depending on the coexisting constituents such as hydroxide, iron and copper. cr(iii) is considered non-toxic, a micro-nutrient in mammalian diets and aids in the metabolism of glucose and lipids. both anthropogenic activity and natural occurrence lead to cr(vi) in the aquatic environment, and natural occurrence plays a more substantial contribution. the occurrence of cr(vi) in the aquatic environment was historically driven by anthropogenic release from chemical and manufacturing industries, including paints, pigments, paper, electroplating and leather tanning (jacobs and testa, ) . one notable example is the disastrous cr(vi) release from an industrial site in hinkley, calif. between s and s , which garnered an international attention four decades later through the blockbuster oscar-winning movie erin broachvich (izbicki and groover, ) . however, as the manufacturing industry sets more stringent standards on wastewater treatment in recent decades, the contribution of industrial wastewater discharge to cr(vi) occurrence becomes less of a routine concern. in contrast, natural release of cr(vi) driven by geochemical sources makes a more substantial contribution to the wide occurrence of cr(vi) in drinking water (coyte et al., ; oze et al., ; vengosh et al., ) . one major source of natural-occurring cr(vi) is from groundwater and aquifer materials. chromium is widely embedded in aquifer minerals as the st most abundant element in the earth's crust (oze et al., ) . the naturally geological weathering of aquifer materials releases cr(vi) via a slow oxidation of cr(iii)-containing iron minerals fe x cr ( -x) (oh) (s) in the aquifer by co-existing manganese (iv) minerals (eary and rai, ; pan et al., ) : where x is the molar fraction of chromium in the mixed phase of iron-chromium hydroxide solid, and its value varies between and . an analysis of a recent us epa database on chromium -unregulated contaminant monitoring rule round (ucmr ) showed that hexavalent chromium is widely present in drinking water sources across the us . drinking water systems using groundwater as the main source water report much higher percentages of cr(vi) occurrence than those using surface water as the main source, especially for cases with elevated levels of cr (vi) higher than µg/l (fig. a) . this dependence clearly indicates the strong influence of naturally geological weathering process in the natural occurrence of cr(vi) in source waters, which preferentially impacts groundwater sources. furthermore, chromium accumulated in urban drinking water infrastructure, namely the drinking water distribution systems risks to become another geochemical source of hexavalent chromium. recent surveys have discovered that chromium accumulates in the corrosion scales of drinking water distribution systems. the level of chromium observed in the corrosion scales has exceeded the level observed in earth crust (peng et al., ) . under oxidative conditions in drinking water and in scenarios with abrupt changes of source water and associated water chemistry, the accumulated chromium can release back into drinking water and increased cr(vi) level at the tap. for example, an analysis of the us epa ucmr database showed that the hexavalent chromium level at the exit point of the drinking water distribution systems was statistically higher than the level at the entrance to the distribution system . a synthesis of the database also showed that a considerable percentage of public water utilities in the us detects cr(vi) at the exist point to the drinking water distribution systems, range from sub-µg/l to higher than µg/l (fig. b) . us epa currently regulates total chromium, i.e., a combination of cr(iii) and cr(vi), at µg/l in treated drinking water. a new standard specifically for cr(vi) was established at µg/l in california in but was subsequently withdrawn (us epa, ) . considering the public health risks of cr(vi) and a tightening regulatory perspective in the future, it is urgent and important to understand chromium chemistry that drives cr(vi) occurrence in drinking water, and develop efficient treatment technologies to minimize risks. because cr(vi) is redox reactive, its redox chemistry drives the fate of cr(vi) in drinking water, and dictates the development of effective treatment processes. consequently, it is imperative to examine the redox chemistry of chromium in typical drinking water conditions. from a chemical speciation perspective, cr(vi) can undergo protonation and bimolecular combination via chronic acid h cro , and exists in different soluble species depending on the solution ph (fig. ) . within the typical ph range of drinking water, i.e., neutral to slightly basic (ph of - ), cr(vi) predominately exists as the deprotonated oxyanion chromate cro -, the protonated hcro only accounts for a small fraction, and other soluble species including cr o are negligible. in contrast, trivalent cr(iii) exists as different solid phases with a low low solubility in typical drinking water chemical conditions. for example, various cr(iii) minerals exist in corrosion products in drinking water distribution systems liu, , ; choi et al., ) . the redox potential of these cr(iii) solids coupled with cro ranged between - . and . v in typical drinking water chemical condition (fig. ) . meanwhile, common disinfectants used in drinking water, e.g., free chlorine (hocl), possess a higher redox potential (e = . v) than any of the cro -/cr(iii) solid couple; therefore, chlorine can oxidize cr(iii) and affect the mobility of cr in drinking water liu, , ) . taking cr(oh) (s) as an example: the reactions of cr(iii) solids oxidation by free chlorine residuals can take place in drinking water distribution systems, as supported by statistical analysis of epa ucmr database. the accumulation of even non-toxic cr(iii) in water distribution systems may be inadvertently converted to cr(vi) by residual chlorine in the distribution systems. this transformation could seriously jeopardize the benefits of upstream treatment. meanwhile, several reductants exists a redox potential lowered than that of cr(vi)/cr(iii) couple and can serve as a reductive reagent to convert toxic cr(vi) to benign cr (iii) end products. for example, ferrous ion with the fe (oh) (s) /fe + redox couple has a redox potential of . v, and divalent tin sn(ii) with the sno (s) /sn + redox couple also has a very low redox potential of - . v in typical drinking water conditions (fig. ) . these reductants provides possibilities for reductive treatment of cr(vi) in drinking water. cr(vi) drinking water treatment technologies . traditional drinking water treatment technologies traditional cr(vi) treatment technologies that have been applied at full scale drinking water treatment plants include ion exchange and ferrous iron fe(ii)-based reduction coagulation (seidel et al., ) . the treatment principle of ion exchange is based on the physical separation of cr(vi) anions using quaternary amino functionalized ion exchange resins, and has been employed in small drinking water treatment systemsmostly groundwater based systems. the handling and disposal of concentrated cr (vi)-containing ion exchange brine is very challenging and costly, which hinders the wide application of ion exchange to large-scale drinking water treatment. in addition, the application of effective ion exchange treatment needs to avoid the competitive exchange in co-existing anion in the source drinking water sources, including sulfate, nitrate, arsenic and radioactive uranium, which can potentially decrease the cr(vi) treatment efficiency or increases the operational complexity (plummer et al., ) . fe(ii)-based reductive cr(vi) removal is another existing technology that has been applied at pilot-scale for cr (vi) drinking water treatment in california (blute et al., ) . this process applies ferrous chloride as a reductant to convert cr(vi) to non-toxic cr(iii) particles, followed by a coagulation step to promote the formation of cr(iii) and fe(iii) particles, and a final filtration step to remove the particles, therefore achieving a total chromium removal. this technology is proven viable at full-scale; however, it also brings operational challenges to deal with, including a long treatment time, high chemical dosage requirement and large secondary solid waste. for example, to ensure a complete conversion of cr(vi) to cr(iii) particles via fe(ii) reduction, this process requires a large over-stoichiometric molar ratio of fe(ii)-to-cr(vi) (between and ) to achieve a desirable reaction kinetics in a continuous treatment mode. the long reaction time for reductive reaction requires min or more. in addition, the over-stoichiometric dosage of fe(ii) requires a subsequent re-oxidation stepachieved by either aeration or chlorinationto convert residual fe(ii) to fe (iii) particles prior to filtration step. this results in the formation of a large quantity of iron sludge waste that is operationally complicated to handle (seidel et al., ) . redox-driven cr(vi) treatment technologies are more promising and cost-effective than physical separation technologies (for example, ion exchange), because cr (vi) is chemically reduced to non-toxic cr(iii) solids in the presence of a reductant and subsequently removed as cr (iii) particles, eliminating the need for additional brine disposal (eary and rai, ) . the challenges to overcome for a full-scale application in drinking water treatment include operational complexity, high chemical dosage requirement and secondary waste generation. to overcome the disadvantages of over-stoichiometric addition of chemicals and secondary waste generation, a new chemical reductive treatment has recently been developed based on divalent tin sn(ii) addition as the chemical of stannous chloride sncl . the following redox reaction takes place (henrie et al., ; kennedy et al., ) : upon the completion of reaction, divalent tin is oxidized to tetravalent tin solid sno (s) and removed together with cr(iii) solids by a filtration unit. compared to fe(ii), using sn(ii) as the reductant is advantageous in multi-fold. first, the reaction kinetics between sn(ii) and cr(vi) is extremely fast, i.e., reaction in typical drinking water condition typically completes within seconds, whereas the reaction kinetics between fe(ii) and cr(vi) is much slower and takes more than min to completion. this inherent advantage of fast reaction kinetics enhanced the easiness of continuous sn(ii)-based treatment operation. second, stannous addition requires a much lower stoichiometric molar ratio of sn(ii)-to-cr(vi) compared to fe(ii)-to-cr (vi) in ferrous-based approach ( - vs. - ). therefore, the formation of solid residual waste is minimized and the secondary waste generation is not a concern when sn(ii) is employed as the reductant. due to the small amount of solid formation as the end product, additional coagulation and flocculation are not required. instead, a simple treatment step utilizing cartridge filtration or porous- medium sand filtration typically follows the addition of sn (ii) to remove residual particles (fig. a) . for example, a pilot-testing conducted in coachella valley, calif. showed that the cost of sn(ii) reductive treatment would substantially decrease capital and operation/maintenance costs, as well as minimizing waste residuals, compared to fe(ii) reductive treatment. third, residual tin in the treated drinking water acts as a corrosion inhibitor and benefits drinking water distribution systems. a recent study has demonstrated that residual tin is sequestered in the corrosion scales of distribution systems. cr(iii) solidsthe end products of sn(ii) reductive treatmentalso exhibited a low risk in re-oxidation to cr(vi) when the treated drinking water enters a distribution system in the presence of chlorine as the residual disinfectant. another promising reductive treatment technology harnesses electrons as a strong reductant for cr(vi) removal via photocatalysis. differing from other chemical reductive treatment technologies, photocatalytic reduction does not require consumable chemicals, and converts photon energy to reactive electrons via catalysts to drive the reduction of cr(vi) to cr(iii). titanium dioxide (tio ) photocatalysts are popular materials because of its high stability, low toxicity, low cost, large surface areas and good optical properties (loeb et al., ) . upon uv irradiation by photons with energy equal or greater than its bandgap, tio undergoes a hole-electron separation process, generated holes on the valence band (vb) and electrons on the conduction band (cb), and induces reductive conversion of cr(vi) when electrons migrate to the surface of tio catalyst (fig. a) . thermodynamically, cb-associated electrons (e = - . v) can be utilized to reduce cr(vi) to cr(iii). however, with conventional tio (e.g., commercial p tio ), the holes on the valence band quickly oxidize surface adsorbed h o to hydroxyl radicals (ho ) and creates a highly oxidative environmental that is detrimental to reductive cr(vi) treatment. in addition, the migration of the electrons takes random pathway, which make it a high probability for electrons to recombine with holes, and interfacial electron transfer is much slower than holeelectron recombination rate and interfacial hole transfer. these facts make electrons from conventional tio materials unavailable for cr(vi) reduction. to fully utilize photogenerated electrons and make tio reductively reactive, polyol molecules as hole scavengers are applied to covalently bond onto the tio surface via bridged o atoms is a promising strategy (fig. b ). this surface modification can be realized simply via thermal hydrolysis, which induces the occupied energy state inside the tio band gap, and significantly improve the hole trapping efficiency (chen et al., ) . polyols reacts with holes, prevents ho generation and effectively releases electrons for cr(vi) reduction. a recent study has shown that using diethylene glycol (deg) as the polyol molecule, the surface modified tio photocatalyst exhibited a much faster kinetics on reductive cr(vi) conversion in comparison to the non-reactive conventional tio material (chen et al., ) . the tio -based photocatalytic cr(vi) treatment process is also cost-effective and safe. conventional tio photocatalysts such as degussa p @ is widely available, and food-grade tio has been frequently used as a pigment for food production. these low-cost traditional tio materials can undergo a simple surface fabrication with polyols and convert into reductive tio tailored for cr(vi) treatment. in addition, the modified tio can be recycled for multiple rounds of treatment without losing its reductive capacity. pilot-scale photocatalytic water treatment reactors are already available in the market (e.g., photocat system @ ) and a full-scale application is possible. both sn(ii)-based and photocatalytic reductive cr(vi) treatments hold extreme promise as well as challenges in the future. future research focused on solving these challenges is necessary. to moving forward in the right direction to minimize the occurrence of chromium in drinking water, the following three important questions needs to be considered: ) what are the challenges on residual waste management during treatment? ) are there hidden risks of cr(vi) re-occurrence downstream of drinking water treatment plant? ) what are the socioeconomic drivers for cr(vi) control in the future? . what are the challenges on secondary waste management during treatment? all reductive cr(vi) treatment inevitably generates cr(iii) solids as the end product of chromium. because cr(iii) is also redox reactive and can participate in an oxidative reaction to re-generate hexavalent chromium, the handling of cr(iii) residual is a major challenge that dictates the success of the treatment. although cr(iii) itself is nontoxic at low dose, an excessive presence of cr(iii) in the treated water can violate total chromium regulatory standard in the us at µg/l. the presence of dissolved oxygen does not pose the risk of re-oxidizing cr(iii) solids. instead, the addition of a secondary disinfectant (e.g., free chlorine) in the treated water can induce the oxidation. therefore, the removal of cr(iii) solids as a subsequent treatment step should be integrated into the cr(vi) treatment train. during the sn(ii)-based treatment, cr(iii) solids are precipitated out together with sno (s) and generate a mixed phase solid product. a cartridge filtration unit with pore sizes of . µm has been demonstrated to successfully remove cr(iii) residual solids. in the final treated drinking water coming out the filtration unit, the addition of free chlorine with a contact time of days did not generate cr(vi), indicating the combination of sn(ii) reductive treatment and subsequent microfiltration or porous media filtration is effective in removing cr(vi) and total chromium (henrie et al., ) . regarding the photocatalytic cr(vi) treatment option, the separation of cr(iii) solids and regeneration of tio photocatalysts are critical to the success of the technology. the reductive tio photocatalysts are typically in nano-to micro-size range. as cr(iii) solids are generated, they deposited on the surface tio particles and results in a composite particle size in the range of - nm (chen et al., ) . accordingly, a membrane ultrafiltration unit can be added to remove tio and cr(iii), and produce crfree drinking water (fig. b) . the combination of a photochemical reactor and an ultrafiltration unit as a treatment train has been commercialized (e.g., photocat @ system by purifics), and the process can be readily scaled up into the design of future full-scale treatment. furthermore, the reductive tio recovered by membrane separation can undergo mild acid washing to dissolve cr(iii) solids and renew its surface (fig. b) . although the polyols coated on the catalyst surface are eventually consumed during the cr(vi) reduction, the catalysts can be re-dispersed in polyol solutions, subsequently heated to replenish the hole scavengers, and regenerated for the next cycle of treatment. in addition to the implementation of treatment technologies, any risks involving a potential release of cr(vi) downstream of the treatment needs to be considered and minimized. the average residence time of water staying in the downstream distribution systems can be up to several days compared to hours in the upstream treatment facility, thus providing ample time for reactions. as an in situ source, cr can potentially be leached from plumbing materials (e.g., cast iron and stainless steel) in water distribution systems (choi et al., ; gonzalez, et al., ; percival, et al., ) . in addition, pipe corrosion scales can accumulated chromium from its trace level in drinking water over decades (hering and harmon, ; peng et al., ) , thus creating in situ sources of chromium in drinking water distribution systems. theoretical redox potential calculation suggests the oxidation of common cr(iii) solids, including chromium oxides, copper chromite and iron chromite by free chlorine are all thermodynamically favorable (fig. ). recent kinetics model also predicts that cr(iii) oxidation by free chlorine can result in a cr(vi) concentration at the tap (i.e., the exit of the distribution system) exceeding µg/l, a possible benchmark for future cr(vi) regulatory standard (henrie et al., ) . in addition, the kinetics of cr(iii) oxidation process driven by free chlorine is catalyzed by bromide, a conservative anion typically existing in source water originated from seawater desalination, water reuse or impacted by wastewater discharge from hydraulic fracking. water utilities need to incorporate source water protection and distribution system corrosion control as part of the mitigation strategy for cr(vi) control in drinking water. . what are the socioeconomic drivers for cr(vi) treatment in the future? the incentive for future cr(vi) treatment and control will first comes from regulatory perspective. currently only total chromium in drinking water was regulated in the us without differentiating cr(iii) and cr(vi); therefore, no mandate to specifically treat cr(vi) exists in the us in the state of california, a recent drinking water standard specifically targeting cr(vi) at µg/l was established in but was subsequently challenged in the court and invalidated in . the withdrawal of the new cr(vi) regulation was partially driven by the huge economic cost of upgrading treatment to comply with the new regulation. however, the economic cost calculation was based on traditional cr(vi) treatment options available back then, i.e., ferrous reductive treatment or ion exchange treatment. in contrast, the newly developed sn(ii)-based or photocatalytic treatment would significantly reduce the cost of water utilities and become economically feasible. combined with the convincing evidence of the toxicological effects of cr(vi), it is reasonable to expect that a new cr (vi) regulatory standard specifically on cr(vi) will be reintroduced in the future. furthermore, the importance of redox processes taking place in drinking water distribution systems should be considered when establishing new cr(vi) standards in drinking water. in the future, climate change adaptation strategies (including water reuse, desalination and water conservation) and associated changes on water chemistry including bromide enhancement will pose challenges for water distribution systems, alter redox chemistry at the pipe-water interface, affect the corrosion behaviors of piping materials, and change the stability of cr-containing corrosion scales. to address this cascade of changes on water distribution infrastructure, future regulatory standards on cr(vi) should incorporate drinking water distribution systems into considerations. for example, in an approach similar to the us epa lead and copper rule, samplings of cr(vi) at the tap should be considered and distribution system corrosion control strategiesespecially for water systems with cast iron pipes and using groundwater as the source watershould be part of the mitigation. more broadly speaking, more cities in the future will deal with aging water infrastructure. although distribution systems might be functional when operating as they have been for decades, the risks are going to come when source waters are abruptly switched in response to droughts or a decision to use a new water supply. these universal challenges needs to be addressed and to prevent another flint crisis with hexavalent chromium from distribution systems. . what is the future outlook of cr(vi) drinking water treatment technologies? sn(ii)-based reductive technology can offer immediate benefit to water systems that are experiencing elevated cr (vi) levels in their source water, and an implementation of this technology will be a cost-effective and easy retrofitting to the existing water treatment infrastructure. although questions regarding the stability of sn and cr residual products need to be better understood through more careful laboratory-scale research and pilot-scale testing, this technology is expected to be a widely considered candidate in the future when new cr(vi) drinking water regulatory standard is established, and used as a benchmark to compare against other new technologies. meanwhile, recent advances in nanotechnology lead to the development of new photocatalysts with tailored surface reactivity for cr(vi) treatment. new photocatalysts that are based on semiconductor materials exhibited a high reduction efficiency for cr(vi). there are ample promising opportunities to further optimize these photocatalysts in areas including enhanced stability, materials longevity, and ease to separate and recover catalysts. in addition, advancement in cr(vi) removal technologies tested at laboratory-scale or tailored for industrial wastewater treatment and environmental remediation, including electrochemistry, nanomaterials and photochemistry can guide the future design for drinking water treatment (chen and liu, ; pan et al., ; pan et al., ) . for example, the introduction of magnetic property and doping of cocatalyst can offer enhanced catalytic capacity and easy reuse after treatment. however, challenges need to be overcome prior to their wide application associated with cost, complex fabrication process and release of nanomaterials to the environment. despite the unresolved issues, these nanomaterial based cr(vi) treatment technologies are promising next-generation technologies for cr(vi) removal in drinking water treatment. water chemistry hexavalent chromium treatment implementation in glendale, calif frontier review: occurrence and speciation of chromium in drinking water distribution systems kinetics and mechanisms of cr(vi) formation via the oxidation of cr(iii) solid phases by chlorine in drinking water oxidation of cr(iii)-fe(iii) mixed-phase hydroxides by chlorine: implications on the control of hexavalent chromium in drinking water photocatalytic removal of hexavalent chromium by newly designed and highly reductive tio nanocrystals photochemical removal of hexavalent chromium and nitrate from ion-exchange brine waste using carbon-centered radicals corrosion behavior of low alloy steels containing cr, co and w in synthetic potable water occurrence and distribution of hexavalent chromium in groundwater from north carolina kinetics of chromium(iii) oxidation to chromium(vi) by reaction with manganese dioxide chromate removal from aqueous wastes by reduction with ferrous iron presence of metals in drinking water distribution networks due to pipe material leaching: a review full-scale demonstration testing of hexavalent chromium reduction via stannous chloride application geochemical controls on chromium occurrence, speciation, and treatability natural and man-made hexavalent chromium, cr(vi), in groundwater near a mapped plume, hinkley, california-study progress as of may , and a summative-scale approach to estimate background cr(vi) concentrations stannous chloride reduction-filtration for hexavalent and total chromium removal from groundwater the technology horizon for photocatalytic water treatment: sunrise or sunset? genesis of hexavalent chromium from natural sources in soil and groundwater chromium geochemistry in serpentinized ultramafic rocks and serpentine soils from the franciscan complex of california rates of cr(vi) generation from cr x fe -x (oh) solids upon reaction with manganese oxide dynamics of chromium (vi) removal from drinking water by iron electrocoagulation cr (vi) adsorption on engineered iron oxide nanoparticles: exploring complexation processes and water chemistry occurrence of trace inorganic contaminants in drinking water distribution systems biofilms, mains water and stainless steel optimization of strong-base anion exchange o&m costs for hexavalent chromium treatment national and california treatment costs to comply with potential hexavalent chromium mcls unregulated contaminant monitoring rule (ucmr ) origin of hexavalent chromium in drinking water wells from the piedmont aquifers of north carolina she joined the department of biochemistry at uc riverside as a postdoctoral researcher in . she is interested in environmental sustainability, bioremediation and state-of-the-art detection technologies including biosensors he completed his b.s. at sichuan university, china, m.s. and ph.d. at university of washington, usa, and a postdoctoral training at uc berkeley, usa. his research group focuses on water chemistry acknowledgements this work was supported by the us national science foundation career program (cbet- ). we thank current and former members of the liu group at uc riverside who have contributed to this effort, as well as the emotional support from jacob y. liu to complete this article during the covid- pandemic. key: cord- -risqto authors: chu, ellen w.; karr, james r. title: environmental impact, concept and measurement of date: - - journal: encyclopedia of biodiversity doi: . /b - - - - . - sha: doc_id: cord_uid: risqto environments on earth are always changing, and living systems evolve within them. for most of their history, human beings did the same. but in the last two centuries, humans have become the planet's dominant species, changing and impoverishing the environment for all life on earth and even decimating humans' own cultural diversity. contemporary cultural worldviews that have severed humans' ancient connections with the natural world, along with consumption and population growth, have deepened this impoverishment. understanding, measuring, and managing human environmental impacts – the most important of which is the impoverishment of living systems – is the st century's greatest challenge. all organisms change their environment as they live, grow, and reproduce. over millennia, organisms evolve to contend with changes in their environment. those that do not adapt go extinct. those that survive are molded by evolution and biogeography as the environment changes. even unusual or seemingly catastrophic events, like tidal waves from earthquakes, are an integral part of the ecological contexts to which organisms adapt over long time spans. some organisms, like beavers and elephants, change their surroundings so dramatically that they have been called ecosystem engineers. beaver dams alter the flow of rivers, increase dissolved oxygen in downstream waters, create wetlands, and modify streamside zones. african elephants convert wooded savanna to open grassland by toppling trees as they browse. change brought about by living things, including ecosystem engineers, has been slow and incremental in evolutionary terms. ecosystem engineers evolve along with other inhabitants of their environments, developing ways to coexist; like other environmental components, ecosystem engineers, and their effects are part of an evolving ecological context. in contrast, human effects since the industrial revolutionincluding many that may be invisible to a casual observeroutpace the capacity of living systems to respond. in evolutionary terms, these effects are recent and outside the experience of most organisms. over the past two centuriesbarely more than two human lifetimes -humans have profoundly altered living and nonliving systems everywhere. for the first time in the earth's history, the environmental impact of one species, homo sapiens, is the principal agent of global change. understanding the environmental impacts of human actions is one of modern science's greatest challenges. understanding the consequences of those impacts, and managing them to protect the well-being of human society and other life on earth, is humanity's greatest challenge. the human evolutionary line began in africa about million years ago (ma). it took some or million years (my) for protohumans to spread from africa to asia and then to europe. these early humans, like other primates, made their living by seeking food and shelter from their environment, gathering plant foods, and hunting easy-to-kill prey. sometimes, they also experienced threats from their environment, including accidents, droughts, vector-borne diseases, and attacks from predators. at this stage, with relatively low population densities and limited technologies, humans were not ecosystem engineers. by some , years ago, however, humans had learned to use fire; developed complex tools, weapons, and language; and created art. on local scales, these modern humans were very much ecosystem engineers. sometimes their enhanced abilities to make a living outstripped their local environment's capacity to provide that living, and they caused local ecological disruptions. on several continents, for example, humans hunted large mammals to the point where many, such as the marsupial lion of australia, went extinct. as humans became more efficient at exploiting their local environments, they spread farther. by , years ago, modern humans had spread to all continents and many islands across the globe. then, about , years ago, people began to domesticate plants and animals. instead of searching for food, they began to produce food. food production changed the course of human and environmental history. domestication of plants and animals enabled people to adopt a sedentary lifestyle. as detailed by geographer and ecologist diamond ( diamond ( , , populations grew as agriculture developed, because larger sedentary populations both demanded and enabled more food production. local ecological disruptions became more numerous and widespread and more intense. with animal domestication, contagious diseases of pets and livestock adapted to new, human hosts. diseases spread more quickly in crowded conditions; inadequate sanitation compounded the effects. from agriculture, civilization followed and with it, cities, writing, advanced technology, and political empires. in just , years, these developments led to nearly billion people on earth, industrial societies, and a global economy founded on complicated technologies and fossil fuels. humans have emerged as ecosystem engineers on a global scale. the ecological disruptions we cause are no longer just local or regional but global, and we have become the principal threat to the environment. yet despite today's advanced technologies, humans are as dependent on their environments as other organisms are. history, not just ecology, has been very clear on this point. from the old kingdom of egypt more than years ago to the culture that created the huge stone monoliths on easter island between and ad to the s dust bowl of north america, civilizations or ways of life have prospered and failed by using and (mostly unwittingly) abusing natural resources. in old kingdom egypt, the resource was the valley of the nile, richly fertilized with sediment at each flooding of the river, laced with canals and side streams, blessed with a luxuriant delta. agriculture flourished and populations swelled, until unusually severe droughts brought on the civilization's collapse. on easter island, the resource was trees, which gave polynesians colonizing the island the means to build shelter, canoes for fishing the open waters around the island, and log rollers for moving the ceremonial stone monuments for which the island is famous. deforestation not only eliminated the humans' source of wood, but also further deprived the already poor soil of nutrients and made it impossible to sustain the agriculture that had sustained the island's civilization. on the dry great plains of north america, settlers were convinced that rain would follow the plow, and so they plowed homestead after homestead, only to watch their homesteads' soils literally blow away in the wind. in these cases and many others, human civilizations damaged their environments, and their actions also worsened the effects on their civilizations of climatic or other natural cycles. yet in each case, a human culture was operating precisely the way it had evolved to operate: the culture of old kingdom egypt enabled its people to prosper on the nile's natural bounty, but prolonged, unprecedented drought brought starvation and political disorder. easter islanders thrived and populated the island until its resources were exhausted. dust bowl farmers lived out their culture's view of dominating and exploiting the land for all it was worth. the inevitable outcome for all three cases was a catastrophe for the immediate environment and the people it supported -not only because the people were unprepared to cope with dramatic natural changes in their environments but because their own actions magnified the disastrous effects of those changes. quoting an apt bit of cynical graffiti, historical philosopher wright ( , p. ) sums up what he calls the hazards of human progress this way: ''each time history repeats itself, the price goes up.'' indeed, as the second decade of the st century begins, humans are ecosystem engineers on a planetary scale, and our global civilization threatens the life-sustaining capacity of all of earth's environmental ''spheres'': • geosphere (lithosphere): earth's crust and upper mantle, containing nonrenewable fossil fuels, minerals, and nutrients that plants require. the activities of plants, animals, and microorganisms weather mineral soils and rocks, create organic soils, and alter erosion and sedimentation rates. humans mine minerals, metals, and gems; extract fossil fuels including coal, oil, and natural gas; and increase erosion and sedimentation by removing or altering natural plant cover through agriculture, logging, and urbanization. • atmosphere: the thin envelope of gases encircling the planet. living systems modify the atmosphere, its temperature, and the amount of water it contains by continually generating oxygen and consuming carbon dioxide through photosynthesis and affecting the amount and forms of other gases. humans release toxic chemicals into the air and alter the climate by raising the atmospheric concentration of greenhouse gases, such as carbon dioxide and methane, through the burning of fossil fuels in motor vehicles, ships, airplanes, and power plants. • hydrosphere: earth's liquid surface and underground water; its polar ice caps, oceanic icebergs, and terrestrial permafrost; and its atmospheric water vapor. living systems alter the water cycle by modifying the earth's temperature and the amount of water plants send into the atmosphere through a process called evapotranspiration. humans build dams, irrigation canals, drinking-water delivery systems, and wastewater treatment plants. they use water to generate electricity; they mine groundwater from dwindling underground aquifers for farming as well as drinking; they alter the flows of surface waters for everything from transportation to the mining of gold; they drain wetlands to gain land area and abate waterborne diseases. modern human interference in global climate is likely to disrupt the entire planetary water cycle. • biosphere: the totality of earth's living systems, that part of the earth inhabited by living organisms. life on earth emerged . billion years ago and has sustained itself through changes in form, diversity, and detail since then. no planet yet discovered supports complex life as we know it on earth. as predators, humans have decimated or eliminated wild animal populations worldwide. as domesticators of animals and plants, humans have massively reshaped landscapes by cutting forests, burning and plowing grasslands, building cities, desertifying vast areas, and overharvesting fish and shellfish. human actions have precipitated a spasm of extinctions that today rivals five previous mass extinctions caused by astronomical or geological forces, each of which eliminated more than % of species then existing. humans themselves may be thought of as a sphere within the greater biosphere: the ethnosphere, or the sum total of all thoughts and intuitions, myths and beliefs, ideas and inspirations brought into being by the human imagination since the dawn of consciousness. in the words of anthropologist davis ( , p. ) , who coined and defined the term in , ''the ethnosphere is humanity's greatest legacy. it is the product of our dreams, the embodiment of our hopes, the symbol of all we are and all that we, as a wildly inquisitive and astonishingly adaptive species, have created.'' but, davis notes, just as the biosphere is being severely eroded, so too is the ethnosphere, but at a much faster pace. today, the scientific consensus is that h. sapiens -a single species -rivals astronomical and geological forces in its impact on life on earth. the first step in dealing with the present impact of human activity is to correctly identify the nature of humanity's environmental impact, concept and measurement of relationship with the environment and how human actions affect that relationship. many people still see the environment as something people must overcome, or they regard environmental needs as something that ought to be balanced against human needs (for example, jobs vs. the environment). most people still regard the environment as a provider of commodities or a receptacle for waste. when asked to name humanity's primary environmental challenges, people typically think of running out of nonrenewable raw materials and energy or about water and air pollution. environmental research and development institutions focus on ways technology can help solve each problem, such as fuel cells to provide clean, potentially renewable energy or scrubbers to curb smokestack pollution. even when people worry about biodiversity loss, they are concerned primarily with stopping the extinction of species, rather than with understanding the underlying losses leading up to species extinctions or the broader biological crisis that extinctions signal. these perspectives miss a crucial point: the reason pollution, energy use, extinction, and dozens of other human impacts are important is their larger impact on the biosphere. ecosystems, particularly their living components, have always provided the capital to fuel human economies. when populations were small, humans making a living from nature's wealth caused no more disruption than other species. but with nearly billion people occupying or using resources from every place on earth, humans are overwhelming the ability of other life-forms to make a living and depleting the planet's natural wealth. at this point in the planet's history, one species is compromising earth's ability to support the living systems that evolved here over millions of years. the systematic reduction in earth's capacity to support life -which woodwell ( ) termed 'biotic impoverishment' -is thus the most important human-caused environmental impact. at best, the ethics of this impact are questionable; at worst, it is jeopardizing our own survival. the connection between biotic impoverishment and extinction is intuitively obvious. by overharvesting fish, overcutting forests, overgrazing grasslands, or paving over land for cities, we are clearly killing other organisms outright or eliminating their habitats, thereby driving species to extinction and impoverishing the diversity of life. but biotic impoverishment takes many forms besides extinction. it encompasses three categories of human impacts on the biosphere: ( ) indirect depletion of living systems through alterations in physical and chemical systems, ( ) direct depletion of nonhuman life, and ( ) direct degradation of human life (table ) . identifying and understanding the biological significance of our actions -their effects on living systems, including our own social and economic systems -is the key to developing effective ways to manage our impacts. humans affect virtually all the physical and chemical systems life depends on: water, soils, air, and the biogeochemical cycles linking them. some human-driven physical and chemical changes have no repercussions on the biota; others do, becoming agents of biotic impoverishment. humans probably spend more energy, money, and time trying to control the movement and availability of water than to manage any other natural resource. in the process, we contaminate water, move water across and out of natural basins, deplete surface and groundwater; modify the timing and amount of flow in rivers, straighten or build dikes to constrain rivers, and alter natural flood patterns. we change the amount, timing, and chemistry of fresh water reaching coastal regions, and we dry up wetlands, lakes, and inland seas. our demands are outrunning supplies of this nonrenewable resource, and the scale of our transformations risks altering the planetary water cycle. physical alterations of the earth's waters, combined with massive industrial, agricultural, and residential pollution, have taken a heavy toll on nonhuman aquatic life. by as much as one-fifth of the world's coral reefs had been destroyed, and only % remained healthy. globally, the number of oceanic dead zones, where little or no dissolved oxygen exists, tripled during the last years of the th century. the biota of freshwater systems has fared no better. a -year survey of the freshwater fishes inhabiting malaysian rivers in the late s found only % of known malaysian species. some % of north america's freshwater fishes are at risk of extinction; two-thirds of freshwater mussels and crayfishes and one-third of amphibians that depend on aquatic habitats in the united states are rare or imperiled. humans use at least % of the earth's accessible water runoff, a figure that is likely to grow to % by . by then, more than a third of the world's population could suffer shortages of fresh water for drinking and irrigation. groundwater aquifers in many of the world's most important cropproducing regions are being drained faster than they can be replenished: a study published in found that the rate of groundwater depletion worldwide had more than doubled from to . natural flood regimes, as in the nile river basin, no longer spread nutrient-rich silt across floodplains to nourish agriculture; indeed, the high dam at aswan traps so much silt behind it that the nile delta, essential to egypt's present-day economy, is slumping into the sea. whole inland seas, such as the aral sea in uzbekistan, are drying up because the streams feeding them contain so little water. in addition to eliminating habitat for resident organisms, the sea's drying is bringing diseases to surrounding human populations. indeed, diseases caused by waterborne pathogens are making a comeback even in industrialized nations. in the past five or six decades, the number of large dams on the world's rivers grew more than seven times, to more than , today. the mammoth three gorges dam across china's yangtze river, completed in , created a -km-long serpentine lake behind it. the dam displaced more than million people and may force the relocation of another million from the reservoir region, which, at , km , is larger than switzerland. the dam has greatly altered ecosystems on the yangtze's middle reaches, compounding perils already faced by prized and endemic fish and aquatic mammals. the sheer weight of the water and silt behind the concrete dam raises the risk of landslides and strains the region's geological structure, while water released from the dam eats away at downstream banks and scours the bottom. and by slowing the flow of the yangtze and nearby tributaries, the dam drains the river's ability to flush out and detoxify pollutants from upstream industries. not just dirt, soil is a living system that makes it possible for raw elements from air, water, and bedrock to be physically and chemically assembled, disassembled, and reassembled with the aid of living macro-and microorganisms into the green cloak of life above ground. accumulated over thousands of years, soil cannot be renewed in any time frame useful to humans alive today, or even to their great-grandchildren. humans degrade soils when they compact it, erode it, disrupt its organic and inorganic structure, turn it too salty for life, and cause desertification. urbanization, logging, mining, overgrazing, alterations in soil moisture, air pollution, fires, chemical pollution, and leaching out of minerals all damage or destroy soils. thanks to removal of vegetative cover, mining, agriculture, and other activities, the world's topsoils are eroded by wind and water ten to hundreds of times faster than they are renewed (at roughly tons ha À year À ). soils constitute the foundation of human agriculture, yet agriculture, including livestock raising, is the worst culprit in degrading soils. agricultural practices have eroded or degraded more than % of present cropland. over the last half century, some , villages in northern and western china have been overrun by the drifting sands of desertification. besides topsoil erosion, the damage includes salting and saturation of poorly managed irrigated lands; compaction by heavy machinery and the hooves of livestock; and pollution from excessive fertilizers, animal wastes, and pesticides. living, dead, and decomposing organic matter is the key to soil structure and fertility. soil depleted of organic matter is less permeable to water and air and thus less able to support either aboveground plants or soil organisms. the linkages between soil's inorganic components and the soil biotanaturalist wilson's ( ) ''little things that run the world''are what give soil its life-sustaining capacity. a clear-cut forest patch whose soil biota has been damaged beyond recovery can no longer sustain trees, no matter how many are planted; another clear-cut patch whose soil community is intact -especially the close associations among fungi and tree rootswill support new tree growth. destroying soil biota unleashes a whole series of impoverishing biotic effects both below and above ground. in , rachel carson's landmark book, silent spring, alerted the world to the pervasiveness of synthetic chemicals produced since world war ii. as many as , synthetic chemicals are in use today. true to one company's slogan, many of these have brought ''better living through chemistry,'' providing new fabrics and lighter manufacturing materials, antibiotics, and life-saving drugs. but industrial nations have carelessly pumped chemicals into every medium. chemicals -as varied as prescription drugs flowing out of sewage plants, pesticides, heavy metals, and cancer-causing by-products of countless manufacturing processes -now lace the world's water, soil, and air and the bodies of all living things, including humans. chemicals directly poison organisms; they accumulate in physical surroundings and are passed through and, in many cases, concentrated within portions of the food web. chemicals cause cancer, interfere with hormonal systems, provoke asthma, and impair the functioning of immune systems. they have intergenerational effects, such as intellectual impairment in children whose mothers have eaten contaminated fish. what's more, over half a century of pesticide and antibiotic overuse has bred resistance to these chemicals among insects, plants, and microbes, giving rise to new and reemerging scourges. many chemicals travel oceanic and atmospheric currents to sites far from their source. sulfur emissions from the us midwest, for example, fall to earth again as acid rain in europe, killing forests and so acidifying streams and lakes that they, too, effectively die. china's burning of soft coal sends air pollution all the way to northwestern north america; the heavy haze hanging over china's chief farming regions may be cutting agricultural production by a third. chlorofluorocarbons (cfcs), once widely used as refrigerants, have damaged the atmospheric ozone layer, which moderates how much ultraviolet radiation reaches the earth, and opened ozone holes over the arctic and antarctic. but even more alarming is an unprecedented acidification of the oceans that has only recently attracted the attention of major scientific research consortiums. acid added to the world ocean by human activity has lowered the ocean's ph; it is lower now than it has been in my, which translates into a % increase in sea-surface acidity since industrialization began. the future of marine life in an ocean acidifying at this speed and intensity looks bleak. as the concentration of hydrogen ions rises, the calcium carbonate in the shells or skeletons of organisms such as tropical corals, microscopic foraminifera, and mollusks begins to dissolve; further, more hydrogen ions combine with the calcium carbonate building blocks the organisms need, making it harder for them to extract this compound from the water and build their shells in the first place. although many of the most obviously deadly chemicals were banned in the s, they continue to impoverish the biosphere. polychlorinated biphenyls -stable, nonflammable compounds once used in electrical transformers and many other industrial and household applications -remain in the environment for long periods, cycling among air, water, and soils and persisting in the food web. they are found in polar bears and arctic villagers; they are implicated in reproductive disorders, particularly in such animals as marine mammals, whose long lives, thick fat layers where chemicals concentrate, and position as top predators make them especially vulnerable. the agricultural pesticide ddt, sprayed with abandon in the s and s, even directly on children, had severely thinned wild birds' eggshells by the time it was banned in the united states. populations of birds such as the brown pelican and bald eagle had dropped precipitously by the s, although they have recovered enough for the species to be taken off the us endangered species list, the bald eagle in and the brown pelican in . reproduction of central california populations of the california condor, in contrast, continues to be threatened by ddt breakdown products, which, years after the pesticide was banned, are still found in the sea lion carcasses the birds sometimes feed on. carson's book revealed the real danger of chemical pollutants: they have not simply perturbed the chemistry of water, soil, and air but harmed the biosphere as well. the list of chemicals' effects on living things is so long that chemical pollution equals humans' environmental impact in most people's minds, yet it is just one form of biotic impoverishment. all the substances found in living things -such as water, carbon, nitrogen, phosphorus, and sulfur -cycle through ecosystems in biogeochemical cycles. human activities modify or have the potential to modify all these cycles. sometimes the results stem from changing the amount or the precise chemistry of the cycled substance; in other cases, humans change biogeochemical cycles by changing the biota itself. freshwater use, dams, and other engineering ventures affect the amount and rate of river flow to the oceans and increase evaporation rates, directly affecting the water cycle and indirectly impoverishing aquatic life. direct human modifications of living systems also alter the water cycle. in south africa, european settlers supplemented the treeless native scrub, or fynbos, with such trees as pines and australian acacias from similar mediterranean climates. but because these trees are larger and thirstier than the native scrub, regional water tables have fallen sharply. human activity has disrupted the global nitrogen cycle by greatly increasing the amount of nitrogen fixed from the atmosphere (combined into compounds usable by living things). the increase comes mostly from deliberate addition of nitrogen to soils as fertilizer but also as a by-product of the burning of fossil fuels. agriculture, livestock raising, and residential yard maintenance chronically add tons of excess nutrients, including nitrogen and phosphorus, to soils and water. the additions are often invisible; their biological impacts are often dramatic. increased nutrients in coastal waters, for example, trigger blooms of toxic dinoflagellates -the algae that cause red tides, fish kills, and tumors and other diseases in varied sea creatures. when huge blooms of algae die, they fall to the seafloor, where their decomposition so robs the water of oxygen that fish and other marine organisms can no longer live there. with nitrogen concentrations in the mississippi river two to three times as high as they were years ago, a gigantic dead zone forms in the gulf of mexico every summer; in summer this dead zone covered , km . the burning of fossil fuels is transforming the carbon cycle, primarily by raising the atmospheric concentration of carbon dioxide. with other greenhouse gases, such as methane and oxides of nitrogen, carbon dioxide helps keep earth's surface at a livable temperature and drives plant photosynthesis, but since the industrial revolution, atmospheric carbon dioxide concentrations have risen nearly % and are now widely thought to be disrupting the planet's climate. in addition, the effects of catastrophic oil spills like the one that followed the april explosion of the deepwater horizon drilling rig in the gulf of mexico -and the effects of the chemicals used to disperse the resulting plumes of oil -may reverberate for decades. in its report, written and reviewed by more than scientists, the intergovernmental panel on climate change (ipcc) concluded that climate warming was ''unequivocal'' (p. ), that most of the average global warming over the previous years was ''very likely due to anthropogenic ghg [greenhouse gas] increases'' (p. ), and that this warming has likely had ''a discernible influence at the global scale on observed changes in many physical and biological systems'' (p. ). the concentrations of heat-trapping gases in the atmosphere are at their highest level in more than , years. the th century in the northern hemisphere was the warmest of the past millennium; the first decade of the st first century, and the first months of , were the warmest on record. higher global temperatures set in motion a whole series of effects, making the study of climate change, and of humans' role in it, complex and controversial. spring arrives at least week earlier in the northern hemisphere. polar glaciers and ice sheets are receding. the arctic is warming twice as fast as the rest of the planet, and arctic sea ice melted at a near-record pace in . with the sun heating the newly open water, winter refreezing will take longer, and the resulting thinner ice will melt more easily the following summer. the large-scale circulation of global air masses is changing and, with them, the large-scale cycles in ocean currents, including the periodic warming and cooling in the tropical pacific ocean known as el niñ o and la niñ a. as a result, the distribution, timing, and amount of rain and snow are also changing, making the weather seem more unpredictable than ever. unusually warm or cold winters, massive hurricanes like those that devastated the us gulf coast in , and weatherrelated damage to human life and property are all predicted to increase with global warming. in the united states in , weather-related damage totaled nearly $ billion. where other nutrients are not limiting, rising carbon dioxide concentrations may enhance plant photosynthesis and growth. rising temperatures may shift the ranges of many plants and animals -both wild and domestic -rearranging the composition and distribution of the world's biomes, as well as those of agricultural systems. the resulting displacements will have far-reaching implications not only for the displaced plants and animals but also for the goods and services humans depend on from these living systems. from their beginnings as hunter-gatherers, humans have become highly efficient, machine-aided ecosystem engineers and predators. we transform the land so it produces what we need or want; we harvest the oceans in addition to reaping our own fields; we cover the land, even agricultural land, with sprawling cities. all these activities directly affect the ability of other life-forms to survive and reproduce. we deplete nonhuman life by eliminating some forms and favoring others; the result is a loss of genetic, population, and species diversity. we are irreversibly homogenizing life on earth, in effect exercising an unnatural selection that is erasing the diversity generated by millions of years of evolution by natural selection. one species is now determining which other species will survive, reproduce, and thereby contribute the raw material for future evolution. in the s, so many sardines were scooped from the waters off monterey's cannery row in california that the population collapsed, taking other sea creatures and human livelihoods with it; after rebounding somewhat in the first decade of the s, the species has still not recovered fully. according to the us national marine fisheries service, nearly % of commercially valuable fish of known status were overfished or fished to their full potential by . atlantic commercial fish species at their lowest levels in history include tuna, marlin, cod, and swordfish. overfishing not only depletes target species but restructures entire marine food webs. marine mammals, including whales, seals, sea lions, manatees, and sea otters, were so badly depleted by human hunters that one species, steller's sea cow (hydrodamalis gigas), went extinct; many other species almost disappeared. in the th century, russian fur traders wiped out sea otters (enhydra lutris) along the central california coast. with the otters gone, their principal prey, purple sea urchins (stronglyocentrotus purpuratus), overran the offshore forests of giant kelp (macrocystis pyrifera), decimating the kelp fronds and the habitat they provided for countless other marine creatures, including commercially harvested fishes. thanks to five decades of protection, marine mammal populations began slowly recovering -only to face food shortages as regional marine food webs continue to unravel because of fishing, changing oceanic conditions, and contamination. timber harvest has stripped vegetation from the amazonian rainforest to mountainsides on all continents, diminishing and fragmenting habitat for innumerable forest and stream organisms, eroding soils, worsening floods, and contributing significantly to global carbon dioxide emissions. in the northern hemisphere, % or less remains of old-growth temperate rainforests. the uniform stands of trees usually replanted after logging do not replace the diversity lost with the native forest, any more than monocultures of corn replace the diversity within native tallgrass prairies. a great deal of human ecosystem engineering not only alters or damages the habitats of other living things but often destroys those habitats. satellite-mounted remote-sensing instruments have revealed transformations of terrestrial landscapes on a scale unimaginable in centuries past. together, cropland and pastures occupy % of earth's land surface. estimates of the share of land wholly transformed or degraded by humans hover around %. our roads, farms, cities, feedlots, and ranches either fragment or destroy the habitats of most large carnivorous mammals. mining and oil drilling damage the soil, remove vegetation, and pollute marine areas. grazing compacts soil and sends silt and manure into streams, where they harm stream life. landscapes that have not been entirely converted to human use have been cut into fragments. in song of the dodo, writer quammen ( ) likens our actions to starting with a fine persian carpet and then slicing it neatly into equal pieces; even if we had the same square footage, we would not have nice persian rugs -only ragged, nonfunctional fragments. and in fact, we do not even have the original square footage because we have destroyed an enormous fraction of it. such habitat destruction is not limited to terrestrial environments. human channelization of rivers may remove whole segments of riverbed. in the kissimmee river of the us state of florida, for example, channelization in the s transformed km of free-flowing river into km of canal, effectively removing km of river channel and drastically altering the orphaned river meanders left behind. wetlands worldwide continue to disappear, drained to create shoreline communities for people and filled to increase cropland. the lower united states lost % of their wetlands between the s and mid- s. such losses destroy major fish and shellfish nurseries, natural flood and pollution control, and habitat for countless plants and animals. the mosaic of habitats in, on, or near the seafloor -home to % of all marine species -is also being decimated. like clear-cutting of an old-growth forest, the use of large, heavy trawls dragged along the sea bottom to catch groundfish and other species flattens and simplifies complex, structured habitats such as gravels, coral reefs, crevices, and boulders and drastically reduces biodiversity. studies reported on by the national research council of the us national academy of sciences have shown that a single tow can injure or destroy upward of two-thirds of certain bottom-dwelling species, which may still not have recovered after a year or more of no trawling. habitat fragmentation and destruction, whether on land or in freshwater and marine environments, may lead directly to extinction or isolate organisms in ways that make them extremely vulnerable to natural disturbances, climate change, or further human disturbance. ''the one process ongoing y that will take millions of years to correct,' ' wilson ( , p. ) admonishes us, ''is the loss of genetic and species diversity by the destruction of natural habitats. this is the folly our descendants are least likely to forgive us.'' both deliberately and unwittingly, humans are rearranging earth's living components, reducing diversity and homogenizing biotas around the world. the present, continuing loss of genetic diversity, of populations, and of species vastly exceeds background rates. at the same time, our global economy is transporting species worldwide at unprecedented scales. the globe is now experiencing its sixth mass extinction, the largest since the dinosaurs vanished ma; present extinction rates are thought to be on the order of - times those before people dominated earth. according to the millennium ecosystem assessment, a -year project begun in to assess the world's ecosystems, an estimated - % of the world's species will be committed to extinction over the next years. approximately % of all vertebrates, including % of sharks and rays, are at risk of extinction. at least one of every eight plant species is also threatened with extinction. although mammals and birds typically receive the most attention, massive extinctions of plants, which form the basis of the biosphere's food webs, undermine lifesupport foundations. the mutualistic relationships between animals and plants, particularly evident in tropical forests, mean that extinctions in one group have cascading effects in other groups. plants reliant on animals for pollination or seed dispersal, for example, are themselves threatened by the extinction of the animal species they depend on. not surprisingly, some scientists view extinction as the worst biological tragedy, but extinction is just another symptom of global biotic impoverishment. ever since they began to spread over the globe, people have transported other organisms with them, sometimes for food, sometimes for aesthetic reasons, and most often inadvertently. with the mobility of modern societies and today's especially speedy globalization of trade, the introduction of alien species has reached epidemic proportions, causing some scientists to label it biological pollution. aliens -zebra mussels (dreissena polymorpha) and tamarisks, or saltcedar (tamarix spp.), in north america; the red sea sea jelly rhopilema nomadica and the common aquarium alga caulerpa taxifolia now choking the mediterranean sea; and leidy's comb jelly (mnemiopsis leidyi) of northeastern america in the black sea, to name just a few -are present everywhere, and they usually thrive and spread at the expense of native species. on many islands, for example, more than half the plant species are not native, and in many continental areas the figure reaches % or more. the costs of such invasions, in both economic and ecological terms, are high. in the united states, for example, annual economic losses due to damage by invasive species or the costs of controlling them exceed $ billion per year -$ billion more than the nation's losses from weather-related damage in . furthermore, alien invasions cause extinctions and, when added to other extinctions and the deliberate monocultures of agricultural crops, homogenize biotas still more. introduced species are fast catching up with habitat fragmentation and destruction as the major engines of ecological deterioration. humans have been manipulating their crop plants and domesticated animals for , years or so -selecting seeds or individuals and breeding and cross-breeding them. the goal was something better, bigger, tastier, hardier, or all of the above; success was sometimes elusive, but the result was crop homogenization. of the myriad strains of potatoes domesticated by south american cultures, for example, only one was accepted and cultivated when potatoes first made it to europe. the new crop made it possible to feed more people from an equivalent area of land and initially staved off malnutrition. but the strain succumbed to a fungal potato blight in the s. had more than one strain been cultivated, the tragic irish potato famines might have been averted. in the last few decades of the th century, people began to manipulate genes directly using the tools of molecular biotechnology, even cloning sheep and cows from adult body cells. us farmers routinely plant their fields with corn whose genetic material incorporates a bacterial gene resistant to certain pathogens. more than genetically altered crops have been approved for sale to us farmers since , with genes borrowed from bacteria, viruses, and insects. the united states accounts for nearly two-thirds of biotechnology crops planted globally. worldwide in , . million hectares in countries on six continents were planted with genetically modified crops, as compared with . million hectares in six countries in -a -fold expansion in years. biotechnologists focus on the potential of this newmillennium green revolution to feed the growing world population, which has added almost billion people in the past decade alone. but other scientists worry about unknown human and ecological health risks; these concerns have stirred deep scientific and public debate, especially in europe, akin to the debate over pesticides in rachel carson's time. one worrisome practice is plant genetic engineers' technique of attaching the genes they want to introduce into plants to an antibiotic-resistant gene. they can then easily select plants that have acquired the desired genes by treating them with the antibiotic, which kills any nonresistant plants. critics worry that the antibiotic-resistant genes could spread to human pathogens and worsen an already growing antibioticresistance problem. another concern arises from allergies humans might have or develop in response to genetically modified foods. although supporters of genetic engineering believe that genetically altered crops pose few ecological risks, ecologists have raised a variety of concerns. studies in the late s indicated that pollen from genetically engineered bt corn can kill monarch butterfly caterpillars. bt is a strain of bacterium that has been used since the s as a pesticidal spray; its genes have also been inserted directly into corn and other crops. ecologists have long worried that genetically engineered plants could escape from fields and cross-breed with wild relatives. studies in radishes, sorghum, canola, and sunflowers found that genes from an engineered plant could jump to wild relatives through interbreeding. the fear is that a gene conferring insect or herbicide resistance might spread through wild plants, creating invasive superweeds that could potentially lower crop yields and further disturb natural ecosystems. in fact, herbicide-resistant turf grass tested in oregon in did escape and spread, and transgenic canola has been appearing throughout the us state of north dakota, which has tens of thousands of hectares in conventional and genetically modified canola. according to the scientists who discovered the transgenic escapees growing in north dakota -far from any canola field -the plants are likely to be cross-pollinating in the wild and swapping introduced genes; the plants' novel gene combinations indicate that the transgenic traits are stable and evolving outside of cultivation. genetically engineered crops do confer some economic and environmental benefits: for farmers, higher yields, lower costs, savings in management time and gains in flexibility; for the environment, indirect benefits from using fewer pesticides and herbicides. but it is still an open question whether such benefits outweigh the potential ecological risks or whether the public will embrace having genetically modified foods as staples in their diet. human biotic impacts are not confined to other species; human cultures themselves have suffered from the widening circles of indirect and direct effects humans have imposed on the rest of nature. over the past hundred years, human technology has cut both ways with regard to public health. wonder drugs controlled common pathogens at the same time that natural selection strengthened those pathogens' ability to resist the drugs. reservoirs in the tropics made water supplies more reliable for humans but also created ideal environments for human parasites. industrialization exposed human society to a remarkable array of toxic substances. although man's inhumanity to man has been both fact and the subject of discourse for thousands of years, the discussions have mostly been removed from any environmental context. few people today regard social ills as environmental impacts or humans as part of a biota. but diminished societal well-being -whether manifest in high death rates or poor quality of life -shares many of its roots with diminished nonhuman life as a form of biotic impoverishment. the intersection of the environment and human health is the core of the discipline known as environmental health. among the environmental challenges to public health are the direct effects of toxic chemicals; occupational health threats, including exposures to hazardous materials on the job; and sanitation and disposal of hazardous wastes. exploitation of nonrenewable natural resources -including coal mining, rock quarrying or other mining operations, and petroleum extraction and refining -often chronically impairs workers' health and shortens their lives. farmworkers around the world suffer long-term ills from high exposures to pesticides and herbicides. partly because of increased air pollution, asthma rates are rising, particularly in big cities. synthetic volatile solvents are used in products from shoes to semiconductors, producing lung diseases and toxic wastes. nuclear weapons production starting in world war ii, and associated contamination, have been linked to a variety of illnesses, including syndromes neither recognized nor understood at the time and whose causes were not diagnosed until decades afterward. the grayish metal beryllium, for example, was used in weapons production and was found decades later to scar the lungs of workers and people living near toxic waste sites. infectious diseases have challenged human populations throughout history, playing a significant role in their evolution and cultural development over the past , years, with % of human diseases linked to wildlife or domestic animals. the th century brought major successes in eradicating such infectious diseases as smallpox, polio, and many waterborne illnesses. but toward the century's end, emerging and reemerging diseases were again reaching pandemic proportions. infectious diseases thought to be on the wane -including tuberculosis, malaria, cholera, diptheria, leptospirosis, encephalitis, and dengue fever -have begun a resurgence. in addition, seemingly new scourges -ebola virus, hantavirus, hiv/aids, lyme disease, and west nile fever -are also spreading, often, it appears, from wild animal hosts to humans as people encroach further upon previously undisturbed regions. a number of studies over the decade before show complex connections between biodiversity and disease: biodiversity loss often increases disease transmission, as in lyme disease and west nile fever, but diverse ecosystems also serve as sources of pathogens. overall, however, the studies indicate that preserving intact ecosystems and their endemic biodiversity often reduces the prevalence of infectious diseases. human migrations -including their modern incarnation through air travel -also accelerate pathogen traffic and launch global pandemics, such as the outbreak of severe acute respiratory syndrome and the swine flu outbreak caused by the h n virus. even something as simple and apparently benign as lighting can become an indirect agent of disease. artificial lighting, especially in the tropics, for example, can alter human and insect behavior in ways that speed transmission of insect-borne diseases, such as chagas's disease, malaria, and leishmaniasis. in addition, especially in highly developed countries such as the united states, diseases of affluence and overconsumption are taking a toll. heart disease is the number one cause of death in the united states; overnutrition, obesity, and diabetes due to sedentary, technology-driven lifestyles, particularly among children, are chronic and rising. one estimate put the share of us children considered overweight or obese at one in three. this rise in obesity rates has been stunningly rapid. as recently as , just % of adults were obese; by the rate had hit %, and two-thirds of americans are now considered either overweight or obese. although not conventionally regarded as elements of biodiversity, human languages, customs, agricultural systems, technologies, and political systems have evolved out of specific regional environments. like other organisms' adaptive traits and behaviors, these elements of human culture constitute unique natural histories adapted, like any natural history, to the biogeographical context in which they arose. yet modern technology, transportation, and trade have pushed the world into a globalized culture, thereby reducing human biological and cultural diversity. linguists, for example, are predicting that at least half of the languages spoken today will become extinct in the st century. with the spread of euro-american culture, unique indigenous human cultures, with their knowledge of local medicines and geographically specialized economies, are disappearing even more rapidly than the natural systems that nurtured them. this loss of human biodiversity is in every way as troubling as the loss of nonhuman biodiversity. the effects of environmental degradation on human quality of life are another symptom of biotic impoverishment. food availability, which depends on environmental conditions, is a basic determinant of quality of life. yet according to the world health organization, nearly half the world's population suffers from one of two forms of poor nutrition: undernutrition or overnutrition. a big belly is now a symptom shared by malnourished children, who lack calories and protein, and overweight residents of the developed world, who suffer clogged arteries and heart disease from eating too much. independent of race or economic class, declining quality of life in today's world is manifest in symptoms such as increased asthma in the united states caused by environmental contaminants and the high disease rates in the former soviet bloc after decades of unregulated pollution. even with explicit legal requirements that industries release information on their toxic emissions, many people throughout the world still lack both information and the decision-making power that would give them any control over the quality of their lives. aggrieved about the degraded environment and resulting quality of life in his homeland, ogoni activist ken saro-wiwa issued a statement shortly before he was executed by the nigerian government in saying, ''the environment is man's first right. without a safe environment, man cannot exist to claim other rights, be they political, social, or economic.'' kenyan maathai ( , p. ) , winner of the nobel peace prize, has also written, ''[i]f we destroy it, we will undermine our own ways of life and ultimately kill ourselves. this is why the environment needs to be at the center of domestic and international policy and practice. if it is not, we don't stand a chance of alleviating poverty in any significant way.'' having ignored this kind of advice for decades, nations are seeing a new kind of refugee attempting to escape environmental degradation and desperate living conditions; the number of international environmental refugees exceeded the number of political refugees around the world for the first time in . environmental refugees flee homelands devastated by flooding from dam building, extraction of mineral resources, desertification, and unjust policies of national and international institutions. such degradation preempts many fundamental human rights, including the rights to health, livelihood, culture, privacy, and property. people have long recognized that human activities that degrade environmental conditions threaten not only the biosphere but also humans' own quality of life. as early as years ago in mesopotamia and south asia, writings revealed an awareness of biodiversity, of natural order among living things, and of consequences of disrupting the biosphere. throughout history, even as civilization grew increasingly divorced from its natural underpinnings, writers, thinkers, activists, and people from all walks of life have continued to see and extol the benefits of nature to humans' quality of life. contemporary society still has the chance to relearn how important the environment is to quality of life. it is encouraging that the united steelworkers of america in released a report recognizing that protecting steelworker jobs could not be done by ignoring environmental problems and that the destruction of the environment may pose the greatest threat to their children's future. it is also encouraging that the nobel peace prize was awarded to a political figure and a group of scientists for their work on climate change. making a living from nature's wealth has consistently opened gaps between haves and have-nots, between those who bear the brunt of environmental damage to their home places and those who do not, and between the rights of people alive now and those of future generations; these disparities too are part of biotic impoverishment. inequitable access to ''man's first right'' -a healthy local environment -has come to be known as environmental injustice. environmental injustices, such as institutional racism, occur in industrial and nonindustrial nations. injustice can be overt, as when land-use planning sites landfills, incinerators, and hazardous waste facilities in minority communities, or when environmental agencies levy fines for hazardous waste violations that are lower in minority communities than in white communities. less overt, but no less unjust, is the harm done to one community when unsound environmental practices benefit another, as when clear-cut logging in the highlands of northwestern north america benefits logging communities while damaging the livelihoods of lowland fishing communities subjected to debris flows, sedimentation, and downstream flooding. the plight of the working poor and the disparities between rich and poor are also examples of biotic impoverishment within the human community. according to the united nations research institute for social development, the collective wealth of the world's billionaires equaled the combined income of the poorest . billion people in . forbes magazine put the number of billionaires in early at , with a total worth of $ . trillion. in the united states during the last decade of the th century, the incomes of poor and middle-class families stagnated or fell, despite a booming stock market. the center on budget and policy priorities and the economic policy institute reported that between and , earnings of the poorest fifth of american families rose less than %, while earnings of the richest fifth jumped %. by the middle of the first decade of the st century, americans' income inequality had become the widest among industrialized nations, with the wealthiest % of the population holding % of the wealth. the wealthiest americans continued to prosper even during the global recession late in that decade, while the less well-off kept losing ground. but perhaps the grossest example of human and environmental domination leading to continued injustice is the creation of a so-called third world to supply raw materials and labor to the dominant european civilization after and the resulting schism between today's developed and developing nations. developing regions throughout the world held tremendous stores of natural wealth, some of it -like petroleum -having obvious monetary value in the dominant economies and some having a value invisible to those economies -like vast intact ecosystems. a united nations study (teeb) estimated that even today, earth's ecosystems account for roughly half to % of the source of livelihoods for rural and forest-dwelling peoples; the study calls this value the gross domestic product (gdp) of the poor. dominant european civilizations unabashedly exploited this natural wealth and colonized or enslaved the people in whose homelands the wealth was found. but the dominant civilizations also exported their ways of thinking and their economic models to the developing world, not only colonizing places but also effecting what wangari maathai has called a colonization of the mind. although dominant st century society tends to dismiss ancient wisdom as irrelevant in the modern world, perhaps the cruelest impoverishment of all is the cultural and spiritual deracination experienced by exploited peoples worldwide. exploitation of poor nations and their citizens by richer, consumer countries -and in many cases by the same governments that fought for independence from the colonists while adopting the colonists' attitudes and economic models -persists today in agriculture, wild materials harvesting, and textile and other manufacturing sweatshops. in the mid- s, industrial countries consumed % of the globe's aluminum, % of its paper, % of its iron and steel, % of its energy, and % of its meat; they are thus responsible for most of the environmental degradation associated with producing these goods. most of the actual degradation, however, still takes place in developing nations. as a result, continuing environmental and social injusticeenvironmental and social impoverishment perpetrated by outsiders and insiders alike -pervades developing nations. such impoverishment can take the form of wrenching physical dislocation like the massive displacements enforced by china's three gorges dam. it can appear as environmental devastation of homelands and murder of the people who fought to keep their lands, as in the nigerian government-backed exploitation of ogoniland's oil reserves by the shell petroleum development corporation. after saro-wiwa's execution, the ogoni were left, without a voice, to deal with a scarred and oilpolluted landscape. despite great advances in the welfare of women and children over the past century, poverty still plagues both groups. children from impoverished communities, even in affluent nations, suffer from the lethargy and impaired physical and intellectual development known as failure to thrive. poverty forces many children to work the land or in industrial sweatshops; lack of education prevents them from attaining their intellectual potential. this impoverishment in the lives of women and children is as much a symptom of biotic impoverishment as are deforestation, invasive alien organisms, or species extinctions. little by little, community-based conservation and development initiatives are being mounted by local citizens to combat this impoverishment: witness maathai's green belt movement, which began with tree planting to restore community landscapes and provide livelihoods for residents, and the rise of ecotourism and microlending (small loans made to individuals, especially women, to start independent businesses) as ways to bring monetary benefits directly to local people without further damaging their environments. ultimately, one could see all efforts to protect the ethnosphere and the biosphere as a fight for the rights of future generations to an environment that can support them. only during the last two decades of the th century did environmental issues find a place on international diplomatic agendas, as scholars began calling attention to -and governments began to see -irreversible connections between environmental degradation and national security. british scholar myers ( ) , noting that environmental problems were likely to become predominant causes of conflict in the decades ahead, was one of the first to define a new concept of environmental security. national security threatened by unprecedented environmental changes irrespective of political boundaries will require unprecedented responses altogether different from military actions, he warned. nations cannot deploy their armies to hold back advancing deserts, rising seas, or the greenhouse effect. canadian scholar homer-dixon ( ) showed that environmental scarcities -whether created by ecological constraints or sociopolitical factors including growing populations, depletion of renewable resources such as fish or timber, and environmental injustice perpetrated by one segment of a population on another -were fast becoming a permanent, independent cause of civil strife and ethnic violence. he found that such scarcity was helping to drive societies into a self-reinforcing spiral of dysfunction and violence, including terrorism. environmental and economic injustices worldwide leave no country immune to this type of threat. typically, diplomacy has stalled in conflicts over natural resources: arguments over water rights have more than once held up israeli-palestinian peace agreements; fights over fish erupted between canada and the united states, spain, and portugal. in contrast, in adopting the montreal protocol on substances that deplete the ozone layer in , governments, nongovernmental organizations, and industry successfully worked together to safeguard part of the environmental commons. the treaty requires signatory nations to curb their use of cfcs and other ozone-destroying chemicals and has been, according to former united nations secretary general kofi annan, perhaps the most successful international agreement to date. if scientists have learned anything about the factors leading to biotic impoverishment, they have learned that the factors' cumulative effects can take on surprising dimensions. as scholars like fagan ( ) and diamond ( ) have chronicled, the multiple stresses of global climatic cycles such as el niñ o, natural events like droughts or floods, resource depletion, and social upheaval have shaped the fates of civilizations. societies as far-flung as ancient egypt, peru, the american southwest, and easter island prospered and collapsed because of unwise management of their environments. the city of ubar, built on desert sands in what is now southern oman, literally disappeared into the sinkhole created by drawing too much water out of its great well. in modern sahelian africa, a combination of well digging and improved medical care and sanitation led to a threefold population increase; sedentary ways, heavy taxes imposed by a colonial government, and an impoverished people took the place of a nomadic culture evolved within the desert's realities. during the first decade of the st century, numerous natural disasters befell nations around the world: wildfires in australia, bolivia, canada, and russia; flooding in china, india, romania, and west africa; devastating hurricanes and typhoons in the caribbean, philippines, taiwan, and southeastern united states; catastrophic landslides and floods in china, guatemala, pakistan, and portugal; and destructive earthquakes in chile, china, haiti, indonesia, and pakistan. neither the rains nor the earthquakes were caused by human activity, but the cumulative effects of human land uses and management practices -from dikes separating the mississippi from its floodplain to deforestation in haiti -made the losses of human life and property much worse than they might have been otherwise. the ultimate cause of humans' massive environmental impact is our individual and collective consumptive and reproductive behavior, which has given us spectacular success as a species. but the very things that enabled humans to thrive in nearly every environment have magnified our impacts on those environments, and the technological and political steps we take to mitigate our impacts often aggravate them. too many of us simply take too much from the natural world and ask it to absorb too much waste. for most of human history, people remained tied to their natural surroundings. even as agriculture, writing, and technology advanced, barriers of geography, language, and culture kept humans a diverse lot, each group depending on mostly local and regional knowledge about where and when to find resources necessary for survival. their worldviews, and resulting economies, reflected this dependency. for example, in northwestern north america starting about years ago, a native economy centered on the abundance of salmon. at its core was the concept of the gift and a belief system that treated all parts of the earth -animate and inanimate -as equal members of a community. in this and other ancient gift economies, a gift was not a possession that could be owned; rather, it had to be passed on, creating a cycle of obligatory returns. individuals or tribes gained prestige through the size of their gifts, not the amount of wealth they accumulated. this system coevolved with the migratory habits of the salmon, which moved en masse upriver to spawn each year. because the indians viewed salmon as equals to themselves, killing salmon represented a gift of food from salmon to people. fishers were obligated to treat salmon with respect or risk losing this vital gift. the exchange of gifts between salmon and humans -food for respectful treatment -minimized waste and overharvest and ensured a continuous supply of food. further, the perennial trading of gifts among the people effectively redistributed the natural wealth brought each year by fluctuating populations of migrating fish, leveling out the boom-and-bust cycles that usually accompany reliance on an uncertain resource. in modern times, the gift economy along with the egalitarian worldview that accompanied it, has been eclipsed by a redistributive economy tied not to an exchange of gifts with nature but to the exploitation of nature and to the technologies that enhance that exploitation. nature became a resource for humans, rather than an equal to humans. in economic terms, natural resources fell under the heading of 'land' in an economic trinity comprising three factors of production: land, labor, and capital. land and resources, including crops, became commodities -expendable or easily substitutable forms of capital -whose value was determined solely by their value in the human marketplace. in adam smith published his famous inquiry into the nature and causes of the wealth of nations, in which he argued that society is merely the sum of its individuals, that the social good is the sum of individual wants, and that the market (the so-called invisible hand) automatically guides individual behavior to the common good. crucial to his theories were division of labor and the idea that all the factors of production were freely mobile. his mechanistic views created an economic rationale for no longer regarding individuals as members of a community linked by ethical, social, and ecological bonds. about the same time, fueling and fueled by the beginnings of the industrial revolution, the study of the natural world was morphing into modern physics, chemistry, geology, and biology. before the mid- century, those who studied the natural world -early century german biogeographer baron alexander von humboldt and his disciple charles darwin among them -took an integrated view of science and nature, including humans. both scientists regarded the understanding of the complex interdependencies among living things as the noblest and most important result of scientific inquiry. but this integrated natural philosophy was soon supplanted by more atomistic views, which fit better with industrialization. mass production of new machines relied on division of labor and interchangeable parts. like automobiles on an assembly line, natural phenomena too were broken down into their supposed component parts in a reductionism that has dominated science ever since. rushing to gain indepth, specialized knowledge, science and society lost sight of the need to tie the knowledge together. disciplinary specialization replaced integrative scholarship. neoclassical economics, which arose around , ushered in the economic worldview that rules today. a good's value was no longer tied to the labor required to make it but derived instead from its scarcity. a good's price was determined only by the interaction of supply and demand. as part of 'land,' natural resources therefore became part of the human economy, rather than the material foundation making the human economy possible. because of its doctrine of infinite substitutability, neoclassical economics rejects any limits on growth; forgotten are the classical economic thinkers and contemporaries of von humboldt, including thomas malthus and john stuart mill, who saw limits to the growth of human population and material well-being. consequently, the th and th centuries saw the rise to dominance of economic indicators that fostered the economic invisibility of nature, misleading society about the relevance of earth's living systems to human well-being. among the worst offenders in this regard are gross national product (gnp) and its cousin, gdp. gnp measures the value of goods and services produced by a nation's citizens or companies, regardless of their location around the globe. gdp, in contrast, measures the value of goods and services produced within a country's borders, regardless of who generates those goods and services. in effect, both gnp and gdp measure money changing hands, no matter what the money pays for; they make no distinction between what is desirable and undesirable, between costs and benefits. both indicators ignore important aspects of the economy like unpaid work or nonmonetary contributions to human fulfillment -parenting, volunteering, checking books out of the library. worse, the indicators also omit social and environmental costs, such as pollution, illness, or resource depletion; they only add and do not subtract. gdp math adds in the value of paid daycare or a stay in the hospital and ignores the value of unpaid parenting or being cared for at home by friends. it adds in the value of timber sold, but fails to subtract the losses in biodiversity, watershed protection, or climate regulation that come when a forest is cut. efforts have been made in the past few decades to create less blinkered economic indicators. social scientists herman daly and john cobb in developed an index of sustainable economic welfare, which adjusts the united states' gnp by adding in environmental good things and subtracting environmental bad things. public expenditures on education, for example, are weighted as ''goods'' while costs of pollution cleanup, depletion of natural resources, and treating environment-related illnesses are counted as 'bads.' unlike the soaring gdp of recent decades, this index of sustainable economic welfare remained nearly unchanged over the same period. still other work aims to reveal nature's worth in monetary terms by assigning dollar values to ecological goods and services. a study by ecologist david pimentel and colleagues calculated separate values for specific biological services, such as soil formation, crop breeding, or pollination; by summing these figures, these researchers estimated the total economic benefits of biodiversity for the united states at $ billion - % of us gdp at the time -and for the world at $ billion. a analysis by pimentel and colleagues reported that the approximately , nonnative species in the united states cause major environmental damage and reparation costs amounting to $ billion a year. as part of the united nations international year of biodiversity in , several studies have translated the value of the world's ecosystems into dollar values. one report estimated the worth of crucial ecosystem services delivered to humans by living systems at $ - trillion per year -comparable to a world gross national income of $ trillion in . another study reported, among other things, that as many as million people worldwide depend on coral reefs -valued between $ billion and $ billion a year -for fisheries, tourism, and protection from ocean storms and high waves, services threatened by warmer and more acidic seas. although a monetary approach does not create a comprehensive indicator of environmental condition, it certainly points out that ecological values ignored by the global economy are enormous. the us census bureau predicts that by , the global human population will top billion -having grown by nearly billion people during the decade between the first and second editions of this encyclopedia. from the appearance of h. sapiens about , years ago, it took humans until to reach their first billion, years to double to billion, and years to achieve billion. human population doubled again from to billion in about years -before most post-world war ii baby boomers reached retirement age. even with fertility rates declining in developed countries, china, and some developing countries where women are gaining education and economic power, and with pandemics like aids claiming more lives, the census bureau predicts that world population will reach billion by . humans appropriate about % of global plant production, % of earth's freshwater runoff, and enough of the ocean's bounty to have depleted % of assessed marine fish stocks. in energy terms, one person's food consumption amounts to - calories a day, about the same as that of a common dolphin. but with all the other energy and materials humans use, global per capita energy and material consumption have soared even faster than population growth over the past years. now, instead of coevolving with a natural economy, global society is consuming the foundations of that economy, impoverishing earth's living systems, and undermining the foundations of its own existence (figure ). for most of the th century, environmental measurements, or indicators, tracked primarily two classes of information: counts of activities directed at environmental protection and the supply of products to people. regulatory agencies are typically preoccupied with legislation, permitting, or enforcement, such as the numbers of environmental laws passed, permits issued, enforcement actions taken, or treatment plants constructed. resource protection agencies concentrate on resource harvest and allocation. water managers, for example, measure water quantity; they allocate water to domestic, industrial, and agricultural uses but seldom make it a priority to reserve supplies for sustaining aquatic life, to protect scenic and recreational values, or simply to maintain the water cycle. foresters, farmers, and fishers count board-feet of timber, bushels of grain, and tons of fish harvested. governmental and nongovernmental organizations charged with protecting biological resources also keep counts of threatened and endangered species. as in the parable of the three blind men and the elephanteach of whom thinks the elephant looks like the one body part he can touch -these or similar indicators measure only one aspect of environmental quality. counting bureaucratic achievements focuses on actions rather than on information about real ecological status and trends. measurements of resource supply keep track of commodity production, not necessarily a system's capacity to continue supplying that commodity. and measuring only what we remove from natural systems, as if we were taking out the interest on a savings account, overlooks the fact that we are usually depleting principal as well. even biologists' counts of threatened and endangered species -which would seem to measure biotic impoverishment directly -still focus narrowly on biological parts, not ecological wholes. enumerating threatened and endangered species is just like counting any other commodity. it brings our attention to a system already in trouble, perhaps too late. and it subtly reinforces our view that we know which parts of the biota are most important. society needs to rethink its use of available environmental indicators, and it needs to develop new indicators that represent current conditions and trends in the systems humans depend on ( table ) . it particularly needs objective measures more directly tied to the condition, or health, of the environment so that people can judge whether their actions are compromising that condition. such measures should be quantitative, yet easy to understand and communicate; they should be cost-effective and applicable in many circumstances. unlike narrow criteria tracking only administrative, commodity, or endangered species numbers, they should provide reliable signals about status and trends in ecological systems. ideally, effective indicators should describe the present condition of a place, aid in diagnosing the underlying causes of that condition, and make predictions about future trends. they should reveal not only risks from present activities but also potential benefits from alternative management decisions. most important, these indicators should, either singly or in combination, give information explicitly about living systems. measurements of physical or chemical factors can sometimes act as surrogates for direct biological measurements, but only when the connection between those measures and living systems is clearly understood. too often we make assumptionswhen water managers assume that chemically clean water equals a healthy aquatic biota, for example -that turn out to be wrong and fail to protect living systems. as environmental concerns have become more urgent -and governmental and nongovernmental organizations have struggled to define and implement the concept of sustainable development -the effort has grown to create indicator systems that explicitly direct the public's and policymakers' attention to the value of living things. moving well past solely economic indexes like gdp, several indexes now integrate ecological, social, and economic well-being. the index of environmental trends for nine industrialized countries, developed by the nonprofit national center for economic and security alternatives, incorporates ratings of air, land, and water quality; chemical and waste generation; and social system economic system natural system figure relationships among the natural, social, and economic systems on earth. human economies may be thought of as icing atop a two-layer cake. the economic icing is eroding the human social and natural layers beneath it, threatening the foundation and sustainability of all three systems. modified from karr jr and chu ew ( ) ecological integrity: reclaiming lost connections. in: westra l and lemons j (eds.) perspectives in ecological integrity, pp - . dordrecht, netherlands: kluwer academic. energy use since . by its rankings, environmental quality in the united states had gone down by % since , while denmark had declined by %. in , world leaders, supported by the united nations development programme, defined a set of eight millennium development goals to be attained by , which combine poverty, education, employment, and environmental sustainability. they include human rights and health goals -such as universal primary education, gender equality, and combating aids and other diseases -as well as ensuring environmental sustainability. in the decade since the program began, progress has been made in reducing poverty; expanding educational opportunities for children, especially in africa; slowing deforestation; and providing better water for many people, among others. but progress controlling climate change, halting biodiversity loss, and achieving gender equality has lagged. a later environmental performance index spearheaded by yale and columbia universities ranks countries on performance indicators in well-established policy categories to determine whether and how well countries are meeting established environmental goals. this index incorporates measurements addressing environmental stresses to human health plus ecosystem health and natural resource management, collectively referred to as ecosystem vitality. the categories representing environmental health include the environmental burden of disease and the effects of air and water quality on human health. those representing ecosystem vitality include air and water resources for ecosystems; biodiversity and habitat; and climate change, among others. although hampered by data gaps and the inability to track changes in countries' performance through time, the index provides a sense of countries' relative performance with regard to the environmental challenges faced by all. top performers in the rankings included iceland, switzerland, costa rica, and sweden. the united states ranked st well below japan and nations in the european union. a resource-accounting approach pioneered in the s by geographers rees and wackernagel ( ) translates humans' impact on nature, particularly resource consumption, into a metaphorical ecological footprint. this accounting estimates the area required by a city, town, nation, or other human community to produce consumed resources and absorb generated wastes; it then compares the physical area occupied by a city or country with the area required to supply that city or country's needs. the largest cities of baltic europe, for example, appropriate areas of forest, agricultural, marine, and wetland ecosystems that are at least - times larger than the areas of the cities themselves. according to the global footprint network, national ecological footprints in ranged from a high of . hectares per person for the united arab emirates to . hectares per person for timor-leste and . for afghanistan and bangladesh. the united states' ecological footprint - . hectares per person -tied for fourth among nations with populations of at least million. one-hundred and four of these nations operate under ecological deficits; that is, their . cumulative effects (frequency of catastrophic natural disasters; costs of weather-related property damage; human death tolls; government subsidies of environmentally destructive activities such as fishery overcapitalization, below-cost timber sales, water projects, and agricultural supports; replacement costs for ecological services; pricing that reflects environmental costs; ''green'' taxes; rise in polycultural agricultural practices; number of organic farms) a these indicators have been or could be used to monitor status and trends in environmental quality, including dimensions of biotic impoverishment. without a full spectrum of indicators, however, and without coupling them to direct measures of biological condition, only a partial view of the degree of biotic impoverishment will emerge. consumption exceeds the biological capacity of their lands and waters to provide needed resources and absorb their wastes. at their present rates of consumption, these nations are therefore overexploiting either their own resources or those of other nations. by ecological footprint accounting, raising the nearly billion people on earth in the early st century to living standards -and thus ecological footprints -equal to those in the united states would require four planets more than the only one we have. clearly, humans are consuming more resources, and discarding more waste, than earth's living systems can produce or absorb in a given time period. this gap is the global sustainability gap the world now faces. most environmental indexes and accounting systems are still human centered; they still do not measure the condition of the biosphere itself. we may know that biodiversity's services are worth huge sums of money and that our hometown's ecological footprint is much bigger than the town's physical footprint, but how do we know whether specific actions damage living systems or that other actions benefit them? how do we know if aggregate human activity is diminishing life on earth? to answer this question, we need measures that directly assess the condition of living systems. biological assessment directly measures the attributes of living systems to determine the condition of a specific landscape. the very presence of thriving living systems -sea otters and kelp forests off the central california coast; salmon, orcas, and herring in pacific northwest waters; monk seals in the mediterranean sea -says that the conditions those organisms need to survive are also present. a biota is thus the most direct and integrative indicator of local, regional, or global biological condition. biological assessments give us a way to evaluate whether monetary valuations, sustainability indexes, and ecological footprints are telling the truth about human impact on the biosphere. biological assessments permit a new level of integration because living systems, including human cultures, register the accumulated effects of all forms of degradation caused by human actions. direct, comprehensive biological monitoring and assessment has been done for many aquatic systems; measures are less developed for terrestrial systems. the index of biological integrity (ibi), for example, was developed in to assess the health of streams in the us midwest and has since helped scientists, resource managers, and citizen volunteers understand, protect, and restore rivers in at least countries worldwide (karr ) . borrowing from the same page as more recent sustainability indexes, ibi takes the concept behind economic indexes like gdp or the consumer price index -that of multiple indicators integrated into a multimetric index -and applies it to animals and plants in bodies of water or other environments. the specific measurements ( table ) are sensitive to a broad range of human effects in waterways, such as sedimentation, nutrient enrichment, toxic chemicals, physical habitat destruction, and altered flows. the resulting index combines the responses of biological parts such as species, as well as processes such as food web dynamics, to human actions. indexes of biological integrity have been developed for a number of aquatic and terrestrial environments; the most widely used indexes for assessing rivers examine fishes and benthic (bottom-dwelling) invertebrates. these groups are abundant and easily sampled, and the species living in virtually any water body represent a diversity of anatomical, ecological, and behavioral adaptations. as humans alter watersheds and water bodies, changes occur in taxonomic richness (biodiversity), species composition (which species are present), individual health, and feeding and reproductive relationships. sampling the inhabitants of a stream can tell us much about that stream and its landscape. biological diversity is higher upstream of wastewater treatment plants than downstream, for example, whereas, at the same location, year-toyear variation is low (figure ) . biological sampling can also reveal differences between urban and rural streams. for instance, samples of invertebrates from one of the best streams in rural king county, in the us state of washington, contain kinds, or taxa, of invertebrates; similar samples from an urban stream in the city of seattle contain only . the rural stream has taxa of mayflies, stoneflies, and caddisflies; the urban stream, only or . when these and other metrics are combined in an index based on invertebrates, the resulting benthic ibi (b-ibi) ranks the condition, or health, of a stream numerically ( table ) . a benthic ibi can also be used to compare sites in different regions. areas in wyoming's grand teton national park where human visitors are rare have near-maximum b-ibis. streams with moderate recreation taking place in their watersheds have b-ibis that are not significantly lower than those with no human presence, but places where recreation is heavy are clearly damaged. urban streams in the nearby town of jackson are even more degraded, yet not as bad as urban streams in seattle. nation-specific biological assessments also can be and are being done. the us environmental protection agency ( ), for example, performed a nationwide survey of stream condition using an ibi-like multimetric index. the survey found that % of us stream miles were in good condition in table biological attributes in two indexes of biological integrity comparison with least-disturbed reference sites in their regions, % were in fair condition, and % were in poor condition ( % were not assessed). the agency has been expanding this effort to include other water resource types, including coastal waters, coral reefs, lakes, large rivers, and wetlands. since , the heinz center ( ) has published two editions of its report on the state of us ecosystems, which seek to capture a view of the large-scale patterns, conditions, and trends across the united states. the center defined and compiled a select set of indicators -specific variables tracking ecosystem extent and pattern, chemical and physical characteristics, biological components, and goods and services derived from the natural world -for six key ecosystems: coasts and oceans, farmlands, forests, fresh waters, grasslands and shrublands, and urban and suburban landscapes. among the many conclusions of the report were that the acreage burned every year by wildfires was on the increase; nonnative fish had invaded nearly every watershed in the lower states; and chemical contaminants were found in virtually all streams and most groundwater wells, often at levels above those set to protect human health or wildlife. on the plus side, ecosystems were increasing their storage of carbon, soil quality was improving, and crop yields had grown significantly. the massive international un millennium ecosystem assessment ( ) remains the gold standard for synthesizing ecological conditions at a variety of scales. from through , the project examined the full range of global ecosystems -from those relatively undisturbed, such as natural forests, to landscapes with mixed patterns of human use to ecosystems intensively managed and modified by humans, such as agricultural land and urban areas -and communicated its findings in terms of the consequences of ecosystem change for human well-being. the resulting set of reports drew attention to the many kinds of services people derive from ecosystems, specifically, supporting services, such as photosynthesis, soil formation, and waste absorption; regulating services, such as climate and flood control and maintenance of water quality; provisioning services, such as food, wood, and nature's pharmacopoeia; creek near portland, oregon (united states), taxa richness differed little between years but differed dramatically between sites upstream of a wastewater outfall and sites downstream. (b) taxa richness also differed between two creeks with wastewater outfalls (tickle and north fork deep) and one creek without an outfall (foster). all three streams flowed through watersheds with similar land uses. fig. for graphs of selected b-ibi metrics at these sites. and cultural services from scientific to spiritual. in addition, the reports explicitly tied the status of diverse ecosystems and their capacity to provide these services to human needs as varied as food and health, personal safety and security, and social cohesion. even while recognizing that the human species is buffered against ecological changes by culture and technology, the reports highlighted our fundamental dependence on the flow of ecosystem services and our direct responsibility for the many faces of biotic impoverishment. among other findings, the assessment found that % of the services provided by ecosystems are being degraded at levels that derail efforts to stem poverty, hunger, and disease among the poor everywhere. government subsidies that provide incentives to overharvest natural resources are a leading cause of the decline in renewable natural resources. such declines are not limited to coral reefs and tropical forests, which have been on the public's radar for some time; they are pervasive in grasslands, deserts, mountains, and other landscapes as well. moreover, the degradation of ecosystem services could grow worse during the first half of the st century, effectively blocking achievement of the united nations' eight millennium development goals. the core message embodied in ecological, especially biological, assessments is that preventing harmful environmental impacts goes beyond narrow protection of clean water or clear skies, even beyond protecting single desired species. certain species may be valuable for commerce or sport, but these species do not exist in isolation. we cannot predict which organisms are vital for the survival of commercial species or species we want for other reasons. failing to protect all organisms -from microbes and fungi to plants, invertebrates, and vertebrates -ignores the key contributions of these groups to healthy biotic communities. no matter how important a particular species is to humans, it cannot persist outside the biological context that sustains it. direct biological assessment objectively measures this context and is fundamental to advancing the kinds of syntheses like those of the millennium ecosystem assessment. every animal is alert to dangers in its environment. a microscopic protozoan gliding through water responds to light, temperature, and chemicals in its path, turning away at the first sign of something noxious. a bird looking for food must decide when to pursue prey and when not, because pursuit might expose the bird to predators. the bird might risk pursuit when it is hungry but not when it has young to protect. animals that assess risks properly and adjust their behavior are more likely to survive; in nature, flawed risk assessment often means death or the end of a genetic line. humans, too, are natural risk assessors. each person chooses whether to smoke or drink, fly or take the train, drive a car or ride a motorcycle and at what speeds. each decision is the result of a partially objective, partially subjective internal calculus that weighs benefits and risks against one another. risk is a combination of two factors: the numerical probability that an adverse event will occur and the consequences of the adverse event. people may not always have the right signals about these two factors, however, and may base their risk calculus on the wrong clues. city dwellers in the united states generally feel that it is safer to drive home on a saturday night than to fly in an airplane, for example. even though the numerical odds of an accident are much higher on the highway than in the air, people fear more the consequences of an airplane falling out of the sky. human society also strives to reduce its collective exposure to risks. governments seldom hesitate to use military power to defend their sovereignty or, albeit more reluctantly, their regulatory power to reduce workplace risks and risks associated with consumer products like automobiles. but people and their governments have been much less successful in defining and reducing a broad range of ecological risks, largely because they have denied that the threats are real. policies and plans generated by economists, technologists, engineers, and even ecologists typically assume that the lost and damaged components of living systems are unimportant or can be repaired or replaced. widespread ecological degradation has resulted directly from the failure of modern society to properly assess the ecological risks it faces. like the fate of old kingdom egypt or easter island, our civilization's future depends on our ability to recognize this deficiency and correct it. risk assessment as formally practiced by various government agencies began as a way to evaluate the effects of toxic substances on human health, usually the effects of single substances, such as pollutants or drugs, from single sources, such as a chemical plant. during the s, the focus widened to encompass mixtures of substances and also ecological risks. ecological risk assessment by the us environmental protection agency ( ) asks five questions: is there a problem? what is the nature of the problem? what are the exposure and ecological effects? (a hazard to which no one or nothing is exposed is not considered to pose any risk.) how can we summarize and explain the problem to affected parties, both at-risk populations and those whose activities would be curtailed? how can we manage the risks? even though these are good questions, ecological risk management has not made any visible headway in stemming biotic impoverishment. its central failing comes from an inability to correctly answer the second question, what is the nature of the problem? our present political, social, and economic systems simply do not give us the right clues about what is at risk. none of society's most familiar indicatorswhether gdp or number of threatened and endangered species -measure the consequences, or risks, of losing living systems. if biotic impoverishment is the problem, then it only makes sense to direct environmental policy toward protecting the integrity of biotic systems. integrity implies a wholeness or unimpaired condition. in present biological usage, integrity refers to the condition at sites with little or no influence from human activity; the organisms there are the products of natural evolutionary and biogeographic processes in the absence of humans. tying the concept of integrity to an evolutionary framework provides a benchmark against which to evaluate sites that humans have altered. directing policy toward protecting biological integrity -as called for in the united states' clean water act, canada's national park act, and the european union's water framework directive, among others -does not, however, mean that humans must cease all activity that interferes with a ''pristine'' earthly biota. the demands of feeding, clothing, and housing billions of people mean that few places on earth will maintain a biota with evolutionary and biogeographic integrity. rather, because humans depend on living systems, it is in our interest to manage our activities so they do not compromise a place's capacity to support those activities in the future; that capacity can be called ecological health. ecological health describes the preferred state of sites heavily used for human purposes: cities, croplands, tree farms, water bodies stocked for fish, and the like. at these places, it is impractical to set a goal of integrity in an evolutionary sense, but we should avoid practices that damage these places to the point where we can no longer derive the intended benefits indefinitely. for example, agricultural practices that leave behind saline soils, depress regional water tables, and erode fertile topsoil faster than it can be renewed destroy the land's biological capacity for agriculture; such practices are unhealthy in both ecological and economic terms. biological integrity as a policy goal redirects our focus away from maximizing goods and services for the human economy toward ways to manage human affairs within the bounds set by the natural economy. it begins to turn our attention away from questions such as, how much stress can landscapes and ecosystems absorb? to ones such as, how can responsible human actions protect and restore ecosystems? in contrast to risk assessment, striving to protect biological integrity is more likely to lead away from mandated corrective actions in the form of technological fixes for environmental problems and toward practices that prevent ecological degradation and encourage ecological restoration. leopold ( , pp. - ) , in a sand county almanac, was the first to invoke the concept of integrity in an ecological sense: ''a thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community. it is wrong when it tends otherwise.'' managing for biological integrity requires the kind of ethical commitment inherent in leopold's words. we are called to curb consumerism and limit population size, to embrace less-selfish attitudes toward land stewardship, and to understand that the biosphere matters. instead of calling on human technical and spiritual wellsprings to manage resources, we have to call on them for managing human affairs. we have to find and use appropriate measurements for all the factors contributing to biotic impoverishment, be they climate change, overharvesting, agriculture, or environmental injustice. measurement of environmental impact founded on the evolutionary idea of integrity allow us to directly assess biotic condition and to compare that condition with what might be expected in a place with little or no human influence. at least then we can make an informed choice: continue with activities that degrade biotic condition or think of an alternative. the ecological world is a complex, variable, and often unpredictable system. when managing for ecological risks, people and their governments need to expect the unexpected and develop formal, yet flexible means of coping with environmental surprises. rather than plunge ahead with projects entailing ecological risks because they can be done, decision makers should follow the precautionary principle, which holds that regulators should act to prevent potential environmental harm even in the absence of certainty. it acknowledges the existence of uncertainty rather than deny it, and it includes mechanisms to safeguard against potentially harmful effects. although inappropriate ecological risk assessment and management are more often the norm today, modern institutions are capable of recognizing ecological threats correctly and responding to them in time, as they did with the montreal protocol. a decade after the agreement's adoption, satellite measurements in the stratosphere indicated that ozone-depleting pollutants were in fact on the decline. given the treaty's success, some policy experts have suggested using it to help curb global warming. specifically, negotiators at the annual meeting of signatory parties were considering a proposed expansion of the ozone treaty to phase out the production and use of industrial chemicals called hydrofluorocarbons, which have thousands of times the global warming potential of carbon dioxide. even though the montreal protocol was not designed to fight climate change, policymakers in favor of the new proposal say that it could and should be used to achieve broader environmental objectives. early in the th century, two sciences of ''home maintenance'' began to flourish: the young science of ecology (from the greek oikos, meaning home) and a maturing neoclassical economics (also from oikos). ecology arose to document and understand the interactions between organisms and their living and nonliving surroundings -in essence, how organisms make a living in the natural economy. in fact, ernst haeckel, who coined the term in the s, defined ecology in an article as the body of knowledge concerning the economy of nature. neoclassical economics, in contrast, reinforced humans' self-appointed dominion over nature's wealth. it brought unparalleled gains in societal welfare in some places, but it also divorced the human economy from the natural one on which it stands (see figure ) . by now it is clear to economists and ecologists alike that human actions and their effects have reached scales unprecedented in the history of life. we have altered earth's physical and chemical environment, changed the planet's water and nutrient cycles, and perturbed its climate. we have unleashed the greatest mass extinction in my and distorted the structure and function of nonhuman and human communities worldwide. in trying to make our own living, we have jeopardized the earth's capacity to sustain other species and our own species as well. we are losing the bio in biosphere. now, faced with these unprecedented losses, we need to understand -not deny -the ecological consequences of what we do. we urgently need a new science and art of home maintenance, one that sees the human species' role as ecosystem engineer for what it has become and reconnects human and natural economies. this new science can help us understand and correctly interpret the consequences of human-driven change. by using indicators that measure what matters for sustaining living systems, this new science can make nature visible again and shed new light on the value of the ancient heritage we share with the larger biosphere. it can help us reunite the fragments of our worldview and re-create ethical, social, and ecological bonds that were put aside two centuries ago in the name of progress. and such a science can enable us to reengineer our own social, political, and economic institutions instead of ecosystems. this we must donow -before we impoverish the biosphere and ourselves for all time. the wayfinders: why ancient wisdom matters in the modern world guns, germs, and steel: the fates of human societies evolution, consequences and future of plant and animal domestication collapse: how societies choose to fail or succeed floods, famines, and emperors: el niño and the fate of civilizations the state of the nation's ecosystems: measuring the lands, waters, and living resources of the united states environment, scarcity, and violence intergovernmental panel on climate change (ipcc) ( ) climate change seven foundations of biological monitoring and assessment a sand county almanac: and sketches here and there the challenge for africa. new york: pantheon books. millennium ecosystem assessment ( ) ecosystems and human well-being: synthesis ultimate security: the environmental basis of political stability song of the dodo: island biogeography in an age of extinction the economics of ecosystems and biodiversity: mainstreaming the economics of nature: a synthesis of the approach, conclusions and recommendations of teeb. united nations environment programme guidelines for ecological risk assessment wadeable streams assessment: a collaborative survey of the nation's streams, epa -b- - our ecological footprint: reducing human impact on the earth the little things that run the world the earth in transition: patterns and processes of biotic impoverishment a short history of progress key: cord- -e le oh authors: hamada, kazuhiro; kaneko, tatsuo; chen, ming qing; akashi, mitsuru title: one-step nanomorphology control of self-organized projection coronas in uniform polymeric nanoparticles date: - - journal: polymer doi: . /j.polymer. . . sha: doc_id: cord_uid: e le oh abstract uniform polymeric nanoparticles with various morphologies of projection coronas like the viruses in the coronavirus group have been formed by the self-organization of macromolecular chains polymerizing in a dispersion system of styrene (st), acrylonitrile (an) and poly(ethylene glycol) monomethoxymonomethacrylate (pegm) in a polar solvent (water/ethanol). an increase in the water composition reduced the crystallization degree of an units, resulting in a variety of the nanoparticle morphology such as the increased particle size, the reduced projection size, the increased projection number, and the decreased inter-projection distance. the difference in the projection morphology strongly affected a dispersibility in water. polymeric particles of nano-and micro-scale have an extremely large specific surface area giving an excellent material sorbability, which attracts broad attention in the fields of drug delivery [ , ] , chromatography and catalyst [ ] . the morphological control of particles is one method for creating new functions and improving the performance of polymeric nanoparticles. since it is expected that non-spherical nanoparticles can have a larger specific area and higher material sorbability than nanospheres of equal size, particles of various morphologies, such as raspberry-like [ , ] , snowman-like [ , ] , and virus-like [ , ] have been prepared by multistep preparation techniques such as hetero aggregation [ , ] and seed polymerization [ ] [ ] [ ] [ ] . in our previous works, polymeric nanoparticles on which nano-projections were uniformly distributed over the whole surface have been prepared using one-step preparation by a dispersion radical copolymerization of styrene, acrylonitrile and poly(ethylene glycol) monomethoxymonomethacrylate [ ] . a similarity to the virus arises since these nanoparticles are formed by the self-organization of the macromolecular chains. another similarity is based on the morphology. tomato bushy stunt virus has a diameter of nm and projections with a length of nm on the surface [ ] , while the viruses in coronavirus group has a diameter range - nm and many projections with a length range - nm [ ] . most viruses have the projections with a bigger or smaller size on the surface to a greater or less extent; the viruses have a variety of projection morphology in terms of the size, the number, and the inter-projection distance, showing individual living characteristics. on the other hand, the morphology and number density of projection coronas of the present synthetic nanoparticles have never been controlled although the growth of the projections was observed by a change in the polymerization time [ ] . in this paper, we aim to give a variety of the morphology of the nanoparticle by controlling the particle size, the projection size, the projection number, and the inter-projection spacing. we find that the morphology is successfully controlled by changing the solvent for self-organization, and the morphology control is very effective on the water-dispersibility of the nanoparticles. ltd) which was used as a radical initiator was recrystallized from ethanol (wako pure chemical ind. ltd) before use. a mixture of distilled water and ethanol was used as the polymerization medium. dispersion polymerization of st, an, and pegm were carried out batchwise in a glass tube. the general procedure used to prepare all of the nanoparticles was as follow: st ( . mmol), an ( . mmol), pegm ( . mmol; . mol% to st and an), and aibn ( mol% of total monomers) were added to the ethanol/water (z / , / , / , / , / , / , / , / , / , / , / (v/v)) mixture (table ) . each polymerization batch was prepared in a glass tube and was repeatedly degassed by freeze-thaw cycles in a vacuum apparatus, sealed off, and then placed in an incubator at c for h. the resulting solutions were dialyzed in ethanol and distilled deionized water using a cellulose doalyzer tube to remove unreacted monomer. the ratex particles were then centrifugated and redispersed in water. scanning electron microscopy (sem) images were obtained with a jsm- f microscopic operated at an acceleration voltage of . kv. specimens were prepared by slow evaporation of a drop of approximately diluted solution deposited onto a brass stage followed by osmium spattering. transmission electron microscopy (tem) images were obtained with a hitachi h- h microscopic operated at an acceleration voltage of kv at a magnification of , !. specimens were prepared by slow evaporation of a drop of approximately diluted solution deposited onto a collodioncoated copper mesh grid followed by carbon spattering. dynamic light scattering (dls) measurement was performed with a beckman coulter laser diffraction particle size analyzer ls - . viscosity measurement was performed on peg solution at a temperature of c by means of standard ostwald viscometers. before measuring viscosity, the samples were stabilized for sufficient time at the given temperature. electron spectroscopy for chemical analysis (esca) was performed with a shimadzu esca apparatus employing mg ka radiation ( . ev) and a pass energy of . ev. the higher resolution utility scans were used to determine the atomic concentrations of carbon, nitrogen, and oxygen. x-ray diffraction patterns were taken with a rigaku x-ray generator (ultrax ; thin-film out-of-plain system) emitting ni-filered cu ka radiation ( kv, ma) at scanning angles ranging from to at a scanning rate of min k . the terpolymers were synthesized by dispersion copolymerization of st, an, and pegm (number-average molecular weight m n z ) in the presence of aibn (radical initiator) in a water/ethanol mixed solvent with various compositions at c for h (scheme and table ). the reaction solution was transparent in the early stages but became cloudy with the progress of polymerization in the water composition range - vol%. on the other hand, the reaction solution was cloudy before polymerization in a water composition range of more than % presumably due to the poor solvent affinity of the st monomer and an monomer. fig. shows scanning electron microscopy (sem) images of the nanoparticles. when the water composition was and vol%, nanoparticles with projection coronas were not formed (not shown). as shown in fig. (a) , nanoparticles prepared in the solvent with a water composition of vol% had projection coronas similar to a coronavirus in morphology. in solvent compositions of , , and vol%, projection coronas were also confirmed distinctly ( fig. (b)-(d) ). however, this kind of nanoparticle morphology was not formed when polymerization was performed in a water composition of more than vol%. this may be due to a difference in the polymerization system; the dispersion system the polymerization was carried out in the solvent ( ml) at c for h. is indispensable for projection formation. when the water composition increased, the particle morphology changed. the effects of water composition on particle morphology are shown in figs. - . fig. shows the particle size measured by dynamic light scattering (dls), which is close to that estimated from the sem images. the particle size increased with an increase in water composition. the same phenomenon was observed in the nanoparticles composed of st and pegm [ ] . the hydrophobic st formed a larger nanoparticle core under conditions of higher water content. fig. shows the average volume of a projection (v p ) calculated from fig. using an image data processing tool (win roof) based on the assumption of a hemispherical projection; the average number of projections (n) on a particle was also counted. when the water composition was vol%, v p was . ! nm which was about % of the particle volume, v. increasing the water composition decreases v p and increases v, and v p became about . % of v. when the water composition was vol%, n was which increased up to with an increase in water composition. fig. shows the average inter-projection distance (d) and the specific surface area (s) of the nanoparticles. d was measured directly from the sem images. when the water composition was vol%, d was nm; d decreased down to nm with an increase in the water composition. figs. and show that a larger number of smaller projections appeared on the nanoparticle surface with a higher water composition. when the water composition was vol%, s was . ! k nm /nm which increased to . ! k nm /nm with an increase in water composition. consequently, s was controlled by a change in solvent composition and s then was about two times larger than that of nanospheres of equal size. validity of all the calculated values were confirmed by recalculation using transmission electron microscopic images (not shown). since the projection morphology may be related to the solvent affinity of the particle surface, i.e. peg chains, we investigated the solvent affinity of peg in terms of reduced viscosity behavior. fig. shows changes in the reduced viscosity as a function of the water composition. peg with a molecular weight of , was used as a sample because a polymer chain with a larger molecular weight shows a larger change in reduced viscosity by a conformation change. the reduced viscosity increased with increasing water composition showing that the peg chain took an extended conformation because of increased solvent affinity when the water composition increased. this finding suggests that pegm graft chains existing on the nanoparticle surface take an extended conformation in a high water composition environment. elemental analysis on the surface of the nanoparticles was performed by x-ray photoelectron spectroscopy (xps). we show o/c and n/c on the surface of the particles prepared under different water compositions together with the bulk composition of an units to the total units in fig. . the o/c ratio slightly decreased with an increase in water composition while the n/c ratio increased. this result shows that the amount of an increased on the particle surface as the water composition increased in spite of a decrease in the bulk composition from . to . unit mol%. this phenomenon indicated the excess surface accumulation of an units, which may be associated with the fact that pan has some solubility in water/ethanol solvent [ ] . since the number of projections also increased, we can support the previously-proposed hypothesis that the projection was mainly comprised of an [ ] . the slight decrease in o/c may result from a decrease in the relative amount of peg to an units. we previously hypothesized that the projection formed on the particle surface was caused by the crystal growth of an. using the present samples with various morphologies, we attempted to investigate the relationship between the nanoparticles crystallinity and the projection size. the main peak of the crystal of pan is detected in qz . ( ) according to the literature [ ] . fig. (a) shows xrd patterns of the nanoparticles showing a sharp peak at qz . (q, diffraction angle) and large amorphous peak at around . crystallization degree of an units to total nanoparticles were shown as a function of the projection volume in fig. (b) . the crystallinity of nanoparticle linearly increased with an increase in projection volume. as a consequence, the projection on the particle surface can be formed by the crystal growth of an units. based on these results, we discussed the reason for the change in the arrangement of projection coronas on the nanoparticles. we previously proposed a hierarchical structure for the nanoparticles with projection coronas: peg grafts exist on an an-rich shell covering the st-rich core [ ] . since the nanoparticle created from st and pegm formed no projections, an is indispensable in the formation of projection coronas [ ] . since pan has some solubility in water/ethanol solvent [ ] , pan chains having pegm grafts may be capable of solvating. the shell in pan and pegm may be swollen in solvent with connecting to a pst core. according to a previous study, the morphology was spherical at the beginning of polymerization, and the projection grew with polymerization time but the number of projections was constant regardless of polymerization time [ ] . namely, the number of projections is determined at the early stages of projection formation where pegm chains first stick out from the shell dragging out pan units with them. in this case, the solvent affinity of a pegm may be an important factor for projection formation. if the water composition is higher, a larger number of an units exist on the particle surface (fig. ) in the presence of bettersolvated pegm chain yielding a larger number of projection nuclei. the solvation can give a chain mobility to make a crystallization of an units and the projection is formed. if the an composition is high, the crystal growth happens in a lot of parts because a number of crystal nuclei were formed on the particle surface. the higher projection population might make its size (fig. ) . thus, solvent affinity is a crucial factor in controlling particle morphology. finally, we investigated the relationship between the nanoparticle morphology and the water-dispersibility. although the phenomenon that the smaller nanoparticles have the higher water-dispersibility was widely-confirmed, the effects of their morphology were never investigated as far as we know. we performed a simple test of the waterdispersibility of the present nanoparticles; after the waterdispersion solutions of the nanoparticles ( mg/ml) whose morphologies are shown in fig. (a)-(d) were centrifuged at rpm for min at c, their opaqueness was observed. fig. shows the representative photograph of the dispersions (the morphology of right side and left side was shown in fig. (a) and (d), respectively) after the centrifugation. while the nanoparticles with the smallest particle size and the largest projections whose morphology is shown in fig. (a) was precipitated, the nanoparticles with the largest particle size and the smallest projections ( fig. (d) ) kept the water-dispersibility well. the water-dispersibility increased with increasing the particle size, contrarily to the widely-confirmed phenomenon in this size range of nm- mm. in the present case, the projection morphology may affect the water-dispersibility more effectively rather than the particle size. the increase in the number of the projection with the reduced size increased s, which should strongly enhance the water-dispersibility of the nanoparticles. in summary, the morphology of nanoparticles, on which nano-projections were distributed over the entire surface prepared by a dispersion copolymerization of an, st, and pegm in water/ethanol mixed solvent was successfully controlled by changing the solvent composition. when the water composition was increased up to vol% to total solvent volume, the particle size increased, the projection size decreased, the number of projections increased, and the spacing between projections decreased. on the other hand, nanoparticles with projection coronas were not formed when the water composition was higher than vol% since the dispersion system was not created during polymerization. the morphology control was based on the solvent affinity of pegm graft chains, which extended better in higher water compositions and concentrate the an units in the shell layer to form larger amount of crystalline projections. the ratio of the specific surface area to the particle volume was from . ! k to . ! k nm /nm , which is twice as large as that of nanospheres of equal size. the increased number of the projection with the reduced size was effective on increasing the water-dispersibility of the nanoparticles. as a result, we prepared the nanoparticles with various morphologies similar with the viruses in the coronavirus group. the study is currently continuing to investigate the morphology-specific catch-up behavior of the nano-sized materials such as viruses using the inter-projection gap on the nanospheres. introduction to protein structure classification and nomenclature of viruses. fourth report of the international committee on taxonomy of viruses polymer handbook water-dispersion behavior for the nanoparticles of poly{styrene-coacrylonitrile-co-poly(ethylene glycol) monomethoxymonomethacrylate} after centrifugation ( rpm for min, c); (right) the nanoparticles prepared in the solvent with a water composition of vol%. the morphology was shown in fig. (a), (left) the nanoparticles prepared in the solvent with a water composition of vol% this research was partly supported by a grant-in-aid for tokuyama science foundation. we show an appreciation to mr. kakoi, a technician of kagoshima university, and dr okunishi of jeol for tem studies, and mr ohzono a technician of kagoshima university for xps studies. key: cord- -a wl f authors: lawler, j. j.; spencer, b.; olden, j. d.; kim, s.-h.; lowe, c.; bolton, s.; beamon, b. m.; thompson, l.; voss, j. g. title: . mitigation and adaptation strategies to reduce climate vulnerabilities and maintain ecosystem services date: - - journal: climate vulnerability doi: . /b - - - - . - sha: doc_id: cord_uid: a wl f abstract increasing temperatures and altered precipitation regimes associated with human-caused changes in the earth s climate are having substantial impacts on ecological systems and human well-being. maintaining functioning ecosystems, the provision of ecosystem services, and healthy human populations into the future will require integrating adaptation and mitigation strategies. adaptation strategies are actions that help human and natural systems accommodate changes. mitigation strategies are actions that reduce anthropogenic influences on climate. here, we provide an overview of what will likely be some of the most effective and most important mitigation and adaptation strategies for addressing climate change. in addition to describing the ways in which these strategies can address impacts to natural and human systems, we discuss the social considerations that we believe must be incorporated into the development and application of mitigation or adaptation strategies to address political situations, cultural differences, and economic limitations. adaptation adjustment in natural or human systems in response to actual or expected climatic stimuli or their effects, which moderates harm or exploits beneficial opportunities. various types of adaptation can be distinguished, including anticipatory and reactive adaptation, private and public adaptation, and autonomous and planned adaptation (ipcc ) . climate change a significant and lasting change in the statistical distribution of weather patterns over periods ranging from decades to millions of years. we focus largely on changes in temperature and precipitation, as they are changing in many regions and have direct impacts on ecosystems and human well-being. mitigation actions that reduce the effects of humans on the climate. resilience amount of change a system can undergo without changing state (ipcc ) . sensitivity it is the degree to which a system is affected, either adversely or beneficially, by climate-related stimuli. the effect may be direct (e.g., a change in crop yield in response to a change in the mean, range, or variability of temperature) or indirect (e.g., damages caused by an increase in the frequency of coastal flooding due to sealevel rise) (ipcc ) . vulnerability the degree to which a system is susceptible to, or unable to cope with, adverse effects of climate change, including climate variability and extremes. vulnerability is a function of the character, magnitude, and rate of climate variation to which a system is exposed, its sensitivity, and its adaptive capacity (ipcc ) . virtually all regions of the earth are experiencing rapid changes in temperature, precipitation, and/or other aspects of climate. human-caused changes in the earth's climate are greatly altering ecological systems, with consequences for human well-being. for example, recent studies have documented shifts in the distribution of plants and animals, advances in the timing of key ecological processes, and extinctions of wildlife populations and species that are likely linked to recent increases in temperature (parmesan ) . such changes are creating regional combinations of environmental conditions that, within the next - years, may have no current-day analog. the resulting changes in ecosystem function will undoubtedly affect the provision of ecosystem services for human communities. maintaining or increasing ecosystem services into the future will require integrating adaptation strategies (actions that help human and natural systems accommodate changes) and mitigation strategies (actions that reduce anthropogenic influences on climate) ( figure ). mitigation strategies are those that reduce the magnitude of climate change that will occur. the vast majority of these strategies involve either reducing greenhouse gas (ghg) emissions or sequestering carbon, although they can address any human climate forcing. by contrast, adaptation can be defined as alterations to human or natural systems that are designed to reduce the negative impacts ofor exploit the opportunities created byclimate change (ipcc a) . adaptation strategies are much more varied than mitigation strategies, as they are more often specific to particular systems or locations. mitigation and adaptation strategies designed to address changes in climate may have additional positive or negative effects on ecosystem functioning, ecosystem services, and human well-being. for example, replacing fossil fuels with renewable energy sources reduces ghg emissions while simultaneously reducing air pollution, acid rain, and smog, as well as asthma and respiratory ailments of humans. conversely, coastal armoring, the building of seawalls, breakwaters, and jetties, may reduce damage to built structures from the combined effects of sea-level rise and storm surges but such actions can negatively affect coastal ecosystems. in this chapter, we provide an overview of what will likely be some of the most effective and most important mitigation and adaptation strategies for addressing changes to the climate system stemming from increased ghg emissions. we attempt to cover the entire spectrum of life, from plants and animals to humans, as well as the geophysical properties of our planet that support these organisms. in relation to human populations, we focus primarily on human health as an index of well-being. we also introduce concepts of climate justice. many of our examples are taken from the united states, although we also include other regions of the world in our analysis as appropriate. we discuss various ways in which mitigation and adaptation strategies can help reduce the magnitude and the impacts of the changes we are likely to experience, as well as improve human health and directly or indirectly affect ecosystem functions and services. finally, although our analysis sets priorities and orders of importance for an ideal world unimpeded by political considerations, cultural differences, or economic limitations, we include a discussion of the social considerations that we believe must be incorporated into the development and application of mitigation or adaptation strategies. in the following sections, we briefly discuss what will likely be some of the most effective mitigation strategies for addressing climate change, largely focusing on strategies to reduce co and other ghg emissions ( table ) . we have grouped these strategies into five categories: transportation, shelter, food, energy, and carbon storage and bioengineering. although we focus on mitigation strategies that will reduce the magnitude of future climatic changes, we also discuss how these strategies can improve ecosystem functioning and, in some cases, human health. transportation, the movement of people and goods, significantly impacts climate through the emission of ghgs. in , transportation accounted for % of the world's energy-related ghg emissions and road transportation represented % of the total carbon dioxide (co ) emissions from transportation (ipcc b) . in addition to being a major source of emissions, transportation also significantly affects ecosystem functions and services. pollution from vehicles affects ecosystem services such as clean air and water directly, and indirectly through impacts on the plants and animals that regulate ecosystem function. as species are lost from or move into ecosystems the functioning of those systems can change, resulting in altered ecosystem services. human health is likewise affected by pollution from vehicles (e.g., through chronic lung disease). transportation networks and moving vehicles also fragment landscapes, reducing the flows of energy and biota resulting in isolated populations and potentially making these populations vulnerable to extirpation and impairing ecosystem functions. mitigation strategies that address transportation can be placed into three basic categories: those that increase the efficiency of particular transportation approaches, those that result in a shift in the mode of transportation, and those that reduce the amount of transportation that is needed. here, we briefly table examples of mitigation strategies aimed at reducing ghg emissions or, in the case of the last category, sequestering carbon to reduce co concentrations in the atmosphere mitigation strategies barriers to implementation a large reduction in ghg emissions can be accomplished by increasing the fuel efficiency of vehicles. for example, increasing the efficiency of light-duty vehicles (cars and small trucks) has the potential to reduce their carbon emissions by up to % by (ipcc b) . such efficiencies can be attained by a combination of reducing vehicle weights, increasing aerodynamics, increasing engine efficiency (particularly diesel engine efficiency), and increasing the use of biofuels and electric and hybrid vehicles. in , the international energy association projected that the proportion of transportation energy supplied by biofuels could be increased to % (iea a) . although there is good evidence of successful biofuel markets and programs (e.g., ethanol from sugarcane in brazil, goldemberg ) , there are also negative consequences of increased biofuel production for biodiversity and carbon storage (fargione et al. ; fletcher et al. ) . one opportunity for transportation mitigation strategy is the promotion of behavioral changes of drivers. efficiency of road vehicles could be improved by - % by changes in driving styles, improved maintenance, more efficient tires, reduced idling, and traffic design and maintenance (ipcc b). a second approach to reducing emissions and many of the other effects of transportation involves changing the ways in which people and goods are moved. one such approach is to increase the use of public and human-powered transportation. increasing the amount of walking, biking, and use of public transit will depend largely on the available infrastructure and the capacity to provide it. supporting the development of 'walkable' cities and neighborhoods, bike-friendly roadways and bike paths, carpooling and car-sharing programs, and efficient and safe mass transit are all approaches for shifting the mode of urban transport. present transportation alternatives vary by country, with europe, japan, and many developing countries having the highest levels of public transportation use, and the united states having the greatest use of personal automobiles. in some developing countries, such as vietnam or china, personal automobile use is a recent phenomenon, and personal motorized transport is a high aspiration because it conveys increased social status. this disparity in the historical development of the transportation sector will mean that mitigation strategies, barriers, and solutions will have to be location specific and may be dynamic over time. in addition to the reduction in emissions and the other environmental benefits of shifts to more human-powered transportation, such changes also have clear human health benefits resulting from increased exercise, weight management, and decreases in air pollution (nemet et al. ) . simultaneously improving the quality of public transport and leapfrogging transport-intensive stages of economic growth is needed in many countries to increase higher levels of mass transit use and human-powered transportation (wright and fulton ) . further reductions in emissions can be made by changing modes of transport for other transportation such as moving more freight by train and ship and less by truck. the third way to reduce transportation-related ghg emissions is to reduce the amount of transportation that is needed. decreasing the demand for transportation largely involves changes in human behavior, urban and landscape planning, and changes in technology. sprawling, low-density urban neighborhoods, such as those found in the united states, perpetuate automobile dependency and transform ecologically functional landscapes into subdivisions. compact, highdensity communities with local urban centers allow residents to work and live in close proximity, and help build social capital among residents and protect natural areas. in conjunction with human-centered transportation infrastructure such as sidewalks, bike lanes, and public transportation, compact neighborhoods reduce car dependency while simultaneously improving the physical fitness of residents. public green space and urban forests sequester carbon while improving air quality and mental well-being. increased vegetation, shade trees, and less asphalt combat urban heat-island effects and reduce energy demand for cooling buildings. advances in communication technologies also have the potential to reduce the need for transportation. the internet, online meeting, document sharing, and conference tools have led to an increase in telecommuting and web-based meetings. similarly, online meetings have begun to replace some inperson meetings and have the potential to dramatically reduce air travel, which can account for a significant portion of an individual's carbon footprint (fox et al. ). the most significant barriers to implementing mitigation strategies in the transportation sector are long-established personal transportation behaviors and the lack of government interest or capacity to invest in new technologies. changing the way and the amount that people move around will be very difficult. in the united states and elsewhere, people's relationships with the automobile involve issues of convenience, independence, safety, and social status. changing these relationships will involve making forms of public transportation more convenient, more effective, and more appealing. rising fuel costs and commute times may provide some opportunity for implementing transportation-based mitigation strategies. failing automobile industries and economic crises may provide another. similar to the projects undertaken by the civilian conservation corps in the united states in the depression of the s, modern-day work relief programs could be used to transform transportation systems. short of such crises, major changes in transportation will take a combination of regulatory action, education, and incentive programs. housing is one of the most idiosyncratic and regionally variable aspects of human culture. the design of structures is dependent on local climate, land access, available materials, and regional architectural histories. much of the recent human-caused change in climate stems, in large part, from the way we design, construct, and inhabit the built environment. the following section focuses on a representative selection of design strategies that mitigate the built environment's contribution to carbon emissions and, in many cases, provide cobenefits to human health and local/regional ecological resilience. building material extraction can have profound impacts on ecosystems and ecosystem services. mining degrades natural landscapes, destroys habitat, pollutes freshwater environments, and can expose human populations to toxins. poorly managed lumber extraction leads to deforestation, eliminates suitable habitats, erodes soils, and diminishes the earth's carbon sequestration capacity. mines that observe best management practices such as land reclamation have fewer negative impacts on climate, local ecosystems, and human health. sustainably harvested lumber can act as a long-term carbon sink and contribute to the productive and ecological resilience of forests. rapidly renewable lumber alternatives such as bamboo can offset the demand for slow-growing trees species, rebuild soils, and set the stage for reforestation. however, as with any renewable resource, expanding the production of bamboo in monocultures has the potential to eliminate and/or degrade existing ecosystems. eight percent of annual carbon emissions worldwide can be attributed to building material extraction, production, transportation, and installation (mazria and kerschner ) . choosing building materials with low embodied energy (the cumulative energy of extraction, production, transport, installation, and disposal) and/or that use alternative energy sources reduces the building sector's carbon footprint. locally sourced materials produce fewer carbon emissions than those transported long distances and both durable materials and those with recycled content help reduce demand for virgin materials. fully recyclable materials can be reprocessed and repurposed with low energy inputs indefinitely. removing toxic compounds from material production processes and building products facilitates the recycling or biodegradation of building components. it also helps prevent air and waterborne industrial pollution and associated human illness. in some cases, it may be possible to choose building materials that actually sequester more carbon than is emitted in their production. the production of portland cement, the most commonly used cement in construction, is a relatively large source of co emissions. magnesium oxide-based cement is as strong as normal portland cement, but can be manufactured at low temperatures using renewable energy sources. magnesium oxide is an abundant mineral and is easily recyclable (smith ) . as it cures, it absorbs more co than is released during its production, resulting in a net negative carbon budget. the average lifespan of buildings in the united states is less than years (o'connor ) . design for adaptationdesign that easily accommodates changes in configuration and useextends the life of buildings and stems demand for new construction and virgin materials. when buildings are ultimately decommissioned, design for disassembly facilitates the breakdown and reuse of their constituent components. both design for adaptation and design for disassembly act upstream from recycling as part of a multicyclic system that repositions well-weathered buildings and building materials as resources rather than wastes, reducing carbon emissions in the process. building operations such as heating, cooling, and lighting account for % of electricity consumption in the united states (mazria and kerschner ) . passive design strategies can drastically reduce a building's dependence on carbon-intensive energy production. in cold climates, airtight, well-insulated building envelopes can work in tandem with building orientation, thermal mass, and passive solar radiation to provide thermal comfort and reduce heating bills. in hot climates, external shading, light colored/reflective roofs and walls, and passive ventilation strategies can reduce cooling loads. daylighting strategies such as light shelves and shallow floor plans bring natural light into interior spaces, reducing the demand for electrical lighting while, at the same time, contributing to the well-being and productivity of building occupants. similar to the use of building materials to sequester more carbon than they produce, buildings can generate more energy than they consume. buildings equipped with photovoltaics, wind turbines, and fuel cells are some of the energy producing technologies that make this possible. recent advances in computer-aided design software help designers address building performance issues early in the design process and optimize building design for energy use and production. improvements in digital monitoring and analysis allow occupants to scrutinize building performance and optimize energy use once buildings are operational. in the future, smart buildings and systems will likely respond dynamically to human occupation, weather, daily cycles, and seasonal change, further reducing their energy footprint. compact communities (discussed in the transportation section, above), land-use planning, and high-density zoning have the potential to reduce co emissions, reduce sensible and latent heat fluxes, and improve human health and ecosystem function. centralized infrastructure and zoning laws that segregate industrial, commercial, agricultural, and residential land use can be beneficial to public health within localities. however, these practices also create a cognitive disconnect between human action and its environmental consequences. they facilitate the transfer of local environmental burdens to regional and global scales, contribute to climate change, and undermine the health of vulnerable populations. integrated land use and responsibly managed distributed ecological infrastructure have the potential to safeguard public health without the transference of environmental burdens. synthesizing commercial, industrial, agricultural, and residential precincts, reimagining landscape as infrastructure, and bringing utilities into the public realm raises awareness about the causal relationships that link human activities, environmental degradation, and human health. urban agriculture, for example, reduces dependence on carbonintensive industrial food production, minimizes the carbon footprint of food transport, teaches urbanites how to grow their own food, and provides nutrition to communities. carbon-conscious materials, regenerative buildings, compact communities, and integrated landscapes have the potential to mitigate the worst effects of climate change. their success will depend on the ability to implement them not only in developed countries but also in the burgeoning cites of the global south. we must adapt these strategies to diverse climates, contexts, and cultures and pursue them in tandem with issues of economic development and social equity. in modern history, land distribution and regulation have always been the prerogative of governments. for this reason, national, regional, and local governments have a large role to play in housing mitigation strategies. governments control land-use planning, zoning, and building-code regulations. the ability to act on the mitigation strategies identified above, therefore, is largely dependent upon the regulatory and enforcement environment in a region. in a democratic society, these will in turn be dependent upon the inclination and ability of the state to assume progressive and proactive attitudes toward mitigation. agriculture releases considerable amounts of ghgs into the atmosphere. in , it accounted for - % of the total global anthropogenic ghgs including approximately % of nitrous oxide (n o) and % of methane (ch ) of the anthropogenic totals (rosenzweig and tubiello ; smith et al. ; burney et al. ) . land-use change, nitrogenous fertilizer applications, livestock production, rice farming, and biomass burning are among the major direct sources of ghg emissions. indirect sources of ghgs from agriculture include the production and/or applications of fertilizers and pesticides, the operation of farm machinery, and the transportation of agricultural products (burney et al. ). there are multiple opportunities for mitigating climate change impacts through modifications in agricultural practices and food consumption. with respect to agricultural production, mitigation strategies include soil carbon sequestration and the reduction of ghg emissions through altered production methods. with respect to food consumption, society can mitigate the impacts of climate change through changes in diet and by reducing the amount of transportation needed to deliver food (e.g., increasing local food production). below, we discuss several of these mitigation strategies. the topic of increasing local food production is covered in previous sections on transportation and shelter. unlike housing, food is a sector that is potentially much more amenable to individual decision-making and personal choice. this will depend on the relative proportion of food in a household budget. while americans spend only % of their incomes on average on food, the figure for indonesia is %. this indicates that people from wealthy countries would have greater flexibility to support alternative and nonindustrial agriculture through consumer preferences than those from countries where food is a larger part of household budgets. agricultural and degraded soils can act as large carbon sinks with the potential to sequester - gt of carbon globally (lal ) . soil organic carbon (soc) is the major component ( %) of this large soil carbon pool (lal ) . soc accumulation is facilitated by adding biomass to the soil and minimizing soil disturbances. the increased soc improves the physical, chemical, and biological properties of the soil and in turn enhances crop productivity, water holding capacity, and land sustainability under a low-input cropping system. several agricultural practices facilitate accumulation and retention of soc in cropland soils, e.g., conservation tillage, mulches, cover crops, manuring, crop rotations, and agroforestry (lal ) . the benefits of these agricultural practices go beyond carbon sequestration. they have the potential to reduce erosion, limit positive feedbacks in drought cycles, and benefit human health. over the past years, % of the world arable land has been taken out of production as a result of erosion (pimentel ) . conventional agricultural practices involving frequent tillage result in soil erosion, which in turn increases concentrations of airborne particulates. these particles can carry pathogens causing infectious diseases and can provide positive feedbacks to drought conditions. recent climate modeling has demonstrated a positive feedback in the 'dust bowl' drought of the s in the united states (schubert et al. ; cook et al. ). cook et al. ( ) discovered that a reduction in vegetation cover and the addition of eroded soil dusts to the air resulted in a high temperature anomaly over the northern united states and intensified the drought. as a result of the dust bowl, more than % of the surveyed farms comprising . million acres were seriously eroded, and the economic loss was valued close to us$ million (aaas ) . a combination of warming, drought, and conventional agricultural practices together have the potential to re-create the dust bowl of the s in the great plains of the united states (rosenzweig and hillel ) . the livestock industry is a main contributor to three major climate-related problems associated with global food systems: ghg emissions, reactive nitrogen mobilization, and plant biomass appropriation (pelletier and tyedmers ) . increases in livestock production and meat consumption are also blamed for the evolution of new and virulent emergent diseases. any improvement in the environmental sustainability of livestock production will concurrently have a benefit for human health. pelletier and tyedmers ( ) estimated that globally the livestock sector contributed % of anthropogenic ghg emissions, % of reactive nitrogen mobilization, and consumed % of human-appropriated biomass as of . reduction in ghg emissions in agriculture can be achieved by developing strategies involving improved production efficiency, land-use changes, cropland and rangeland management, and livestock and manure management (rosenzweig and tubiello ; smith et al. ; burney et al. ; pelletier and tyedmers ) . for example, enhancing production efficiency through agricultural intensification has been credited with the avoidance of gt of carbon emission globally since (burney et al. ). this figure highlights that the investment in agricultural research to improve the environmental sustainability of global food production systems (i.e., livestock, crop, and aquatic systems) should be a prioritized mitigation strategy (lobell et al. ; burney et al. ; steinfeld and gerber ) . another mitigation strategy to reduce emissions involves shifting livestock production regimes from ruminants to more efficient and lower impact monogastric species (e.g., poultry), well-managed fisheries and aquaculture, and promoting plant-based protein sources (pelletier and tyedmers ; steinfeld and gerber ) . changing livestock production from ruminants to monogastric animals and reducing livestock production overall both require significant changes in diet in many parts of the world. this is, of course, where tradition and culture play an important role, yet it is not impossible to induce dietary changes. initiatives that reduce the consumption of meat could have dramatic effects on ghg emissions particularly in developed countries (mcmichael et al. ) . such policies would also reduce impacts on ecosystems and promote other ecosystem services such as a reduction in the clearing of amazonian rain forests, reduced nitrogen deposition, and a reduction in the application of high-phosphate fertilizers used to grow feed. reduced meat consumption could also improve human health in developed countries particularly when coupled with incentive structures and educational measures to replace high-fat, sugar-rich foods with more complex diets based on plant proteins. reduced red meat consumption will likely lower the risk of obesity, diabetes, and several types of cancer (and specifically colorectal cancer), and may reduce the risk of ischemic heart disease (mcmichael et al. ) . a shift from meat-based proteins to plant-based proteins has the potential to substantially increase the total food calories available for consumption worldwide. worldwide educational efforts, altered trade policies, shifts in farm subsidies, public health campaigns and new environmental stewardship programs will be needed to achieve a fundamental change in the consumption of meat to achieve large-scale cobenefits for the environment and human health. energy consumption and production affect carbon emissions, which in turn affect human health and ecosystem functioning and services. these effects can be divided into the influence of energy consumption and production. while human well-being is in many ways positively affected by the increased availability of energy, there are also negative health effects resulting from the energy consumption and production. below, we describe potential mitigation strategies for the energy sector. the us energy information administration reports on energy consumption in the united states in four sectors: residential, commercial, transportation, and industrial. in , consumption across the four sectors accounted for , , , and % of overall energy consumption, respectively (us energy information administration ). overall energy consumption, as well as co emissions from energy consumption, continues to rise. total us co emissions from energy consumption were million metric tons of carbon in and million metric tons in (research and innovation technology administration ). reducing ghg emissions through declining energy consumption can be accomplished through a combination of technological advances and changes in human behavior (including the adoption of new or alternative technologies). several mitigation strategies for addressing energy consumption have been provided in the previous sections on transportation and shelter. in addition to those strategies, increasing the efficiency of appliances and lighting is likely to provide significant reductions in ghg emissions. the most efficient appliances in use today use half to one-fifth of their most inefficient counterparts currently in use (ipcc b) . in developed countries, substituting smaller and more efficient refrigerators, washing machines, and other appliances for less efficient ones can substantially reduce energy use. in addition, in developed countries, improvements in the efficiency of electronic devices with inefficient standby modes and power supplies (or more simply unplugging these devices when not in use) can significantly reduce energy consumption. in developing countries where biomass (e.g., wood, dung, and charcoal) is used for cooking fuel, substantial reductions in ghg emissions and improvements in human health may be attained by increasing the efficiency of biomass stoves, fostering a shift to cleaner burning liquid and gaseous fuels, and improving access to electricity (ipcc b). significant reductions in emissions can also be made through the use of more efficient lighting. the emissions from electric lighting worldwide are equivalent to roughly % of those generated by light passenger vehicles (iea b). substituting more efficient lighting technology (e.g., compact fluorescent bulbs and occupancy detectors) can reduce residential energy use by a factor of four or five (ipcc b). programs designed to increase the energy efficiency of appliances, vehicles, and lighting (e.g., the energy star and vehicle fuel economy (cafÉ)) also have the potential to reduce ghg emissions. although such programs have not decreased energy use in the united states, they may be partially responsible for halting the growth of per capita energy consumption (us energy information administration ). methods for reducing the impacts of energy production on atmospheric co concentrations, human health, and the environment focus primarily on improvements in an increased generation of renewable energy. renewable energy sources include those based on waves and tides, biofuels, solar, wind, and hydropower. a shift to renewable energy sources has great potential to reduce ghg emissions, reduce impacts on ecosystems and ecosystem services, and improve human health. nonetheless, the production of renewable energy will have adverse effects on some ecosystems. below, we discuss some of the benefits and potential side effects of some sources of renewable energy. in regions of large tides and tidal currents, such as the united kingdom, tidal energy could supply as much as % of the country's energy supply (blunden and bahaj ) . two types of systems are in use or are proposed, the tidal barrage and tidal turbines. tidal barrages allow water to flow into a bay or estuary during flood tide, then they release the water back during ebb tide. as water is released it flows through turbines generating energy. the impact of barrages on fish may be substantial (aprahamian et al. ) . power can also be extracted in the marine environment by turbines in regions of high wave and current energy. however, the ecological impact of the extraction of energy in highly energetic environments is not clear (shields et al. ) . the effects of such installations include alteration of benthic (ocean bottom) environments through changes in sediment transport and detrimental impacts on intertidal species through altered currents and nutrient flows. biofuels (discussed briefly in section . . . transportation) include corn-and sugarcane-based ethanol, woodbased methanol, biodiesel from soy, rapeseed oil, and switchgrass. biomass is also used in cofiring (combining with coal or other fossil fuels in the burning process) or burned alone as a fuel. crops can be grown explicitly for biofuels, or residues from farming or forestry can be used as biofuels. biofuels can also be converted into gasses and then used to fuel gas engines. nonetheless, many biofuels require significant amounts of land and water to grow them, which brings fuel production into conflict with food production as well as potentially result in a climate effect from the alteration in the surface fluxes of heat and moisture into the atmosphere. in addition, fuel plants may not be as effective as others at absorbing ghgs, and more natural lands converted to agriculture for biofuels will result in increased erosion, decreased water quality, and a loss of wildlife habitat and biodiversity. solar energy can be used to generate electricity, heat water, and heat buildings and can be a highly effective form of renewable energy in some regions. although photovoltaic power generation only accounted for . % of world power production as of , expansion is occurring at roughly % per year, largely in developing countries (ipcc b) . hot water production is the second single largest use of energy in residences in both the united states and china. solar water heating is a cost-effective alternative to other fuel sources in many locations. similarly, passive solar heating of buildings (mentioned in the section . . . shelter) is an effective heating measure in many regions. finally, solar energy can be used to generate power with solar thermal electric plants that focus solar rays to heat a liquid, which is then used to generate electricity. passive solar space heating and hot water production likely have the lowest environmental costs. decentralized photovoltaic use on residences and buildings incurs environmental costs in terms of the production process, but likely have few other negative effects. conversely, photovoltaic fields and solar thermal electric plants can have large footprints, adversely affecting plant and animal habitat. like solar power, wind-generated power is more effective in some areas than others. wind produced . % of global electricity in but has been growing at a rate of approximately % per year since (ipcc b ). there are potential adverse effects on birds, through collision, displacement due to disturbance, barrier effects, and habitat loss, with more research needed in all areas (drewitt and langston ; fox et al. ). in addition, the impact of wind turbines on views is perceived to be a substantial barrier to implementation in some communities (wolsink ) . as of , hydropower accounted for % of global electricity (ipcc b) . together, large hydropower plants and small and micro hydropower systems can supply power to large municipalities and isolated rural communities with relatively few emissions. hydropower is not carbon neutral, however, and some studies have estimated significant emissions from hydropower reservoirs (ipcc b) . in addition, dams and reservoirs fragment river networks, alter natural flows of water and sediments, and change stream temperatures. these effects are discussed in section . . . human-dominated systems. there are several other major renewable energy sources including nuclear, hydrogen, and geothermal. as with the renewable sources described above, there are both benefits and costs to using these other renewable energies. most of the mitigation strategies discussed above are focused on reducing ghg emissions. additional strategies can be used to increase carbon sequestration, removing co from the atmosphere. below, we discuss a few strategies that have been proposed for increasing carbon sequestration and storage as well as much riskier geoengineering proposals. deforestation is one of the largest sources of emission accounting for over gt co per year in (stern ) . although there are many other uses of natural resources that result in carbon emissions, here we focus on mitigation strategies that focus on deforestation. given the slow rate at which trees absorb carbon dioxide, simply planting new trees in place of those removed does not immediately offset the removal of a tree. a more rapid approach to mitigation is to reduce deforestation. unlike the majority of emissions that emanate from industrialized countries, most emissions from deforestation are generated by developing countries. deforestation also itself alters the climate system (pielke et al. ). reduced emissions from deforestation and forest degradation (reddþ) is a program of the united nations that seeks to mitigate climate change by providing incentives to conserve forest cover. given recognized challenges in redd (dickson et al. )including country by country differences in who owns the forest, who should receive the monetary benefits of preserving the forest, and the reliance of various indigenous groups on forests for livelihood and cultural identitythe program was modified to reddþ in to minimize negative consequences and respect the rights of indigenous peoples and members of local communities (kanninen et al. ) . assumed cobenefits include the suite of ecosystem services such as watershed protection; reduction of soil erosion; flood control; provision of fuel, timber, and foods; and cultural and spiritual amenities for local communities. biodiversity is presumably also enhanced by protecting natural systems. it is possible that forest-dependent communities will be able to maintain access to forests through this initiative. potential coharms are increased demand for conversion of other ecosystems if forests are protected, replacement of mixed stands with plantations, and benefits accruing to those not responsible for care and maintenance of the forests. prevention of deforestation mitigates atmospheric co concentrations by preventing the release of stored carbon that occurs when forests are harvested and protecting forest stands that absorb carbon, as well as limits changes in surface energy and water fluxes. as of , south america and africa continue to have the largest net loss of forests and large decreases in carbon storage (fao ) . although tropical forests have been the focus of early redd efforts, recent evidence indicates that a large amount of carbon can be stored in mangrove forests (donato et al. ) , peatlands (dise ) , and boreal forests (carlson et al. ) , making these potential targets for conservation efforts. urban forests have also been largely neglected as a potential carbon sink. escobedo et al. ( ) review the services and disservices of urban forests combined with human health and pollution mitigation as well as carbon sequestration. in some locations, urban forests are on par with other techniques for carbon sequestration and reduction. in addition to capturing carbon in plants, it may be possible to increase carbon storage in the water and in the earth's crust with various methods. these proposed approaches are still on the scientific fringe and considered to be highly unproven and/or potentially quite risky. one such approach is to capture carbon in the oceans through iron fertilization. in regions of the ocean where there are ample nutrients to drive primary production (photosynthesis) but observations show that chlorophyll is relatively low, the lack of iron is thought to limit the productivity. over the last two decades, several experiments have attempted to determine whether the addition of elemental iron into the water could cause an increase in algal production and ultimately, as photosynthesis fixes carbon into organic molecules that then sink, the sequestration of carbon in deep ocean sediments. however, the results of these experiments are mixed (de baar et al. ) , with the export of carbon being difficult to assess and likely quite modest. at the same time, the impacts on local ecosystems and thus ultimately fisheries in potentially highly productive regions of the subpolar oceans are not yet known. other carbon sequestration approaches have also been proposed including pumping air down into the oceans, injecting carbon into the ground, and incorporating biochar into soils. given the magnitude of projected changes in climate and the difficulty in implementing meaningful mitigation measures, some scientists have argued for large-scale efforts to reduce the amount of incoming solar radiation. these approaches are collectively referred to as geoengineering. at present, these approaches are unproven, risky, and, at best, will only address some of the impacts of the human influence on climate. one such geoengineering approach would inject sulfate aerosols into the stratosphere. these aerosols would reflect incoming solar rays and reduce warming. although such an approach might reduce average global temperatures, it would also likely decrease average global precipitation (bala et al. ) as well as alter atmospheric and ocean circulation patterns. for a given change in radiative forcing, solar forcing is more effective at changing the hydrological cycle than forcing from carbon dioxide owing to differences in the surface latent heat (and thus evaporation) response. while the impact of carbon dioxide fertilization could increase net primary production in terrestrial ecosystems (govindasamy et al. ) , ecosystems could continue to be vulnerable due to changes in water resources. in addition, solar radiation management would not address the potentially significant problem of ocean acidification to marine ecosystems. the majority of both natural and human systems will have to adapt to climate change in some form or the other. a broad array of adaptation strategies has been proposed for reducing the impacts of climate change in both more natural systems and human communities (ipcc a; heller and zavaleta ) ( table ) . many of these strategies are either general concepts or strategies that apply to most ecosystems and regions. we begin by describing general concepts and strategies. we then describe some of the strategies that have been proposed for four broad types of ecosystems including freshwater, terrestrial, coastal, and human-dominated systems. for the freshwater, terrestrial, and coastal systems, we focus on strategies that would lessen negative impacts of changing climate on ecosystem functioning and ecosystem services. for the human-dominated systems, we discuss the ways that some of the most often discussed strategies for climate adaptation in these systems will impact (either negatively or positively) ecosystem functioning and the provision of ecosystem services in these and in surrounding areas. developing adaptation strategies to address future shifts in climate will require expanding the temporal and spatial scales of plans, policies, and management (kareiva et al. ; lawler three-year, -year, and even -year plans will fail to address many climate impacts. future planning will need to address both short-term and long-term changes in systems. to address long-term changes, plans and policies will need to look at least years into the future and will be more useful if they look ahead or more years for many ecosystems and ecosystem services. it will often also be necessary to expand the spatial scale at which systems are managed and policies and plans are developed. as species move and ecosystems change, it will be necessary to think well beyond neighborhood, park, refuge, state, or even national boundaries. planning will need to occur at multiple, integrated scales and will need to involve local, regional, and sometimes national participants (kareiva et al. ) . while expanding the temporal and spatial scale of climate adaptation strategies, it will be necessary to keep in view local histories, politics, and economies. scale and locality also play a formative role for issues of 'environmental' or 'climate' justice. environmental justice describes the tendency for poor communities to be more vulnerable to environmental degradation. poor communities tend to bear not only the unequal burden of polluting, destructive, and destabilizing industries but also a disproportionate share of the blame for environmental destruction and responsibility for ameliorating environmental change. for example, polluting industries are often located in poor communities due to cheap land values and the inability of poor communities to politically resist their presence, or a desire to develop jobs at any cost. for instance, swidden agriculturalists in southeast asia have been blamed for forest fires despite satellite data pinning large fires to governmentsponsored oil palm plantations (harwell ) and southeast asian fishers living near toxic riparian runoff from mining have been blamed for polluting local waters by cyanide fishing (lowe ) . the broad spatial scales that are required to address the effects of a changing climate necessitate cooperation and coordination among managers, planners, and policy makers from different jurisdictions, agencies, and management units (kareiva et al. ) . they require agencies with diverse mandates and cultures to work together in ways that they have not done in the past. as an example, the us climate change science program is a federal organization that reaches across federal agencies. the broad scale of climate impacts also requires cooperation across sectors of society and different types of organizations including nongovernmental organizations, local and regional governments, citizen groups, and industry. the us fish and wildlife service's landscape conservation cooperatives are an example of a system designed to bring together diverse groups of scientists, stakeholders, policy makers, and managers to, in part, address climate change. in addition, as emissions from one locale affect the climate in other locales, addressing the global nature of climate change will involve cooperation at an international level (hannah ) . although, as the above examples demonstrate, some regional, national, and international efforts have been put in place to foster collaboration, more such efforts will need to be made to adequately address climate change. in addition to cooperation among scientific and political leaders, local and regional stakeholders need to work together as a unified force for social and environmental change. representatives from the global north need to appreciate perspectives emanating from the global south and vice versa. climate adaptation can be brought into many other environmental and social justice agendas among community advocacy groups and should not remain the purview of the community of knowledge leaders with scientific expertise. there are three fundamental approaches to managing systems in a changing climatepromoting resistance, resilience, and transition. resistance-based adaptation strategies focus on maintaining the current state of a system in the face of climate change or climate-driven changes in the environment. building a seawall to prevent erosion as sea levels rise and storm surges intensify is an example of a resistance-based adaptation strategy. resilience can be defined as the ability of a species or system to return to its current state following a perturbation (e.g., holling ) . resilient systems will be able to maintain ecosystem functions and processes and avoid a transition to a new state as climates change. most of the discussion of climate adaptation to date has focused on managing ecosystems for resistance or resilience. most of the general adaptation strategies discussed below and the more specific strategies discussed for each of the four types of ecosystems are intended to increase resilience. the magnitude and rate of projected climatic changes over the coming century will likely make both resistance-and resilience-based strategies insufficient or ineffective in the long run. managing many systems will require promoting change to a new state. strategies for promoting change in human-dominated systems may include planting new crops, redesigning road networks, and relocating people. strategies for fostering change in more natural systems include assisted colonization of threatened plants and animals (see below) and shifting management efforts from one species to another. the outcomes of these more aggressive and forward-looking strategies will be more uncertain, and their implementation will be more controversial. change is especially hard in social contexts where it is uncomfortable, disadvantageous, expensive, or where the old system is remunerative or beneficial to elites, decision makers, or other small influential groups. adaptive management is a flexible approach to managing a system in which the management actions are treated as largescale experiments (holling ; walters and hilborn ) . it is an iterative process in which the system is monitored following the initial management action and new actions (again treated as experiments) are designed and implemented in response to the results of the initial actions. adaptive management was conceived for managing highly uncertain systems, and thus it has great potential for managing systems in a changing climate (arvai et al. ) . adaptive management approaches designed to address climate change will likely have four basic steps (kareiva et al. ) : ( ) assessing the potential impacts of climate change on the system in question, ( ) designing management actionsin the form of experiments with testable hypotheses or predictionsto address the potential impacts, ( ) monitoring the system for climatic changes and system responses, and ( ) using the results of the monitoring to evaluate the effectiveness of the management actions and redesign them as necessary before repeating the four steps. it is generally accepted that more diverse systems are more resilient to environmental change than less diverse systems. thus, restoring or protecting the biological diversity of a system will likely increase resilience of that system to climate change (glick et al. ). this diversity can be in the form of species diversity, environmental diversity, or genetic diversity (schindler et al. ) . a second, general approach to increasing resilience of species and systems involves reducing the impacts of other factors that reduce the overall resilience of the species or system. for individual species, this might mean reducing harvest, restoring or protecting habitat, eliminating diseases, or reducing human interactions. for ecosystems, this may mean reducing pollutants, restoring vegetation, reducing human use, and controlling invasive species. as climates change, many species need to move to track suitable climatic conditions and changing habitats. in some cases, it may be possible to facilitate movement by increasing connectivity of freshwater and terrestrial landscapes (see sections . . . freshwater systems and . . . terrestrial systems). however, many species with limited dispersal abilities or that face significant barriers to dispersal will be unable to respond to changing climates. assisted colonization is one strategy that has been proposed for facilitating climate-driven range shifts (hunter ; mclachlan et al. ); also referred to as managed relocation and assisted migrationassisted colonization is the translocation of species outside their native range to facilitate movement in response to climate change. the concept is controversial for both ethical and ecological reasons. some researchers have highlighted the potential for negative, ecological, evolutionary, and economic impacts, as well as ethical concerns (ricciardi and simberloff ; sax et al. ). advocates argue that assisted colonization will be essential for cases in which species will have to move to find suitable environments but will be unable to do so, that many of the potential risks are overblown, and that guidance can be developed for the strategic and informed use of assisted colonization. several decision-making frameworks and prioritization strategies for applying assisted colonization have been proposed (hoegh-guldberg et al. ; richardson et al. ). although assisted colonization will likely be a useful strategy in many systems, there will be specific factors and risks that will need to be considered for particular systems (e.g., in freshwater systems, olden et al. ). virtually all freshwater systems are already impacted by human activities (e.g., vörösmarty et al. ) . under current climate projections, virtually all freshwater ecosystems will face significant impacts by the middle of this century. most of these impacts will be detrimental from the perspective of existing freshwater ecosystems and the human livelihoods and communities that depend upon them (poff et al. ) . discussions have broadly focused on rising water and air temperatures, increasing and decreasing precipitation, increasing evapotranspiration, and changing regimes of groundwater and surface runoff. there are four basic types of adaptation strategies that have been proposed for addressing these impending impacts. we define these as ecological water management, connectivity, protected areas, and restoration strategies. below, we briefly describe some of the specific strategies that have been proposed in each of these areas. increased water scarcity associated with decreased precipitation and increased warming in some regions is likely to prompt the modification of dams and reservoirs to improve storage capacity (see section . . . . managed aquatic systems). traditional water management has generally sought to dampen the natural variability of river flows to attain steady and dependable water supplies for domestic and industrial uses, irrigation, navigation, and hydropower, and to moderate extreme water conditions such as floods and droughts (postel and richter ) . however, the extraordinary species richness and productivity characteristic of freshwater ecosystems is strongly dependent upon, and attributable to, the natural variability of their hydrologic conditions. thus, large changes to natural flow regimes can have large impacts on species, ecosystems, and society (postel and carpenter ; iucn ; wcd ) . the water needs of humans and natural ecosystems are commonly viewed as competing. however, water managers and political leaders increasingly recognize that there are limits to the amount of water that can be withdrawn from freshwater systems before their natural functioning and productivity, native species, and the array of goods and services they provide are severely degraded ). this has led to a growing realization that society derives substantial benefits both from out-of-stream extractions of water as well as by maintaining adequate flows of water within freshwater ecosystems. in recent years, the water needs of river ecosystems are receiving increasing attention in water-supply planning, offering hope that many rivers can be protected before their health and ability to provide important goods and services is seriously compromised by water development. restoring or protecting some semblance of the natural river flow conditions necessary to support ecosystem function (called environmental flows) into the future is one of the most important climate change adaptation strategies for flowing waters. recent regulatory mandates and policy decisions calling for restoring and preserving environmental flows have led to a reexamination of dam operations for better protection of river health (watts et al. ) . for example, the south african national water act ( ) calls for the creation of a reserve of water in each river basin to meet both basic human needs and protect river ecosystem health. similarly, other national directives and international agreements such as the water framework directive of the european union are providing mechanisms for river protection, including the provision of adequate environmental flows (postel and richter ) . maintenance of environmental flows (flows that sustain river ecosystems) is likely to be the highest priority adaptation response for freshwater ecosystems in most contexts. this will require policies and implementation mechanisms to protect (and, if necessary, restore) flows now, and to continue to provide environmental flow regimes under changing patterns of runoff associated with climate change. as air and water temperatures warm, many freshwater species will be forced to rapidly adapt to their new environments, to migrate to more suitable habitats, or face extinction (heino et al. ). responses of aquatic organisms to climate change will be constrained because they are limited to dispersal along pathways of connected water. consequently, the linear nature of river systems makes them highly prone to fragmentation from waterfalls, dams, and water withdrawals. such fragmentation can not only limit dispersal and the ability of species to track climate change but also disrupt the completion of life cycles for many freshwater organisms. for many streams, especially in arid and semiarid ecosystems, there is concern that more frequent and severe droughts will lead to the building of more dams and water diversions, further fragmenting stream networks (seager et al. ) . increasing the connectivity of freshwater systems is one key way to help aquatic systems adapt to climate change. species responses to climate change will be limited by human-engineered structures, including hundreds of thousands of dams, diversions, and impassable road culverts that exist globally (nilsson and schopfhauser ) . in the united states alone, there are over large dams and an estimated . million or more small impoundments that disrupt the dispersal of aquatic organisms. in recent decades, however, new water laws and programs have helped to increase habitat connectivity by removing or modifying human barriers to movement in river systems. increasingly, small dams and diversions are being removed for a number of ecological, social, and economic reasons, including the enhancement of longitudinal connectivity for fish migration (stanley and doyle ) . similarly, temporary barriers such as impassable culverts under roadways are being modified to allow for easier recolonization and movement of fishes (roni et al. ) . although this management strategy may promote the adaptive capacity of species to respond to climate change, the removal of such obstructions can simultaneously increase the risk of invasion by nonnative species and associated diseases, which are major threats to ecosystem structure and function. in fact, the conversion of culverts into permanent barriers has been a common management approach to protect isolated native populations from invasive species spreading upstream (fausch et al. ). given the complexities of promoting the movement of native species while concurrently limiting the spread of invasive species, it is unlikely that the removal of barriers to enhance habitat connectivity will be a successful adaptation strategy in all areas for reducing the impacts of climate change on ecosystem function. in addition to restoring or protecting environmental flows and increasing connectivity within stream networks, it may be necessary to protect the species themselves to allow them to move through or persist in more intensely managed landscapes. in aquatic ecosystems, this may involve increasing the size of existing reserves or creating new protected areas that encompass important freshwater resources and allow for native species and assemblages to persist into the future (abell et al. ) . such acquisitions and agreements ideally would capture a large portion of the geographical, ecological, and geophysical range of multiple species, thus increasing opportunities for organisms to adapt and evolve to changing environmental conditions. protected areas have historically been a cornerstone of terrestrial conservationand are discussed in much more detail in that section, below. nonetheless, protected areas will likely play a major role in protecting aquatic species as climate change and areas could easily be prioritized to provide protection for both terrestrial and aquatic systems in a changing climate (abell et al. ). projected increases in air temperatures, combined with reduced snowpack, earlier onset of spring peak flows, and lower summer baseflows, will have direct implications for the thermal regimes of streams and rivers (poff et al. ) . the ecological implications of stream warming are significant. water temperature directly influences the metabolic rates, physiology, and life histories of aquatic species and helps determine rates of important ecological processes such as nutrient cycling and productivity. in general, warming will produce a general shift in species distribution poleward with extinctions and extirpations of cold-water species at lower latitudes and range expansion of warm-water (often invasive species) and cool-water species into higher latitudes. restoring and protecting riparian vegetationthe trees and shrubs that grow along the banks of rivers and streamshas the potential to reduce stream temperatures in certain places. recent modeling suggests that riparian habitat restoration can play an important role in offsetting the effects of climate change. battin et al. ( ) used a series of linked models of climate, land cover, hydrology, and salmon population dynamics to investigate the impacts of altered stream flow and temperature on the effectiveness of proposed habitat restoration efforts designed to recover chinook salmon populations in the pacific northwest of the united states. this study showed that in the absence of habitat restoration, basin-wide spawning populations would decline by an average of - % by . by contrast, they found large increases in juvenile rearing capacity associated with proposed riparian habitat restoration, which limited climate-induced declines to % according to one climate model and increased salmon abundance by % according to another model. however, the model results showed that not all expected climate impacts could be mitigated entirely. in relatively narrow streams, reforestation was predicted to decrease water temperatures by increasing shading, but in wide, main-stem reaches where most chinook salmon spawn, riparian vegetation had a minor effect on water temperature. in addition to restoring riparian areas, targeted protection of forested riparian buffers using fencing to stop livestock from entering the stream and trampling/grazing vegetation or conservation easements to eliminate farming activities may also help to reduce stream temperatures. the many adaptation strategies that have been proposed for addressing climate change in terrestrial systems can be loosely grouped into three categoriesthose that involve protected lands and conservation planning, those that address connectivity, and those that are more species or site specific in nature. protecting land is arguably one of the most effective ways of conserving biodiversity and terrestrial ecosystem services. it is generally assumed that lands that are selected to protect today's species and ecological systems will protect those species and systems into the future. climate change brings this assumption into question. as climates change and species distributions shift, species will move into and out of reserves. changes in species composition, the timing of ecological events, and the rates of ecosystem processes will result in changes in the functioning of ecosystems and the services they provide. many proposed adaptation strategies address these challenges by augmenting current protected areas with additional reserves, parks, and preserves (heller and zavaleta ; lawler ). these strategies range from broad calls for more protected lands to more sophisticated recommendations about how to select new lands or design new reserves to better protect biodiversity and ecosystem services as climates change. a number of strategies have been proposed for determining where to put new protected lands to best address the effects of climate change. some of the simplest strategies involve placing reserves at the poleward or elevational boundaries of the range limits of specific species (peters and darling ) , at major transitions between vegetation formations (halpin ) , or at the core of species environmental distributions (araújo et al. ) . others have proposed placing reserves between existing reservesin the hopes that those new reserves will act as stepping stones allowing species to better move from reserve to reserve. yet another recommendation involves placing reserves across strong environmental gradients. reserves that span many different environments will potentially allow species to shift their distributions without leaving the reserve. in addition to these simple rules of thumb for placing reserves to address climate change, many more sophisticated approaches to selecting areas for new reserves have been proposed. for example, modeled climate-driven shifts in species distributions can be used to select new reserves that will protect biodiversity today and into the future (hannah et al. ; vos et al. ; hole et al. ). although this approach may prove useful for some species and systems, care must be taken in the use of the models developed to project species range shifts. there are many uncertainties associated with the models themselves and the climate projections on which they rely. another proposed approach to building a reserve network that will be resilient to climate change involves protecting environments in lieu of protecting species and systems. this approach has been referred to as 'protecting the ecological stage.' this analogy implies that the species and the ecological systems are players and that the soils and topography of a region form the stage on which they interact and evolve. by protecting a suite of diverse environments, it is assumed that one will protect the areas where new ecological systems can develop as climates change (anderson and ferree ; beier and brost ) . similarly, others have suggested that more resilient reserve networks will be those that include a diversity of current climates (pyke and fischer ) . although climates will change, many of the climate gradients generated by latitudinal and topographic patterns will remain. thus, a set of reserves that protects a diversity of climates today will likely protect a diversity of climates in the future. at least one recent study has applied the concept of protecting environments (topographies, soils, and climate gradients) to a reserve-selection problem (schloss et al. ) . one last approach to building a resilient reserve network in a changing climate involves placing reserves in areas that are likely to serve as climate refugia. refugia are areas that are projected to experience minimal climatic changes and/or have cooler microclimates (saxon et al. ; hansen et al. ; shoo et al. ) . one potential opportunity for climate change adaptation is to consider people as an essential component of species conservation and climate adaptation. while reserve and park systems have a long history of alienating local people, using violence and other coercive measures to separate people and wildlife, in this case, local livelihoods are also threatened by climate change. this provides an opening for a new approach to conservation and a new discourse on the relationship between local people and wildlife. as for freshwater systems, improving the connectivity of the terrestrial landscape will be critical for addressing climate change. in response to historical climatic changes, many species shifted their rangesmoving toward the equator as ice sheets advanced and moving poleward as the ice sheets retreated. today's species will have a much harder time responding to current and impending climatic changes. present-day landscapesunlike the landscapes of the distant pastare fragmented by roads, agricultural fields, and residential development. given that many species will have trouble moving across these human-dominated landscapes, it will be essential to reconnect fragmented landscapes to allow species to respond to shifting climates. not surprisingly, improving landscape connectivity was the most often recommended climate change adaptation strategy in a recent review of the literature (heller and zavaleta ). many of the recommendations for increasing connectivity involve designing corridors that connect current protected lands. traditionally, such corridors have been designed for particular focal species, and they have connected reserves or remaining fragments of habitat. however, corridors that facilitate species ranges shifts and the reshuffling of ecosystems will need to connect what is habitat for a species today with an area that may become habitat in the future. in this way, corridor design will need to evolve to address climate change. one such approach to developing corridors for addressing climate change involves basing the corridors on environmental gradients instead of on the current vegetation (which is often used as a measure of habitat) (beier and brost ) . another approach uses projected changes in climate to orient corridors (ackerly et al. ) . a third method involves using modeled shifts in species distributions to identify potential routes that species would need to take to move from currently suitable climates to places where climates will likely be suitable in the future (williams et al. ; rose and burton ) . these three approaches are based on different assumptions and have different levels of associated uncertainty. in general, the uncertainty increases with the intensity of modeling required to identify the corridor, and thus the first of the three approaches has the least inherent uncertainty and the last of the three has the most. although corridors may be useful for some species, they will not be feasible or effective in all places or for all species. in addition to designing corridors to facilitate species movements, it will likely be necessary to improve the permeability of the landscape in general (franklin and lindenmayer ). increasing landscape permeability will involve changing the way we use and manage the landscape. changes in forest harvest practices from clear-cuts to selective, retention harvests can increase the connectivity of forested landscapes. landscape planning and zoning for residential development can reduce landscape fragmentation. windbreaks, fencerows, and crop choices can be used to increase the connectivity of agricultural lands. at a finer scale, choices of landscaping plants, road placement, and wildlife over-and underpasses can increase landscape permeability. protected lands and connectivity increase species ability to adapt to changing climates at relatively large spatial scales. in general, they address species movements. adaptation strategies are also needed at finer scales. in particular, strategies are needed that address individual species and ecosystems. many of these finer scale approaches tend to involve reducing temperatures or otherwise offsetting the exposure to the changes in climate or climate-driven changes in environmental conditions. for example, it may be possible to reduce temperatures by restoring vegetation to provide shade (wilby et al. ; wilby and perry ) . it may be possible to reduce the impacts of changing fire regimes through forest management practices. fire suppression policies, may, for example, have particularly severe ramifications in a climate in which fires will generally be larger and more frequent. it may also be feasible to provide supplemental water for particularly vulnerable plants or animals in times of drought or during heat waves. similarly, shade shelters could be provided to help some animals weather heat waves in extreme climates. although some of these strategies may prove useful in the short term, many will be neither cost-effective nor long-term solutions. another system-specific approach to addressing climate change involves altering species composition or community structure in a way that allows a system to better cope with climatic changes. for a forested system, this may mean planting new species or planting seeds of the same species that have a different genetic makeup (millar et al. ; glick et al. ). likewise, for grasslands and shrublands, this may mean choosing different species or strains for restoration purposes. moreover, it may mean facilitating the transformation of an ecosystemfor example from a shrubland to a woodland or from a shrubland to a grassland. in many cases, the first species to move into these transitioning systems may be invasive species. facilitating the transition to a new state using species native to the region may, in many cases be preferable to having new systems dominated by invasive species. these extreme cases will require ex situ conservation strategies, including seed banking and captive breeding to ensure the long-term survival of the species. the interface between the land, sea, and air found in coastal areas is a high-energy system subject to storms, winds, and tides. it is also where the majority of protein comes from in many regions of the world. many vital ecosystem services are provided along coastal margins of continents and islands. fringing coral reefs and mangrove forests buffer wave energy, which reduces erosion along the coast. these systems also provide critical habitat for many species, especially nursery areas for provisional services such as fisheries. in many areas, sea-level rise and associated storm surges will make it difficult to protect these systems and the services they provide. nonetheless, there are two main approaches that will likely prove most useful in coastal systems: building resilience through protection and restoration and facilitating the transition to new ecosystems. one of the more beneficial ways, from both an environmental and economic standpoint, to adapt to predicted changes from climate change is by protecting and restoring natural systems that provide multiple ecosystems services. this would include protection of human infrastructure from sea-level rise and increased inundation. protection and restoration of natural buffers such as barrier islands, mangroves, and coral reefs not only require little financial construction and maintenance but also maintain the wealth of ecosystem services associated with these systems, which include nursery and rearing habitat for many commercial seafood stocks such as fish and shrimp, recreational and cultural opportunities for humans, and protection from inundation. some natural protection features such as sea ice cannot be maintained in the face of increasing temperatures. in alaska, the loss of sea ice and melting of permafrost due to warming ocean and air temperatures has exposed the shore to wave energy that used to be absorbed or damped by sea ice or frozen soils. undoubtedly, ecosystem services that depended on that sea ice protection have also been lost. many regions have seen a huge decrease in the area covered by mangroves as coastal areas are drained and mangroves removed for aquaculture. worldwide, it is estimated that more than % of mangrove forests have been destroyed or converted to other uses. on a local scale, many areas have lost all of their mangroves. this loss destroys a wide range of ecosystem services provided by these forests, which range from carbon sequestration to flood protection, spawning, and rearing grounds for many fish and other marine species. restoring mangrove forests results in increased storm surge protection for natural and human coastal systems, provides nursery habitat for many commercially important fisheries, and stores sediments that may damage offshore coral reefs. worldwide, corals exhibit well-documented environmental adaptability over time scales of weeks to thousands of years. corals are actually two creatures that live together in a symbiotic relationship. zooxanthellae are the algal component of corals that provide nutrients generated from photosynthesis to the animal component. different zooxanthellae species and different species of corals in general have differential environmental tolerances. for example, some corals near the arabian peninsula exist under conditions of salinity and temperature that are greater than near-term predicted future average salinities and temperatures resulting from climate change. these adaptive changes likely operate on a decadal time scale. thus, in some cases, it may be possible to identify particular species or zooxanthellae and algal combinations that are more resilient to warming. protecting coral reefs from other stressors such as excess sediment and/or nutrient and pollutant inputs from terrestrial sources and unregulated diving can increase the coral's ability to adapt to increased water temperatures. technological adaptation measures include using sprinklers to create roughened sea surface to decrease light penetration or covering reefs with shade cloth to limit bleaching of corals. as sea levels rise, many coastal systems will need to migrate inland. when there are extensive human uses of coastal space or urban buildup, space for ecosystem recovery and migration of species will not be available. providing space for the migration of biotic elements of the coastal system at the same time as when human populations are forced to move would provide cobenefits for coastal life and human communities. ecosystems will usually adjust to changing salinities and water levels, and the services provided by the system will be maintained where conditions are suitable. for example, in , a subdivision east of houston in baytown, texas, was bought out, the structures removed, and the entire area restored to coastal wetlands. a more extreme example of planned retreat is the maldive islands. their current plan is to exploit the current coastal amenities, primarily beaches and coral reefs; secure capital with which to train younger citizens; and prepare them for relocation to other countries such as india, sri lanka, and australia. in the short term, there are negative consequences to ecosystem services due to commercial and touristic exploitation, but in the long run, by abandoning the islands, the system will be able to come to a new ecological state. in some cases, removing human infrastructure will be a necessary but insufficient means of fostering landward shifts in coastal systems. in such cases, it may be necessary to facilitate the shift in natural systems and stabilize soils by planting more coastal species inland as water levels rise. such actions have been taken as part of the alligator river climate change adaptation project on the north carolina coast in the southeastern united states poulter ) . here we describe some of the adaptation strategies that have been proposed for built environments, agricultural systems, and managed freshwater systems. we explicitly focus on how the most commonly proposed strategies could affect associated ecosystems. the severity of climate change impacts will depend, in large part, on our ability to transform the built environment in response to changing conditions. the following section discusses some of the climate change adaptation strategies that will likely take place in built environments and the effects that these strategies may have on ecosystem functions and services. we briefly discuss human migrations, flood control, sea-level rise, increased water storage, and urban heat-island effects. perhaps the most basic form of climate change adaptation involving the built environment is human migration. vulnerable populations will be forced to abandon their homes, relocate, and rebuild. they will displace resident populations and open up the possibility of conflict in the process. the bulk of climate change-related human migration that is already occurring involves movement from rural areas to cities in developing countries. unplanned urban growth will fuel the expansion of informal urban settlements, and by ,  people, one-third of the world's population, may inhabit urban slums. in both rural and urban environments, proactive planning and resettlement strategies that identify populations at risk and relocate them stand to drastically reduce long-term human hardship stemming from climate change. they can also protect vital ecosystems from the degradation associated with unplanned development. however, resettlement can have profound negative impacts. poorly executed, it can degrade indigenous culture, undermine traditional livelihoods, destroy social networks, and deepen poverty by distancing poor communities from employment opportunities. given these dangers, in some cases, it is preferable to fortify communities against climate change impacts in place. this approach introduces improvements to infrastructure and housing, builds upon the prior efforts of communities, and preserves social networks. in many regions, climate change will result in increased rainfall and flooding. conventional approaches to storm water management rely on defensive hard structures. water runs from impermeable concrete and asphalt surfaces into storm drains, culverts, and concrete embankments. rivers are straightened, channelized, and isolated from their natural floodplains to accommodate human settlement. as flood events become more intense and frequent with climate change, storm water management strategies must evolve. rather than take a defensive stance, they should promote the coexistence of humans, habitat, and water. soft infrastructure or low-impact development strategies, such as permeable paving, green roofs, green walls, rain gardens, bioswales, and constructed wetlands offer a promising alternative to conventional storm water infrastructure. they promote groundwater recharge, reduce flooding, and improve water quality with downstream benefits to aquatic habitats. in areas of severe flood risk, resettling residents on higher ground and reintroducing rivers to their floodplains protects human populations, dissipates flood energy, and creates wetland habitat. through temporal landuse practices such as intermittent farming, floods become an asset rather than a threat, depositing sediments that enrich agricultural lands. by , rising sea levels may displace as many as million people (myers ) . like conventional storm water infrastructure, conventional seawall infrastructure relies on defensive hard structures. as sea levels rise, existing levees will become obsolete. here again, soft-infrastructure strategies offer a promising alternative. graduated transitions from sea to land including manmade offshore islands, expansive shallows, and coastal marshes act as buffers for storm surge better equipped to absorb the impacts of extreme weather events. they provide habitat for marine species and, in coastal cities, public space for recreation when conditions permit. in places like coastal peru and sub-saharan africa, disappearing glaciers and reduced precipitation will increase the frequency, duration, and impacts of drought, undermining food and water security. a number of interventions in the built environment can help communities cope with water scarcity. increasing water storage at scales ranging from dams to urban reservoirs and household rainwater barrels helps bolster water supplies during prolonged dry periods. in coastal areas, desalination plants and, in select localities of south america and africa, fog harvesting may prove to be viable alternative water sources. ecological sanitation technologies such as dry toilets and gray water wetlands conserve potable water for drinking and, as part of a network of integrated ecological infrastructure systems, irrigate and provide nutrients for urban farms and forests. they also help prevent contamination of local water bodies, preserving their viability as sources of potable water and ecological integrity. further impacts of, and opportunities associated with, these various adaptation strategies are discussed in the sections . . . . and . . . . . cities in developed countries are on average - c hotter than the surrounding countryside (un human settlements programme ). higher urban temperatures increase energy use, degrade urban air quality, raise the temperature of urban storm water runoff, and contribute to general discomfort, respiratory illnesses, heat stroke, and other heat-related illnesses. warming in conjunction with increased urbanization will amplify urban heat-island effects and their associated impacts. several strategies can help reduce urban temperatures. at the scale of the site, urban trees with high, dense canopies, strategically planted around buildings, in parking lots, and along streets permit the passage of air, shade heat-absorbing surfaces, and cool the atmosphere through evapotranspiration. they also reduce the temperature of runoff water. green roofs perform a similar function on roof surfaces. buildings and landscapes constructed from materials with high solar reflectance or albedo absorb less heat and reduce urban temperatures. at a larger scale, streets oriented parallel (or up to ) to prevailing winds, permeable networks of public open space, strategically situated low-rise buildings, and gaps between high-rise structures increase the air permeability of urban environments. increased temperatures and changes in precipitation regimes are likely to have negative impacts on global food security by worsening the existing regional disparities between developing and developed countries (easterling et al. ; rosenzweig and tubiello ) . this is supported by the prediction that a moderate warming accompanied with elevated co concentrations is likely to lead to an increase in crop productivity of developed countries in temperate mid and high latitudes, whereas even a slight increase in temperature could damage crops that are already growing at supraoptimal temperatures in countries located in tropical, low latitudes (easterling et al. ; easterling ) . as in other systems, even with significant mitigation efforts, adaptation strategies will be necessary to address the impacts of climate change on agriculture. adaptation strategies for more resilient agricultural systems may include altering the timing and location of cropping, switching crop species and cultivars, selecting livestock breeds, crop rotations, and efficiently using and managing water resources. these types of adaptation strategies have been referred to as autonomous strategies (easterling ) . implementing some of these strategies, such as changing the type of livestock that is grazed or changing crop rotations could have profound effects on ecosystem functioning. however, in general, these types of adaptation strategies will have environmental impacts smaller than more intensive strategies that are designed to transform systems. for many agricultural systems, the magnitude of climatic changes will necessitate more intensive planned adaptation strategies that foster significant transformations of systems or infrastructures (easterling ) . such intensive strategies will be particularly important for sub-saharan africa and south asia where climate will have major impacts on agriculture (lobell et al. ; nelson et al. ). these planned adaptation options include developing new infrastructure (e.g., irrigation infrastructure, transport and storage infrastructure, and efficient water use and reuse technologies), investing in new technical and management options (e.g., crop and livestock improvement, enhancing germplasm), shifts in the distribution of agriculture (e.g., abandoning some areas and farming new lands), and building the capacity to make continuing adjustments. here, we discuss two of these strategiesimproving crop performance and water conservationin more detail. stresses on crops due to extreme heat and pest and pathogen pressures are likely to increase with climate change (deutsch et al. ; battisti and naylor ) . multiple adaptation options are available for improving crop performance in the face of such climate-driven stresses. first, in conjunction with conventional breeding, crop improvement through genetic engineering is seen as an effective adaptation tool in a changing climatealthough public opinion is often divided on the use and consequences of genetically engineered crops (macdiarmid ; jones ) . a second approach involves maintaining crop genetic diversity by, for example, collecting and storing the diversity of current cultivars or local landraces. a third adaptation strategy involves exploiting symbiotic relationships between crops and other organisms. for example, it has been shown that numerous fungal and bacterial endophytes confer stress tolerance to heat, drought, salinity, and pathogen attacks (rodriguez et al. ; rodriguez et al. ), and others promote growth and fix atmospheric nitrogen xin et al. ). other strategies include the application of kaolin particle films to reduce heat stress and photoinhibition and to mitigate pest damage (glenn et al. ; glenn ) as well as the incorporation of biochar into soils to confer numerous agronomic benefits that enhance crop performance and stress tolerance (marris ) . irrigated agriculture accounts for about % of the world's freshwater withdrawals (rosegrant et al. ). with increasing global population, the demand for irrigated agriculture will continue to increase globally because realized crop yields normally increase considerably with irrigation. adaptation strategies that foster more efficient use of water in agriculture will be critical for achieving global food security while conserving water in a changing climate. these strategies will include institutional water management reform, economic incentives for efficient water use, and infrastructure investment for efficient irrigation and water delivery systems (rosegrant et al. ). the 'virtual water' trade through crop products from water-abundant to water-deficit areas should be considered as one of the key adaptation options to climate change (rosegrant et al. ; easterling ) . improving crops for higher water-use efficiency (i.e., greater yield with less water use) should be a goal for genetic, ecological, and agronomic options of adaptation discussed above. alternative strategies of increasing water storage capacity with dams and increasing the number of stream diversions for agriculture will, in contrast, have significant impacts on aquatic ecosystems (see the section . . . . managed aquatic systems). as discussed above, changes in temperature and precipitation are likely to lead to changes in the demand for water. projected climatic changes will likely lead to increased demands for residential use, irrigated agriculture, hydropower production, and carbon capture and storage technologies. in some regions, current water management may not adequately cope with the impacts of changing precipitation regimes and increased evaporation on the reliability of water supply, flood risk, health, agriculture, energy generation, and freshwater ecosystems (palmer ) . adaptation strategies designed to address water management for human uses will either directly or indirectly affect ecosystem functioning and ecosystem services. in addition to the adaptation strategies for addressing water demands for agriculture and residential use (addressed to some extent in sections . . . . and . . . . ), other important adaptation strategies for managed aquatic systems that will impact ecosystem functions and services include increased extraction of groundwater, improving storage capacity by building or modifying dams and reservoirs, desalination of seawater, and expansion of rainwater storage and water transfer (bates et al. ; hirji and davis ) . improving storage capacity by building or modifying dams and reservoirs is likely to be one of the most common regional responses to decreased water availability and increased demand for water. the great utility of running water has resulted in the extensive exploitation of streams and rivers by humans throughout the world, a process greatly facilitated by the construction of hundreds of thousands of dams globally (nilsson et al. ) . by capturing high river flows and releasing the water in a carefully controlled manner, dam managers can deliver steady and dependable water supplies to downstream areas, protect settlements from floods, or generate power. although human manipulation of the world's river flows has provided many societal benefits, it has also caused considerable ecological damage and the loss of important ecosystem services valued by society (baron et al. ) . flow regulation for hydropower and storage, diversion for human use, and extraction of groundwater connected to stream systems often significantly affect the natural seasonal and interannual variation in river flow, commonly referred to as the natural flow regime (poff et al. ) . a natural flow regime is needed to sustain the complex riverine and riparian ecological functions and processes that support native biodiversity and provide myriad ecological services including maintaining water quality, recharging groundwater, and nutrient cycling (naiman et al. ) . facing an ominous future of increasingly severe watersupply shortages in many areas of the world, social planners and government leaders are exploring strategies for ensuring that enough water of sufficient quality is available for use by future generations. human demands for water are expected to increase under a warmer climate, exacerbating current management problems. increasing demands for irrigation and industrial cooling water could conflict with the increasing demands for municipal water supplies resulting from urban growth. climate-induced changes to water availability and increasing human demand for water are likely to prompt the construction or modification of dams and reservoirs to improve storage capacity and enhance water security (wcd ; palmer ). for instance, by storing water in reservoirs, water managers capture high flows during wet years or seasons to supplement water supplies at drier times, thereby maximizing the reliability of water supplies and certain economic benefits each year. for regions where increases in runoff variability or more prolonged drought conditions are associated with climate change, the pressure to build watersupply reservoirs will be great. although the construction of additional dams and reservoirs will undoubtedly provide many societal benefits, a rich body of scientific knowledge has shown that river regulation also causes considerable ecological damage and the loss of important ecosystem services valued by society (baron et al. ; postel and richter ) . river ecosystem health deteriorates when natural flows of water, sediments, and organic materials through a river system are substantially disrupted or modified by human activities (poff et al. ; richter et al. ) . river damming and associated environmental alteration is now widely recognized as a leading cause of declines in freshwater biodiversity globally (bunn and arthington ) . dams have also been implicated in the loss of commercial fisheries in many estuaries and coastal areas, and in the degradation of other natural ecosystem products and services worldwide (wcd ) . in addition, reservoirs increase the loss of water due to evaporation. finally, as mentioned above, reservoirs emit substantial quantities of carbon dioxide and methane into the atmosphere (st. louis et al. ) , and therefore, the creation of additional reservoirs is likely to further promote climate change. in many regions, impending water shortages will also lead to greater interest in desalination as a technique for tapping into the vast and infinitely tempting water supplies of the sea. recent improvements in reverse osmosis technology, coupled with the rising cost and increasing unreliability of traditional water supplies both currently and projected in the future, are making desalination a water-supply option with major plants in operation, in planning, or under consideration in europe, north africa, north america, australia, china, and india. like most large-scale industrial processes, making water has a number of actual or potential impacts on aquatic goods and services (wwf ) . emerging issues include significant cost and pollution emitted by desalination plants and the mostly unknown, but potentially substantially, effects on freshwater and coastal ecosystems. intake pipes can remove larvae and small marine organisms, and discharge from desalination plants can elevate levels of salt and other constituents of seawater such as boron; dead sea life, which consumes oxygen while decomposing; chemicals added to change the composition of the water for processing, as well as reducing contamination and clogging of filters and membranes; corrosion byproducts; and the heat added for or during processing. finally, water manufacturing is an energy-intensive process. although increasing ghg concentrations and the resulting increasing temperatures are global phenomena, the globe will often be an inappropriate scale for developing mitigation and adaptation strategies. human activities generate differential risks and vulnerabilities that vary with location due to differences in social, political, cultural, and economic conditions (beck ) . although, in one sense, all people are vulnerable to catastrophic climate change, at another more fundamental level, the risk of climate change impacts (along with blame and responsibility) are unevenly distributed. we can begin to think of the unequal distribution of the vulnerability to climate change as a scale issue. mitigation and adaptation strategies can be applied at multiple, and often overlapping, scales. at the finest scale, the development of mitigation strategies will depend on local leadership and the attitudes of local populations and individuals toward climate change. at national and international levels, mitigation efforts will be organized through large and formal programs and institutions (e.g., un-redd). although some adaptation strategies will be national and international in nature, much adaptation will take place at finer scales of communities and regions. a hallmark of climate change as a global phenomenon is that leadership and coordination across local, national, and international scales is important for ameliorating its effects. in turn, coordination across scales will entail understanding local climate risks and local and regional understandings of the causes of climate change and the costs and benefits of different mitigation and adaptation strategies. the effectiveness of any mitigation or adaptation strategy (building codes, population planning, dietary changes, etc.) depends on its acceptance in local, regional, and national communities. social impediments to adoption of climate change strategies include ideological outlooks, structural constraints, and cultural barriers. for example, free-market ideologies can impede the ability of governments to invest in alternative energies; agricultural subsidies and corporate monopolies on agricultural inputs may create structural barriers to alternative agriculture; and cultural norms for relative water and energy use, family size, or the flavor and texture of food can inhibit conservation of resources and changes in diet. there is a clear disparity between societies that have contributed the most to ghg emissions and those that will likely suffer the direst consequences of climate change. at the international level, efforts to shift blame from the activities of the rich to the actions of the poor have been used to shirk the responsibility to mitigate climate change and to impede the development of global mitigation strategies. the dispute between the global north and south over who caused climate change and who must now take responsibility for mitigation is by now well known. countries in the global south argue that northern countries have already developed their economies relying on fossil fuel consumption and now, when it is their turn to develop, they are asked to put restrictions on their energy consumption and natural resource use. meanwhile, the united states has used the reluctance of developing countries to cap emissions as an excuse for inaction on its part. mitigation and adaptation activities that do not take globally inequalities in wealth, resources, and adaptive capacity into account, or insist on 'equality' in action in a demonstrably unequal world cannot be effective. addressing population growth clearly plays a key role in both mitigation and adaptation strategies. however, focusing on population size while ignoring global patterns in the consumption of resources and the distribution of wealth will result in failed policies and strategies. the world's human population is approaching  , and a growing population inevitably means greater demand for the numerous goods and services supported by terrestrial and aquatic ecosystems. while the availability of resources has seemingly stretched beyond the direst predictions of several decades ago, the crisis of overconsumptionnamely, of coal and other fossil fuels that produce ghgs and contribute to climate changeis simultaneously becoming clearer. while trends over the past several decades suggest that population growth is a significant driver of co emissions (bongaarts ; dietz and rosa ) , it is also true that urbanization, aging, and changes in household size similarly affect energy use and emissions (mackeller et al. ; cole and neumayer ) . particular point sources of ghg emissions from energy consumption in developed countries, like the internationally managed oil fields of nigeria or emissions from the recent iraq war, which since has released over million metric tons of carbon dioxide, the equivalent of adding million new cars to american roads (reisch and kretzmann ) , should bear at least as much scrutiny as the desires of third world women and men to bear children. an overarching emphasis on population detracts from a nuanced understanding of the multiple drivers of ghg emissions. it will be ineffective to focus on the effects of climate change on ecosystems without considering the concurrent effects on human well-being and vice versa. disparities in climate change risk and human health often overlap, and human health and environmental health are intricately intertwined. although diseases are often approached as singular phenomena, good models exist that demonstrate the interactive effects of multiple morbidity factors on health outcomes and disease trajectories. for example, interactions between acquired immune deficiency syndrome and tuberculosis, or diabetes and severe acute respiratory syndrome, have synergistic impacts on patients that must be understood together. likewise, social attributes, like poverty or exposure to racism, interact with disease conditions to increase the damaging effects of disease. such interactions have the potential to influence the effects of climate change on disease movement and clustering, disease interactions, and the impact of climate change on global health (baer and singer ) . human action and human vulnerability to such interactions between climate, disease, and the environment are structured by social and economic inequality, and thus mitigation and adaptation strategies will need to take these inequalities into account. what is clear from a social and political perspective on mitigation and adaptation is that no one set of mitigation or adaptation strategies will be applicable to all people in all places. suggested approaches to either mitigation or adaptation will involve costs and benefits that are unequally distributed across nations, populations, and societies. there will, for example, always be those who can escape pollution with air filters and private vehicles, use air conditioning to survive heat waves, have access to global food markets and multiple water sources, and those who cannot. the most effective mitigation and adaptation strategies, therefore, will address both climate change and environmental justice. the dust bowl area unlocking the potential of protected areas for freshwaters the geography of climate change: implications for conservation biogeography conserving the stage: climate change and the geophysical underpinnings of species diversity climate change and the green energy paradox: the consequences for twaite shad alosa fallax from the river severn would climate change drive species out of reserves? an assessment of existing reserveselection methods adaptive management of the global climate problem: bridging the gap between climate research and climate policy global warming and the political ecology of health impact of geoengineering schemes on the global hydrological cycle meeting ecological and societal needs for freshwater climate change and 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decision-making climate change, biodiversity and the urban environment: a critical review based on london statistical downscaling of general circulation model output: a comparison of methods planning for climate change: identifying minimum-dispersal corridors for the cape proteaceae wind power implementation: the nature of public attitudes: equity and fairness instead of 'backyard motives climate change mitigation and transport in developing nations making water -desalination: option or distraction for a thirsty world? world wildlife fund a diazotrophic, indole- -acetic acid-producing endophyte from wild cottonwood we thank the global health and environment faculty fellows at the university of washington as well as thomas hinckley, richard fenske, judith wasserheit, and tikvah weiner for valuable discussions and support. key: cord- -wtu r j authors: goddard, frederick g. b.; ban, radu; barr, dana boyd; brown, joe; cannon, jennifer; colford, john m.; eisenberg, joseph n. s.; ercumen, ayse; petach, helen; freeman, matthew c.; levy, karen; luby, stephen p.; moe, christine; pickering, amy j.; sarnat, jeremy a.; stewart, jill; thomas, evan; taniuchi, mami; clasen, thomas title: measuring environmental exposure to enteric pathogens in low-income settings: review and recommendations of an interdisciplinary working group date: - - journal: environ sci technol doi: . /acs.est. c sha: doc_id: cord_uid: wtu r j [image: see text] infections with enteric pathogens impose a heavy disease burden, especially among young children in low-income countries. recent findings from randomized controlled trials of water, sanitation, and hygiene interventions have raised questions about current methods for assessing environmental exposure to enteric pathogens. approaches for estimating sources and doses of exposure suffer from a number of shortcomings, including reliance on imperfect indicators of fecal contamination instead of actual pathogens and estimating exposure indirectly from imprecise measurements of pathogens in the environment and human interaction therewith. these shortcomings limit the potential for effective surveillance of exposures, identification of important sources and modes of transmission, and evaluation of the effectiveness of interventions. in this review, we summarize current and emerging approaches used to characterize enteric pathogen hazards in different environmental media as well as human interaction with those media (external measures of exposure), and review methods that measure human infection with enteric pathogens as a proxy for past exposure (internal measures of exposure). we draw from lessons learned in other areas of environmental health to highlight how external and internal measures of exposure can be used to more comprehensively assess exposure. we conclude by recommending strategies for advancing enteric pathogen exposure assessments. exposure to enteric pathogens is associated with a heavy disease burden, largely born by young children living in lowincome settings. globally, enteric infections represent the third leading cause of death among children under five, accounting for approximately deaths in . methods to characterize human exposure to enteric pathogens have not been advanced in the same way as they have in other areas of environmental health, such as exposure to air pollution or industrial chemicals, perhaps in part because the resources are not available in the settings where enteric diseases carry a disproportionally high burden of disease. exposure science was born out of a need to measure exposures to industrial and occupational air pollution and chemical toxicants. this need arose largely in high-income countries following the industrial revolution, which ushered in a period of rapid urbanization and proliferation of factories in urban centers exploiting processes involving fuel combustion. perhaps one of the most notable examples of this sudden increase of population exposure to high levels of air pollution is the london smog incident, which caused an estimated casualties in . thereafter, air pollution exposure scientists developed methods that advanced exposure assessments, ranging from stationary equipment to measure aggregate population-level exposures and pollutant trends, to portable air pollution monitors that provide personal data in real time. chemical exposure science has since evolved to include both external and internal biological measures of several hundred toxicants in large, diverse populations. there are a number of factors that contribute to the difficulty of characterizing exposure to enteric pathogens; some of these factors are associated with the microbial agent, others with the environment and yet others with the host response. first there are technological difficulties of detecting and quantifying a wide range of microbes whose pathogenicity, transmissibility, and fate and transport can vary under different environmental conditions. , even identifying the agents is challenging, as there is still considerable uncertainty about whether and under what conditions detectable microbes are pathogenic, specific pathogens can be present at low concentrations in the environment, and due to frequent discoveries of new and emerging pathogens or known organisms that have acquired disease causing genes. while the source of enteric pathogens is mainly from fecal contamination, there are mixed correlations between the presence of indicators of fecal contamination and enteric pathogens in the environment. , second, environmental enteric pathogen exposure assessments need to consider multiple potential fecal−oral transmission pathways, commonly represented by the f-diagram: fingers, flies, food, fluids (water sources), fields (soil), and fomites. − of increasing interest is the role of zoonotic transmission of enteric pathogens via animal excreta. third, the biological relevance of enteric exposures is complicated by responses to exposure, which is affected by various host susceptibility factors that mediate dose−response relationships, including those engendered by vaccines or previous exposures. response to exposure may also be influenced by gastrointestinal health, such as the diversity of the gut microbiome or increased risk due to enteropathies such as environmental enteric dysfunction. external versus internal measures of exposure to enteric pathogens. exposure to an environmental contaminant is conditional on a complex source-to-host pathway that includes ( ) the release of a contaminant from its source; ( ) the fate and transport of the contaminant in the environment leading to a specific concentration in different environmental media; ( ) the host interaction with those media, ( ) a portal of entry into the human body (i.e., an exposure route), and ( ) uptake of the contaminant into the body. traditional exposure science uses numerous approaches for measuring human exposure along this pathway continuum. a recent national research council report, exposure science in the st century, presented a summary of these approaches, ranging from those that measure environmental concentrations of contaminants to predict exposures before the contaminant reaches the human boundary, to those that estimate a dose after the contaminant has been taken up into the body. here, we propose a similar approach to defining exposure to enteric pathogens, by differentiating between external and internal exposure assessments. we define enteric pathogens as microorganisms transmitted via the fecal−oral route that can cause gastrointestinal infections, leading to acute (e.g., diarrheal disease, gastroenteritis, enteric fevers) and chronic infectious disease outcomes (e.g., environmental enteric dysfunction, growth faltering, impaired cognitive ability). enteric pathogens include bacterial, viral and protozoan pathogens, with fungi and helminths increasingly receiving more attention as causes of neglected tropical infectious disease. , a common approach for external exposure assessments is to detect indicators of fecal contamination or specific pathogens in a known size of environmental sample. environmental measures such as these can act as proxies for external exposure as they assess specific fecal−oral transmission pathways. measuring host-specific microbial source tracking markers in environmental samples can also provide information on the source of fecal contamination (i.e., animal versus human). however, environmental measures on their own do not provide precise measures of the magnitude of exposure (i.e., how much contaminated water one actually ingests) which must be estimated or imputed, so can only be considered as an indirect proxy for actual ingestion of pathogens. augmenting environmental data with data on human interactions with their environment, such as those published by the united states environmental protection agency in the exposure factors handbook, can help provide actual estimates of exposure (i.e., pathogen ingestion). while the handbook is focused on chemical exposures in the united states, it demonstrates the types of frameworks that can be employed to inform assumptions for external exposure assessments, such as ingestion rates (drinking water, soil, and food) and object mouthing. in contrast, internal enteric pathogen exposure assessments using human biological specimens may estimate the actual exposure to enteric pathogens after crossing the human body envelope, typically via oral ingestion. in this respect, they address the main shortcoming of the external exposure assessment, i.e., whether a pathogen has actually been ingested. on the other hand, they provide little information about the source or transmission pathway that external exposure assessments offer. they can, however, provide some information on the presence, types, and frequency of past exposure to enteric pathogens as well as indications of potential health impacts. while the ingested dose of enteric pathogens is not typically measured, past exposure events can be inferred from serology, detection of pathogens in feces, and other biomarkers as proxies of internal exposure. the need for improved exposure methods: the wash case. systematic reviews of water, sanitation, and hygiene (wash) evaluations, conducted to identify the health effects of interventions designed to reduce enteric pathogen exposure, have generally found improved wash to be protective against diarrhea, soil-transmitted helminthiasis, and malnutrition. , however, much of the evidence is from observational studies or short, smaller-scale trials. recent experimental field evaluations of some of these interventions found either no evidence of health benefits, − a reduction in diarrhea but no improvement in child growth, or improved growth but no impact on diarrhea. , these mixed results have focused greater attention on the need for more rigorous exposure assessment, in part to explain why effects of wash interventions are realized in some trials and not others. a recent consensus statement recommends that interventions need to radically reduce fecal contamination in the environment to achieve more consistent child health benefits, though which pathogens and pathways are most important and the necessary reductions needed to achieve health impactsmay be highly context-specific. filling these gaps requires greater attention on more rigorous exposure assessment, as few studies have attempted to directly measure exposure along the various transmission pathways, and those that do normally rely on quantifying indicators of fecal contamination in the environment as a proxy for pathogens. moreover, these approaches are largely confined to assessments of external exposure, sometimes combined with observations or modeling to estimate ingested dose of enteric pathogens. the theory of change underlying the impact of a wash intervention on enteric health outcomes is that an intervention will prevent disease if the intervention (a) is capable of but only a few have assessed the impact of an intervention on exposure to fecal contamination along the transmission pathways targeted by the interventions, in part because there is no consensus methodology for how to measure exposure. some of the null findings from recent wash interventions are consistent with the wash theory of change, reporting null effects from potentially effective interventions delivered to a vulnerable population when coverage and uptake were low. , , , however, other studies have reported null effects on diarrhea , and/or stunting , , even with higher levels of coverage and use; others have reported protective effects on stunting (but not diarrhea) with high levels of coverage and use, especially from reductions in open defecation. , , some wash evaluations, identified from a systematic review investigating the relationship between indicators of fecal contamination and child health outcomes, included exposure assessments that raised questions about the manner in which they characterized exposure (figure ). in a recent trial of individual and combined wash interventions, luby and colleagues reported reduced diarrhea from wash interventions, except for water treatment using chlorine, even though reductions in fecal indicator bacteria were found in both stored drinking water and food in households that received the water treatment intervention compared to control households. this suggests perhaps that the chlorine-susceptible indicator bacteria used to estimate fecal contamination were not representative of chlorine-resistant pathogens, such as protozoa that may have been contributing to diarrhea symptoms. this is consistent with the trial's findings of reductions in giardia infection in all wash arms except for the water treatment arm. the same evaluation also reported reduced diarrhea and prevalence of infections with protozoa and soil-transmitted helminths in the sanitation-only arm , even though there was no evidence of a change in fecal indicator bacteria in water, food, soil, on hands, or a change in fly density in this arm compared to controls, suggesting reductions in disease transmission not captured by the fecal indicator bacteria measurements. reese and colleagues reported no impact on diarrhea but reduced stunting from a water supply and sanitation intervention despite no evidence of a reduction in fecal contamination of drinking water or hands. pickering and colleagues also found no effect of a community-led sanitation intervention on diarrhea but an improvement in child growth, despite no reduction in fecal contamination in drinking water; however, latrine fly presence and observed human and animal feces did significantly decrease in the treatment group. collectively, these studies illustrate the need for improved characterization of external exposures to enteric pathogens. similarly, limited tools currently exist for assessing internal exposures. as described below, some studies combine external assessment with behavioral observations to estimate actual ingestion (e.g., measuring pathogens in soil and frequency of geophagia, or measuring fecal indicators deposited by flies when alighting on food and the number of fly landings). however, these methods rely heavily on assumptions about conditions and behaviors that vary significantly within and between individuals, and over time. others have begun using proxies of internal exposure as an indicator of past exposure, such as measuring enteric pathogen shedding in child stool or serology from dried blood spots as indicators of past exposure. , while investigators have sought to reconcile the inconsistent relationship between exposure measurements and health with accepted theories of change, the lack of a clear and consistent progression between these proxies of exposure and health outcomes raises fundamental questions about current exposure assessment methods. in addition, they raise questions concerning "how clean is clean enough" for realizing reductions in rates of infection and disease. for program implementers to optimize interventions that improve health, rapidly identifying the leading sources of exposure in a community can help develop and test context-specific interventions that address them. epidemiologists are seeking reliable data on which to build models to ascertain determinants of disease risk and prioritize disease control strategies. public health officials are seeking data on exposure to implement cost-effective population-based strategies to mitigate health burdens. all of these demands rest on our ability to generate better, cheaper, faster, field-deployable approaches that can be used in low-resource settings with minimal training. with this as background, an interdisciplinary group of environmental health researchers convened at emory university in atlanta, georgia (u.s.a.) in september for a workshop aimed at identifying priorities for improved approaches to measuring enteric pathogen exposure. while much of the discussion focused on exposure assessment in household-level wash studies, the group agreed that the challenges extended to exposure to enteric pathogens in lowand middle-income countries more generally that are transmitted via the fecal−oral pathway, including exposures from agricultural, commercial and recreational activities. the objectives of the workshop were to (a) identify applications and criteria under which to compare exposure assessment approaches, (b) explore potential lessons in exposure science from other areas of environmental health, (c) critically review current and emerging enteric pathogen exposure assessment practices across a range of applications, and (d) define research priorities to help move the field of enteric pathogen exposure science forward. participants in the workshop were u.s.-based experts purposely selected from a variety of disciplines who were available and willing to meet and explore the issues presented. such expert gatherings present the potential for selection bias in the opinions expressed. we identified four primary applications that could benefit from further resources dedicated to improving enteric pathogen exposure methods. . identifying primary sources and modes of transmission: improved measures to identify the leading sources and modes of transmission of enteric pathogens in a given setting could inform programs and investments designed to improve health by reducing fecal exposure. specific pathogens are differentially mediated by various fecal−oral transmission pathways, and the importance of these pathways may differ between settings (e.g., urban vs rural), subpopulations (e.g., children in different age groups) or time points (e.g., season). improved external exposure measures would contribute to a better understanding of what exposure pathways need to be interrupted in a given setting to achieve consistent child health benefits. . evaluating interventions: better tools to estimate enteric pathogen exposure could be leveraged to evaluate the extent to which interventions are able to reduce exposure, and could have implications for the sample sizes needed to detect differences between groups. these tools could include measuring pathogens or proxies along specific pathways that interventions are designed to interrupt, and measuring pathogens in human biological samples to characterize past exposure. using exposure assessments to evaluate interventions would potentially enable a faster and more objective evaluation of interventions designed to improve health by reducing exposure compared to studies that focus on health end points. . surveillance: novel approaches to characterizing exposure to enteric pathogenssuch as monitoring emerging agents in wastewatercould be applied for surveillance purposes. these purposes could range from monitoring human infection prevalence, rapid assessments of environmental threats, such as potential infectious disease outbreaks or bioterrorism, to monitoring the progress of regional or national efforts toward reducing infections. . exploratory: there are broader research applications that could benefit from improved measures of enteric pathogen exposure. examples include previously understudied links between enteric pathogen exposure and its effects on the gut microbiome and malnutrition, as well as its relationship with comorbidities such as respiratory and vector-borne diseases. improved exposure assessments can also be linked to important subclinical conditions, as well as to a better understanding of inflammatory responses that are increasingly associated with a wide range of health effects. there is also a need to better understand the underlying mechanisms of transmission and pathogenesis associated with environmental exposures to enteric pathogens. when considering different approaches to measuring exposure to enteric pathogens for each of these applications, there are a number of criteria to consider. these include the following: • external vs internal: is exposure characterized in the environment as a proxy for external exposure and does it provide data on the source of exposure, or is it measured after the contaminant has crossed the human boundary (i.e., internal exposure)? for external exposure assessments, is exposure characterized proximal to the human boundary or is it a more distal measure that requires modeling to estimate more proximal exposure? for internal exposure assessments, are the measures mediated by host susceptibility to infection? • pathway-specific: can the exposure assessment quantify the relative contribution to total exposure by different transmission pathways? deployment in the field in low-resource or emergency settings? does the assay require cold-chain transport or a consistent energy source? are the required materials bulky or dangerous to handle? how much training and material is required to collect samples, conduct analyses, and interpret results? is it fast enough to provide actionable feedback to reduce exposures in a population of interest? • ethics: are exposure assessment methods potentially burdensome on the communities and individuals where they are conducted? do they require respondents to provide a substantial amount of resources (e.g., large volume water samples) and time? do they involve an invasion of the respondents' privacy? do they provide interpretable information for end users and how do users respond to that information? ■ lessons from other areas of environmental health air quality. in other areas of environmental health, such as air and chemical pollution epidemiology, moving from ecologic or population-level measures of exposure to measures that better reflect exposure at the individual level has been a focus of research for the past two decades. in , for example, the national research council committee on research priorities for airborne particulate matter outlined the quantification of the difference between proxy and personal measures of exposure as a key research priority for better understanding differences in observed health risk estimates across individuals and locations. since then, new approaches have been designed to measure an individual's inhalation exposure to particulate matter in air, using personal breathing zone samplers. for characterizing exposures to cookstove emissions, in particular, new devices, such as the exposure child monitor (ecm) and the ultrasonic personal air sampler (upas), provide more precise measurements for use in public health research applications. personal samplers for enteric exposure would face significant additional barriers given the diverse agents, the fate and transport of those agents in the environment, their infectious doses, and the myriad of exposure pathways. measuring long-term exposures to air pollution, a known driver for a range of chronic adverse health effects, necessitates alternative approaches for characterizing exposure, and often employ hybrid methods that combine both modeling and personal monitoring. a promising, and increasingly common approach for estimating spatiotemporally resolved long-term exposures to particulate matter and several gaseous pollutants comes from satellite remote sensing. these methods have the ability to use satellite optical instrumentation, calibrated with ground-level ambient monitoring data, to create long-term global exposure surfaces. some water quality parameters can be measured via remote sensing (e.g., chlorophyll-a), but these methods apply more to large water bodies than specific volumes of water consumed by humans. other methods integrate human activity patterns, questionnaires related to sources of exposure, and measurements conducted within defined settings (i.e., microenvironments) to predict individual level air pollution exposures over short-and long-term periods. the air pollution exposure model (apex) is an example of this class of air pollution exposure model, which was developed in response to prior limitations related to air pollution exposure and which may offer insights for novel, combined approaches for characterizing exposures to enteric pathogens as well. similarly, there are analogous hydrological water quality models that have been developed for estimating exposures to water pollution for surface water bodies. chemical toxicants. chemical toxicant exposure assessments, the measurement of a chemical, or its metabolite, degradate, reaction product or surrogate, include external and internal exposure assessments. external chemical exposure assessments can be pathway-specific (e.g., water, air) and route-specific (e.g., ingestion, inhalation) while internal assessments integrate all pathways and routes of exposure through which a chemical has entered the body. for example, assessment of dermal chemical exposures includes the use of hand wipes, patches, and body suits as dosimeters of exposure. the dosimeters are removed after exposure and chemical concentrations are measured in them, estimating dermal exposure. similarly, personal air space pumps or patches are used to estimate inhalational exposures and duplicate diet or water measurements may be used to estimate ingestion exposures. emerging techniques such as the use of silicon wristbands to absorb airborne contaminants have also been used as efficient means to capture exposure to up to air contaminants including polychlorinated biphenyls, pesticides, flame retardants, polycyclic aromatic hydrocarbons and volatile organic chemicals. internal chemical exposure assessments (i.e., biomonitoring) have burgeoned over the last two decades and are often the "gold standard" methods for assessing exposures in relation to adverse health outcomes. biomonitoring of chemical contaminants in urine and serum with mass spectrometry-based methods have resulted in many population-based data reports on human internal exposure in the united states, canada, and korea. − advances in exposomics using high-resolution metabolomics allow for the detection of numerous endogenous and exogenous chemical metabolites simultaneously. biomonitoring is also seeing application in air pollution exposure assessments in low-income settings, for example with the detection of polycyclic aromatic hydrocarbons in urine to quantify household air pollution exposure. in comparison, internal enteric pathogen exposure assessments face the difficulties of pathogens as dynamic biological organisms that can amplify and die-off inside the host as well as acquired immunity and other modifiers of dose−response relationships. measurement error. analytical frameworks have been introduced to assess the impact of exposure measurement error, i.e., the difference between the measured and the true exposure, in health effects models. they have shown that some forms of error may lead to biases and greater uncertainties estimating exposure-outcome relationships. although the nature of measurement error in enteric pathogen exposure assessments may be complex and multifactorial, quantifying sources of error can enable prioritization of how to eliminate the greatest sources of error and better capture biologically relevant exposures. prior research on the effects of measurement error on waterborne disease epidemiology has shown that enumeration methods for indicators of fecal contamination can attenuate the relationship between indicator levels and diarrhea by up to %. another source of error occurs when true exposure variability is not sufficiently reflected when using proxy indicators. for example, spatiotemporal rainfall variability can attenuate associations between heavy rainfall and diarrhea by single measures of water quality can attenuate the relationship between fecal indicator concentrations in water and child linear growth by up to % compared to repeated measures that account for temporal variability in water quality. the same study also reported that neglecting exposures to water sources outside of the household can attenuate the water quality-diarrhea associations by up to %. other examples of sources of measurement error include sampling protocol design introducing data collector error (i.e., through nonspecific sampling instructions), assigning household-or community-level exposure measures to individuals, and environmental sample processing errors (i.e., during sample collection, transport, or analyses) that can lead to under-or overestimates of concentrations of enteric pathogens or fecal indicator organisms in environmental media or to false negative or false positive results. as part of this workshop, participants collectively identified a set of current and emerging methods, some of which have been widely used in other environmental health sectors, that may contribute to improved measures of enteric pathogen exposure. we describe those methods here and summarize them against the criteria listed above in supporting information tables s and s . measuring enteric pathogens in the environment. studies investigating the association between enteric pathogen exposure and disease, or evaluations assessing the effectiveness of wash interventions that include an exposure assessment, have mainly attempted to characterize fecal contamination in different environmental media as a proxy for enteric pathogen exposure, neglecting to characterize human interaction with those media. a systematic review of the effects of sanitation interventions on fecal−oral transmission pathways identified the following approaches used: enteric pathogens or indicator bacteria in environmental samples (drinking water, hands, sentinel toys, food, household and latrine surfaces, and soil); the presence or abundance of flies; and observations of human and animal feces. there are a number of factors to consider when measuring enteric pathogen prevalence in the environment, including environmental sampling strategies, the use of indicators as proxies for enteric pathogens, differentiating between human and animal sources of contamination, detection limits, and selecting which specific pathogens to target. sampling strategies. the vast majority of studies that have measured environmental fecal contamination coupled with child health outcome data have focused on drinking water and, to a smaller extent, fly density, hands, and fomites, with limited data on food and soil contamination. drinking water samples can be collected directly from the source, from the household storage container or from the utensil used to drink water, each progressively capturing additional pathways of contamination between the source and the point of consumption. often a choice has to be made between multiple sources of water used by the household, only some of which are dedicated for drinking. hand contamination has been assessed through rinsing hands in sterile water and analyzing the rinsewater. food samples can include items prepared and stored at home or bought outside the home (e.g., produce typically eaten raw or streetfood). , surfaces can be sampled by swabbing, and soil by scraping topsoil from a designated area. the site of collection of surface swabs or soil is a key decision in sampling protocols. a tool for capturing overall contamination in the domestic environment is sentinel toys, a nonporous object (e.g., plastic ball) that is left in the household for a prespecified amount of time for household members to interact with and then rinsed. fecal contamination in all of these domains is highly variable temporally, − seasonally, , and spatially, , and additional variability can be introduced by the methods used to analyze samples. some strategies to address this variability include selecting sampling locations relevant for exposure for the target population (e.g., soil in areas where children spend time), focusing on key times of exposure (e.g., measuring hand cleanliness before eating) and collecting longitudinal samples. additionally, when the goal of sampling is to assess the relationship between pathogen exposure and health outcomes, environmental samples can be collected prior to ascertaining health end points, allowing an appropriate incubation period before onset of health outcomes. − the common practice of collecting samples and health data during the same household visit, typically for logistical convenience, risks capturing contamination levels at a time that is not relevant for disease transmission. as a result, this practice is vulnerable to introducing reverse causation (e.g., water boiled in response to illness, fecal contamination in the environment due to diarrhea), obscuring the true relationship between exposure and disease. indicators of fecal contamination. fecal contamination in the environment is commonly estimated by using indicators of fecal contamination. these indicators have the advantage that they are easier and less expensive to measure compared to multiple specific pathogens and they can be indicative of a range of enteric pathogens. they provide an indication of the presence of fecal matter in a sample, but do not confirm the presence or absence of pathogens, nor do they provide any indication on the infectivity or diversity of enteric pathogens in a sample. there are a number of indicators of fecal contamination, including chemical indicators (e.g., fecal sterols, caffeine, estrogen hormones) , as well as microbial indicators. fecal indicator bacteria are often grouped into the coliform and streptococcal bacterial groups. total coliforms include a broad spectrum of bacteria occurring in feces, but can also be found in nonfecal matter. fecal coliforms or thermotolerant coliforms are a group of bacteria that are more specific to fecal contamination, with the exception of klebsiella. escherichia coli (e. coli) is the most commonly found fecal coliform bacteria and is more specific to human and animal fecal matter, though genome comparisons suggest that some clades of e. coli primarily originate from nonhost natural environments, and there are reports of e. coli detection in pristine areas of tropical and even temperate environments. − in recent years, several laboratory-grade products have been validated in the field as predictive of e. coli. , however, these instruments require frequent cleaning, are not intended for long-term autonomous operation, are subject to the drawbacks of methods measuring indicators of fecal contamination and are expensive. recent efforts are focused on reducing these product costs and enabling continuous in situ water quality monitoring. fecal streptococci were identified as an alternative to total coliform in the s when it became clear that total coliform was a nonspecific indicator for fecal contamination, but the use of this indicator diminished once more specific methods for e. coli culturing were established. enterococci are a subset of environmental science & technology pubs.acs.org/est critical review species of the fecal streptococci group, more specific to fecal contamination than fecal streptococci and more persistent in the environment. coliphages and crassphages, types of bacteriophages (viruses that infect bacteria), are also used as indicators of fecal contamination because of their similar morphological characteristics and ability to mimic the persistence of viral pathogens in the environment. , the use of fecal indicators was historically established to measure fecal contamination in drinking water and recreational waters and to monitor the performance of water treatment processes. in the united states, total coliform is still used to monitor treatment efficacy and post-treatment contamination of drinking water supplies in accordance with the total coliform rule, which requires the monitoring for the presence of total coliform in public water systems at a frequency proportional to the number of people those systems serve. e. coli and fecal coliform are presently the most commonly used fecal indicator bacteria for monitoring fecal contamination in water. the world health organization (who) uses levels of e. coli and fecal coliform to define microbial quality of drinking water. meta-analyses of the association of drinking water quality measured using fecal indicator bacteria and diarrheal disease in low-income settings have presented mixed results. gundry et al. found no significant association between e. coli or fecal coliform in household water and diarrhea, gruber et al. found a significant association between levels of e. coli in water and diarrhea but not for fecal coliform, whereas hodge et al., using an individual participant data (ipd) meta-analysis, showed a higher risk of diarrhea with increasing levels of fecal coliform in water. a recent ipd meta-analysis found evidence of association between both e. coli and fecal coliform levels in household drinking water and diarrhea and impaired linear growth. fecal indicator bacteria are increasingly used to characterize fecal contamination along other fecal−oral transmission pathways. the shortcomings related to indicators of fecal contamination, when used for purposes other than their original intended use of monitoring water supply systems, have been well documented, including that e. coli has been found in environments where fecal contamination is improbable. these shortcomings are amplified by the limited association between indicators and the presence of enteric pathogens. , however, measurement of fecal indicator bacteria may be valuable as an indication of "total fecal load" in environmental samples and to provide a common metric for comparison with previous studies. source tracking. fib used in solitude and some other indicators of fecal contamination do not distinguish between different sources of contamination. fecal source tracking aims to provide data on the source of fecal contamination by detecting signatures of specific sources (e.g., particular animals or particular geographies). in addition to humans as a source of fecal loading in the environment, animal fecal contamination is of particular interest in low-income settings where households often cohabitate with animals in confined spaces. a recent systematic review outlined the importance of animals as a source of fecal contamination to human health, suggesting an association between animal feces exposure and diarrhea, soil-transmitted helminth infection, environmental enteric dysfunction, growth faltering, and trachoma. a variety of source tracking methods have been described in the literature, ranging from fecal coliform/fecal streptococcus ratios to host-specific molecular markers. host-specific bacteroidales are commonly used in high-income countries to complement the use of fecal indicators, and are seeing increased use in low-income settings to distinguish between human and animal sources of fecal contamination. − bacteroidales can distinguish between sources of fecal contamination, because they adapt to their host differentially, allowing for the identification of host-specific fecal contamination. however, fecal source tracking assays developed in one geographic location must be validated to be used in additional locations, due to geographic variation in human and animal fecal microbiomes, and the molecular methods used to detect bacteroidales provide no viability or infectivity data. host-specific bacteroidales have been measured in tanzania to characterize human-specific fecal contamination on hands and in drinking water, and test the relationship between humanspecific fecal contamination and diarrhea. a nested study within a cluster-randomized controlled sanitation trial in odisha, india utilized fecal source tracking to discern the effectiveness of a sanitation intervention on the reduction of human-specific bacteroidales compared to animal-specific bacteroidales. phage-based methods are a rapid and low-cost approach for distinguishing between human and animal feces. gb- phages that infect human bacteroides fragilis have been used in a number of countries to identify human fecal contamination. − like the molecular bacteroidales method, these phage-based methods also need to be validated in each geographic location. other methods use next generation sequencing data to distinguish between different host microbial communities within a given household or community, , and use that information to attribute sources of contamination. , however, these library-dependent methods are still under development, can be expensive and difficult to standardize, and require shipping samples to sequencing facilities. currently, reference databases used to ascribe taxonomy in these studies are biased toward bacterial communities associated with persons living in high-income communities, but there are continuing efforts to expand these reference databases. enteric pathogen detection with traditional methods. specific pathogen occurrence or concentration, instead of the use of indicators as proxies, are less commonly measured in the environment although they may be more representative of the actual health risk associated with enteric pathogen exposure. exposure assessments that measure specific pathogens need to consider not only the diversity of potential pathogens occurring in the environment but relevance of each included pathogen for health outcomes of interest, which is highly context specific. the possibility for improved specificity from measuring specific pathogens instead of indicators of fecal contamination may come at a loss of sensitivity, since selected pathogens may not be representative of all possible pathogens in the environment and are typically present in lower concentrations in the environment. monitoring water supplies for pathogens can be prohibitively resource intensive due to the array of pathogens and the low concentrations of specific pathogens in water, requiring sensitive assays. the who estimates that concentrations of pathogens in water corresponding to − disability-adjusted life years (dalys) per person per year are typically less than organism per − liters. testing for pathogens in water is achieved by concentrating the water sample, such as filtering large quantities of water, on the order of − l, environmental science & technology pubs.acs.org/est critical review followed by using culture-dependent or culture-independent methods to enumerate the density of pathogens. however, ultrafiltration using kidney dialysis filters is a low-cost method to efficiently and simultaneously concentrate viruses, bacteria, and protozoa from large volumes of water and wastewater. − culture-dependent methods are also limited by their low sensitivity and their resource intensity. furthermore, some enteric pathogens (salmonella typhi, vibrio cholerae, campylobacter spp., and others) can enter a viable but nonculturable state in the environment that may require special resuscitation steps or molecular methods to detect. while resuscitation or enrichment steps increase the probability of pathogen detection from an environmental sample, they only confirm the presence of the pathogen and do not provide quantitative data. enteric pathogen detection with molecular methods. culture-independent molecular methods have been developed for many enteric pathogens, but these have traditionally required extensive laboratory equipment and highly skilled technical staff. as molecular methods, such as as polymerase chain reaction (pcr) based assays have become lower-cost, easier to multiplex, and more robust to inhibitors in environmental samples, a number of studies have successfully detected bacterial, viral, helminth, and protozoan pathogens in drinking water, on hands, and in soil in low-resource settings. , , − liu et al. developed the customizable taqman array card (tac), an emerging method for quantitative detection of multiple enteric pathogens encompassing viral, bacterial, protozoal, and helminth targets for enteric infections. tac is a taqman probe based real-time pcr assay which can test up to eight samples with individual singleplex (could be expanded to duplex) reaction wells per sample in a -well format. these tac assays were validated and field tested in many low-and middle-income country settings. examples of recent environmental applications of the tac method include the simultaneous detection of a number of enteric pathogens in surface water, soil, and infant weaning food in kenya. , pholwat et al. developed a custom environmental surveillance tac to identify antimicrobial resistance genes, poliovirus, and enteric pathogens in environmental samples such as fecal sludge, food, sewage, soil, and water in low resource settings. advantages of tac are the ability to test + targets/pathogens simultaneously, to obtain quantitative results relatively quickly, and to utilize the same method for both environmental and human samples. an important step in detecting a broad spectrum of targets is to extract high quality total nucleic acid (both dna and rna) from the sample. unlike culture-based methods where pathogen growth is dependent on many factors such as antibiotic history, culture media, and temperature, high throughput molecular diagnostics such as tac can detect many pathogens quantitatively and be used across wideranging sample types. however, molecular methods cannot be used to establish viability of the organism and can be prohibitively costly. resource-intensive field collection and lab processes mean these methods are challenging to deploy in low-income settings where resources are limited. environmental metagenomics. metagenomics, the sequencing and analysis of all dna in environmental samples, circumvents the problem that many enteric pathogens cannot be easily cultured and metagenomic data can provide information on the abundance and diversity of microorganisms in environmental samples. unlike many molecular methods, it does not require prespecification of targets, allowing the user to probe for all potential enteric pathogens present in a sample. while it has been historically difficult to identify specific enteric pathogens with amplicon sequencing or low coverage metagenomic approaches (i.e., capturing s rrna genes), shotgun metagenomic approaches (i.e., capturing all metagenomic dna) can be used to distinguish between specific enteric pathogen strains. reduced sequencing costs over time as well as recent advances in sequencing technologies and bioinformatics pipelines will continue to open up opportunities for enteric pathogen detection. long read sequencing technologies, such as oxford nanopore technologies (ont), enable pathogen identification through assembly or direct alignment of long dna reads to published pathogen genomes. metagenomics can be used to characterize both pathogens in environmental samples and in human stool samples. environmental metagenomics has recently been used to profile viral pathogen diversity in environmental waters, examine antibiotic resistance diversity in wastewater, and to demonstrate exchange of antibiotic resistance genes between soil bacteria and clinical pathogens. limitations of metagenomics include poor sensitivity if enteric pathogens are at low prevalence in the microbial community or when sequencing depth is low, it does not confirm viability or infectivity of the pathogens, high cost, required bioinformatics expertise, and the need for improved analysis pipelines for identifying pathogens. wastewater surveillance. wastewater monitoring is an emerging approach to surveillance and has the potential to deepen our understanding of community health. wastewater has been shown to provide useful community-level information regarding illicit drug use, antimicrobial resistance, , signals of chronic disease, and advanced warning of viral outbreaks, , including recent efforts to detect sars-cov- in wastewater. most prominently, wastewater is used for poliovirus detection in global eradication efforts. identification of enteric pathogens in wastewater can indicate downstream exposure potential, as well as suggest past exposure to that pathogen among those contributing waste. however, there are challenges with wastewater surveillance: ( ) large dilution from other households in the catchment area of pathogens shed by a small number of infected people; ( ) need for concentration methods and sensitive detection methods compared to stool samples; ( ) need to understand the wastewater collection network, flow, and catchment area in order to identify strategic sample collection points, and ( ) differential pathogen transport, die-off or regrowth by wastewater network need to be considered. dynamic models can be valuable for optimizing the development of a sensitive environmental surveillance system. , wastewater surveillance may not capture feces from the poorest populations who are most likely to have the highest burden of infection and exposure because these populations may practice open defecation or use on-site sanitation, and as a population-level approach limits individual-level scientific inference. biosensors. instead of using laboratory intensive approaches to estimating pathogen prevalence in the environment, there is room for innovation to detect enteric pathogens on-site in environmental media by using biosensing technologies. biosensors isolate dna encoded biological responses to an electrical signal that can be logged digitally. such methods have the advantage that they have the potential to be deployed in resource-constrained and remote settings, because they environmental science & technology pubs.acs.org/est critical review remove the need for sample collection, transport, and intensive laboratory processing. bioreceptors can include tissues, microorganisms, enzymes, antibodies, and other biologically derived elements, although most current applications are limited to indicators of fecal contamination rather than specific pathogens. a recent application of biosensors to detect fecal contamination in drinking water used an odorant-binding protein derived from mosquitos to test for the presence of coliform bacteria. bacteriophage with a broad host range specificity for e. coli have been combined with magnetic beads to capture and then separate e. coli in drinking water. measuring enteric pathogens in humans. as a proxy measure of internal exposure, infection with enteric pathogens can provide confirmation of actual exposure following ingestion, thus potentially providing important data on past exposure for the evaluation of interventions and for surveillance. findings from these measures are complicated by host immunity, and absence of infection or serological indication of prior exposure cannot confirm that exposure did not occur. more specifically, these data can be employed to evaluate how well interventions reduce exposure to specific pathogens and which pathogens humans are exposed to in their community. data generated from surveillance and epidemiological studies has benefited from advances in surveillance capacity and detection methodologies for estimating the burden of disease, disease severity, attributing health outcomes to pathogens, and pathogens to exposure pathways. these data facilitate hazard characterization, the first step of risk assessment. dose response data are highly variable by pathogen and are limited because human challenge studies are difficult, expensive, usually single-pathogen focused, primarily performed among adults in high-income countries, cannot control for previous exposures and differences in immune responses to infection, and may be at odds with acceptable ethical standards. furthermore, little data is available on how much of an ingested dose passes through specific and nonspecific host defense barriers and reaches the target cells and is capable of inducing infection. animal models often do not exist for enteric pathogens or do not cause the same health outcomes as in humans. exposure assessments can take into account the hazard characterization information derived from epidemiologic and dose−response studies to focus on pathogens that cause the greatest disease burden in the region of interest, taking a more narrowed approach to exposure assessments by focusing on specific pathogen-source pairs. pathogen shedding in stool. methods to detect enteric pathogens in stool samples range from using microscopy or enzyme-linked immunosorbent assays to detect single pathogens, to using molecular or metagenomics methods to characterize multiple pathogens in a sample. multiplex pcr is a technique that has been widely employed in enteric disease surveillance (sporadic and outbreak) and epidemiological studies such as in multicountry case-control and longitudinal birth cohort studies, as well as in recent studies measuring health impacts of wash interventions. , shotgun metagenomic approaches have also been employed, for example to distinguish between foodborne disease outbreak strains of salmonella, and to identify the likely causes of diarrheagenic e. coli in ecuador. the limitation of these techniques is not only the intensive resources they require, but it can also be difficult to attribute a specific pathogen to a disease outcome when multiple enteric pathogens are detected in stool simultaneously or when asymptomatic infections are common. additionally, the sensitivity of these methods can vary. these methods identify infections by detecting pathogens in human stool samples, and pathogens may only be shed by an infected person for a short period of time (days, weeks) or shed intermittently, so infections between sampling events may be missed. furthermore, the duration of shedding after infection is highly pathogen-specific, so these methods can be biased toward persistent pathogens that are shed for a longer period of time compared to more transient pathogens. pathogen-specific immunoassays. another way of estimating past exposure to enteric pathogens is through immunological assays detecting pathogen-specific antibodies in serum or saliva, which can be multiplexed to detect exposure to multiple enteric pathogens. for these immunological methods, the timeline of exposure can be difficult to ascribe as low levels of pathogen-specific antibodies (immunoglobulin (ig)a, and particularly igg) can be present in saliva and serum for weeks to years after infection. this can also be an advantage, as the methods can be used to integrate prior exposure over longer periods of time, rather than relying on pathogen shedding in stool. exposure data without regard to history of infection can be useful for some applications, such as to determine if a population has been exposed to a rare or emerging pathogen or particular microbial strain, which could be important for focusing exposure assessment approaches. sero-epidemiology is a promising approach to measurement of force of infection of enteropathogens across entire populations. − however, one's immune response depends on a number of host-specific factors including history of previous exposure (acquired immunity ), nutritional status, genetics, composition of the gut microbiome, underlying disease such as hiv infection, and age (antibodies can appear in low concentrations in young children, particularly in saliva). measuring interaction with the environment. exposure to enteric pathogens is not only conditional on pathogen presence in the environment, but also on host interaction with that environment. collecting these data can complement exposure assessments and the analysis of exposure data, as has been done in estimating human exposure to other environmental pollutants. survey data on self-reported behaviors or observational data on practices can enable the targeting of environmental media and locations where the study population is predominantly exposed. quantitative observational data can be combined with environmental measurements of enteric pathogens to estimate pathogen ingestion rates. the sanipath tool, an example of such an approach implemented in nine countries to date, combines environmental sample collection and analyses for e. coli with surveys of behavior to estimate exposure. , here we describe current methods that have been used to characterize host interaction with the environment and summarize those methods in supporting information table s . when using these methods, it is important to consider the collection of behavior data disaggregated by gender and age group because of the differences in behavior between men and women, boys and girls, and adults and children. surveys and self-reports. surveys have been used as a rapid and cost-effective tool to collect information on a range of self-reported behaviors that serve as proxies for exposure patterns. while surveys carry the risk of various types of bias, such as recall bias, courtesy bias, and reporting bias associated with self-report of socially desirable behaviors, , surveys are nonetheless a useful tool to obtain information on neutral behaviors that do not trigger these biases. there are multiple approaches for collecting self-reported data. many studies have used household surveys with the head of household or primary caregiver for young children. one major limitation of this approach is that the reported behavior for the respondent is often incorrectly seen as a proxy for behaviors for the entire household. community participatory surveys and surveys in school classrooms, that combine some discussion of the behavior with a method for the participants in the group to confidentially report their own behavior, such as pocket voting, have also been used to identify high-risk behaviors. surveys and self-reports can also be used to inform sampling locations (e.g., where a household obtains their drinking water, prepare their food etc.). observations. spot-check observations can capture wash infrastructure and behaviors that result in high risk of exposure (e.g., latrine cleanliness, presence of a handwashing station, handwashing at key moments, washing raw produce before consumption) that can be difficult to elicit by self-report due to biased reporting. structured observations, , including the use of videography, , offer an opportunity to gather information on complex behaviors, including recording the frequency, duration, and type of interaction with the environment, which could subsequently be used to estimate ingestion rates. these tools are resource intensive at scale due to high heterogeneity of behavior within-host, i.e., observations of individuals, and between-hosts, i.e., observations of public domains. observations can also cause reactivity in participants where the presence of an outside observer leads individuals to alter their behaviors while observed. sensors have been used to compare observed behaviors to reported behaviors with some studies indicating that reported behaviors are inconsistent with sensor measured use. − sanitary surveyssurvey-based inspections of water systems or sanitation facilitieshave been designed and promoted, often in combination with periodic water quality testing, as screening and risk assessment tools for fecal contamination exposure. however, recent studies have shown a poor correlation between sanitary inspection scores and fecal contamination in drinking water supplies. − location tracking. location tracking, for example using personal global positioning system (gps) tracking, can inform where a host is spending time and can thus provide data on where to collect environmental samples, for example by identifying potential hot spots in communities where community members may be experiencing frequent exposures. the use of gps devices has broadly been validated for exposure assessments, and has been used to inform air pollution and chemical exposure assessments. a study in brazil found that gps tracking was an effective tool to quantify personal movements of urban slum residents and evaluate exposure sources of environmental leptospirosis transmission. tracers. tracers, substances introduced into the environment so that their distribution can be detected from their distinctive properties, can provide data on where and how hosts are interacting with their environment. they have been used in air pollution epidemiology to differentiate between indoor and outdoor exposures and a study in china estimated child soil ingestion by measuring concentrations of tracer elements in soil. challenges with using tracers include that seeding several common fecal−oral transmission pathways simultaneously to quantify relative exposure contributions from different pathways may be impractical beyond certain microenvironments, and tracers are needed that do not degrade in the environment and pose no risk to human or environmental health. modeling tools can complement emerging pathogen diagnostics to provide a more nuanced understanding of both pathogen dynamics in the environment as well as infection transmission dynamics. these tools are used to interpret data, are an inherent part of scientific inquiry and can take on many forms. statistical models describe the data in the context of sampling variation and to examine associations that inform environmental determinants of disease risk. for example, they are used to estimate pathogen prevalence, persistence and how that may vary in time and space. conceptual models, sometimes implicit and sometimes explicit, are distinct from statistical models because they represent a hypothesis of the causal relationship between exposure and disease outcome, and are an important driver of how we conduct studies and interpret data. these conceptual models can be codified using mathematics, allowing simulation of different intervention scenarios to conduct thought experiments. such models are complementary to epidemiological analysis. their benefits include lower cost than conducting epidemiology studies that involve recruitment of human subjects. they are also mechanistic, reflecting hypothesized or measured quantitative relationships between key variables that describe complex transport (in the environment) and transmission dynamics (from and between individuals, including via animals and the environment), and have clearly articulated assumptions about these relationships that can be evaluated with real-world data. to study enteric pathogen exposures and risk, there are various types of mathematical models described in the literature. these models allow for in silico experiments and we mention two here. first, quantitative microbial risk assessment (qmra) models estimate the infection or disease risk from a single pathway as a function of pathogen exposure measurements in the environment. for example, by measuring the contamination in surface water, qmra can be used to estimate the risk associated with the drinking water pathway and examine the potential for disease reduction given different mediation efforts. − importantly, the uncertainty in the exposure measurements, or any other data measurement used in the model, can be propagated and reflected in the risk estimate. whether the variance of the risk estimate is too high depends on how this estimate is used; in any case, the variance can be lowered through collection of more precise data. second, infection transmission models dynamically estimate the infection risk associated with multiple pathways of transmission. these models can test scenarios, for example, to understand causes of disease outbreaks from contaminated drinking water, or to explain the interdependency of water, sanitation, and hygiene pathways. , in general, these pathways are often interdependent; i.e., an increase in soil contamination due to poor sanitation can result in increases in contamination of drinking water sources due to runoff events, or poor hygiene can increase the person-to-person transmission rates that result in more people contaminating water sources used for bathing and washing clothes. dynamic infection transmission models can explicitly account for these environmental science & technology pubs.acs.org/est critical review interdependencies to gain an understanding of how mediation in one or more pathways may affect overall infection rates. the power of these tools is that they can generate hypotheses without the need for collection of empirical data, and can then be tested through empirical studies. thus, empirical study design can be driven by model simulation results, and models can be further refined by better parametrization with empirical data in an iterative process. a number of challenges remain in effectively applying these modeling approaches to the study of enteric pathogen exposures. first, modeling results are only as good as the data used to inform parameter estimates. for example, enteric pathogen transmission dynamics are complex and influenced by a range of variables not always measured in field studies. second, modeling results assume that the model structure is correct. for example, dose−response models are highly uncertain and variable, given the limited data available for deriving these relationships, the artificial setting in which dosing trials are conducted and the many variables that may influence the dose−response relationship. more broadly, a transmission system is complex and assumptions are implicit. for example, our models often assume that humans are the most important source of enteric pathogens, that we have accurately identified all relevant pathways of potential exposure, or that the data we have available for use are representative over space and time. many of these limitations apply to science in general and reflect the limitations of the scientific process. modeling approaches bring a number of advantages to understanding enteric exposures. models yield data that may be compared with empirical observationincluding epidemiological data and can therefore help elucidate important mechanistic processes at play, potentially explaining the "why" questions that contextualize epidemiological findings. modeling approaches allow us to interrogate theories of change and identify which interventions have the potential to reduce risks of exposure, and under what conditions. such methods may allow for more rapid development of technologies and approaches that have the greatest potential for improving public health. we have presented the need for new approaches to improve measures of enteric pathogen exposure, identified principal applications that would benefit from better measures, and have considered the merit of current and emerging methods to be employed for these applications as well as lessons learned from exposure assessments for other environmental contaminants. based on this review, we have identified a suite of recommendations that we believe are critical to moving enteric pathogen exposure method development forward: . outline the benefits and potential health impacts of improved enteric pathogen exposure assessments. identify the entities that may harness improved exposure assessments to positively impact health (e.g., governments, international organizations, donors, and funders). emphasize the role of exposure monitoring with respect to other threats to public health, including climate change and antimicrobial resistance. catalog opportunities to link improved exposure assessments with other strategies and campaigns, including sustainable develop-ment goal (sdg) six targets on safely managed drinking water and sanitation services. . create a path forward for exposure assessment methods across the principal applications. develop a vision and the innovations that meet a broad range of applications and criteria necessary to radically improve measures of enteric exposure. consider novel technologies for use in the field and laboratory. collaborate across sectors to comprehensively measure exposure by combining modeling, observational, microbiological, epidemiological, and statistical tools. explore opportunities for integration with one health, planetary health, climate action, nutrition programs, and other initiatives. . dedicate research to defining biologically relevant exposure assessments. consider biological relevance in exposure assessments as it pertains to health outcomes. use archived specimens from previous studies to test new methods, where possible, and implement longitudinal studies to provide rigorous evidence on relative contributions from specific transmission pathways and pathogens to adverse health outcomes. leverage external and internal measures of exposure as complementary approaches for more holistic assessments of exposure. consider how varying approaches reduce differences between measured and true exposure and their subsequent cost-effectiveness in reducing exposure measurement error to design more biologically relevant exposure assessments. . compile exposure data and fill evidence gaps. define and standardize best practices for microbiological and observational methods used for exposure assessments and develop a database framework with uniform data reporting standards to enable better comparisons across different studies and settings. promote the archiving of data and banking of samples for further analysis. identify and fill evidence gaps, including generating data on the temporal and spatial variability of enteric pathogens in environmental matrices, and neglected fecal−oral transmission pathways such as food and soil, to provide a more complete picture of exposure. . demonstrate the potential market and economic drivers of improved exposure methods. model the size of the market across various sectors, and describe how exposure monitoring products could be commercialized. describe the value of these improved methods in higher income settings where there is greater potential to attract investment into research and development while delineating the benefits for improving health in resource-challenged environments. explore the potential use of exposure data in results-based financing, i.e., payfor-performance by defining performance as reducing exposure. in this review, we outlined a number of approaches used to characterize exposure to enteric pathogens. exposure to enteric pathogens is highly context-specific, the density and diversity of pathogens in the environment as well as human interaction with that environment is highly variable over space and time. we presented emerging methods and through lessons learnt from other areas of environmental health distinguished between external and internal measures of exposure. we provided a set of recommendations to narrow the gap between environmental science & technology pubs.acs.org/est critical review the measured and the true exposure, including more holistic exposure assessments that consider approaches across sectors to provide more complete measures of exposure. ■ associated content the supporting information is available free of charge at https://pubs.acs.org/doi/ . /acs.est. c . summary of methods to measure enteric pathogens and indicators of fecal contamination (table s ); summary of methods to characterize interaction with the environment (table s ) (pdf) the global burden of diarrhoeal disease, as estimated from studies published between global burden of disease collaborative network. global burden of disease study exposure science: a view of the past and milestones for the future a retrospective assessment of mortality from the london smog episode of : the role of influenza and pollution the changing paradigm of air pollution monitoring towards a unified system for detecting waterborne pathogens exploring the evolution of diversity in pathogen populations are 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title: safe sanitation in low economic development areas date: - - journal: treatise on water science doi: . /b - - - - . - sha: doc_id: cord_uid: xxov x this chapter presents the advances in sanitation for developing countries as well as the needs in terms of goals, standards, technology, and policy. to understand why a special chapter for low-income areas is needed, it describes the main types of pollutants, their origins, and treatment options. as part of the text and to face the requirements for poor rural and urban areas and water-scarce regions, basic sanitation, wastewater treatment, and water reuse alternatives are described with emphasis on their constraints and limitations for developing countries. some aspects concerning the development of policies, costs, and funding to fulfill the millennium development goals are also included. the importance of developing innovative solutions that can be sustainable, affordable, and progressively implemented is discussed. emerging chemical pollutants . . . . slow filtration . . . . waste stabilization ponds . . . . wetlands . . . . land treatment . . . . reservoirs and water storage tanks . . . . upflow anaerobic sludge blanket before reading this chapter, it should be considered whether it is justifiable to have a specific section dealing with sanitation for low economic development areas (developing countries). evidently, the editors of this book think so. the reasons include • an increasing evidence that wastewater quality in high and low economic areas is different regarding some parameters that determine treatment options and • differences in economic conditions necessitate alternative solutions not only at the technical level but also in terms of the ways to implement them. to protect health, raise the quality of life, and increase the economic level, a good sanitation service is required in developing countries. while in developed countries, sanitation coverage is almost % as a result of a clear commitment of governments to provide it as part of the public services, in developing ones it is only around % (who-unicef, ). in addition, in the developed countries, the term sanitation applies not only to the installation of sewers but also to the full implementation of systems for the safe disposal and reuse of treated wastewater, sludge, and septage. in contrast, in developing countries, the term sanitation mostly applies to the use of sewers not always ending in treatment plants. in fact, reported sanitation figures frequently do not reveal the disposal of wastewater or excreta uncontrolled into the environment, the existence of malfunctioning wastewater treatment plants, or the use of rudimentary and inefficient basic sanitation facilities sometimes contributing to increased environmental pollution rather than to control it. as a result, waterborne diseases affect millions of people in the developing world, and the water quality of surface and groundwater bodies is increasingly deteriorating. the aim of this chapter is to assist the process of increasing sanitation in low-income regions by contrasting the differences in needs and solutions' options with high-income regions. most technical publications have traditionally grouped developing countries together as low-income societies without considering that in them there are high-and low-income areas and that among the latter ones there are several factors that create differences that need to be taken into consideration to provide suitable solutions, that rarely fall under the logic used in developed countries to provide sanitation. most people lacking sanitation include the millions of poor people ( figure ) living under precarious institutional conditions and under an economical and social situation that avoids the use of conventional solutions. this renders the provision of sanitation in low-income areas a major challenge. the history of sanitation is mainly about three aspects: toilets, sewers, and final disposal. as sanitation is a broken subject in developing countries, the story of these three is also the same. when mankind was nomadic and lived in very small communities, sanitation was not an issue. nature could absorb human wastes. later, when villages grew, there was the need to set up special practices and facilities. in ancient egypt (b bc), each household had the responsibility to dispose of their garbage and excreta at the communal dump, in irrigation canals, or in open fields. irrigation canals were the first drainage and waste disposal systems. at that time, toilets were a luxury that only the wealthier people could afford in cities. toilets were carved of limestone, and the used water was disposed of into pits in the streets (msu, ) . flushing toilets -some of them communal -existed in india since the twenty-sixth century bc. reports on the use of toilets and other safe sanitation practices in ancient civilizations from asia, latin america, and africa were common in places where nowadays lack of sanitation is a problem. the earliest covered sewers reported are from the indus civilization ( ( - where pakistan is located today. cities used sewers to control inundations caused by pluvial water. the cloaca maxima or roman sewer dates from around bc. initially, it was an open drain that was covered and left below the urban level, as the city building space became costly (wikipedia, ) . later, when water began to be supplied in large quantities to households, getting rid of the used water became a problem and water was considered as a waste. it was then when sewers were found to be a useful infrastructure to convey wastewater out of the city in addition to stormwater. concerning disposal, land application of wastewater and excreta has a long tradition in many countries. for centuries, farmers in china used human and animal excreta as fertilizers. the oldest references to the use of excreta in aquaculture come from some asian countries, where it was employed to increase fish production (who, ) . further, even now in china, mexico, peru, egypt, lebanon, morocco, india, and vietnam wastewater is used as a source of crop nutrients (jiménez and asano, ) . according to rusong ( ) , in contrast to the 'mechanical' ideas predominant in industrial societies, human ecological thoughts in ancient china emphasized the use of systems advocating 'man and nature as one' . this principle is considered as equivalent to the sustainability principle and is based on terms describing concepts that are dissociated in modern civilizations, such as • tian -heaven or nature; • di -earth or resources; • ren -people or society; • wuxing -the five fundamental elements and movements within any ecosystem, that need to be in equilibrium by promoting and restraining each other; and • zhong yong -describing that things should never go to their extremes but should be kept at equilibrium. for several centuries, based on these ecological principles, china has developed and supported % of the world's population with only % of the world's arable land and less than % of the world freshwater resources (rusong, ) . once again, similar conceptions can be found in ancient civilizations from asia, africa, and latin america, in the same places where there are environmental crises now. the urban water cycle is a relatively new concept used to analyze water quality problems in cities (jiménez, b) , which is depicted in figure . it is useful in identifying conventional and nonconventional sources of pollution, in particular those that are specific to developing countries. it is important to understand the difference in order to be able to apply proper solutions to sanitation that go beyond the simplistic approach of merely installing wastewater treatment plants. a similar analysis could be made for rural areas. the urban water cycle is important because of the large increase in urban population that is being experienced worldwide. by , the urban proportion of the global population is expected to be around %. over the next years, in developing countries, most of the population growth will occur in urban and periurban areas. furthermore, most of the cities with the most rapid growth are located in chronically water-short regions in the developing world (un-habitat, ) . providing water sources to urban areas from the developing world is a challenge because nearly one-third of the population ( . % compared to a % in developed countries in ) are poor people living in slum areas. the slum growth rate is of . %, a value significantly higher than the average world urban growth rate of . %. traditionally, pollution sources are classified as point and nonpoint sources. municipal and industrial wastewater discharges are considered to be point sources, while agriculture (considered as the surface return flow from irrigation), storm runoff, and a wide variety of others are considered as nonpoint sources (jiménez, a) . municipal discharges are those produced by cities and small towns. they are considered to be point sources of pollution where they are produced and collected in sewers and thus disposed of as a well-identified source. when not treated, the main environmental concerns relate to conventional pollutants, such as biological, biodegradable, and nonbiodegradable organic matter, and heavy metals, in that order of importance. the content of almost all these of pollutants is similar around the world, tending to be more concentrated in arid and semiarid areas because of lack of water. in some cases, higher concentrations of pollutants result from increased industrialization of cities. unfortunately, even when treated, municipal discharges introduce used water containing used compounds, some of which are pollutants, to water bodies. municipal wastewater is never treated to recover its original quality (the one it had at the water source) as the selfcleansing and dilution capability of nature is used to complete the task. this is confirmed by the increasing amount of trace pollutants, such as endocrine disrupters, found in water sources. the presence of these compounds might be considered as an indicator that we have surpassed the natural depollution capability of the environment. despite this, the idea of using water bodies or soil to depollute wastewater is still very common, and it could be reduced in water bodies as the depollution capability is lost as result of the water temperature increase due to climate change. in developing countries, the environment is frequently used to depollute wastewater, included when not treated at all, explaining the low quality of water bodies and the widespread presence of diarrheic diseases. industrial wastewater has very variable quality and volume depending on the type of industry producing it. it may be highly biodegradable or not at all, and may or may not contain compounds recalcitrant to treatment. these include organic synthetic substances or heavy metals whose content in developing countries' wastewater may be considerably different (in quantity and quality) from that of developed ones. the main concern with industrial wastewater is the increasing amount (in quantity and variety) of synthetic compounds contained in and discharged to the environment. a list of the most common pollutants in industrial discharges can be found in jiménez ( a) . due to the difficultly in tracking toxic compounds and their fate, combined with the need to use complex and costly treatment methods to remove them from wastewater, it is advisable and cost effective to consider the implementation of cleaner production methods in industries (such as the replacement of toxic recalcitrant compounds with others that are less harmful or not harmful at all) and, also to raise awareness of society to reduce the use of such types of compounds (jiménez, b) . water pollutants come not only from urban and municipal wastewater discharges, but also from nonpoint sources, some of which are not perceived as such. most of the nonpoint sources have been initially recognized as such by groundwater experts (foster et al., ) who realized that soil (urban or rural) was an important means of transporting pollution to ground and surface water through complex interactions. a list of such pollutants is presented in table and a detailed description of some of the pollution sources can be found in jiménez ( a) . in this section, the types of different pollutants are reviewed, emphasizing those of special interest in developing countries. biological pollutants are the major threat to low-income countries as diseases caused by them are rapidly manifested and have important effects on children and the elderly, sometimes even resulting in fatalities. according to who ( ) , diarrheal diseases accounts for an estimated . % of the total daily global disease burden and is responsible for . million deaths every year. it is estimated that % of that burden is attributable to unsafe water supply, sanitation, and hygiene. biological pollutants cause hydraulic diseases that are frequently divided into three categories: . waterborne diseases that are caused by pathogenic organisms ingested when consuming water polluted with fecal contamination or food irrigated with polluted water. examples of these types of diseases are giardiasis and amebiasis. . water-washed diseases that are caused by the lack of safe water or simply any water for hygiene purposes. disease transmission is linked to skin or eye contact. an example is trachoma, a disease that causes blindness. some million people have been blinded by trachoma. another million need treatment, and an estimated million are at risk. the disease is endemic in countries, with only china and india accounting for million cases. productivity losses caused by trachoma are estimated to be us$ . billion (who, ) . . water-based diseases that are caused when water accumulates and stagnates, promoting the breeding of vectors such as mosquitoes that cause dengue or malaria. there are four groups of organisms that can be found in waste and polluted water: viruses, bacteria, protozoa, and helminths (in the form of eggs, jiménez ( ) ). the general characteristics of these organisms can be found in specialized literature. in the following sections, properties relevant to developing countries will be highlighted for each type of group. a list of pathogens that have been detected in wastewater is presented in annex . the main aspect to highlight is the notable difference in the quantity and variety of pathogens found in wastewater between developed and developing countries ( table ) . viruses are the smallest ( . - . mm) infectious agents. there are more than types of enteric viruses capable of producing infections or illnesses that multiply in the intestine and are expelled in feces. unlike bacteria, pathogenic viruses are found in wastewater and feces when people are infected, independently of whether they display symptoms. in regions where viral diseases are endemic, they are constantly isolated from wastewater. the presence of viruses and their concentration in wastewater is linked to the season of the year and the age distribution of the population. concentrations are usually higher during summer and lower in the autumn months. the composition, type, and especially the content of viruses contained in wastewater are poorly known, particularly in developing countries, as a result of the complex and costly analytical techniques required to identify them (jiménez, ) . the enteric viruses most relevant to man are enteroviruses (polio, echo, and coxsackie viruses), norwalk, rotaviruses, reoviruses, caliciviruses, adenoviruses, and hepatitis a viruses. rotaviruses are responsible for between . and billion cases of diarrhea per year in children under years of age in africa, asia, and latin america and up to . million deaths. usually, between % and % of the cases of children with gastroenteritis that are hospitalized are caused by rotaviruses. reoviruses and adenoviruses are the main causes of respiratory illness, gastroenteritis, and eye infections and have been isolated from wastewater. to date, there is no evidence that the human immunodeficiency virus (hiv) causing the acquired immunodeficiency syndrome (aids) can be transmitted via a waterborne route. it is recognized that low virus levels may cause infection or illness; wastewater contains thousands of them, some of which are much more resistant to chlorine disinfection than bacteria (jiménez, ) . viruses discharged in polluted water can migrate long distances in soil and groundwater. the reported horizontal migration varies between and m, while vertical migration ranges from . to m depending on soil conditions. industrial sources industries located in urban or rural areas, in general variable, mostly synthetic compounds bacteria are single-celled microorganisms ranging from . to m in size with different shapes. they reproduce and grow in an appropriate environment at defined ranges of temperature, salinity, ph, etc. they may or may not be encapsulated. the environmental distribution of bacteria is ubiquitous and has different nutritional requirements. many species of bacteria are not harmful to man. in fact, some even live inside humans forming intestinal colonies. bacteria are expelled in feces at high concentrations (jiménez, ) . table shows some characteristics of pathogenic bacteria that can be found in the feces of infected people. in wastewater, pathogenic bacteria are always present but at a variable concentration, depending on the local health conditions. as shown in table , due to the high rate of diseases caused in developing countries, salmonella, shigella, and helicobacter pylori are bacteria of importance as agents causing endemic diseases. in contrast, vibrio cholerae is present only when an epidemic exists. protozoa are the group of parasites most closely associated with diarrheas. they are single-celled organisms ( - mm in size) that develop in two ways: as trophozoites and as cysts. infections are produced when mature cysts are consumed. cysts are resistant to gastric juices and transform themselves into trophozoites in the small intestine, lodging in the wall where they feed on bacteria and dead cells. in time, table characteristics of some bacteria frequently found in wastewater (with information from jiménez ( ) and lenghton et al. ( ) ) escherichia coli is commonly found in wastewater at high concentrations. different e. coli strains can cause gastroenteritis in both animals and humans and pose a high risk to newborns and children under years of age. e. coli strains implicated with human diseases are: ( ) enteropathogenic e. coli ; ( ) e. coli that is the common cause of traveler's diarrhea, which provokes a liquid and profuse diarrhea with some mucosity, nausea, and dehydration; ( ) enteroinvasive e. coli that invades the intestinal mucus lining like shigella spp., and ( ) e. coli (ehec) that produces a similar toxin to shigella causing hemorrhagic colitis. infective doses are relatively low ( organisms). salmonella spp. is frequently present in wastewater at content always lower than that of fecal coliforms by - log. there is a wide variety of strains capable of infecting humans and animals. the incidence in humans is lower than in animals and has a seasonal variation. the most severe form of salmonellosis is typhoid fever caused by salmonella typhi. typical symptoms are chronic gastroenteritis with diarrhea, stomach cramps, fever, nausea, vomiting, and headache. in severe cases, collapse and death might occur. transmission is through ingestion of polluted water or food, and is very common in developing countries. infective dose is of the order - microorganisms, but for salmonella typhi doses as low as - have been reported. shigella is similar to salmonella spp. but less frequent in wastewater. there are more than strains, but s. sonnei and s. flexeneri represent almost % of total wastewater isolations. it rarely infects animals and lives for a shorter period in the environment. one route of transmission is through swimming in polluted water. shigella spp. produces bacillary dysentery or shigellosis. this is light watery diarrhea that can develop into full-blown dysentery. the symptoms are fever, nausea, vomiting, abdominal pain, migraine, and myalgia. the classic form of dysentery is characterized by the expulsion of feces containing blood with or without mucus. the infective dose is less than microorganisms. helicobacter pylori is found in wastewater. its major habitat is the human gastric mucosa. three species are human pathogens: h. pylori, h. fennelliae, and h. cinaedi. the pathway of transmission is not entirely clear but water could be involved. in developing countries, h. pylori is acquired early in childhood, and up to % of children are infected by the age of . this contrasts with the low infection rate during childhood observed in developed countries ( . - %). campylobacter jejuni usually is a pathogen to animals but it can cause severe gastroenteritis in humans. the main source of infection is nonchlorinated water supplies. mycobacterium tuberculosis along with m. balnei (marinum) and m. boris causes pulmonary diseases and tuberculosis. for m. tuberculosis, contaminated water is the main source of infection. vibrio cholerae is the cause not only of epidemic but also eight pandemics, the last one between and . cholera epidemics are caused by v. cholerae group o and some non-o . symptoms are abundant liquid diarrhea with significant loss of hydro-electrolytes and severe dehydration associated with vomiting. v. cholerae is rare in developed countries but frequent in poor ones. humans are the only known hosts. the most frequent pathway of transmission is water, either through direct consumption or when used to irrigate produce that is consumed uncooked. fish grown in polluted water are another source of transmission. since , there have been outbreaks of cholera in india, iraq, congo, vietnam, and zimbabwe. in , west africa suffered more than cases of cholera, leading to deaths. - - fecal streptococci, no. ml À (u, b, k) - - protozoan cysts, organisms l À (u, m) giardia lamblia, cysts l À (u, e, k) - - cryptosporidium parvum, oocysts l À (u, e) - nd helminth ova, egg l À - - trophozoites become once again cysts that are expelled in feces. infected persons may or not display symptoms. protozoa do not reproduce in the environment, only in their host. however, they are able to survive in the environment and remain active for periods ranging from some months to up to several years, depending on the environmental conditions. most intestinal protozoa are transmitted through polluted water and food contaminated with polluted water or unsanitary handled (jiménez, ) . table shows the characteristics of some protozoa. in the developing world, the more relevant protozoa because of their effects on humans are giardia and amoeba. cryptosporidium is a threat to developed countries, as was unfortunately demonstrated in milwaukee, us, when people became ill and more than died after an infection was transmitted through the drinking water supply (hrudey and hrudey, ). helminths are worms some of which are parasites in humans. where helminths are the origin of waterborne diseases, they are mainly transmitted through the consumption of contaminated food (crops, meat, or fish). helminths can also be transmitted through the oral-fecal route and, therefore, hygiene is important as a factor in their control. as helminths are associated with turbid water, they normally are not a concern in drinking water. helminths are pluri-cellular worms and because of this they are poorly addressed in environmental microbiology books. the eggs -their infective form -are microscopic and travel along with wastewater. helminths occur in different types and sizes (from mm to several m in length), and have diverse and complex life cycles compared to most of the microorganisms known in the sanitary field (jiménez, a) . before infecting humans, in some cases, they may have an intermediary host as is the case for schistosoma spp. that temporarily lives in snails. there are three different types of helminths: ( ) plathelminths or flat worms, ( ) nemathelminths, nematodes or round worms, and ( ) annelids. if plathelminths have their body formed by segments, they are called cestodes; if not, they are then called trematodes. only the first two types are of sanitary importance. although common in sanitary engineering literature, it is improper to use the terms nematodes, ascaris, and helminths as synonyms. this misunderstanding comes from the fact that ascaris (a nematode) is the most common helminth egg in wastewater and sludge. a list of helminth eggs found in wastewater and sludge and its classification can be found in jiménez ( a) . helminthiases are diseases of high incidence in developing countries compared with developed ones. globally, there are around - thousand million people suffering of helminthiases but most of them are from developing countries where it affects up to % of the population. the incidence rate may reach % in regions where poverty and poor sanitary conditions prevail. in contrast, in developed countries, helminthiases' incidence is at the most . % and affects mainly poor immigrants (jiménez, a) . helminthiases have different manifestations but, in general, they cause intestinal wall damage, hemorrhages, deficient blood coagulation, and undernourishment. they can degenerate into cancer tumors. helminthiases affect mainly children, the elderly, and poor people . around % of the more than billion cases of diarrhea in the world are caused by helminths (murray and ló pez, ) . there are several kinds of helminths with different local names (annex ). this along with the fact that it is hard to properly identify them clinically unless a costly laboratory analysis is performed, makes it difficult to track the actual incidence of all the table protozoa related to sanitation problems and that are of interest for developing countries (with information from jiménez ( )) entamoeba histolytica is one of the most important parasites detected in municipal wastewater and is commonly known as amoeba. trophozoites measure - mm and t cysts - mm. amoebae usually lodge in the large intestine; occasionally they penetrate the intestinal wall, traveling and lodging in other organs. they are the cause of amoebic and hepatic dysentery. entamoeba histolytica infects % of the world's population -mostly in the developing world -resulting in approximately million infected persons; there are between and million cases of invasive amebiasis per year resulting in up to annual deaths (placing it second after malaria in mortality caused by protozoan parasites). ninety-six percent of these cases occur in poor countries, especially on the indian subcontinent, west africa, the far east, and central america. giardia spp. are common in wastewater as it frequently causes endemic diseases. it especially affects children under suffering from malnutrition. the total number of sick people is of the order . billion, % of whom live in poor countries. giardia spp. is the most common parasite of humans but water is not necessarily the main pathway of transmission. cysts (that are - mm long and - mm wide) can survive in water bodies for long periods, especially in winter. giardia lives in the intestines of a large number of animals as trophozoites. the disease is characterized by very liquid and smelly explosive diarrhea, stomach and intestinal gases, nausea, and loss of appetite. cryptosporidium spp. is a parasite widespread in nature. oocysts are resistant to chlorine and due to their small size ( - mm) are difficult to remove from water, as many other protozoan. cryptosporidium spp. infects a large spectrum of farm animals and pets and was recently recognized as a human pathogen that is why it is considered as an emerging pathogen. cryptosporidium spp. is capable of completing a life cycle within the same host and causing reinfection. once an individual has been infected, the person carries the parasite for life and can be reinfected. the disease rate in developing countries has been poorly studied, in particular due to the higher occurrence of other types of diseases. cryptosporidiasis in developing countries has shown a greater incidence among immune depressed people and in rural areas (snelling et al., ) . the main symptoms of cryptosporidiasis are stomach cramps, nausea, dehydration, and headaches. although it is known that the infectious dose varies between and , outbreaks have always been associated with large concentrations in water. helminthiases. that is why frequently figures are underestimated. technically, helminthiases take their name from their causative agent. for instance, trichuriasis is named after thrichuris. ascariasis, affecting nearly million people, is the most common of the helminthiases and is endemic in africa, latin america, and the far east. even though the mortality rate is low, most of the people infected are children under years of age with problems of faltering growth and/ or decreased physical fitness. around . million of these children will probably never bridge the growth deficit, even if treated (silva et al., ; jiménez, a) . the helminthiases' infective agents are the eggs, not the worms. actually, worms cannot live either in wastewater or in sludge because they need a host. helminth eggs are transmitted through ( ) the ingestion of crops polluted with wastewater or sludge, ( ) direct contact with polluted sludge or fecal material, and ( ) the ingestion of polluted meat or fish (jiménez, a) . each type of helminth has its own pathways of infection. eggs of different helminths generally occur in different shapes, sizes, and resistances ( figure ) . as a result of the higher incidence of ascariasis, in wastewater and sludge, these examples of local names given to helminth and helminthiases diseases are the eggs found in the highest concentrations ( figure ) . the percentage of types of helminths might vary from one region to another following the disease's pattern. due to differences in health conditions in developed and developing countries, their helminth eggs content is very different in wastewater and sludge ( table ) . eggs contained in sludge are not always viable and infectious. to be infectious, the larvae need to develop, and, for that, a certain temperature and moisture are needed. the necessary conditions are frequently met in soil or crops, where eggs are deposited when polluted wastewater, sludge, or excreta is used as fertilizer. under such conditions, the larvae develop in days. according to previous information (that has not been updated using better analytical techniques), ascaris eggs remain viable - months in crops and many months in soil, freshwater, sewage, feces, night soil, and sludge -periods which are much longer than those for microorganisms (jiménez, a, figure ). this high resistance is due to a cover composed of - layers that gives mechanical resistance to eggs and protects them from desiccation, strong acids and bases, oxidants, reducing agents, detergents, and proteolytic compounds (jiménez, a) . the resistance of different helminth eggs genera under environmental conditions has not been reported in literature. to inactivate helminth eggs, it is recommended to raise the temperature above c for - days for ascaris or to reduce moisture levels below %. these conditions are not ease of use during wastewater treatment; thus, helminths are usually removed from wastewater to be subsequently inactivated in sludge. helminth ova of interest in the sanitary field figure continued. measure - mm, have a specific density of . - . , and are very sticky. these properties are used to remove eggs from wastewater (jiménez, a) . helminth ova criteria. as shown in table , not all wastewater and sludge contain significant amounts of helminth ova. for this reason, they are not included in all countries' wastewater, sludge, or fecal sludge norms, as is the case with biochemical oxygen demand (bod) or fecal coliforms, which are universal parameters used to design wastewater treatment (jiménez, a) . based on toxicological and epidemiological studies, the world health organization who ( ) suggested a value of r egg l À in wastewater intended for the irrigation of crops that are eaten uncooked. wastewater used for the culture of fish should contain egg l À , since trematode eggs (schistosoma spp., basically) may multiply in an intermediary host (a snail) before infecting fish and humans. for excreta, the recommended criterion is of egg g À total solids (ts). thermotolerant coliform bacteria (commonly referred as fecal coliforms) are the group most frequently used as indicators of fecal pollution because they behave in a similar way to most pathogenic bacteria in the environment, and, during treatment, they are abundant and easy to determine. hy me no lep is sp p. to xo ca ra sp p. tri ch os om oid es sp p. en ter ob ius sp p. ta en ia sp p. un cin ari a thermotolerant coliforms are less specific indicators of fecal contamination than escherichia coli, since they may sometimes arise from nonfecal sources, especially in tropical climates (who, ) . however, it is becoming increasingly evident that they are not useful to simulate the behavior of all enteric viruses, protozoa -in particular with regard to giardia and amoeba -and helminth eggs that are of concern in low-income regions. despite this, it is frequently, but wrongly, assumed that fecal coliforms are indicators of all kinds of biological pollution. even though they can be useful indicators of fecal pollution in developed countries' drinking water, this is not always the case for water and wastewater from developing ones, owing to the presence of a wider variety and larger quantities of microorganisms (jiménez, ). this does not mean that fecal coliforms are not useful for developing countries; it simply means that care must be taken to select additional indicators for specific purposes, such as for wastewater and sludge reuse in agriculture and aquaculture. in these cases, the helminth egg content (who, ) needs also to be specified. it is worth mentioning that the treatment procedures to inactivate helminth eggs are frequently developed using ascaris eggs as models as they have been informally considered as indicators for all helminth eggs, although this has not been fully proven experimentally. in other cases, taenia saginata or ascaris galli, types of eggs that are rarely present in wastewater, are used to test treatment procedures. some pathogens that are not usually followed during conventional monitoring have been linked to outbreaks in developed countries. these pathogens have been called 'emerging' pathogens. they have led to new regulations as well as to improvements in water and wastewater treatment procedures. some of the microorganisms considered as emerging pathogens are giardia lamblia, cryptosporidium parvum, cyclospora cayetanensis, blastocystis hominis, legionella pnuemophila, e. coli h , campylobacter, mycobacterium, and norovirus (jiménez, b) . in developing countries, some of these pathogens are endemic, while others have either not been studied or not reported as disease-causing agents. assessing the biological quality of water is always a challenge due to the diversity of organisms and the need for different and proper methods to identify and enumerate them, some of which are complex, time consuming, and costly. in the following sections, a short description on the techniques used for different type of organisms is described. viruses. identification and quantification of viruses in wastewater, sludge, or excreta is complicated due to the low level of recovery from wastewater and the need to use complex and costly techniques to analyze them. a laboratory requires days, on average, to determine the presence or absence of a virus in water and another days to identify them, using conventional procedures. polymerase chain reaction (pcr) techniques have considerably speeded up the process, as they can be used to determine viruses online. these techniques are based on the amplification of a single or few copies of a piece of dna allowing the identification of different types of viruses. however, quantification with the precision required in the sanitary field remains a challenge. in addition, the method is sophisticated, and requires highly specialized equipment and highly trained personnel. due to these difficulties, it is sometimes preferred to detect bacteriophages, that is, bacteria infected by viruses. bacteriophages are used as informal indicators of viruses and not been linked to human diseases; therefore, their presence has no health significance (jiménez, ) . bacteria. as mentioned previously, thermotolerant bacteria are the common accepted indicator of bacterial fecal pollution. they are detected by using a selective medium and incubating it after inoculation at or . c and/or or . . c, depending on the medium used. the materials and equipment used for this analysis are very common in most wastewater laboratories. pcr techniques to detect e. coli are useful as well. protozoa. there are enough accessible techniques to determine the presence of the main protozoan pathogens in wastewater and sludge; however, fewer techniques are available to quantify them with the required precision for the sanitation field. the presence of protozoa on samples does not necessarily always imply a risk, since this requires them to be also viable. to determine the viability, several days are required. pcr techniques for protozoa are not as well developed as they are for bacteria and viruses. helminth eggs. helminths eggs require laborious techniques to detect them and even more so to enumerate them. fortunately, the technique is readily available and does not use complex equipment, although it does require well-trained laboratory personnel. currently, there is no standardized method and most of the few laboratories trained to detect them are using either different analytical procedures or similar ones with modifications. moreover, most of the laboratories, instead of reporting the total content of helminth eggs, only report the ascaris content, as is done in developed countries where it is frequently the single type of helminth eggs present (jiménez, a) . analytical techniques for quantifying helminth eggs can be divided into two: direct and indirect techniques (jiménez, a) . the first consists of separating helminth ova from the other particles contained in wastewater or sludge (where there are many) and then identifying and counting different genera using a microscope. some examples of these techniques used the us-epa (united states-environment protection agency), the membrane filter, the leeds i and leeds ii, and the faust techniques. the most widely used technique seems to be the us-epa ( ) . a comparison of the performances of the above-mentioned methods has been made by maya et al. ( ) . the recovery rate among them varies from % to %. sensitivity for each notably varies as well and not all are capable of measuring the criteria values set by who ( ) of egg l À for wastewater and egg g À ts for sludge. the second types of techniques are indirect ones, and these have been applied only for wastewater. they are based on measuring either the total suspended solids (tss) content or the particle size distribution (psd), and then correlating the concentration to the helminth egg content. calibration curves need to be established for each type of wastewater and treatment process. nevertheless, it is a worthwhile method because the helminth egg determination costs us$ - if tss are used, and us$ with the psd, instead of us$ , which is the cost of direct methods. it is important to distinguish between fertile viable and nonfertile eggs as only the viable eggs are infectious. this can be done visually using stains or by incubation at c for - weeks (jiménez, a) . conventional parameters as understood in this text are those commonly used to design or select wastewater and sludge treatment processes worldwide, and they refer mainly to the organic matter content (measured as bod or codbiological or chemical oxygen demand), or suspended solids. in general, they are similar worldwide except for the heavy metals content that in general -and especially for sludge -is notably lower in developing countries than in developed ones (leblanc et al., ) as result of the difference at the industrialization level. however, at a local level, metal content in some industrialized areas of developing countries, notably where metal or tanning industries are placed, may be high. a detailed description of conventional parameters and their significance can be found in jiménez ( a) . the term (chemical) 'emerging pollutant' is used to describe a wide variety of complex organic chemical compounds that are candidates for future regulation and that have not usually been monitored. to detect them, complex and costly analytical equipment is needed, such as gc-ms or gc-ms-ms (gas chromatography coupled with one or two mass spectrometers) as these are the only ones capable to measure the very low concentrations at which the pollutants are present (in the order of micro-or ng l À ) and to identify them. emerging pollutants have been detected in untreated wastewater, treated wastewater, surface water, groundwater, and even in drinking water of both developed and developing countries (some). among the countries that have measured and detect emerging pollutants, the following can be cited: austria, brazil, canada, finland, germany, italy, japan, mexico, the netherlands, spain, switzerland, uk, and usa (jiménez, b) . the sources of emerging pollutants are diverse. they come from nonpoint sources, municipal wastewater (treated or nontreated), and industrial discharges. they are also the result of the improper disposal of solid wastes. two groups of compounds that are considered as emerging pollutants are: endocrine disrupter compounds (box ) and personal care and pharmaceutical products (pcpps). wastewater treatment processes have not been designed to remove them; thus, they are randomly removed during conventional treatment. from the limited literature currently available, emerging pollutants -as other organic compoundsare concentrated in sludge during wastewater treatment. initial risk studies suggest minimal ecological and health effects through biosolids recycling to soils (leblanc et al., ) . as most of these pollutants have only been recently studied, the knowledge of their fate, transport, behavior during treatment, and risks is still poor in the sanitary engineering field. chemical emerging pollutants, in general, are not considered at the moment as a priority for the developing world as there are more pressing health and environmental pollutants of concern. it is important to bear in mind that the simple presence of a pathogen or a toxic chemical in wastewater, sludge, or excreta does not necessarily mean that a negative effect will occur. for that, several other things need to happen. these include ( ) the need for a compound/pathogen to reach a certain concentration; ( ) the existence of a pathway for transmission to human or the environment; ( ) the ingestion or presence of a certain dose to cause long-or short-term effects; ( ) sufficient exposure times to the pollutant; and ( ) sufficient sensitivity of a person or of the environment to pollutants. in addition, it should be remembered that, for humans, water is not the only source of risk, as food and air are also sources of pollutant ingestion and, in some cases, they may be the main ones. in terms of the differences of biological risks to humans in developing and developed countries, there are additional aspects to consider as humans develop immunity to pathogens depending on the type of environment they are exposed to, and thus infectious doses may be higher. genetic history, nutrition, and the combination of social patterns also intervene. for these reasons, data developed for developed countries are not always applicable to developing ones to perform risk analysis. in order to quantitatively assess risks, it is necessary ( ) to establish the type and quantity of given microorganisms in a region, ( ) to know the actual infectious dose, and ( ) to define and evaluate the possible infection route. to box endocrine compounds. from jiménez b ( b) wastewater risks in the urban water cycle. in: jiménez b and rose j (eds.) urban water security: managing risks, p. . paris: unesco leiden: taylor and francis group. endocrine disruptors are chemicals that mimic hormones or have antihormone activity interfering with the functioning of endocrine systems in various living species. they derive from many sources including pesticides, persistent organic pollutants, nonionic detergents, and human pharmaceutical residues. some of them have been identified in municipal wastewater and many of them may persist in the environment for some time. endocrine disruptors have been also found in drinking water. their presence in recycled waters also raises broader questions about the risks and benefits of water recycling and our approaches to anticipating the emergence of new contaminants. human health effects potentially linked to exposure to these chemicals include breast, prostate, and testicular cancer; diminished semen quantity and quality, and impaired behavioral, immune or thyroid functions in children. although direct evidence of adverse health effects in humans is lacking, reproductive abnormalities, altered immune function, and population disruption potentially linked to exposure to these substances has been observed in amphibians, birds, fish, invertebrates, mammals, and reptiles. notably, feminization or masculinization on male or female animals, respectively, has been reported. quantitatively evaluate the risk from a chemical or microbial pollutant, several methodologies are available in literature, but the data needed to apply them may be lacking for special cases in developing countries. sanitation is a term that has a clear meaning in the developed world. however, for the developing one, there is need to have a better definition. traditionally, sanitation has been reported as the percentage of the population having access to the service. in practice, this service in low-income regions ranges from simple access to sewers that are discharging the wastewater just behind households or into the streets to sewers connected to sophisticated wastewater treatment plants coupled with water reuse projects and comprising safe sludge management practices. for basic sanitation -sanitation provided in rural or poor periurban areas, the term sanitation includes a wide variety of on-site sanitation options going from simple pit to highly comfortable package treatment plants, which may or may not be functioning. to overcome this, the joint monitoring programme (jmp) from who-unicef proposed in to introduce the term 'improved sanitation' . improved sanitation is a system in which excreta are disposed of in such a way that the risk of fecal-oral transmission to users and to the environment is reduced (who-unicef, ). table shows which options qualify as improved sanitation and which do not. in , the world summit on sustainable development (wssd) provided a definition for basic sanitation that, besides considering the service itself, considered its impact on human health. this definition comprises the following: • the development and implementation of efficient household sanitation systems; • the improvement of sanitation in public institutions, especially in schools; • the promotion of safe hygiene practices; • the promotion of education and outreach focusing on children, as agents of behavioral change; • the promotion of affordable and socially and culturally acceptable technologies and practices; • the development of innovative financing and partnership mechanisms; and • the integration of sanitation into water resources management strategies in a manner that does not negatively affect the environment (it includes protection of water resources from biological or fecal contamination). as a result, the wssd's focus is not only on the construction of a particular number of toilets but also on the effective improvement of health and hygiene through basic sanitation. however, still new elements are needed to be added as problems caused by lack of sanitation are combined with those arising from the lack of economic resources and frequently also with lack of water in societies lacking even from social, economical, and political rights (box ). the millennium development goals (mdgs) are drawn from the actions and targets contained in the millennium declaration that was adopted by nations and signed by heads of state and governments during the un millennium summit held in new york city on september (who-unicef, ). they comprise eight goals and quantifiable targets. water is part of the th goal under target c: ''reduce by half the proportion of people without sustainable access to safe drinking water and basic sanitation.'' fulfilling this target represents the challenge of providing safe water supply to . million people and safe sanitation to . million people within years. table improved and unimproved sanitation facilities according to who-unicef ( ) connection to public service or bucket latrine sewer or septic tank traditional latrine pour-flush latrine public latrine or shared toilet pit latrine with slab open pit or pit latrine without a slab vip latrine open defecation in bush or field ecological sanitation box what sanitation should include, with some information from lenghton l, wright a, and davis k (eds.) ( ) health, dignity and development: what will it take? millennium development goals. london: earthscan. reporting figures concerning the state of sanitation in the developing world is a difficult task. first, there is a lack of information; second, the information available is generally presented in a heterogenic way; and third, different sources tend to contradict each other despite national and international efforts to produce consensus. the worsening situation with regard to sanitation in developing countries can be described using different indicators (box ). contaminated water and poor sanitation account for the vast majority of the . million child deaths each year from diarrhea -almost every day -making it the second largest cause of child mortality (undp, ) . the expansion of water services is essential to reduce the burden of waterrelated diseases and to improve the well-being of a large part of the world's population. it is also vital for economic development and poverty alleviation (who, ) . according to the figures presented by who-unicef ( ) , despite the efforts made and due to population growth, between and , the population with access to sanitation services has increased from million to million ( %), while the net number of people without improved sanitation decreased by only million. the difference between the level of sanitation in developed and developing countries is high: % versus % (table ) . however, between and , the percentage of people with access to improved sanitation increased from % to % with countries' variations ranging from % to % (who-unicef, ) . the difference observed between rich and poor countries is also observed between urban ( %) and rural ( %) areas from developing countries and as well between rich and poor people living there following the inequities of wealthy distribution. the sanitation coverage as percent of the population with service per country is presented in the map of figure for the year . annex contains a table with countries with less of % of the total, urban, or rural population. sanitation in developing countries is quite a complex issue, because the lack of it is combined with other several problems, some of which are geographically described on the maps - from annex . by analyzing these maps, the following conclusions may be drawn: . several low-income countries are located in arid or semiarid regions; thus, besides sanitation problems, they face the problem of water scarcity. . many of the areas under greatest stress (where people are already overexploiting rivers by tapping water that should be reserved for environmental flows) coincide with areas that are heavily developed for irrigation to provide water for food, that is, mostly in developing countries. . water withdrawal for agriculture is mainly performed in developing countries as a result of low water availability and the high dependence of agriculture. . areas where poverty and hunger are prevalent coincide with areas lacking sanitation. . in the future, it seems that the situation may worsen as water availability will decrease in the countries already experiencing water-related problems, including lack of sanitation. as result of the past and present situations, sanitation has different aspects on developing countries that cannot be described simply using the percent of population-covered index. in the following, some of these aspects will be described. basic sanitation versus sanitation. providing services for excreta management in poor rural or urban areas is frequently known as basic sanitation. thus, it has to do with excreta management rather than with sewerage and wastewater treatment plants (box and figure ). the quality of the service is frequently associated with peoples' economic level, and thus, is box some figures for global sanitation * for each four persons that do not have access even to a simple pit latrine, six have it. * for each one person that does not have access to sanitation, another one has it. * in rural areas, for each two persons, only one person has access to a sanitation service. * for each l of wastewater that is nontreated, l is treated. also a sign of status. another aspect to consider is that the lack of basic sanitation frequently is associated with lack of water. leblanc et al. ( ) highlights that research and experience suggest the following hierarchy of risk to human health: ''living in a dense community without basic sanitation (is more risky thany) irrigation of crops with untreated, pathogencontaminated wastewater use of untreated, pathogen-contaminated excreta or wastewater sludge on soils use of untreated, pathogencontaminated animal manures on soils use of treated manures, wastewater, or biosolids on crops use of these treated materials in accordance with strict modern regulations that address heavy metal and chemical contaminants.'' differences on sanitation services. possibly, one of the aspects that contributes the most to render sanitation in developing countries a challenge is the variety of needs and circumstances arising from social differences. as shown in figure , for instance, poor people not only are less served but also the quality of the services is lower. one of the deepest disparities is between urban and rural areas as for the former the coverage is twice as much than for the latter in developing countries. traceable differences in sanitation services have been reported as well among indigenous and nonindigenous people and minorities such as castes and women (box ). among these differences, the following common challenges can be identified: • the need to provide the service in poor areas with large population increases. • for urban areas, a very fast service demand growth in slums that are spread out in cities, have high population density, and there is no land to place the infrastructure. • for rural areas, the need to assist a population frequently dispersed and hence at higher cost. • the need to fund projects combining liquid and solid waste collection and treatment infrastructure. • the need to develop new or different management structures to provide services in social and political complex areas. • the need to include health education and awareness programs on sanitation projects. • the need to use public funding to provide services that are to be subsided. • the existence of regions having high income where services can be provided in a similar way to developed countries. sanitation versus wastewater treatment. as described previously, sanitation coverage does not necessarily result in wastewater being treated or safely disposed of. to illustrate this, figures for the situation in some developing countries are provided. two comments on this figure are that ( ) it is really difficult to find data on wastewater treatment, notably for the asian and african regions and ( ) although there should not be a full correspondence between the sanitation coverage and the wastewater treatment -as some people are served using basic sanitation facilities -the figures should not be as different as they are for some countries. in latin-america, for instance, although the sanitation coverage was % in , only % figure ). according to lenghton et al. ( ) , the amount of money needed to fulfill the sanitation mdgs ranges from us$ billion to us$ billion representing, in mean conditions, an annual average investment of us$ . billion. to put these figures in perspective, the above-mentioned authors mention that each year europe and the united states spend us$ billion on pet food and europe spends us$ billion on ice cream. the overall cost estimation of the current water and sanitation deficit is of the order of us$ billion, equivalent to . % of developing countries' gross domestic product (gdp). for each us$ invested for sanitation, the economic even if sanitation represents an economic benefit, its cost is still important to societies in which this is not the only requirement. therefore, it is useful to combine options that involve building infrastructure with others that do not (such as washing or cooking produce that has been irrigated with polluted water) in order to improve health conditions while the sanitation services can be gradually provided. such an approach is described in who ( ) . in the next sections, options to build up wastewater management systems are reviewed. a wastewater management system (wwms) is understood in this chapter as the combination of one or several of the following components: ( ) basic sanitation facilities or toilets; ( ) wastewater collection systems (sewers) or box some challenges to provide basic sanitation in low-income countries * open defecation is practiced by % of the population in southern asia and % in sub-saharan africa. * in ouagadougou, the capital of burkina faso, the access to sanitation facilities is % while the figure for the country is . %, a figure that reduces to only % for rural areas (paskalev, ) . * in yaoundé, cameroon's capital with inhabitants, the available facilities for most people ( %) are external and in shared proprieties (figure ). * basic sanitation and sanitation figures reported are not the same. for instance, for cote d'ivoire, a coverage of % is reported for rural areas, but, in fact, % refers to basic facilities and only % to adequate systems (angoua, ) . ( ) wastewater disposal or reuse facilities. before presenting these components in detail, the two options in which they can be managed (centralized or decentralized) are discussed. conventionally, to handle wastewater, sewers connected to wastewater treatment plants have been used. this is known as a centralized system and is a well-mastered and well-managed technology approach applicable to cities, provided funds for its construction and operation are available. in terms of operation, centralized systems are often cheaper and easier to handle than decentralized ones. for isolated slums and dispersed rural areas and even for cities where new sewerage systems is too costly, it is advisable to use decentralized wastewater management systems. in these, sewers of reduced size result in a lower capital cost (around %) due to the smaller diameter and length of the used pipelines. in addition, they offer the following benefits (lenghton et al., ; correlje and schuetze, ) : ( ) they allow investments to be made stepwise, in line with available funds, local development, and population growth; ( ) they are used in smaller areas of service that are easier to manage; ( ) they allow the use of different technologies to provide services to different socioeconomic groups; and ( ) they facilitate the reuse of water on-site. nevertheless, all these advantages need to be assessed in practice, as they cannot be taken for granted universally. as for many water utilities, decentralized systems represent a higher number of systems to manage, which is difficult and complex; to overcome this limitation, centralized management of decentralized systems is recommended. this way it is possible to ensure high performance and reliable operation, reduce costs, and also ensure the need for specialized operators (hughes et al., ) . from a technical point of view, there are four important components to consider when providing a basic sanitation service: ( ) the type of toilet, ( ) the storage facility for feces which frequently are associated to the toilet, ( ) the way in which feces are extracted from the pit, and ( ) their further management. this section deals with the first two components. their main characteristics are discussed here; for design, it is recommended to consult specialized books. a good option to begin with is the united nations environment programme (unep) website (see section titled 'relevant websites'). latrines are the most widespread type of on-site sanitation facility. they are used in rural settings and deprived areas in cities. they consist of a makeshift pit dug in the ground and box women and sanitation (with information from lenghton l, wright a, and davis k (eds.) ( ) health, dignity and development: what will it take? millennium development goals. london: earthscan.) one explanation for the low effective demand for sanitation is gender inequality. women tend to place a higher value on household toilets than do men for a number of reasons, among them privacy, cultural norms, care-giving responsibilities, and the risk of sexual harassment and assault. in addition, the unique sanitation needs of women and girls (e.g., during menstruation and during and after pregnancy) receive little recognition when discussions about sanitation and hygiene occur. yet, the limited political and personal power of women in many developing countries means that some of sanitation's strongest advocates are virtually absent from decision making and priority-setting processes. generally covered with any material (a wooden, plant, or metallic cover, whichever is available). when latrines are full they can be emptied (this is unpleasant and has an associated cost) or closed to build another one (this requires the availability of land). these latrines, instead of having a single vault, are made up of a shallow pit divided into two - m vaults. their major advantage is that they are a permanent facility due to the alternate use of each pit. the name comes from the inclusion of a properly designed pipe allowing ventilation, which also requires a screen to avoid the accumulation of flies. the pit cover is made of precast concrete, wood, palm leaves, or metallic material, and is removable. emptying is performed manually in low-income areas, but can be done mechanically every - years. the ventilated improved pit (vip) latrine with multiple pits can be built for collective use, such as in schools, markets, fueling stations, and administrative buildings (mamadou, ) . the septic tank is commonly used as primary treatment in rural areas, low-income urban settings, isolated households, or on sites where soil is not suitable for the installation of sewers (jiménez and wang, ) . they are built where a constant water supply is available and are used to partially treat domestic wastewater and to digest the settled sludge. they remove around % of the organic matter and suspended solid content in - days. for sludge digestion, . - year is required; during this time, sludge is mineralized and its volume is reduced. septic tanks are made up of a series of communicating chambers. they must be water sealed to avoid underground infiltration and are built using bricks, mortar, or concrete. a variation of the septic tank is the imhoff tank, having the advantage of a shape that allows the removal of suspended solids and the control of foul odors in a better manner. septic tanks need to be periodically cleaned ( - times per year, leaving % of the mature sludge as inoculum for digestion). this represents an additional cost that cannot always be afforded by poor people. septage (the slurry taken out of septic tanks) is sent to wastewater treatment plants or treated separately. to treat septage, lime is frequently added until a ph of is reached, over a period of min (jiménez and wang, ) . effluents from septic tanks are discharged into trenches for subsoil infiltration or diverted to the sewerage system (when available). septic tanks are widespread sanitation systems but are often responsible for environmental pollution due to poor purification effects and leakages notably affecting groundwater. composting toilets are characterized by the separation of urine and feces. for this reason, they are also referred to as urine diversion (ud) toilets. they are constructed with two vaults or chambers. when the first vault is full, the pedestal is moved over to the second vault, and the first hole is closed. when the second vault is full, the first vault is emptied and so on. the urine is diverted to a soakaway. in comparison to vip latrines, they have a lower cost associated with emptying the pits (snyman, ) . urine is collected in small cans ( - l) and can be used to enrich the soil after a stabilization period of days. feces are treated using an aerobic composting process. to control odors and to assist in the mineralization of feces, materials, such as ashes or pieces of wood, are used daily to raise the ph. the pathogens in fecal matter are inactivated over time through the drying process so they can be safely removed by the owner at no cost to the municipality. once the sludge is digested, disinfected, and removed, it is used as fertilizer. ud toilets are seen as a viable option for rural applications. the main reasons are that they are cost-effective and, since the rural community is accustomed to the use of manure, the ud toilet is socially acceptable. however, its use in periurban areas is more problematic. the emptying of the vaults requires large-scale programs for which small businesses can contribute to the emptying of tanks (from ud or vips) either manually, using appropriate safety equipment, or by the use of a tanker. the disposal of the fecal matter in periurban areas is challenging due to the lack of land. if space allows, fecal sludge is buried on-site. where this is not feasible, the sludge is blended into the waterborne system. this frequently leads to the complete overloading of the wastewater treatment plant (snyman, ) . there are several options of composting toilets (see section titled 'relevant websites'). pour-flush toilets have been developed based on traditional flush toilets, which rely upon a water seal to perform cleansing and to control odors and insect infestations. the system works via a manual flush, where - l of water are poured into the toilet. the water, urine, and excreta are collected in an anaerobic chamber, which works similarly to a septic tank. the chamber needs to be periodically emptied and the partially treated wastewater needs to be disposed of, normally to land (hughes et al., ) . in the context of water-scarce areas, a very interesting option is combining graywater reuse with basic sanitation using pour-flush toilets. this concept was developed by united nations international children's emergency fund (unicef) on a system called the wise water management scheme (godfrey et al., ) . this system was conceived to provide both water supply and sanitation services for water-scarce areas and can be used for both rural and lowincome urban areas. it was conceived in madhya pradesh, india, a densely populated and poor area. the wwms uses groundwater as the primary source of water and also includes rainwater harvesting, used to dilute groundwater when polluted with fluoride to reduce its content for human consumption ( figure ) . first-use water is employed for cooking, handwashing, and bathing. water from these two activities is recovered and properly treated in a sand filter to be used for toilet flushing and kitchen garden irrigation. the graywater reuse system can be installed independently of the rainwater harvesting system. by matching water demands, in quantity and quality, to different conventional and nonconventional water sources, the wwms increases water availability by nearly %. sanitation using low-consumption reused water flush toilets has proven sustainable under the prevailing local conditions and has eradicated open defecation. . . . . additional recommendations to set up basic sanitation facilities one important aspect to keep in mind when selecting the technology is that facilities need to be operational and, to achieve this, there is a need to sustain them under operation from the economical, technical, and cultural perspectives. investment costs are linked to the type of sanitation system selected, the construction materials, and labor. frequently, to reduce costs, cheap materials and the users are employed to build the facilities. however, this may result in failures, as cheap material frequently means low quality and the users are not people experienced enough, even if trained. it is thus preferable to invest in good and durable material and to use experienced workers. in india, for instance, sanitation programs using professional well-trained masons are being implemented in which the same masons for whom sanitation is a source of income become at the same time sanitation promoters. norms and institutional capacity to provide basic sanitation constitute another weak link in the complex chain needed to implement and provide services. how to build institutions, policies, and human resources to provide successful sanitation services is better known in high-income countries than in developing ones. each country/region needs to look for the proper way to solve their problems. finally, concerning basic sanitation, it needs to be considered that in several places, providing basic sanitation means to change open defecation habits and to handle domestic solid wastes (box ). it means as well to properly dispose of the toilet paper. under this section, only the toilets using less water or none at all are described as compared to the others (pour flushing toilets using l of water is a well-known technology widely spread commercially). concerning these toilets, one aspect to highlight is that even if convenient from the point of view of the used water, care must be taken when designing treatment plants as wastewater will be not only lower in volume but also highly concentrated, notably in terms of its organic matter content. these toilets are based on the same working principles as common flush toilets but they are specially designed to fully operate with less water ( - l). in such toilets, it is possible to select either a full flush (with , , or l depending on the model) for solids or a half flush ( - . l) for liquids. box poor people have a globalized attitude towards excreta management as described for senegal by ba ( ) , in most poor areas of the developing world, water from baths and in some cases from showers are routed to septic tanks from which the effluent is sent to infiltration wells or trenches. kitchen and laundry water is generally poured directly into the street, discharge areas in the wild, a well, a nearby river, or riverbed. wastewater and noncollected solids are also frequently mixed creating breeding sites, odor problems, and development of flies. these toilets are also available with separate drainage for urine to reduce the impact of nutrients and pharmaceuticals on the sewage and to facilitate the reuse of urine as a fertilizer. however, most water-saving toilets available on the market are designed to be connected to typical drainage systems. there are several technological options on the market, some of which use a vacuum to transport feces at a much higher cost. the investment cost for low-volume toilets is comparable to high-volume toilets. however, dual flush toilets may cost more than common ones (nearly double). the installation of water-saving toilets must be stimulated by education (e.g., in the form of campaigns to raise awareness concerning watersaving issues), water metering, and pricing. water-saving urinals, using - l, are also available (correlje and schuetze, ) . the idea of dry toilets is not new. they have been used for thousands of years in east asia (china, japan, and korea). dry toilets are available as industrial prefabricated products and can also be constructed in local workshops; however, knowhow for its good operation and to avoid foul odors is required. investment, construction, or installation costs vary significantly and depend on the specific system and design. the cost ranges from low investment for simple dry toilets to comparatively high cost for industrialized composting toilets. due to the large size of the storage and composting chambers, these toilets require a large space underneath; if this is not possible, then they need to be regularly emptied and feces need to be transported to treatment facilities. user acceptance depends on cultural background and awareness. generally, people who are already using flush toilets do not readily switch to dry toilets because the image of dry toilets is less attractive than that of flush toilets. equally important as the type of on-site sanitation system selected is the provision of all the services associated. past experiences (water decade, - ) have shown that massive sanitation infrastructure provision without a proper planning of the whole scheme can be a complete failure (koné, ) . besides the technical aspects that are discussed later, the most worrying aspect is the lack of financial, institutional, and regulatory framework in most of the developing countries to establish the network required. management of on-site sanitation infrastructure comprises on-site sanitation systems emptying, fecal sludge haulage, treatment, and safe reuse or disposal (koné, ) . fecal sludges refer to sludge collected from on-site sanitation systems such as latrines, nonsewered public toilets, or septic tanks. the criteria to select an extraction method -a task that is never pleasant -depend on ( ) the ts content and ( ) the funds available. sludges with less than b % ts, such as those produced in septic tanks, can be pumped; but, for the rest of facilities producing all sludge with % ts, pits need to be emptied using cesspit trucks or manually by laborers (koné, ) . even though when mechanically emptied and water is used for toilet cleansing, - % of the contents in the lower pit part need to be manually emptied to extract the thicker sludge. the use of mechanical equipment allows carrying away the sludge several kilometers for disposal on controlled sites or on treatment facilities, but this is often expensive and needs proper equipment and skilled laborers. in contrast, when sludge is manually emptied, this is deposited in nearby lanes or on open spaces representing a source of risk. according to koné ( ) - % of the on-site sanitation facilities from west african countries are emptied manually. in addition, in almost every developing country, fecal sludge collection and haulage are conducted by private entrepreneurs. however, their important role and responsibilities as key stakeholders are not yet fully recognized and legalized (koné, in many low-income areas, the sanitation problem begins with the lack of sewerage. one option is to build sewers of small extent coupled with on-site sanitation systems. sewers carry the treated effluent to disposal (usually to soil for infiltration, to irrigation canals, or into water receptors), to wastewater treatment plants, and/or to reuse sites located within a short distance. as these sewers frequently convey partially treated wastewater (such as septic tank effluents), they are designed for self-cleaning using a high wastewater velocity and/or a steep slope. this option is applicable for rural areas or urban ones where adequate land is available. another option is to use simplified sewers. these are recommended where an uncertain population increase is occurring, as normally happens in periurban areas or slums. small sewers are built to reduce the infrastructure and maintenance costs, as well to allow high operational flexibility. inspection chambers such as manholes are replaced by inspection cleanout. the life expectancy of such sewers is in the order of years rather than the years quoted for conventional sewers. such sewers are short and shallow (hughes et al., ) . one example of simplified sewers are condominial ones in which pipelines are laid through housing lots instead of on the side street, in a way that allows isolated and stepwise construction (unep, ) . condominial sewers were developed in the s in brazil with the aim of extending sanitation services to low-income communities. this technology has now become a standard sanitation solution for some urban areas in brazil, irrespective of income levels. condominial sewers reduce the per capita costs of service by replacing the traditional model of individual household connections to a public sewer with a model in which household waste is discharged into branch sewers, and eventually into a public sewer through a group (or block) connection (watson, cited in lenghton et al., . these are structures that are bigger and deeper than those previously discussed. details for design can be found in conventional literature on sewers. many developing countries are located within regions subject to tropical storms, or in areas where there are only two seasons per year: wet and dry. therefore, urban hydraulic infrastructure needs to be designed accordingly to have sewers that can handle large peaks of stormwater and the normal wastewater flows (wastewater treatment plants should also be capable of dealing with the varying wastewater characteristics in quantity and quality, at least in large cities). sewers in tropical areas produce a high amount of sediments to be disposed off, which turns out to be a peculiar and difficult-to-solve problem not frequently commented upon in specialized literature but that needs proper methods to extract sludge and handle it. in addition, when conveyed in sewerage systems, stormwater must be treated in treatment plants at the same time as wastewater; but, if transported separately, it can be discharged to surface water or into wells for groundwater infiltration receiving treatment in soil. in this case, it must be kept in mind that stormwater quantity and quality are determined by rainfall, catchment processes, and human activities, which cause its flow and composition to vary in space and time. normally, for the first rains of the year, stormwater has higher suspended solids, heavy metal content, and bacterial numbers than nontreated wastewater, and lower dissolved solids, nutrients, and oxygen demand than secondary-treated sewage effluent. wastewater treatment is the typical method applied for sanitation, and is the predominant option used in developed countries for that purpose. although it cannot be considered a caveat for all the negative impacts produced by wastewater, it is still a very important option, and, in many cases, the only one. there are several steps to treat wastewater. the primary step basically serves to remove easily decantable and floating solids. the secondary one, generally a biological process, is used to remove biodegradable (mostly) dissolved suspended material. the tertiary step is used to refine the quality of the effluent produced by a secondary treatment. it may have different purposes, most commonly being the removal of nutrients (n and p). as the treatment steps were conceived following treatment needs, in practice, they are usually implemented in separate tanks or in well-defined sections of wastewater treatment facilities; however, it is possible to use compact processes eliminating physical separation among steps and thus reducing costs (jiménez, ) . wastewater treatment plants are not common facilities in low-income countries. in contrast to developed countries, in developing ones, the sanitation figure ( % according to who-unicef ( ) ) does not include the treatment of wastewater, which barely reaches % (us-epa, ). moreover, when available, the treatment merely consists of a primary step or including eventually a secondary step that is not always properly functioning. in many developing countries, the main issue concerning treatment is still the proper disposal of feces, particularly in low-income urban or rural areas. this, combined with a high content of pathogens in wastewater, sludge, or fecal sludge, implies the need to properly select the treatment process in order to effectively control disease dissemination. in general, coupling any kind of secondary wastewater treatment process (biological or physico-chemical) with a filtration step before disinfection will considerably reduce the pathogen content. however, this is rarely feasible for economic reasons and therefore it is sensible to consider the use of other technologies alone or combined with other type of intervention methods to build up a multiple barrier system to control wastewater risks (jiménez, b) . in the following sections, guidance will be provided to support the selection for treatment options, based on the type of pollutants. to address problems caused by suspended solids, organic matter, nutrients, and fecal coliforms, there is a wide variety of available technologies supported by literature and practical results. their affordability in economic terms and the suitability of the processes for local conditions are among the important aspects to consider for developing countries. it is beyond the scope of this chapter to provide a full description of treatment technologies for conventional pollutants, which can be found elsewhere in the literature. table shows the removal of pollutants by different processes so that it is possible to identify those acting upon the same type of pollutants. . . . . pathogens treatment table presents organisms' removal or inactivation achieved by different wastewater treatment processes. this table is a guide for selecting a process. however, to design complete treatment schemes, the operating conditions need to be properly selected as well as the pre-and post-treatment. table differs from the one presented by who ( ) in showing the removal efficiency data for helminth eggs in terms of a percentage instead of log removal. this is because helminths eggs' content is by far much lower and log units are meaningless. for developing countries, the removal of protozoa and helminths eggs is the main concern, considering their content and the occurrence of diseases caused by these types of agents. to remove protozoa, filtration is a good treatment option. conditions used to remove cryptosporidium oocyststhe targeted protozoan for developed countries -can be used as well to remove protozoa relevant to developing countries. helminth eggs are not affected by conventional disinfection methods (chlorination, ultraviolet (uv) light, or ozonation); thus, they are first removed from wastewater using sedimentation, coagulation-flocculation, or filtration processes to be subsequently inactivated in sludge (jiménez, a) . removal occurs because eggs are particles - mm in size. it is estimated that for contents of - mg l À of tss in treated wastewater, the concentration of eggs is around - eggs l À , while for values below mg l À it is around egg l À or less (jiménez, a) . however, for a process to be reliable, besides the removal efficiency attained, it is important for it to produce an effluent with constant concentration. the removal efficiency of emerging chemical compounds during conventional treatment can be found in jiménez ( b) . it is recommended that experimental tests be performed under laboratory conditions, prior to treatment selection. in the following, a description of main wastewater treatment processes is made, highlighting aspects that are relevant to developing countries, notably concerning their efficacy to control pathogens. table removal of pollutants by different wastewater treatment process that can be used to buildup a multiple barriers treatment scheme (with information from jiménez ( ) noxious by-products can be formed. if there is no competition with organic matter (bod or cod). doses are several orders of magnitude higher than those used for disinfection. if granular carbon is used. high for nonpolar organic compounds with log k ow > and when there is no competition with organic matter. medium to high depending on the presence of cations and organic matter. high but not for low molecular weight uncharged compounds. effective for several ec but not for carbamazepine, primidone, and iodinated x-ray contrast media. high for some ec, as it depends on the strength of solar irradiation removal will be different for different latitudes, or conditions. can be enhanced with photosensitizers. ? unknown or insufficient information * , can be removed or inactivated. no, not applicable for the pollutant. , . . . . slow filtration slow filtration is recognized in water potabilization as an efficient method to control microbial pollution in rural and low-income communities. the few studies carried out on slow filtration of wastewater have demonstrated a removal range of - % of suspended solids and - e. coli log, with coarse sand (jiménez, ) . in rural areas, it may be coupled with absorption wells, irrigation reuse, or a soil aquifer treatment (sat) system. waste stabilization ponds (wsps) are shallow basins that use natural factors such as biodegradation, sunlight, temperature, sedimentation, predation, and adsorption to treat wastewater (mara, ) . wsps are capable of removing organic matter with efficiencies similar to the activated sludge process and all kind of pathogens. they are easy to design and operate but require long retention times (several weeks). wsp systems comprised several ponds connected in series. lagoons are made through the shallow excavation of around - m, and they are frequently unlined to reduce investment costs. after a period of time, soil percolation and sedimentation form an impermeable barrier. if the water table is very high at the site, ponds need to be impermeable from the beginning. wsps remove up to bacteria log, up to viruses log, and almost all the protozoa and helminth ova. to control cryptosporidium spp., almost days' retention time is needed . in developing countries with wet warm climates, the use of stabilization ponds is recommended if land is available at a reasonable price. for arid and semiarid regions, high evaporation rates limit their application as there is a net loss of water of - % due to evaporation. this, in addition, increases the salinity of the effluent limiting its use for agricultural irrigation . sludge production in ponds is low but if extracted it needs disinfection as helminth ova remain viable in ponds for more than years (nelson et al., ) . wsps can be coupled with aquaculture systems that are shallow ponds or wetlands where fish, duckweed, or aquatic vegetables are produced as is frequently done in indonesia, china, and thailand. ponds can be used to produce only one crop such as duckweed that is used as food for the next pond where grass carp are grown. different species can also be cultured in the same pond, as happens in nature. to operate the system, wastewater is applied to ponds at the required rate (estimated in terms of the organic load applied per hectare of ponds per unit time), and the organic matter and the nutrients contained serve as food for plant and animal production (hughes et al., ) . in order to avoid health problems, wastewater needs to be previously disinfected according to who guidelines ( ) . constructed wetlands are used to naturally remove organic matter, pathogens, and nutrients from wastewater through biodegradation, adsorption, or filtration in a similar way to wsps. nutrients are also removed by plant uptake and pathogens by competition and sun uv-light inactivation (jiménez, ) . wetlands are shallow ponds where aquatic macrophytes are planted in soil, sand, or gravel. there are three main types: surface-flow, horizontal-flow subsurface, and vertical-flow systems. juncus spp. or phragmites are commonly used plants but any local plant can be employed. construction requires expertise and skilled labor. once installed, operation is relatively easy. wetlands remove nitrogen, phosphorus, and heavy metals. up to - % of thermo-tolerant coliforms, - % of ms coliphages, and - % of protozoa are inactivated or removed using hydraulic retention times of - days. in practice, pathogen removal is highly variable and depends on climate, type of wetland, and the kind of plant used. to completely remove helminth ova, it is necessary to couple wetlands with filtration, otherwise effluent with variable content may be produced. breeding of mosquitoes and unpleasant odors can be a problem if wetlands are not operated correctly. subsurface wetlands are used to avoid mosquito breeding (correlje and schuetze, ) . wetlands are a good solution for wastewater treatment in urban or rural areas where space is available; as a rule of thumb, . - . m per person is required for the treatment of graywater and - m per person for domestic wastewater. they are considered environmentally sound technology by unep for the treatment of graywater and stormwater urban runoff. they are used as secondary or tertiary treatment units, in which case, they treat effluents from septic tanks, anaerobic ponds, upflow anaerobic sludge blanket (uasb) reactors, or conventional wastewater treatment plants. treated wastewater can be reused for agricultural irrigation, although its nutrient content is low. wetlands have been used in bangladesh and china to treat wastewater and to cultivate fish and ducks. in addition, they have the advantage of producing a low quantity of sludge. soil can be used to treat wastewater by infiltration. it has a greater depollution capacity than water receptors, as there is no limit for the oxygen transfer needed for biodegradation. land-based treatment is recognized as an environmentally sound technology by unep ( ) that has a low cost when used for primary effluents. among its disadvantages is the high demand for land (jiménez, ) . in the case of land treatment, depollution takes place in the unsaturated zone through biodegradation, adsorption, ion-exchange filtration, and precipitation. for the removal of organisms, in addition to predation and humidity, the temperature also plays a role. heavy metals and trace organic compounds (such as emerging pollutants) are removed mainly by adsorption. to operate, wastewater is to be applied at specific rates; if pretreatment is needed primary sedimentation or sand filtration might be used (brissaud and salgot, ; jiménez, ; bouwer, ) . in developed countries, pre-treatment usually consists of a secondary treatment. wastewater application occurs in cycles at a rate that depends on the soil infiltration characteristics. in a typical situation, the cycle involves week of wastewater flooding where infiltration is reduced by organic buildup, and week of drying where bacteria consume the organic matter and soil drying takes place. there are several types of land treatment options in specialized literature that can be consulted. for efficient functioning, hydraulic loads ( - m m À yr À ) and mass loads should be limited. to avoid aquifer pollution, application of wastewater (preferably partially treated) is restricted to sites where groundwater is a minimum of m in depth. applied as primary or secondary treatment, land treatment produces a consistently high-quality effluent (tss o mg l À , organic carbon mg l À , and total nitrogen mg l À , with a phosphorus removal of almost % with minimal pre-treatment). as tertiary treatment, it removes % of bod, % of cod, % of ts, % of detergents, % of ammoniacal nitrogen, % of total nitrogen, and % of phosphorus. land treatment is effective for the removal and/or inactivation of helminth eggs, protozoa, bacteria, and even viruses (jiménez, ) .treated wastewater can be used for irrigation or any other use and can be collected on the surface or underground. reservoirs or wastewater storage tanks can be used as well to treat wastewater. while wastewater is stored during the wet season to provide water for irrigation during the dry season, pathogens are removed or inactivated via sedimentation, uvsunlight inactivation, predation, and other similar processes, which also occur in wsps. nevertheless, the efficiency is lower. procedures for designing wastewater storage and treatment reservoirs are detailed in juanicó and milstein ( ) and mara ( ) . reservoirs and storage tanks are easy to operate and maintain, and if considered as part of the irrigation system, they result in a low investment cost. however, they facilitate vector breeding if they are not well maintained and operated, and algal development in effluents may interfere with irrigation applications. effluent storage reservoirs remove À -log of viruses, À -log of bacterial pathogens, and À -log units of protozoan (oo)cysts. if treatment reservoirs are operated as batch systems with retention times over days, the complete removal of helminth eggs can be achieved (juanicó and milstein, ) . in addition to large storage reservoirs, small storage ponds can be utilized for pathogen removal when used for urban agriculture irrigation as intermediate water storage reservoirs. such reservoirs reduce the helminth ova content by around % (keraita et al., ) . the uasb is used to remove organic biodegradable matter. a uasb is a kind of attached system where microorganisms adhere to themselves, forming flocs. uasbs are considered as the most successful anaerobic process applied to treat wastewater due to low hydraulic retention time compared to other anaerobic processes thanks to the high density of biomass attained in the blanket (campos, ) . the reactor is designed to not only produce the biological reaction but also to sediment and filter suspended solids from wastewater. in addition, sludge retained in the bottom part of the reactor is anaerobically digested (campos, ) . the uasb produces better results when the wastewater has a high organic matter content. as by-products, it produces methane and partially treated sludge. the gas can be used as a source of energy, while the sludge remaining, after proper treatment to control the pathogen content, can be used to fertilize soil. uasbs remove - % of bod and cod and helminth eggs through filtration in the sludge blanket and through sedimentation. however, their efficiency with regard to the removal of helminth eggs is very variable. from wastewater containing - eggs l À , they produce effluents with - eggs l À ( - % removal). therefore, uasbs are frequently coupled with other treatment process such as stabilization ponds or filtration to completely and reliable remove helminth ova and to inactivate other pathogens. several stand-alone uasb plants or those coupled with wsp are currently under operation in curitiba, brazil. uasb reactors require careful design and operation to avoid bypasses (campos, ). the construction, operation, and maintenance of improved anaerobic technology such as biogas installations require considerable expertise and skilled labor as well as space (correlje and schuetze, ) . uasb reactors have a low capacity for tolerating toxic loads, need several weeks to start up the process, and require a post-treatment step. it is the most common way to treat wastewater in developed countries. compared to other secondary biological processes, activated sludge is effective for pathogen control as it removes % more than trickling filters. both sedimentation and aeration play an important role in this. sedimentation eliminates heavy and large pathogens, while aeration promotes antagonistic reactions between different microorganisms, causing their elimination. as a result of becoming entrapped within the flocs (which are subsequently sedimented), there is fairly good removal of small nonsedimentable microorganisms, such as giardia spp. and cryptosporidium spp., which remain concentrated within the sludge (jiménez, ) . helminths eggs are also removed, but due to continuous difficulties in achieving efficient and reliable sedimentation of suspended solids in secondary decanters, protozoan and helminths eggs may be found in effluents along with flocs. for an initial helminths egg content of - eggs l À , effluents with - eggs l À are produced . other biological secondary treatment options include aerated ponds, oxidation ditches, and trickling filters. much specialized literature exists describing the processes that are used to treat effluents before discharge into water bodies. this is a process that was almost abandoned for the treatment of municipal wastewater in the - s due to the high sludge production, which considerably increased the overall wastewater treatment cost. the introduction of new chemical products, in particular flocculants, combined with the possible reuse of treated effluent for agricultural irrigation and ocean disposal, has been instrumental in its reintroduction. coagulation-flocculation removes helminths eggs while preserving nutrients and organic matter in contents suitable to grow plants. when this process is applied using low coagulant doses combined with a high molecular weight and high charge density flocculants, it is called chemical enhanced primary treatment (cept). if, a high-rate settler is used instead of a conventional settler, it is referred to as advanced primary treatment (apt). as a result, cept has a total hydraulic retention time of - h while, for apt, this is only . - h. among the coagulants that have been used, iron and alum compounds are the most common. apt removes - % of protozoan cysts (giardia, entamoeba coli, and e. histolytica) and - % of helminths eggs. from a content of up to eggs l À , an apt can consistently produce an effluent containing . - eggs l À . this process produces an effluent with a low content of suspended solids or turbidity, which leads to greater disinfection efficiency, either with chlorine or with uv light. likewise, the process allows the use of sprinkler irrigation in high-tech countries or countries where water is scarce. the effluent quality is improved by the soil effect, and aquifers can be used as water supply storage (jiménez, . apt and cept are useful in middle-and high-low-income countries on large urban areas as an economical alternative to an activated sludge process as the treatment cost for apt is one-third of this process when considering sludge treatment and disposal within km. coagulation-flocculation can also be applied as a tertiary treatment after a biological process. this is a very good method to remove enteric viruses (jiménez, ) . rapid filtration (at rates over m m À h À ) is very efficient in removing protozoa and helminth eggs from wastewater, primary effluents, and biological or physicochemical effluents. it removes % of fecal coliforms, salmonella, pseudomonas aeruginosa and enteroviruses, - % of protozoan cysts (giardia, entamoeba coli, and e. histolytica), and - % of helminths eggs. efficiency can be increased to easily reach % if coagulants are added . for helminth ova removal, rapid filtration is performed in silica sand filters with . - . mm media size, a bed depth of at least m and filtration rates of - m m À h À . the helminth ova content in the effluent is constantlyo . ho l À in filtration cycles of - h for primary effluent (jiménez, . the challenge for any disinfection method is that microorganisms respond differently. efficiency depends on the disinfecting agent, the type and content of microorganism, the dosage, and the exposure time. the water matrix has as well a relevant influence, which becomes more important as its concentration and complexity increase. the most common disinfection processes for wastewater are chlorination, ozonation, and uv-light disinfection. . chlorination. it is the most widely used process to control microorganisms. it is effective for the inactivation of bacteria, less so for viruses and protozoa, and not at all for helminth eggs. with regard to virus and bacteria, chlorine has inactivation efficiencies of up to - log. however, chlorine is a very reactive agent and, therefore, before attacking microorganisms, it reacts with many substances contained in wastewater, in particular with organic matter, hydrogen sulfide, manganese, iron, nitrites, and ammonia. as a result, chlorination is a process that, in order to be efficient, needs to be applied at the end of treatment schemes to avoid interferences. if, in treated wastewater, ammoniacal nitrogen and organic matter are still presented, chloramines and organo-chlorinated compounds are formed. these are compounds that increase cancer risks. notwithstanding such risks, it is always preferable to chlorinate wastewater as microbial diseases have faster and often more dramatic health effects (jiménez, ) . . ozonation. ozone is very effective at inactivating viruses and bacteria. it inactivates - log concentration units in a very short time, provided there is a low demand for oxidizing agents by wastewater. there is abundant information in the literature concerning the design and operation of the processes. required ozone doses for several microorganisms are also available in the literature but, frequently, they are not affordable. as happens with chlorine, by-products generated during ozonation are a source of concern as many of them have been reported in the literature as toxic (jiménez, ) . . uv light. nowadays, uv-light disinfection closely competes with chlorination because it does not generate by-products that are too costly to remove from wastewater. besides, compared to chlorination, uv light does not need storage facilities, does not imply the handling of hazardous chemicals, and uses very small-size treatment tanks as disinfection contact times are very small (in the range of seconds or minutes). furthermore, due its simplicity of operation and high adaptive potential, it is suitable for rural and isolated communities. . . . sanitation and wastewater treatment costs figure presents estimated cost for different sanitation options, including from basic sanitation system to wastewater treatment plants. simple services certainly are much cheaper to provide, but they do not necessarily represent what the society wishes to have due to the comfort level. as cost is an important barrier to spread sanitation services, one would expect that these data is a well-known parameter. despite this, in many developing countries there are no reference costs, as exist in developed ones. as result of this situation, in many bids, costs are established using international data that do not necessarily reflect the local conditions (table ) . differences are due not only to build the sanitation facilities but also for the use of fuel and electricity, two important inputs to operate wastewater treatment plants. sludge management and disposal ( figure ) is another source of different affecting costs ( figure ). table also shows that the cost of emptying onsite sanitation systems is not negligible. the selection criteria for wastewater treatment processes are presented in table , emphasizing the needs of developing countries. after treating wastewater, the next step is its disposal. recently, some researchers have suggested (asano, ) to use the term 'dispersion' instead of 'disposal' in order to change the perception of getting rid of used water, but this term has to an extent the connotation of wanting to dilute a problem. in this chapter, the term 'reintegration' is introduced in order to emphasize that water needs to be returned to the environment or used once again (reuse). by reintegrating the water to the environment, the responsibility of using it and then restoring it back to the environment in a proper way may be realized. as, well water can be reintegrated into the hydraulic cycles in which is been used by the society, thus reducing the negative impact of extracting water from the environment beyond the amount needed for ecological use (environmental flow). water can be reintegrated to the environment by discharging it to the soil or into water bodies. in the following, different ways to reintegrate used water are discussed. this is followed by discussing the reintegration of water through reuse. soil reintegration (disposal) consists of discharging treated or nontreated water into land. as discussed in the section . . . the soil may act as a treatment step if a proper management is provided. the options to reintegrate treated wastewater into the environment are presented below. after discharging used water to soil, it will be evaporated, infiltrated, or will percolate to reach surface or groundwater bodies. the extent of each of these will depend on the soil and local conditions. they are used mostly for on-site sanitation effluents. they consist of a trench in which partially treated wastewater is discharged to allow its infiltration to the subsoil. the seepage in the trench allows uniform disposal of the wastewater over a given area. the leach drain is often filled with gravel or highly permeable material and a perforated pipe -from which used water is distributed -is placed in the centre at about . m beneath the soil surface. the perforated pipe is typically around . m in diameter (hughes et al., ) . the size of the trench depends on the wastewater load and the soil type, groundwater depth, and precipitation. leach drains are not recommended disposal options if the groundwater table is close to the surface (e.g.,o . m depth) or the soil has low permeability (e.g.,o mm d À ). they are convenient where soil is highly impermeable (e.g., clay) but can also be used in permeable soil from where water is both evaporated and infiltrated. in each case, plants are positioned to increase evapotranspiration and to remove nutrients from wastewater. if a limited area is available, evapotranspiration beds can be used in conjunction with a seepage trench. to increase dispersal of the wastewater throughout the whole bed, perforated pipes surrounded by gravel are used. the design of the bed should ensure it is large enough to hold wastewater loading and pluvial precipitation while, at the same time, providing sufficient water and nutrients to plants (hughes et al., ) . soil disposal can be coupled with soil treatment in the soil aquifer treatment-aquifer storage recovery system (sat-asr). an aquifer storage recovery system (asr) consists of holding water in an appropriate underground formation, where it remains available in such a way that it can be recycled by extraction when needed. an asr can have several objectives, some of which are (dillon and jiménez, ; jiménez, ) temporary or long-term storage; decrease of disinfection by-products; reestablishment of underground water levels; maintenance or improvement of underground water quality; prevention of saline intrusion; deferment of expansion of water supply systems; aggressive water stabilization; hydraulic control of contaminant plumes; and compensation of soil salinity lixiviation. the major advantages of underground storage is that evaporation losses are considerably lower than dams (b %) and do not have the eco-environmental problems associated with them (dillon and jiménez, ) . aquifers can be an economical option to reintegrate water to the environment in arid and semi-arid countries where it remains available for future use. they are also convenient in densely populated urban areas where, besides storing treated water, aquifiers can store stormwater runoff. effluents from treatment plants can be used for the augmentation of surface water bodies, in which the effluent is diluted with freshwater and reused as a source for water. the water quality of receiving water should be preserved to facilitate a safe water supply. for this, it is important to control pollutant content in the effluent, notably pathogens, organic matter, and nutrients (especially for surface water bodies with slow flow). two aspects need to be monitored: oxygen depletion in rivers and eutrophication in dams and lakes. to avoid oxygen depletion, biodegradable organic matter needs to be removed before introducing the wastewater. there is considerable literature available concerning this aspect as it has been the main target for most wastewater treatment processes. control of eutrophication is achieved by removing n and/or p from effluents; this is an operation costly to perform in wastewater treatment plants for most developing countries. as an alternative, land treatment can be used or treated wastewater used first for agricultural irrigation recovering it from the agricultural drainage before sending it to on lakes. eutrophication of dams and lakes is a frequent problem in developing countries; alternatives for its control are discussed in box . reuse is another option to reintegrate water to the environment but through its use. due to the increase in the human population and the increased use of water for almost all human activities, water is becoming scarce and new tools are needed to use it better. such tools are ( ) the efficient use of water (using less water for the same activity -this is beyond the scope of this chapter) and ( ) water reuse. water reuse is a key component to alleviate the mismatch between water supply and water demand. at the global level, water availability is of around m inhab À yr À but with important variations at a regional, national, and local level. for instance, it is estimated that around million people ( % of the total population) in countries live in areas with less than m inhab À yr À . by the year , % of the total world population will live under such water stress, increasing to % (in countries) by the year (undp, ) . as shown in maps , , and (annex ), most of the affected people live in developing countries. for these countries, three aspects can be highlighted concerning water stress and water demand. first, water is needed for economic development and a better quality of life (even if industrialized countries are not completely making an efficient use of water; they use - times more water than developing ones (un/wwap, ) ). second, agriculture is the dominant user of water worldwide, but, in addition, for developing countries, agriculture is usually the criteria for selecting wastewater treatment operation and processes must be evaluated based on past experience, data from full-scale plants, published data, and from pilot and full-scale plant studies. if few data or unusual conditions are encountered (atypical wastewater characteristics) pilot plant studies are essential. for developing countries: -since much less experience is available, a good wastewater characterization is needed as well as a request during bids that the applicability of the processes should be demonstrated before construction. -bids should encourage operating at lower costs at the same pace the process is optimized. -technology complexity need to be in agreement with the type of community being served: rural areas, rural isolated areas, small urban towns, large towns, and megacities (low-, middle-, and high-income urban and periurban areas densely or dispersed populated). -possibility to combine treatment technologies with soft intervention methods (management). performance needs to be expressed not only in terms of the effluent quality but also on its allowed variability, and both must be consistent with the effluent discharge requirements and the possible use of treated wastewater. performance needs also to be considered in terms of its reliability, as it may vary according to the process type. reliability is very important when the effluent is to be reused or treated water is to be discharged into sensitive aquatic environments. performance should be verified in terms of the disinfection needs locally required. consider wastewater characteristic variations in probabilistic terms. consider wastewater variability in terms of climate change impacts and climate variability. for developing countries: -it is important to have a statistically representative wastewater characterization considering parameters not only defined in norms but also those that might interfere with the treatment processes or the future use of treated water. -design data should not be based on bibliography data, especially that coming from other countries. -since segregation and pretreatment of industrial discharge is not common, there are high chances that the wastewater to be treated will contain inhibiting constituents. an evaluation of these is important but not as intensive as the one required for the characterization of the targeted treatment parameters. -consider wastewater quantity and quality possible variation if programmes to reduce water consumption (such as the use of water less toilets) are to be implemented. achievable performance needs to be expressed in statistical terms and in short and long terms, taking into account water flow and wastewater quality variations. for developing countries: -unusual situations and emergencies are common. selecting robust albeit more expensive processes might be cheaper long term, both economically as well as in terms of the negative effects that malfunctioning can produce. reactor sizing is based on the governing reaction and kinetic coefficients. if kinetic data are not available, process loading criteria are used, but not always with good results, even in developed countries. -most of the available information used in the design of biological process comes from the developed world, where wastewater and climatic conditions, among others, are different, and so bibliographic kinetic data and load criteria use should be avoided as much as possible. -for coagulation-flocculation process doses and mixing conditions determine at laboratory conditions are essential to minimize cost and sludge production. -for disinfection processes conditions need to be determined or checked up using laboratory data -if experimental data are not available, the adjustment of published data to local conditions, such as pressure and temperature, should always be checked in bids. the process should be matched to the expected ranges of flow rates. moreover, whenever possible, considering the presence of stormwater, notably considering impacts of climate change. for developing countries: -for those located in regions with high pluvial precipitation concentrated in short periods of time, treatment processes must be able to deal with flow and major variations in quality. -alternatively, the use of flow equalization tanks and their cost should be considered. -processes that can be operated as modules than can be easy to start should be preferred to match variable influents in terms of quantity and quality. the types and amounts of solid, liquid, and gaseous residuals produced must be estimated. use pilot plant studies to identify and quantify residuals. continued for developing countries: -by-products and wastewater treatment residues are often disregarded in proposals in order to offer a lower operating cost. to avoid this, it is important to clearly state in bids that any residues must be quantified and the management options considered within costs. design, operation, and maintenance must have the same degree of investment and complexity of its management as that of the wastewater treatment. for developing countries: revalorization of sludge as biosolids (treated sludge) for soil fertilization, erosion control, or land remediation are to be considered as a priority. for urban areas, use of biosolids to cover landfill cells can be an interesting disposal option. temperature affects the reaction rate of most chemicals and biological processes; therefore, local water temperature should be taken into account when selecting a processes. for developing countries: -in most developing countries temperature is relatively high, so problems arise due to high temperatures not low ones. high temperature may accelerate odor generation and also limit solubilization of gases such as oxygen. in densely populated urban areas, temperatures may rise even more than expected due to the 'heat islands' phenomena. environmental factors, such as prevailing winds, may restrict or affect the use of certain processes, especially where odors are produced near residential areas. a wastewater treatment plant may have negative impact on the environment if not properly designed. the disposal site restrictions of the treated wastewater need to be considered regardless of the norms to be met. water reuse can be a way of making wastewater treatment more attractive in economic terms. for countries located in water-stressed areas, besides being ecologically sound to reintegrate water to the environment as disposal option, reuse serves to alleviate water scarcity. for developing countries -land degradation is costing - % of their agricultural production (young, ) and fertilizers have often a prohibitive cost for farmers; in both cases, biosolids can be used to remedy these problems. wastewater treatment plants are often accompanied by ancillary (complementary) processes that do not necessarily directly relate to the wastewater treatment process, such as power plants, special storing facilities for reagents, etc. it is important therefore to know, before selecting a process, what are those needs, their cost and viability to obtain them from the local market. the type and amount of chemicals to be used need to be considered as well as their cost and market availability, both now and in the future. if chemicals are added during the treatment of wastewater or sludge and these are to be reused, their selection needs to be compatible. for developing countries: -although the use of chemicals is often prohibited, an economic comparison is worth making, especially if chemicals are locally available. the present and future cost of the energy used is something to consider. in selecting and designing wastewater treatment plants, the location, efficient use of energy, and the possibility of recovering/producing energy for in-plant use must form part of the selection criteria that in the long term will contribute to properly closing the urban water cycle. the energy foot print of the wastewater and sludge treatment plant should be minimized to contribute to the reduction of ghg (greenhouse gases). the amount of people as well as their skill levels need to be well defined. for developing countries -the most common situation is a high availability of low-skilled personnel working for low salaries. thus, selected processes may have a high labor demand but cannot be very sophisticated. alternatively, intense training programs should be considered; nevertheless, high indexes of personal rotation are frequently experienced in developing countries when personnel are trained. define operational needs under routine and emergency conditions. define the type and need for repairs. it is important that the items selected be compatible for efficient operation. for developing countries: -it should be considered that cheap or obsolete equipment may become costly if frequent repair is needed. -equipment and spare parts must be available within an appropriate period of time. obsolete equipment is very difficult to repair. main source of income and the main mean to feed a growing population. third, the increasing demand for water by municipalities and industries is increasing the competition for its use with farmers. it is estimated that, in developing countries, water withdrawals will increase more ( %) than in developed ones (undp, ) . among the uses demanding water, sanitation needs to be considered and, in that respect, water reuse may be a component in some areas to promote it through the alleviation of water demand, saving water for sanitation facilities or through coupling projects to treat wastewater with reclamation ones. two types of water reuse can be distinguished: nonintentional and intentional or planned. as, in several developing countries, lack of sanitation is generating nonintentional reuse, national policy will need to encourage controlled options -normally, few items are produced or available locally, therefore overall equipment selection needs to consider compatibility between different equipment traders. many treatment plants will need to adapt to future conditions and not all systems have the same capability to be adapted. cost evaluation must consider initial capital cost and long-term operating and maintenance costs. the plant with lowest initial capital investment may not be the most effective with respect to operating and maintenance costs. the nature of the available funding will affect the choice of the process. it is important to consider the size of the selected treatment process with respect to available land, including buffering zones for future expansions. for developing countries: -there is not always land or cheap land available, as frequently believed. -considering the fast growth of cities in the developing world and the possibility of building plants in modules, it is very useful to consider buffering zones to increase treatment capacity, complete the treatment process or even to avoid building human settlements near to the facilities. communities reject systems producing foul odors or vector breeding. communities also tend to more readily accept natural process that are integrated with the landscape. low-income communities accept better technologies that are a source of jobs for local people than rich ones. eutrophication is a process in which plants (such as water lilies or hyacinths (eichornia crassipes), hydrilla (hydrilla verticillata), cattail -(thypa sp.), and duckweed (lemna sp.)) proliferate in surface water bodies due to the presence of high concentrations of phosphorus and/or nitrogen that may come from wastewater, treated effluents, or agricultural runoff. it is commonly observed in polluted lakes or dams, but problems in low flow rivers and agricultural canals have also been observed. aquatic plants cover the water surface preventing sunlight and oxygen from entering the water. other negative effects that are provoked are ( ) oxygen depletion in the hypolimnion; ( ) release of fe, mn, nh , and heavy metals from the sediments; ( ) vector breeding, such as schistosomas and mosquitoes; ( ) loss of biodiversity, especially in higher trophic levels; ( ) displacement of native species, ( ) obstruction of hydroelectric plants and irrigation canals and drains; and ( ) restrictions on tourist, recreational, and fishing activities. to reduce aquatic weed density (plants m À ), five methods are available: * biological control. it consists of using living organisms to control weeds. in theory, it is a cheap option as no equipment or chemicals are required but it has an associated labor cost in order to perform maintenance. to be completely effective, the rate of grazing needs to be higher than the plant growth rate, which is very difficult to match in practice. a wide variety of fish, arthropod, fungi, and bacteria have been used for this purpose. * mechanical control. these methods remove or cut weeds into pieces using mechanically or manually operated equipment. it is an expensive option that can play a role in quickly reducing the extent of infested areas prior to the application of another control method. * chemical control. pesticides are also used to control weeds. some substances that have been used are terbutryn, diquat, , -d, glyphosphate, paraquat, and simazine. however, due to their toxicity, they can only be applied under controlled conditions and for a limited period of time. * water level control. in this method, the water level is decreased so the weeds located close to the edges of the water body dry out. the applicability of this method is limited to dams where water levels can be controlled, and to the dry season in which rain would not convey plants once again to the water. * nutrient control. weed growth is caused by high n or p content in water, and so, lowering their concentration through wastewater treatment is another alternative. unfortunately, the cost remains high. due to their low efficiency or cost implications, in practice, two or more methods are often used to control weeds. instead of promoting practices to start up water reuse. this is the biggest difference with developed countries, where reuse is being promoted once wastewater is treated. in literature, water reuse is considered merely as an activity where wastewater is intentionally treated to be used once again. therefore, water reuse is understood as an artificial man-made practice. however, unintentional reuse also exists as part of the natural hydrological cycle, but this is frequently not acknowledged. (jiménez, a) . 'nonintentional', 'nonplanned' or 'incidental' water reuse describe situations where used water is mixed with (or becomes part of) the water supply. in most cases, this unplanned reuse is difficult to identify, although it would be important to acknowledge it in order to properly control it. the nonplanned use of water is at the origin of the presence of emerging chemical pollutants in water sources and the reason why drinking water standards are becoming increasingly comprehensive and stringent and more sophisticated technologies to treat water are needed (jiménez, b) . nonplanned reuse of wastewater is happening for agricultural irrigation, aquifer recharge, and human consumption. . nonplanned reuse for agriculture. three-quarters of the total irrigated area worldwide is located in developing countries, and, as a consequence, there is a high dependence on water for food production. frequently, due to lack of sanitation in these countries, wastewater is used to irrigate land. this is a practice that happens almost naturally because of the combination of the high demand for water for irrigation ( % of total use compared to only % in developed countries, figure ), the availability of wastewater, the productivity boost that the added nutrients and organic matter provide, and the possibility to sow crops all year round (jiménez, ) . it is estimated that at least million hectares in countries (around % of irrigated land) are irrigated with raw or partially treated wastewater (who, ) . approximately one-tenth of the world's population consumes crops irrigated with wastewater, diluted or not. as an example, in hanoi, vietnam, wastewater is used in the production of % of the vegetables consumed locally (ensink et al., ) . the use of nontreated wastewater is also common for urban agriculture, which is practiced in urban and periurban areas of arid or wet countries where there is local demand for fresh food products, and people live on the verge of poverty with no job opportunities (jiménez, b) . for urban agriculture, wastewater flowing in open channels is used to irrigate very small urban plots of land where trees, fodder, or any other product that can be introduced to the market in small quantities (flowers and vegetables) or be used as part of the family diet are grown (ensink et al., ) . in terms of volume, reuse of nontreated wastewater is at least times higher than of treated wastewater (jiménez, ; jiménez and asano, ) . as a consequence, any sanitation project in localities using wastewater should consider its actual use. . unintentional reuse for water recharge. since groundwater is not water that can be observed as in lakes or dams, very often its pollution and nonintentional recharge is not perceived. infiltration may result from agricultural irrigation, leakages from wastewater and water urban networks, unlined dams, tanks or reservoirs, and on-site sanitation systems. little information on the extent of this problem is reported in literature, but some cases (a summary is presented in table ) have been described highlighting the importance of this phenomenon as a source of water supply. for the one referring to the tula valley, it has been the best documented (jiménez, b ) that recharge with wastewater amounts to at least m s À , and the aquifer is used to supply people. infiltration and pollution of groundwater supplies varies from negligible to severe, and the recognition of unplanned reuse is needed in order to advance understanding of how to manage the risks. this may involve continuing groundwater recharge with water of improved quality and/or separating the recharge areas further from points of water abstraction. appropriate monitoring information will allow the most cost-effective investments to be identified (dillon and jiménez, ) . . nonintentional reuse for human consumption. nonintentional reuse for human consumption occurs as described previously, not only through aquifer recharge but also through surface water sources when effluents, treated or nontreated, are discharged into them. this has been documented in developed countries. for instance, in the river thames in england, during dry periods, % of the water used as supply downstream comes from treated effluent. in california's santa ana river, a large part of the supply consists of treated wastewater (gray and sedlak, ) and in berlin, - % of the city's water supply comes from an advanced treated effluent that is discharged to a nearby water supply (jekel and gruenheid, ) . the increasing evidence of the presence of emerging contaminants in water sources is an indication of the nonintentional reuse of water. information on this subject for developing countries is very poor, and possibly only reported as pollution cases. recognizing the nonintentional reuse of water for human consumption will help society to acknowledge that water reuse is unavoidable in the future and also to understand that, to properly reintegrate used water to the environment is needed. for this, tools other than wastewater treatment plants will be needed. according to asano ( ) , wastewater reclamation involves the treatment or processing of wastewater to make it reusable; and wastewater reuse or water reuse is the beneficial use of treated water. planned reuse may be performed for agricultural irrigation, industrial purposes, environment restoration, and municipal uses. . reintegrating water for irrigation. most of the world's poorest people, million to billion rural people, live in arid areas and depend directly on natural resources, including water, for their livelihoods (dobie, ) . in such a context, safe wastewater reuse can be a sanitation option that could also be coupled with food security and economic development goals. under prevailing land and water management practices, a balanced diet represents a depleting water use of m inhab À yr À , which is times more than the l inhab À d À required for basic household water needs (siwi-imwi, ) . for several middle-and low-income countries, agriculture is currently, and will continue to be, a key sector representing % of export earnings. limited and unreliable access to water is a determining factor in agricultural productivity in many regions, a problem rooted in rainfall variability that is likely to increase with climate change (lenghton et al., ) . to feed this sector, water reuse can be one option. planned reuse of water for agricultural irrigation in developing countries is a convenient strategy for many reasons (jiménez and garduñ o, ; jiménez, jiménez, , a who, ; keraita et al., ) , such as • it is an easy option to increase controlled reuse when nontreated wastewater is already in use as it allows more profitable and safe products. • it can be a low-cost option to manage wastewater and to reintegrate water into the environment. • it allows the reclamation of nutrients (n and p, to increase soil fertility) and organic matter (to improve soil characteristics) at no cost. • particularly in (but not limited to) arid and semi-arid areas, it permits higher crop yields, as it allows crops to be sown year-round due to higher water availability. • due to the availability and reliability of water, crops with better profitability can be selected. • it avoids discharging pollutants to surface water bodies (which have a considerably lower treatment capability than soils). • it is possible to recharge certain type of aquifers through infiltration. • it can be part of a strategy to secure food and increase poor people's income in water-scarce areas. to obtain all the advantages from reusing wastewater for agriculture in planned projects, it is important ( ) to control possible negative effects (jiménez, ; who, ) such as those related to health; ( ) to keep in mind that in many cases nontreated wastewater is being reused at low or even no cost by poor farmers and, hence, they will be unable to afford reuse costs; and ( ) from the legal aspect, the historical use of nontreated wastewater by farmers confers riparian rights. . reintegrating water for industrial reuse. industrial reuse (reclamation of wastewater from a different use, i.e., reuse of a municipal effluent for industrial cooling) differs from municipal and agriculture reuse as it involves the private sector that has its own rules and well-defined needs driven by economic factors (jiménez and asano, ) . before reusing water, industries always prefer to implement watersaving projects as these immediately reflect on their budgets; for reusing water, investments to provide proper treatment and monitoring programs are needed. to promote industrial reuse, the best government strategy is to provide incentives rather than setting compulsory regulations (jiménez and asano, ) . among the different industrial reuse options, cooling is the most popular due to its high water demand, and the possibility of using secondary-treated municipal effluents, sometimes coupled with filtration or softening processes. as a consequence, power plants located near urban areas are potential sites of industrial water reuse. . reintegrating water to the environment. more than . billion people live in river basins where the intense use of water threatens freshwater ecosystems (smakhtin et al., ) . reintegrating water to the environment is a practice that is gaining momentum, as it is being recognized that ( ) the environment needs water and ( ) the environment has the same entitlement to water as other uses. unfortunately, these two aspects are better recognized by developed countries than developing ones. overuse of water tends to occur in regions heavily dependent on irrigated agriculture or where there is rapid growth of densely populated areas (undp, ) , two characteristics common in developing countries. among the more prominent examples (undp, ) of water overuse, the exploitation of the yellow river basin, in northern china, can be cited: human withdrawal currently leaves less than % of the flow remaining in the river. the river ran dry km inland for a record days in . the drying up of the river caused a drop in agricultural production averaging . - . million tons a year, with losses estimated at us$ . billion for . the purified effluent from sewage treatment plants can be used for the augmentation of river flows, to raise the level of wetlands or lakes, to recover dried lakes, or even to create new lakes or wetlands. in doing so, biodiversity may recover. care must be taken when restoring water into water bodies to preserve or improve the actual quality of water. used water reclamation can be combined with rainwater reclamation. water reuse with environmental restoration can be coupled with projects of urban image improvement or programs to provide better facilities at recreational areas. . restoring water to aquifers. aquifer recharge is not, itself, a use of reclaimed water but is often part of the pathway to reuse. it is a convenient way to reintegrate water into the environment but can be used only under certain circumstances related, in particular, to the type of soil and groundwater. aquifer recharge can be performed to recover groundwater levels, to control saline intrusion, to augment drinking water sources, to protect and, in some cases, to improve underground water quality, to protect surface water bodies from contamination by effluents, to increase water availability for any use, and simply to store water for the future (dillon and jiménez, ; corrleje et al., ) . intentional recharge with reclaimed water can play a role in providing balanced storage and supplemental treatment for water (bouwer, ; dillon and toze, ) . it also provides low-cost storage that occupies a minimum of valuable urban land, while stored water is protected from pollution and evaporation. there are two methods to recharge aquifers. the first is known as land-spread infiltration where treated wastewater infiltrates through soil by gravity. this option has relatively low operating and maintenance costs. the second method for recharge is direct well injection. in this option, wells are used to convey a highly treated effluent directly to aquifers. regulation to recharge aquifers are very different from one country to another; some are set at a national level while others are defined using a case-by-case approach (jiménez, ) . most of the projects to recharge aquifers are found in developed countries. in developing ones, some examples are found in atlantis, south africa (for drinking and agricultural purposes, using pond infiltration), in windhoek, namibia (for drinking purposes and using injection wells), in new delhi, india (for irrigation using infiltration ponds for treated urban wastewater and stormwater), in beijing, china (for drinking purposes using wells and recharge basins), and in mexico city, mexico (for drinking purposes on a limited scale and using infiltration ponds; dillon and jiménez, ) . in all these cases, wastewater is treated to at least at a secondary level (see section titled 'relevant websites'). . reintegrating water for municipal use. in years, % of the world's population will be living in cities (un, ) . this being the case, more water will be needed for municipal use and, at the same time, more municipal wastewater will be produced. this situation, therefore, represents an opportunity to increase municipal wastewater reuse. water reuse in cities represents an opportunity to conveniently treat wastewater, with environmental and even economic advantages. opportunities to reuse wastewater in cities are classified into two groups: ( ) those demanding relatively low-quality water and involving low health risks, and ( ) those demanding high-quality water where health risks are high. in the first group, there are several types of uses, such as: (a) the filling of recreational lakes or the operation of fountains; (b) car, truck, or street washing; and (c) green area irrigation. options demanding high water quality include reuse for drinking supply. around the world, there are successful examples of both types of reuse, low risk options being the most common. water reuse for human consumption, although less common, is no less important. moreover, the only two examples of the reuse of water for human consumption in the world are notably from two countries from the developing world: namibia and singapore (box ). graywater (i.e., domestic wastewater not containing toilet wastewater) is more accessible for reuse as it is less contaminated than wastewater, notably in terms of (but not limited to) pathogens. typical sources of graywater are bathing, laundry, dishwashing, and food preparation. due to its comparably low and easily degradable contamination, it can be relatively easily treated for reuse. graywater reclamation entails the production of less wastewater to be treated in centralized plants. graywater reuse is performed at the same facilities where it is produced and, as a result, a short storage time is needed ( day retention time). graywater reuse can be performed individually (for a single home) or collectively (several groups of houses or larger buildings). treated graywater may be used for watering plants, kitchen gardens, and for the safe augmentation of ground-or surface water. treatment can be very simple or highly sophisticated, ranging from simple manually operated sand filters to biomembrane reactors, hence, covering the needs for rural areas or buildings located in upmarket areas in megacities. further details on design and operation can be found in correlje and schuetze ( ) . graywater reuse can be as well an important component for basic sanitation, as described in section . . . . as the quantum of wastewater treatment is still low in developing countries, little information is available concerning the actual situation. leblanc et al. ( ) performed a survey in some countries showing that the tendencies are the following: . for middle-income countries. from information coming from middle-income countries, including africa (namibia and south africa), the middle east (iran, jordan and turkey), asia (china and russian federation), and latin america (brazil, colombia and mexico), it is shown that wastewater treatment facilities serve mostly urban areas using preliminary, primary, and, in some cases, secondary processes. for rural or poor periurban areas, basic sanitation facilities are provided. although sludge is produced in these facilities, this is not always managed as part of the sanitation service. the disposal options for the sludge from wastewater treatment plants produced are landfill dumping, dumping into sewers, storage at wastewater treatment plants, land application, and agricultural reclamation. land application and agricultural reclamation are options limited by space problems, while the use of landfills is restricted in densely populated urban areas, where solid wastes compete for space with sludge. as sludge production is still low in the few wastewater treatment plants available, sludge management policies are novel, and are still in a maturation phase. some of these policies offer new approaches different to those used in developed countries (leblanc et al., ) . with regard to fecal sludge, the main constraint for their management is the cost to empty on-site sanitation systems as these are often located in inaccessible areas, are large in number, and are frequently highly dispersed. it is noted that the high cost of latrine emptying is not sustainable, even for large municipalities. extracted fecal sludge is often buried on-site, dumped into landfills or sewers or sent to uncontrolled discharge sites. discharge of sludge and fecal sludge in sewers often lead to surpass the wastewater treatment plants' capacity when available. . for low-income countries. data from different african countries (burkina faso, cameroon, cô te d'ivoire, ethiopia, mali, mozambique, namibia, nigeria, senegal, and south africa) demonstrated a similar situation focused on the need to provide basic sanitation services either in rural or urban areas. few cities have complete sewerage systems and, when available, sewers frequently feed into partially functioning wastewater treatment plants. in these countries, the use of on-site sanitation systems, such as septic tanks, bucket latrines, pit latrines, and dry latrines, produces fecal sludge, which is often 'contaminated' with domestic waste. in dense informal settlements, the challenges to properly handle fecal sludge are significant as besides the technical constraints other factors related to the social, political, and cultural aspects come into play. fecal sludge handling includes the need to provide reliable and low-cost options to emptying the facilities, to provide proper and affordable treatment and transportation, and to have suitable sites for safe disposal. literature exists concerning the alleviation of sludge and fecal sludge disposal and revalorization problems, not all of which is relevant for developing countries. common issues in box reuse of wastewater for human consumption in namibia and singapore windhoek, namibia, has been reusing wastewater for human consumption for more than years (van der merwe et al., ) as result of an original idea in . since its operation, no measurable health risk has been observed and neither have people drinking reused water displayed associated health problems. the reclamation plant has undergone several modifications to improve the technology used. the quality of the water supplied can be consulted every day in the local newspaper. the amount of water reused is around ls À , which is distributed after dilution by a factor of - with first-use water. the monitoring program for the facility represents % of the operating costs, and is performed by the wastewater treatment plant and also by three independent laboratories. the system is operated using a multiple barrier concept that goes beyond the wastewater treatment plant. the astute words ''water should be judged by its quality; not its history'' are attributed to dr. lucas van vuuren (van der merwe et al., ) , one of the pioneers of the windhoek reclamation system. this refers to the fact that fear of reused water should be based on rational aspects. the other example of direct reuse of wastewater for human consumption comes from singapore (funamizu et al., ) and is known as the newater project. it started in and uses a secondary effluent that is further treated with a membrane system (microfiltration (mf) and reverse osmosis (ro)) and uv-light disinfection. the water produced is cleaner than tap water as it fulfills all the requirements set by us-epa and who for drinking purposes. treated water is channeled to a reservoir, from which it is taken as supply after dilution with first-use water. water is distributed through the network for use for domestic and industrial purposes. when the newater project was launched, it operated at a rate of l s À . this will be progressively increased to reach l s À by (b . % and . % of total water consumption, respectively). in both cases, namibia and singapore, before the implementation of the reuse programs, stringent industrial pre-treatment programs and segregation of industrial effluent from the sewer were put in place. properly managing sludge and excreta in developing countries are as follows (leblanc et al., ; jiménez, • conventional sludge and excreta treatment options used in industrialized countries do not necessarily achieve the levels of pathogen inactivation required for its safe reuse. • nutrients, organic matter, and energy are resources available in fecal and wastewater sludge that should be utilized as best as possible. there are examples around the world showing the feasibility and convenience of reclaiming them. • applying properly treated excreta and biosolids to soils in a safe way can contribute to soil fertility and with it to food security; it can also raise income for poor farmers. • proper management of excreta and wastewater sludge can significantly reduce releases to the atmosphere of potent greenhouse gases such as methane and contribute to carbon sequestration in soils. the mdg target stating ''reduce by half the proportion of people without sustainable access to safe drinking water and basic sanitation is considered under goal : ensuring environmental sustainability'' (box ). therefore, sanitation is to be provided in a sustainable framework which, in practice, means to provide a service comprising much more than was expected in the past. to implement it, a proper policy is needed. in order to consistently provide sustainable water services, it is recommended that an integrated water resources management (iwrm) approach is used. this approach is useful to analyze situations such as when • multiple barrier system comprising solutions that go beyond the construction of wastewater treatment plants need to be implemented to protect health and the environment; • sanitation needs to be provided as a tool (sometimes indispensable) to have clean water supplies and to provide a safe water supply (box ); • sanitation is coupled with projects contributing to food security, job opportunities, increases in exportation, soil erosion control, efficient use of water, etc.; • sanitation needs to be provided over a wide area rather than to a single section of it to effectively control negative environmental impacts; • sanitation needs to be part of a three r concept system (reduce, reuse, and recycle); • sanitation is considered as part of a cycle in which wastewater is properly reintegrated to the environment; • sanitation needs to consider the impacts caused by climate change; • projects need to be designed, operated, and/or managed by different institutions, sectors, basin agencies, or even countries; box what does sustainability mean? ''a process that promotes the coordinated development and management of water, land and related resources, in order to maximize the resultant economic and social welfare in an equitable manner without compromising the sustainability of vital ecosystems'', un-water, according to leblanc et al., , elements defining sustainability are * dealing transparently and systemically with risk, uncertainty, and irreversibility; * ensuring appropriate valuation, appreciation, and restoration of nature; * integrating environmental, social, human, and economic goals in policies and activities; * providing equal opportunities and community participation; * conservation of biodiversity and ecological integrity; * ensuring inter-generational equity; * recognizing the global integration of localities; * a commitment to best practice; * avoiding net losses of human or natural capital; * implementing principles for continuous improvement; and * providing good governance. bissau, guinea, in west africa is a city attracting huge numbers of people from the surrounding countryside. most of them have settled in squatter new areas around the old colonial center. during a study performed in the s, it was found that the newly piped water taps ran dry several times per day. as a result, many people returned to the old wells. these were often more contaminated than before because the new pit latrines installed close to the wells polluted the groundwater. groundwater quality was also impacted by solid waste thrown into the pits dug for the production of adobe blocks to build new houses. moreover, the new network of gutters was now efficiently removing most of the clean rainwater that used to recharge the groundwater. the gutters caused an extra problem. on the edge of the settlements, where the gutters ended, storm water peaks caused serious soil erosion. this created problems for a newly developed scheme of vegetable gardens on the urban fringe, and even threatened houses.the original problem -the lack of water in piped water taps -was related to electrical power failures causing water pumps to stop. similar situations can be encountered in many developing countries and they cannot be easily solved as long as their roots are not properly and integrally tackled. • good technical solutions needing proper social, economic, and political policies are to be put in place; and • wastewater, treated or not, is being nonintentionally reused. developed countries, through experience, research, and technological innovations have progressively improved their sanitation services and have developed systems that are what they need. however, as described in this chapter, the problems they have faced and the problems they are now facing, although similar, are not the same as those confronted by developing countries. thus, there is a need for low-income nations to develop their own processes using part of the developed countries' experience. to contribute to this process, a definition of the issues to address and the challenges to face is provided in the following. the issues that need to be addressed are as follows: • low sanitation coverage lagging behind population growth, needing an intense effort in order to be tackled. • need/importance to couple sanitation programs with others addressing problems such as food security, low income, and soil erosion control. in practice, this requires increased efforts of coordination. • lack of sanitation as a component of poverty, and therefore, as a problem that cannot be completely solved if its roots are not properly addressed (box ). • lack of sanitation, particularly in vulnerable groups that, due to their own characteristics, are often more difficult to provide services for. • a growing population, notably in urban areas and, within them, in slums. • higher vulnerability to the negative impacts of economic and climatic change on sanitation needs. • for low-income countries, lack of economic capacity to deal with the cost of covering the sanitation mdg targets and, for middle-income countries, the need to mobilize funding required to put sanitation above other needs. • the proper management of sludge and excreta, two byproducts often not considered as part of sanitation targets of funding programs. the challenges to be encountered are listed below: . the lack of political will and commitment at the highest level (who/unicef, ) is a barrier that is greater than, for instance, the lack of economic resources, the capacity for building, or the acquisition of appropriate technology, since all these may be overcome by a strong political support. in order to develop political will, politicians and society need to appreciate the value of sanitation. an understanding that it is through the provision of water supply and sanitation that industrialized countries build up strong societies with good health and good economic conditions is needed (box ). . the second challenge is to put in place accountability mechanisms to ensure that resources provided to fulfill in cameroon, some houses are equipped with a m-deep hole for a latrine, surrounded by pieces of timber. when the hole is full, it is covered with earth and medicinal or aromatic plants, and another facility is built. if the family has no land to dig another hole (as frequently happens), they call the tanker to empty it at a cost of us$ . sometimes, while the family saves up the money, excreta overflows and pollutes the nearby area where wells and boreholes are located, threatening drinking water quality. when feces are removed by tanker trucks, they are often dumped into rivers or the forest, because there are no treatment facilities. houses in modern residential areas have septic tanks, and their effluents are directed into wells for filtration. often, this does not happen in the correct way because builders have not mastered the technology. some collective residential areas, universities, and hospitals are connected to sewers that convey wastewater to a treatment plant, from where treated water is directed to a river. but still, there are people without access to any of the facilities described above who go into the bush to relieve themselves on the spot. villagers continue to use this practice because they have no choice. box clean meansy yy y healthy? mexico city produces % of mexico's gross domestic product (gpd) (us$ per capita). after the swine flu (h n flu) outbreak in may , a loss of us$ million was experienced solely due to the shutting down of restaurants, and us$ . million were lost due to the closure of public transport for just days. to allow the city to return to normal conditions, health experts advised constant handwashing and the disinfection of school toilets. at this point, politicians realized that public schools had no water at all, had malfunctioning toilets and more had no facilities at all. before the swine flu epidemic, politicians had not understood the link between water, sanitation, and health and had not addressed this problem, although on many occasions parents' associations had requested the services. the president of one parents' association commented on the news that, in contrast to most mexicans, he believed that the swine flu had been a blessing as it was the only way to ensure proper sanitation facilities at schools. the mexico city government invested us$ million on the school program 'clean means healthy'. the mdgs (public and private from donors) will be used wisely and for what they were originally intended for. . the third challenge involves a broader aspect. even if sanitation programs are put in place, if poverty is not properly addressed, most of the solutions provided will be unsustainable. this will possibly lead in the future to adding addressing poverty to the already lengthy list of reasons why sanitation has failed in developing countries (this list already comprises financing, institutions, education, the need for decentralization, and the need for private participation). although there is no recipe for success, strategies that can be considered when developing plans for sanitation include the following (jiménez and garduñ o, ; jiménez, jiménez, , lenghton et al., ; undp, ; who, ; leblanc et al., ; correlje and schuetze, ) : to develop policies: • take time to perform proper planning in order to identify the resources (human and economic) needed to design, build, operate, and maintain facilities, and to develop policies and institutions. do not initiate projects for which this has not been previously defined, otherwise there is a risk of losing any investments made (a case in point is the existence of many facilities installed around the world, which have been subsequently abandoned). • take time to define how much money is needed, supported by experts with no commercial interest, specifically not those from companies that are potential participants in bids. • define needs and priorities using the best available information even if it does not come from the water sector. priorities can be set by using the methodology proposed by lenghton et al. ( ) , which considers actual water service coverage, and mortality due to gastrointestinal diseases and density of settlements, considering urban and rural areas. evaluate risks using quantitative methodologies to properly identify and prioritize problems, and select solutions accordingly (in terms of size, and economic and human resource investments). • as much as possible during the planning stage, involve sectors related to the solutions other than the water sector (e.g., the federal, regional, and local governments, ministers of the environment, urbanism, agriculture, land use, transport, economic development, social development, finance, etc.). • couple sanitation programs with programs related to food security, soil remediation, and economic development. • produce efficient, affordable, and enforceable norms and set goals for them that are easy to understand. • promote innovation at all levels (institutional -box -, financial, regulatory, and technological). • combine different intervention methods to control problems; consider not only of sewers, latrines, and wastewater treatment plants. • consider water reuse and the safe reintegration of sludge and fecal excreta as an important part of the overall sanitation program. • promote the management of the environment in an integrated way, even considering climate change effects. • design monitoring programs that wisely use resources by including information that will be used. use the new information obtained to evaluate and improve the program. • review the program to ensure it covers the specific targeted population sectors (women, the poor, rural areas, etc.) and meet the defined goals. for funding: • be creative in finding solutions to funding needs; • extend financial support to the poorest households to ensure that sanitation is an affordable option; • discern whether there is an absolute lack of resources for expanding water supply and sanitation coverage, or if there is a need to redistribute potentially sufficient existing resources; and • develop and put into practice transparent mechanisms to easily and rapidly transfer monetary resources from central to local institutions. for institutional design: • develop national and local political institutions that reflect the importance of sanitation in terms of social and economic progress. • promote institutions throughout government that use or at least understand concepts of integrated management, not only for water. • develop institutions where innovation and solidarity are considered as a virtue. water and sanitation service agencies are typically modeled after utilities in industrial countries, and as such are organized around the goals of maximizing operational efficiency for public sanitation components (trunk sewers and treatment plants) rather than providing services to poor people, slums, disadvantaged groups, etc. as result, in, in developing countries, experience and institutional structures to provide the type of services needed is deficient. as a result, services are being provided by other means. data from india indicate that as much as % of rural households across the country invest their own money and use small private providers to construct latrines. self-provision accounts for about million privately installed septic tanks in manila and in jakarta. research in africa confirms that the role of the small-scale private sector in sanitation provision is significant. these findings are further supported by data from the jmp (who-unicef joint monitoring programme): between and , the increase in the number of people served by sanitation reported by the jmp was much larger than the expected impacts of the public investment that occurred during this period. the reorientation of public programs to either modify their structures or to promote and assist the provision of sanitation services by small private and even familiar companies is needed. this does not currently occur in developed countries. • consider the need to have as part of the institutions welltrained and highly professional personnel. • identify which problems should be addressed by using norms (compulsory), criteria (recommendations), or other type of tools (such as incentives and education). • set appropriate and affordable sanitation risk-based standards, designed to contribute to solving local problems that can be reviewed over time to integrate experience. these should be able to be adapted to new and better conditions in order to move progressively to an ideal situation. • allow the development of norms that are adapted to local needs and capabilities (table ) . sanitation systems are often adopted from other developed countries without sufficient adaptation and users tend to put in place an idealized solution in which a uniformly high level of service is provided and the technology to be used is already set. • set up regulations that combine different intervention methods to control risks that are not based only on wastewater treatment plants. • keep in mind that parameters selected are to be enforced and they will demand economic and human resources for. • review the whole legal framework related to the standard so they can fit in and be implementable. • set up standards using a participatory approach, which includes stakeholders and expert participation, notably coming from local universities. • where noncontrolled reuse is already in place, regulations need to maintain the benefits already obtained while progressively controlling drawbacks; this can be done by promoting controlled reuse rather than adopting vanishing current practices. • incorporate reuse as part of the sanitation standards. to set up programs: • perform a national inventory of the actual needs and solutions to be implemented to manage wastewater, excreta, and sludge, include a survey on water reuse possibilities to couple them with sanitation solutions when feasible. • implement policies by promoting incentives rather than imposing rules and fines; but when rules are to be observed, be firm on decisions, and inform society in order for it to be perceived that jeopardizing the health of others is important. • as there is no universal solution, support a wide range of sanitation technologies and service levels that are technically, socially, environmentally, and financially appropriate. • promote innovation to have both technically and economically feasible technologies to deal with local pollutants, notably for the high and varied pathogen content. • implement pilot plant programs to test policies and use the information obtained to retrofit your program before scaling it up (box ; spaliviero and carimo, ). • empower local authorities and communities with the authority, resources, and professional capacity required. • in order to fund the maintenance and expansion of services, local governments and utilities should ensure that users who can pay, do so. • carry out training programs addressing all stakeholders needs, from plumbers to politicians. some aspects to consider when setting regulations definition of fixed treatment option(s) to use and inclusion of predefined treatment design and operating criteria. -reduces the need for monitoring and surveillance. -limits innovation -renders project implementation easier. -encourages bias in regulators who will be responsible for both selecting the method of control and meeting objectives. -may lead to nonviable schemes from an economic point of view. selection and use of the best indicators as parameters. reduces monitoring and surveillance cost. -introduces the idea that indicators are the best and ideal parameters to define pollution. -most of the current best indicators have been proven effective for developed countries but have not been tested for all conditions in developing countries. -may give a false impression of safety. selection of normal monitoring parameters and establishment of limits for each one. -facilitates surveillance. -cannot be universal or static over time. -increases supervision costs. use of epidemiological local data. -introduce protection for local problems. -information not always available for all of the diseases currently present. use of toxicological tests. -data available internationally. -often render norms too stringent. -helps to establish cause-effect relationship. -for diseases originating from microbial pollution do not correspond to local conditions when diseases are endemic. use of risk evaluation models. -help governments to make rational decisions. -difficult to explain their meaning to the population. • implement programs to segregate and/or pre-treat industrial discharges to sewers to render municipal wastewater treatment more affordable and to avoid the presence of noxious compounds in treated wastewater and sludge that will limit their revalorization options. • as wastewater, sludge, and excreta management regulation compliance often depend on the work of different ministries, coordinate the work of such institutions taking care that the objectives of each are compatible. • develop public indicators to follow up progress globally and also consider the implementation of indicators to follow specific targets such as wastewater treatment coverage, safe reintegration of treated water to the environment, and sludge and fecal excreta management. attention should also be provided to deprived sectors (women, poor people, slums, dispersed rural areas, etc.) • seek to validate your indicators by a third independent party such as a university or a non-governmental organization (ngo). • verify that the same information is provided international, nationally, and locally. to raise support for the program: • make it understandable to all that lack of sanitation means a barrier for economic development is an unsustainable way to manage the environment, is at the origin of local pollution problems, contributes to water scarcity as it reduces water availability, and increases vulnerability and reduces the capacity to adapt to climate change. all of these issues have broad support among society and different groups, not all of which are concerned by sanitation for the poor. • build community-level initiatives through government interventions aimed at scaling up best practice. • create awareness of the nonplanned reuse of wastewater and the importance of investing in it as an option to make clean water accessible for any use. figure shows the investments made for water supply and sanitation from to ; it can be observed that, in the past, most efforts were orientated to water supply and cities, leaving sanitation (only about one-fourth of investments made for water supply) and rural areas far behind. figure shows the origin of investments. in the case of asia and latin-america, almost all the finances have come from governments, while, for africa, it represented nearly a half. from the previous analysis, it is evident that there is need to invest money to catch up with the level of services needed. before calling for funding, it is convenient to analyze (preferably only within each country, without the input of donors or enterprises) what the money should be used for. to sustainably increase sanitation coverage, economic resources are needed not only to build sanitation infrastructure, but also for planning according to local needs and possibilities, developing research and technology, and developing institutional capacity in a local context. unfortunately, most of the time, funding is provided only for some of these activities (mostly for infrastructure); one major reason being that, often, this is the only type of funding that is sought. there are two funding options: public or private, each of which has different modalities. for public funding, the money comes from federal or local governments either directly from tax revenues or user charges, or, indirectly through crosssubsidies from users who can afford to pay, private-sector investment, or international and national loans. private sector box development of a stepwise program in mozambique (with information from spaliviero m and carimo d ( ) mozambique. in: leblanc rj, matthews p, and richard rp (eds.) global atlas of excreta, wastewater sludge, and biosolids management: moving forward the sustainable and welcome uses of a global resource: unhsp, pp. - . vienna: un.) following mozambique' s independence in , the government identified sanitation as one of the key components to improve health conditions. as such, in , the ministry of health launched an intensive national campaign for the self-help construction of latrines. many thousands of latrines were constructed during a relatively short period. however, there were numerous problems, including insufficient awareness about environmental conditions, a lack of technical guidance in latrine design and construction, and shortages of critical building materials. consequently, many of the latrines became structurally unsafe and unusable. in response, a research project was initiated in to ''identify and develop a suitable technology and method for large-scale implementation of improved sanitation in periurban areas.'' the result was the development and successful pilot testing of an appropriate and cost-effective technology. from to , around improved latrines were produced. in addition, an awareness campaign was carried out on the use of the latrine, hygiene promotion, and capacity building. in , the program was extended to the rural areas. prior to , more than latrines were constructed and installed. in december , the program was formally transferred to the national directorate of water affairs. overall, it has been a long and steady scaling-up process over more than years that ended by ensuring a progressive withdrawal of the government from latrine production. the emphasis now is given to decentralization and privatization for the services, although the responsibility for the program remains with the government. from this experience, some lessons learned, are * although technology must be simple, it is important for massive use to ensure its local production and commercialization. there must be several types of sanitation facilities with different prices in order to commercialize. * a good network needs to be established between users (periurban communities, the government, nongovernmental organizations (ngos), small private companies, and donors) to ensure that the program progressively developed its own dynamism. * latrines need to be emptied and the service needs to be provided. investments and national and international loans are to be paid from taxes, the difference is only that payments differ in time and are used simply because it is very difficult to finance sanitation projects directly from users. as a result, people who pay for the services are not always the same who will be using them. private aid is made available by private enterprises or ngos. private funding is used simply because developing countries have greater needs than economic resources. the participation of private enterprise cannot be taken for granted as there are several factors that actually inhibit their participation. these include low accessibility to loans from towns and municipalities, the need to organize projects that have payback periods of years, and the need to recover costs through water tariffs (lenghton et al., ) . private funding includes not only international or national firms, but also self-provision schemes provided by nonconventional private enterprise. these nonconventional private enterprises have been called by some 'informal' although for several developing countries, they have in many cases proven to be more formal, useful, and to provide more reliable services than formal ones. for example, in india, an ngo named sulabh has installed pour-flush toilets that are operated on a fee-paying basis and are maintained by attendants who live at the facilities. through providing good reliable service, sulabh's facilities have become a model for sustainable public sanitation services. this shows that there is growing knowledge and capacity provided by small and even family-run companies that are capable of producing significant and innovative improvements in access to sanitation. financing strategies are specific for each country and situation and depend on the political will, the compatibility with existing institutional arrangements, the degree of community involvement in decision making, the available economic and financial resources, and the prevailing social and cultural preferences, among other aspects. when either private or public funding is used, some key elements to make a good use of it according to lenghton et al. ( ) are • maximum scalability. the selected financing strategy needs to be one that can be scaled up quickly and in a straightforward manner to allow for rapid increases in the population served. • minimal transaction costs. • full financial accountability. • closed revenue cycle, that is, financially viable in the sense that all capital and operating costs are fully covered -either through user fees, government subsidies, or external finance. sanitation is of public interest (box ) and hence is a public process. in order to implement what needs to be provided is, for the governments, to identify the main requirements, the areas of responsibility, the risks associated, who is responsible for what, the different options to address needs, and the associated costs. once this is performed, it is required to review, set up or adapt the legal and institutional framework, and to educate all the persons involved (from society to politicians, experts, regulators, private companies and functionaries, besides children and women). sanitation management (basic sanitation facilities management, wastewater collection, treatment and reintegration, by-product management, and risk control) requires the coordination of different public institutions, society, academia, private enterprises, and in some cases, even different countries. therefore, the government is needed to set up the programs. today, around the world, it is still mostly government agencies that construct and operate wastewater collection and treatment systems. however, private companies are contracted to conduct operations in many places, and all countries have significant commercial enterprises built around collecting excreta and septage and managing wastewater sludge and biosolids, mostly in cities. theoretically, private companies, if well used by the government, could be useful to increase sanitation coverage if the level of society is raised and private companies are not used to increase the already-considerable differences existing between economic social classes. nevertheless, private participation is not increasing in sanitation. after steadily increasing at a global level between and , it began to decrease (lenghton et al., ) . there are many reasons for this, one of which is that it is not easy to build up successful schemes combining private and public interests. box how industrialized governments approached funding for sanitation (with information from lenghton l, wright a, and davis k (eds.) ( ) health, dignity and development: what will it take? millennium development goals. london: earthscan.) in general, in developed countries, public water infrastructure components have been highly subsidized by governments, reflecting an understanding that the public health benefits of sanitation generate substantial positive external gains that merit public investment. in britain, for example, urban authorities borrowed more than d . million for sewerage work during the period - . eventually, the public provision of sanitation became an uncontroversial and indeed, an expected part of life. similarly, for many municipalities in the united states, public financing of sanitation infrastructure was seen as the only option for ensuring investment adequate to protect public health. in the nineteenth century, boston, for example, had lower-than-expected connection rates among households to the city's new water and sewer network; this prompted the city to cover the cost of service pipes for all unconnected households. in , an influential state sanitary survey concluded that governments must accept responsibility for financing public sanitation infrastructure because, left to their own devices, a large proportion of massachusetts residents would be unable or unwilling to take on personal responsibility to conduct their lives in accord with recommended sanitary principles. until recently, grants of up to % or more were provided for innovative sanitation technologies in the united states. one aspect to keep in mind concerning public and private participation is that for the sanitation field, these funding options combine better with certain type of sanitation systems, characterized in terms of their size and used technology (table ) . low-income countries need to invest - % of their gdp to fulfill their mdgs (lenghton et al., ) . for some, these are figures difficult to reach even if the use of loans is considered. for them, external donors can play an important role. middleincome countries have fewer needs and more economical capacity to meet their mdgs. for some, it is estimated that they could use up to % of their gdp, and hence it is considered that no external finance is needed (lenghton et al., ) . moreover, this situation, from the point of view of some authors, offers to inform the private sector of great opportunities to conduct a business and, as a result, in several middle-income countries private funding is being promoted. one possible risk, which needs to be considered by local government and known by society in general, is that through private participation and international loans, technology and sanitation schemes from other countries are promoted, which do not always effectively solve local problems in the cheapest and most efficient way. another risk is the use of the money for additional purposes. to deal with this, it is important, on the one hand, for the government to be accountable and, on the other hand, for society to demand transparency. in any case, it is certain that developing countries need to be creative to raise funds for sanitation. one option is to raise them as part of other projects in which sanitation can be a component; these include those considering goals for food security, health, land remediation, environmental problems control, and adaptation to climate change, for which several donors may be available. as an example, carbon credits could be used to fund projects to manage sludge and fecal sludge. in developed countries, a complex and complete system of public agencies, private companies, equipment vendors, consultants, scientists, engineers, operators, and supporting professional and educational organizations makes sanitation possible. promoting this organizational and human capacity in developing countries is one of the challenges on the path to increasing adequate sanitation, wastewater reuse, and proper fecal sludge and wastewater sludge management. science and innovation are needed in developing countries to reduce their intense dependence on developed countries. unfortunately, in many situations, technology originating in high-income countries is still preferred and implemented. however, this may not match the actual needs or promote local the brazilian sanitation research programme (prosab) is a public program that has received financial support for different projects since . its goal is to develop and optimize existing technologies for water supply, wastewater treatment, and solid residues management. for that, its objectives are * to establish the state of the art of technology; * to adapt or develop technology to provide sanitation services in local and regional conditions, and to meet the different needs of all population sectors, preserving and restoring the environment; * to make technology and knowledge part of the public domain; and * to support participatory processes, creating cooperative research networks to discuss subjects. the total investment for the three phases listed is around us$ million distributed as shown in table , in which investments made for salaries and scholarships are not considered. both, research papers and technological innovation, were produced from this program. economic development. in some other cases, developing countries are even used as laboratory testing grounds for new magic solutions. in low-and middle-income countries, examples can be found where a significant part of the investment made for wastewater treatment plants is used to pay for the intellectual property rights of the processes, as happens with many other activities. in figure , it is shown that royalties received because of patents in developing countries are nonexistent or low while those for developed countries are high; sanitation could be in the future another source of this dependency and inequity. on the top of this, some of these processes do not solve actual problems and, as a result, around the world, several places can be found where new solutions for providing sanitation to poor people have been installed in series unsuccessfully. this situation has two negative effects: first, it discourages donors from making further investments and, second, it makes local people wary of possible solutions. the only way to prudently overcome this is to promote the development of technology by people immersed in local problems. for this purpose, investment in education and local research is important (box and table ). as presented here, the solution to sanitation problems can be combined with the solutions to other problems. the possibility therefore exists to develop new and individual technologies, to adapt the existing ones, and even to rediscover ancient local solutions. in parallel, the same can be done with policies to manage water. at an international level, there is current mobilization to support and improve sanitation conditions in developing countries. this mobilization is being expressed in terms of donors, private participation, and international aid agencies support. from this chapter, it is concluded that there are many reasons explaining why providing sanitation in developing countries is different to the solutions implemented in developed ones; therefore, care must be taken to not to use the aid to implement projects, which may prove not successful. for this reason, it is important to promote that each country defines first its needs and works defining programs. as the challenges to provide sanitation are many and very complex (policy definition, technologies to be used, education and awareness programs implementation, development of adequate institutional capacity, finding new financing options, etc.) it is important for developing countries to share among them their knowledge and experiences in the framework of the so-called south-south cooperation. sanitation is an important pillar to develop wealthy societies (in terms of health and economic capacity) and, for this reason, governments should promote investments in this field that are to be properly and responsible managed. the only way to assure this is to promote, allow, or to demand a participatory approach. finally, the water situation in developing countries has some bright sides. the first consists in the fact that the wide divisions observed in developed countries within the water sector (water supply and wastewater experts) does not exist or is not so pronounced. this allows easier understanding and promotes the integrated management of the problem. the second has to do with the high degree of solidarity existing among the population, which may play an important role in speeding up a sanitation program proven successful and contributing to raising the quality of life. global atlas of excreta, wastewater sludge, and biosolids management: moving forward the sustainable and welcome uses of a global resource: unhsp global atlas of excreta, wastewater sludge, and biosolids management: moving forward 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health urban wastewater as groundwater recharge evaluating and managing the risks and benefits water reuse: an international survey of current practice, issues, and needs water safety plans for grey water in tribal schools removal of -b-estradiol and -a-ethinyl estradiol in engineered treatment wetlands safe drinking water: lessons from recent outbreaks in affluent nations conventional small and decentralized wastewater systems in developing countries indirect water reuse for human consumption in germany -the case of berlin state of the art report health risk in aquifer recharge using reclaimed water irrigation in developing countries using wastewater water reuse new paradigm towards integrated water resources management unplanned reuse of wastewater for human consumption: the tula valley coming to terms with nature: water reuse new paradigm towards integrated water resources management encyclopedia of biological, physiological and health sciences wastewater risks in the urban water cycle water reclamation and reuse around the world. in: water reuse: an international survey of current practice using ecosan sludge for crop production navigating rough waters: ethical issues in the water industry. american water works association jiménez b and wang l ( ) sludge treatment and management semi-intensive treatment plants for wastewater reuse in irrigation extent and implications of agricultural reuse of untreated, partly treated and diluted wastewater in developing countries. cab reviews: perspectives in agriculture, veterinary science making urban excreta and wastewater management contribute to cities' economic development: a paradigm shift water policy global atlas of excreta, wastewater sludge, and biosolids management: moving forward the sustainable and welcome uses of a global resource un-habitat dignity and development: what will it take? millennium development goals global atlas of excreta, wastewater sludge, and biosolids management: moving forward the sustainable and welcome uses of a global resource: unhsp domestic wastewater treatment in developing countries comparison of techniques for the detection of helminth ova in drinking water and wastewater global atlas of excreta, wastewater sludge, and biosolids management: moving forward the sustainable and welcome uses of a global resource: unhsp sludge accumulation, characteristics, and pathogen inactivation in four primary waste stabilization ponds in central mexico global atlas of excreta, wastewater sludge, and biosolids management: moving forward the sustainable and welcome uses of a global resource: unhsp system consideration of eco-sanitation wastewater irrigation in developing countries: health effects and technical solutions morbidity and mortality due to ascariasis: reestimation and sensitivity analysis of global numbers at risk water -more nutrition per drop. towards sustainable food production and consumption patterns in a rapidly changing taking into account environmental water requirements in global-scale water resources assessments. comprehensive assessment of water management in agriculture cryptosporidiosis in developing countries south africa. faecal sludge management global atlas of excreta, wastewater sludge, and biosolids management: moving forward the sustainable and welcome uses of a global resource: unhsp water for people, water for life. the united nations world water development report united nations development programme human development report beyond scarcity: power, poverty and the global water crisis international source book on environmental sound technologies for wastewater and stormwater management the state of the world's cities report / ; the millennium development goals and urban sustainability: years of shaping the habitat agenda united nations/world water assessment programme guidelines for water reuse targeting sanitation water reuse in windhoek, namibia: years and still the only case of direct water reuse for human consumption guidelines of the safe use of wastewater and excreta in agriculture and aquaculture guidelines for drinking-water quality: recommendations guidelines for the safe use of wastewater global water supply and sanitation assessment report, joint monitoring programme for water supply and sanitation meeting the mdg drinking water and sanitation target: a mid-term assessment of progress meeting the mdg drinking water and sanitation target: the urban and rural challenge of the decade progress on drinking water and sanitation: special focus on sanitation. geneva: who and unicef earth trends: environmental information key: cord- - l gw sk authors: avgoustaki, dafni despoina; xydis, george title: how energy innovation in indoor vertical farming can improve food security, sustainability, and food safety? date: - - journal: nan doi: . /bs.af s. . . sha: doc_id: cord_uid: l gw sk food safety is an important scientific field, but at the same time a discussion topic of modern society that occupies more and more space of our every day time, dealing with the preparation of food, with its nutritious value, and various transportation and storage ways aiming at preventing food-related sickness. this work compares traditional farming with greenhouses and indoor vertical farming focusing on the challenges and the opportunities for each category. the scope of this work was to stress the role of indoor vertical farming towards this direction. indoor vertical farms can produce high quality and virus-free products that can be locally distributed, inside the urban environment that such investments take place, saving annually millions of tons co emissions. beyond that, in this work it was pointed out how energy plays a role in food safety in such systems. it was stressed that indoor vertical farms can act as a demand response aggregator. in large scale units it could play a role to adjust their production according to different electricity prices offered in different time zones throughout the day. this way, the owners under a multi-value business model will create the opportunity to the vertical farm owners not only to improve their production but at the same time absorb inexpensive electricity offered, by creating an additional profit mechanism (multiple revenue streams) under such an approach by entering into contracts with companies in a utility electric region. sustainability of resources and safety in the food production line is a major issue globally. by , it is expected that the global population will reach the . billion people, . billion people more that need to be fed (united nations, department of economic and social affairs, ) . today, agriculture occupies land equal to the size of south america in order to cover the demand of the global population. based on the assumption that the minimum daily demand of a single person is minimum kcal, if we maintain the same agricultural practices, we will need additional land equal advances in food security and sustainability # elsevier inc. issn - all rights reserved. https://doi.org/ . /bs.af s. . . to the size of brazil ( . billion acres) to cover the global food demand (despommier, ). on the other hand, according to lotze-campen et al. ( ) , the land used for agriculture is projected to be transformed for other purposes such as urbanization, energy production, or infrastructure growth. it is worth to mention, that another crucial challenge that will significantly affect agricultural production in the upcoming years is the rapid increase of the global temperature, as per each degree of temperature rise, % of existing agricultural land will be lost (despommier, ) . nowadays, climate change is a huge issue since it is expected that the upcoming years will outstandingly affect the agricultural process. the significant increase of the carbon dioxide emission levels from a global perspective-since it constitutes an important impact factor of agricultural productivity-can influence the global economy via the effects on the agriculture's total production rate. in specific, based on mulatu's et al. ( ) research conducted for ethiopia, indicates that the impact of co emissions will decrease . % to . % the real agricultural gdp since it will lead to lower the agricultural productivity and subsequently reduce the amount of traded and non-traded crops. such population increase certainly indicates a significant rise in the required food production, raising concerns on the deficiency, the quantity, and the quality of future food products. we should also take into account the fact that nowadays food travels daily thousands of miles from the production areas to the urban consumers, in order to meet the demand, releasing huge amounts of co . less developed countries such as ethiopia that were mentioned above, apart from global climate change will have to face and other enlarged problems concerning food safety. for example, human excrements that are used as fertilizers (estimation of % of the global farming) can cause diseases such as cholera, typhoid fever and numerous parasitic infections (despommier, ) . nowadays, even the more developed counties have to face food safety and security problems even if this kind of infectious diseases have been eliminated. it is worth to mention the pandemic of our age, covid- caused by virus sars-cov- that was initially reported in the province of hubei, wuhan in china. the disease is estimated to have originated from a seafood market in wuhan where wild animals were traded such as marmots, bats, snakes and birds (zhou et al., ) . the specific family of viruses, coronaviruses, are known to be transferred from animal to humans. according to zhou et al., , it is mentioned that % of the genetic makeup of covid- is matched with the coronavirus found in bats. the uncertainty that is caused globally via covid- has caused apart from multiple deaths and lockdowns to most of the european countries, will affect significantly the economy and will cost trillions of dollars in the global economy, during and beyond (unctad, ) . food safety is a major issue of our era, as there are multiple reports of cases worldwide over the last years that have caused food recalls due to bacterial infectious diseases leading to loss of billion dollars. why do we seem to have so many outbreaks concerning food production these days? only in the us, despite the attempts to provide a safe food supply, every year are recorded million foodborne illnesses, , hospitalizations and deaths (cdc, ) . in - , e.coli o : h outbreak in the us caused sudden eruption linked to consumption of leafy greens and the romaine lettuce. the pathogen was mainly reported in the regions yuma, az and salinas, california, where greenhouse installations that produce more than % of the leafy vegetables and greens in the united states are based. e.coli contamination in the production line almost all of the times originates from the irrigation water used in the fields. additionally, further risk in the contamination process from various bacteria and pathogens comes from the washing of field-grown products after they are harvested, while this step can spread contamination to the whole production. the most regular technique that outdoor farming applies after harvest is to dunk lettuce heads in water tanks from rainfall or irrigation, while most greenhouses apply triple washes with running water from the local network. vertical farms are a novel type of farming in a controlled-environment with a total replacement of solar radiation with artificial lighting that provides the necessary nanometers of the spectrum for the growth and development of plants. in vertical farms, plants grow in soilless cultivation systems such as hydroponic (roots are immersed in multiple substrates, i.e., perlite, rockwool enriched water with nutrient solution), aeroponic (soilless air/ mist solution) or even aquaponic (co-cultivation of fish and hydroponic plants) systems that allow stacking multiple layers or columns of plants horizontally or vertically. vertical farms are located in completely isolated spaces from outdoor environment with thermally insulated installations (especially when at the top floor of the building) and airtight structures that give the opportunity to the farmers to control the environment in terms of temperature, humidity and co (avgoustaki and xydis, ) . since vertical farms can theoretically be placed anywhere in the urban network, they allow local, nutritious and fresh consumption for consumers. in specific, a study conducted by jill ( ) , mentioned that food sourced from conventional farming uses to times more fuel compared to locally grown food and emits to times more co . meanwhile, vertical farms may be able to increase the productivity rate in highly urbanized areas that can lead to improvements in the food security of the community. the purpose of the following subchapter is to compare the different farming techniques of outdoor farming, greenhouses and vertical farms in between them in terms of input of resources, the final product in terms of safety and the shelve life of the products in terms of nutrient status and freshness. additionally, we will examine the above criteria for lettuce, which is one of the most important cultivated species in vertical farms and will give us access to multiple data. lettuce belongs to the basic daily diet products; its nature is fragile and can be easily contaminated and spread diseases among the population. in order to make more understandable the concept of resource use efficiency, in fig. the essential resources for growing plants under various farming types are shown. the most vital for plant growth is water, co , light, nutrients, electricity (for ventilation purposes) and heating. as shown in fig. , the definition of resource use efficiency (rue) is given by the ratio of the final plants production to the total input. in order to calculate the total input of a system, we have to summarize the input of resources, the environmental pollutants and the production system. in order to evaluate the sustainability and efficiency of a production system in the food industry, we have to assess three key directions of the system. • rue: the amount of necessary resources to produce. • the cost performance: the ratio of the sales amount to the production cost. • the vulnerability of the system, meaning the deviation of the yield production per year and the quality value per product unit. water is absolutely necessary for all food production such as vegetables, fruits, grains, meat etc. based on nederhoff and stanghellini ( ) , the water use for the global food production reaches at km and has a rapid increasing rate. the irrigation water-use efficiency can be researched under different scopes and multiple concepts such as storage, delivery distribution of the water to the farm or out of the farm. additional systems that can affect water use efficiency is the ratio of water that is delivered for irrigation and the water that supplies the system. there are various ways we can calculate water use efficiency as one of the major resource inputs in food production that can be accomplished with agronomic ways, engineering or even economic approaches. more analytically, irrigation efficiency estimates the ratio between the diverted water and the consumed by the cultivation, thus it provides water-use measurements that estimate the performance of the irrigation system. on the other hand, water use efficiency is considered an economic concept that in practice evaluates the farm, as it is calculated by the crop yield unit of water diverted (kg/m ). in terms of energy consumption, it is one of the reasons that causes greenhouse gas emissions (ghgs) contributing at the rising global warming. the main gases released by agricultural production are carbon dioxide (co ), methane (ch ) and nitrous oxide (n o). since the global policy makers, organizations, researchers, retailers and producers try to propose and implement novel techniques that identify and reduce ghgs, it is necessary that we will focus and refer to the status of emissions under each farming type and propose mitigation measures in the sector. in order to describe sustainability in agriculture, it is not enough to relate sustainability with the field only from the resources perspective. understanding and evaluating what constitutes a sustainable farming system, it is of vital importance, to furthermore understand the economic and social terms that influence the contemporary issues, values and perspectives of a unique system. economic efficiency reflects to the value that is relative to the cost. in order a resource to reflect an economic value, has to be rare and difficult to obtain, for the market prices to allocate the use of this resource for competitive purposes. for example, even if air and water are essential resources for life giving them high "intrinsic" value, nevertheless under most circumstances they have no economic value due to their sufficiency levels in the environment (ikert, ) . they only obtain an economic value in cases of scarcity due to, e.g., high levels of pollution or drought. even if is the most ancient way that people use land, over the last decades with the technological breakthroughs and the numerous innovations introduced, outdoor farming has changed. sensors, satellites and advanced machinery allow farmers to apply more targeted (and precision) agriculture to treat the fields individually according to the needs of the crop and the soil, by dividing it in smaller parts in order to take into consideration the variability level of each unit. to complete the whole picture of climate change issues, an additional evolution process that crucially reduce the growth rate of plants is soil degradation due to excessive floods and droughts. traditional food production systems offer food solutions for people from the beginning of human history. over time, additional innovative techniques were applied in traditional farming in order to rise the productivity rate and reduce the cost and the crops overall footprint. in terms of resources, conventional farming seems to have an increase demand for water use (table ) as traditional agriculture uses almost % of the available fresh water globally. furthermore, a very common problem in terms of sustainability in water use efficiency of conventional farming is the limited soil water-holding capacity that results from the limited mulching of the soil and the consistency in the same fertilizers/soil-preparation practices. scientific results (pimentel et al., ) have shown that this maintenance of these practices lead to low soil moisture status and low conservation levels of conventional farming systems. the most used approach for conventional farms is the irrigation efficiency and the water use efficiency. it is worth to mention that the more water applications are applied in a crop, the higher the water delivery losses are. in order to improve the water use efficiency, many farmers apply a combination of hydroponic systems with drip irrigation and smart scheduling of water distribution. hydroponics successfully address the challenge of soil drought and salinity that reduces both yield and crop quality. it should be noted that a decisive factor for the selection of hydroponic systems is the high irrigation water needs that renders the requirement for recirculating water. it becomes apparent that combination of water-saving technologies with limited-water application technologies (such as close-loop hydroponics, drip irrigation, mulching and smart scheduling of water supply) are the most effective solutions for optimizing water use efficiency. regarding land use, growing and producing food to respond to the expanding demand of the world has led agriculture production and food scarcity that can be difficulty bridged. today's farmlands, occupy almost % of the global habitable land (ourworldindata.org). we gathered the footprint of the various resources that meet the demand for lettuce production via traditional farming techniques. worth noticing that deforestation is a major problem, since forests are continuously sacrificed against farmland that leads to climate change acceleration and soil inability to maintain water at lower levels. depending on the cultivated variety, the techniques and the season, traditional farmed lettuce has a cultivation cycle between . and . months. therefore, farmers have the ability to grow multiple successive crops in the same field throughout a yearly cultivation period in order to increase their yield and income. additional techniques that open-field farmers follow in order to increase their yield and income per hectare (ha) of cultivated land is the density of planting, fertigation (combination of fertilization with irrigation) application and the use of healthy transplants grown in nurseries. assuming that romaine lettuce growing in the mediterranean is planted in distances of - cm between the rows and - cm between the plants, then the resulted yield reaches at , - , plants per ha (savvas et al., ) . by increasing the planting distance per row by cm, it can lead to a % reduction of the total production. harvest period vary depending on the type or the variety of the cultivated crop. for the romaine lettuce grown outdoors, the harvest period is between and days with a typical yield of - tons/ha. the energy use in outdoor farming is mainly linked to fossil fuels for operations such as soil plowing, sowing, fertilization, harvesting etc. additionally, further electricity is required for pumping (water irrigation), which in developed countries can reach up to % of the total fossil fuel usage (despommier, ) . conventional farming, unfortunately, is associated to higher emissions in comparison to other types of farming. the majority of the emissions is directly linked to the transportation of the products, also known as food miles. the amount of miles that is required in order for food to travel from the producer to the consumers could release between to kg of co emission depending on the location of the farm (gerecsey, ) . since farmlands are often located many kilometers away from the urban centers, where the majority of the end-user is located. food miles emissions represent on average % of the total emissions released throughout traditional farming. another important source of co emissions that is linked to traditional farming is the significant amounts of food waste. even if food waste is not only linked to traditional farming, maladministration and mismanagement on-farm losses, and non-marketable crops put traditional farming under the spotlight of high shares of carbon footprint. for the estimation and assessment of the economic efficiency of farming, significant role in the calculation, the resources that bear an "economic" value have played a role. in traditional farming, there is limited motivation to protect and evaluate the quality, use and water maintenance, air, solar radiation and in some cases even soil fertility and productivity. the costs of a farm can vary between two main categories: the variable costs (operational expenses-opex) and the fix costs (capital expenses-capex). in the category of variable costs, all the expenses that cover particular farming actions in a specific period of time such as seeds, fertilizers, chemicals, labor are included. on the other hand, in the fix cost category, all the expenses that will be incurred regardless the process and status of production, building expenses (rent, installations, land) and equipment (irrigation system, machinery) are included. thus, the economic efficiency consists from a combination of technical and other components. based on aurangzeb et al. ( ) and a research that conducted to compare the economic efficiency between traditional farming and mechanized farming systems, it is pointed out that the net income in mechanized farms is significantly higher due to the higher yields/ha than the one of traditional farms. this effect of traditional farms could be explained by the longer time periods in soil preparation, limited tillage practices as well as the high cost requirements of labor expenses (specifically in seasonal workers during harvesting and sowing) in comparison to the high technology and mechanization farming systems. last, another factor that highly affects the final quantity of production is biodiversity. for this reason, the selection and maintenance of mono-cropping techniques that provide a uniformity in the applied practices, can reduce the labor costs and make harvesting easier. however, by cultivating only onespecies crops in the entire field, it can highly influence the biodiversity and make crops more susceptible to pathogen infections. to avoid this effect, traditional farmers apply chemicals and genetically modified organisms to maintain a simple farming system. this practice, though, requires a lot of continuous input of resources and energy (cost). the innovative and high quality mechanization and technological innovation can lead to the increase of production and hence income. multiple practices become more and more vital in traditional farming, as they improve the efficiency of resources use in general and can overall enhance sustainability. concerning the water usage, there are several approaches that new farms bring along in the field and can optimize the existing severe water waste situation. common agronomic measures such as improved crop husbandry and changed crop mix driven by the crop selection, can have a huge impact in improvement of water usage. furthermore, there are various cultivation techniques such as modification of the irrigation infrastructure, which can also influence positively the water use efficiency. last, management actions such as optimal irrigation planning and frequent maintenance irrigation system scheduled maintenance can also influence positively the system's efficiency (wheeler et al., ) . due to the growing population, farming has shifted to technologies that enhance significant scale-up of the production via innovative technologies. greenhouses are types of installations, designed to protect and enlarge the cultivation season of various crops. plants growing under greenhouses can grow protected from severe weather conditions such as hail, snow, extreme low temperatures or excessive heat, while at the same time can allow cultivations of out-of-season species. greenhouses first introduced in the th century but only on the th century were commercially applied in the global market. according to their installed area, greenhouses can be presented with various coverage materials such as plastic, glass, polyethylene and rigid that protect crops from the variability of the outdoor conditions, diffuses solar radiation and traps moisture, which contributes to increased plant growth. the coverage system allows farmers to control the cultivation environment according to each crop preference, as they can apply different techniques that will maintain the heating and the cooling requirements to the desired levels. this way, inside the greenhouses, farmers can develop and maintain the desired microclimate and create a more predictable environment that enhances the final plant yield, achieving higher quality and reduced water consumption compared to open field crops. there are different greenhouse systems that are diversified according to the energy flow inside the greenhouse and the resources flow in the production line. in more details, open greenhouses refer to the structure of the irrigation system, meaning that they do not collect the drained water of the crops for reuse (usually have soil-based crops). these systems seem to have low level of water usage efficiency as they are affected by water losses due to soil depletion and constant water drainage, which drains the excess amount of water with fertilizers. this waste of resources cause significant problems to the environment. usually growers can control the amount of drain as part of the management strategy of resources they follow. the percentage of drain can number between % and % of the water supply, but can be improved by reusing this drain in the irrigation system. additionally, open greenhouses use window openings as the only mean of dehumidification and cooling technique. there are also the semi-closed systems of greenhouses that have a smaller cooling capacity and window openings, combined with mechanical ventilation air-cooling systems. the combination use of mechanical systems and window openings depending on the cooling demand. concerning the irrigation systems, semi-closed greenhouses reuse the drained nutrient solution by collecting it to a tank that is constantly topped-up with fresh water. in some cases is followed water disinfection in order the collected drain water to be purified for avoiding diseases spread in the crop. to avoid imbalances in the nutrient solution, farmers use various techniques such as bleeding or dumping. in specific, bleeding techniques remove constantly % of the drain water, while in the dumping technique the mixing tank gets completely emptied and refilled with fresh water enriched with nutrient solution. finally, closed-systems refer to absolute mechanical support of the cooling and dehumidification system by air treatment units. the air treatment unit consists of a heat exchanger that is connected to a ventilator. the purpose of the ventilator is to withdraw air from the interior of the greenhouse, cool it, dehumidify it, and then distribute it back into the greenhouse. furthermore, in closed-systems water usually follows a close loop that allows the collection, recycle and re-distribution of the irrigation water both for irrigation purposes but also for cooling and heating purposes from inside the distribution pipes between the plant lines (qian, ) . concerning the irrigation system in closed-systems of greenhouses, the water does not follow the procedures of bleeding or dumping that are followed in semi-closed systems. on the other hand, the water is constantly recirculated in the mixed tank as it is automatically topped up with the correct and precise amounts of fresh water and each nutrient element. the growers are aware of the status of each nutrient element and are able to adjust it precisely in order not to disrupt the nutrient balance. this process becomes possible because of the high evolvement of automations, sensoring and programming in close greenhouse systems and achieve a - % better water use compared with open greenhouse systems (nederhoff and stanghellini, ) . greenhouses have different techniques for irrigation and water collection and highly depend on if greenhouses use soil based techniques or soilless for crop production. another factor that highly influences the final water use and water use efficiency is the type of the system, meaning it is an open system, a semi-open system or a closed-system. however, as can be retrieved from tables and the big difference in water use efficiency can be explained primarily because of the higher production accomplished in greenhouses compared to traditional farming but also because of the lower transpiration in greenhouses. transpiration is the most important factor that influences the water uptake by %, thus the control and reduction of transpiration rate can have a huge impact on the water use. transpiration is highly affected by the status of humidity and the irradiation levels inside the greenhouse. the higher the humidity inside the greenhouse the lower the transpiration levels are. if growers manage to control these two factors in the optimal levels for each crop, then there is reduced transpiration level per m , which means lower water usage and therefore better water efficiency. the selection of the applied irrigation system, has also a significant influence. drip irrigation is one of the most popular irrigation techniques in greenhouses. water is located at the foot of each plant with the use of a pipe. drip irrigation has the advantage of saving large water amounts and also can control and maintain the humidity levels of the soil or the hydroponic substrate in constant levels. in that way, water stagnation and puddling of the ( ) food miles - km selected substrate mean can easily be avoided. finally, drip irrigation allows the targeted and limited fertilization being dissolved, in the watering system. other irrigation systems are the micro sprinklers that spray water in a range around two meters according to the pressure of the selected nozzle type. this system is mainly used in soil-based greenhouses with sandy soil texture. another very commonly used system is the irrigation with diffusers and is mainly used in narrower areas and the pressure of the diffuser depends on the nozzle that regulates the water supply and flow. finally, other irrigation systems applied in greenhouses are irrigation with hose and underground irrigation mainly found in soil-based greenhouses and present low level of water efficiency. most of the modern greenhouses apply hydroponic solutions that allow plant to grow without soil. in more detail, the word hydroponic comes from the greek words "Ύδωρ + Πονέω" translated as "water + cultivate," meaning that plants do not grow in soil but in mineral nutrient solutions in water solvent. various substrates in the market replace soil such as perlite, rockwool and zeolite. because of the nature of this technology, plants are permitted to dip directly in their roots into the nutrient-rich solution and subsequently plants can absorb faster the nutrients and in an easier way in comparison with soil-based crops. because of this process, plants grown in hydroponics form smaller root system and can divert more energy for growing their leaves and stems. additionally, smaller root allows more plants in the same area to be grown and harvest higher quantities in comparison to the outdoor farming. the above-described capacity of hydroponic systems, boosts the ability of growing food in limited areas as greenhouses can be. hydroponics consist of a total automated system that pumps water, and pipe-system can be completely auto-controlled. under various handlings and monitoring of every aspect that can be practiced in hydroponic systems, the growers can result into optimal food production results. more specifically, this process gives the opportunity to farmers to control the whole irrigation process of the crops according to the demand of each species and the seasonality. in addition, they can have access to data that can optimize the development rate and the resource footprint of the plants such as (a) the quantity of water that is distributed in each plant, and (b) the amount of nutrient solution that was given to the plants. hydroponics offer a big advantage as they are usually installed in close or semi-close loops that return the excessive water with the enriched nutrient solution back to a collective tank in order to re-distribute it back to the cultivation area. in contrast to the hydroponic solutions, traditional farming experiences huge amounts of resource and water waste as farmlands face the negative effects of soil degradation and the harmful effect of eutrophication (when nutrients from agricultural land create massive increase of phytoplankton populations leading to reduction of oxygen and nutrient reduction of from water and suffocation of multicellular water organisms). unfortunately, in traditional agriculture, excess supply of phosphates and nitrates in the soil can cause nutrient run/off and leaches. furthermore, the close or semi-closed loop of hydroponics categorizes them as more efficient in terms of sustainability process for water efficiency in comparison with traditional farming where most of the water is drained to lower levels of soil that plants cannot access. greenhouses consist of air-sealed cultivation rooms where are installed various automations and technologies that can control and provide the optimal environmental conditions for each crop. according to factors such as location, size of installation, height, outdoor climate conditions, greenhouses use different technologies that can properly adjust the indoor environment to the ideal air conditions. heating is one of the most important processes for space heating inside the growing room, when the outdoor conditions and too hostile for the plants' growth. for heating purposes, the technologies that are usually used vary according to the demand of each case. in general, heating systems use the interior hot air of the greenhouse to transfer heat through a heat exchanger to the stored water that is used as a thermal storage medium. a very common and cheap technique is using water heating systems that consist from plastic bags and ground tubes filled with water placed inside and between the rows of the plants. during daytime, this system absorbs and traps the solar irradiation and during nighttime, the stored heat is transferred in the interior of the greenhouse by releasing heat (sethi and sharma, ) . there are electric heaters operated via a thermostat or an automatic timer in order to rise the inside temperature to the desired levels. additional techniques used for heating are rock bed storages, movable insulation and ground air collectors. cooling is a technique of similar importance with heating as it enables to reduce the thermal energy inside a greenhouse and maintain the optimum temperature in each growing stage of the crop. various techniques are used around the world according to the specific climatic conditions, the size and the demand of each case. such techniques can be natural or forced ventilation, fogging and misting, roof cooling and fan-pad systems, as well as shading and reflection systems. the most successful systems are the composite systems since they are giving the opportunity for both heating during the winter period and cooling during the summer period. according to sethi and sharma ( ) , the most promising composite system is the earth-toair-heat exchanger system (eahes) that operates with the underground constant temperature of earth mass and utilize it to transfer or dissipate heat from or to the greenhouse. according to botanists plants are diversified to "long day" plants and "short day" plants based on the photoperiodism needs-meaning on how many hours of light they have to be exposed during the day in order to grow. artificial lighting is a technique that provides greenhouses supplementary lighting in case that the solar radiation does not completely meet the photosynthetic demand of each plant species for optimal growth and development. efficient and proper use of lights in horticulture and with additional boost of reflectors can provide apart from the optimal levels that are required for photosynthesis also can benefit the greenhouses with additional heating (fig. ). heat and energy loss is a common issue in greenhouse and artificial lighting. the latter can become an effective solution that mitigates these losses and add an additional value on the required lighting solutions. the most common types of lamps that are used in greenhouses are high pressure sodium lamps, lighting emitting diodes (led) lamps and ceramic metal halide lamps. energy use into a hydroponic production line is mainly meeting the demand of artificial lighting, heating and cooling loads as well as water pumps. the energy that meets the water pumping needs in a hydroponic system for lettuce is estimated by the average pumping time that is needed to irrigate the plants and the corresponding nominal power of the pump. based on the calculations of kublic et al. ( ) it was estimated that the average irrigation duration for lettuce is four and a half hours of total pumping daily. the energy related to the heating and the cooling loads in a lettuce production greenhouse is estimated by using the following equation the heat transfer coefficient depends on the coverage material of each greenhouse, while the efficiency of cooling and heating systems depends on the height of the greenhouse ceiling. the loss of heat depends on the external climatic conditions and it is a decisive factor of the air technique modification to be used. artificial lighting usage depends on the photoperiod necessary for each species and the active hours of sunlight that plants can absorb for photosynthesis purposes. the active time that lamps have to operate is highly relevant with the location of the greenhouse, meaning that greenhouse areas with limited solar irradiation hours (north part of europe, i.e., netherlands, denmark) have higher demand on artificial lighting in comparison with areas under sunshine (southern part of europe, i.e., spain, greece, italy). furthermore, the duration of the supplementary lighting depends on the nature of the cultivated plants in photoperiodism (if they belong to "long day" or "short day" plants as we mentioned before) . this characteristic can differentiate the need of the plants in total daily radiation and according to the outdoor sunlight, the extra hours that artificial lamps need to operate should be estimated. the ultimate purpose of artificial radiation is to provide to the crop the indispensable photosynthetic active radiation (par) in mol/m /day for optimal yield production. in order to calculate the energy of a mole of photons that reach the canopy the following equation is used: the result value of the above calculation of the energy demand of artificial lighting in the greenhouse is in [kj/kg/year]. food production and consumption is constantly rising, having a significant environmental impact making the implementation of more sustainable practices in food production necessary. in order consumers to satisfy their demand for off-season vegetables and fruits, the necessity of heated greenhouses for production is continually increasing. as it is mentioned in the traditional farming section, food transportation causes huge amounts of ghg emissions. however, this number is lower in comparison to the ghg emissions corresponding to heating hydroponic greenhouses in cold climate areas (ntinas et al., ) that try to meet high yields in order to meet customers demand. when heating of greenhouses is achieved with the use of natural gas, the consumed energy can reach the . mj with . kg of co for the production of kg of tomatoes. since the majority of greenhouses use fossil fuels to meet their heating demand such as natural gas, diesel, fossil fuel and liquid petroleum gas, it is of vital importance to strongly limit the greenhouses heat losses, upgrade the heating systems and to shift in utilization of renewable energy sources . heat losses can be minimized with the use of double glazing coverage material or with the use of multiple screens. the upper goal of these measures is to increase the environmental sustainability of greenhouse production lines. as it has already been mentioned, greenhouses combine different energy technologies, automations and digitalization for plants' monitoring, controlling and harvesting. greenhouses is a type of farming that can provide the option to connect with renewable energy resources in order to increase the sustainability of such systems and the energy efficiency of the various treatments that are necessary for mass food production (manos and xydis, ) . different types of renewable energy sources such as solar, wind, geothermal, hydroelectric, biofuels, biomass etc., are found all over the world bringing the possibility to greenhouse plants to produce yields under a more sustainable, economical and cost-efficient way (xydis, a) . energy policy strategies in a national and a global level, have as a high priority the support of electricity generation and heating from renewable energy and biofuels (xydis, b) . over the last decades significant improvements in a big variety of significant renewable energy systems, which are ground source-based, solar-based energy systems and wind-based energy systems have been made (koroneos et al., (koroneos et al., , . these can be for example electricity-driven heat pumps instead of traditional combustion-based heating systems consumes - % less energy in comparison to a conventional fuel heater (avgoustaki and xydis, ) . another advantage that heat pumps present . - . times higher energy efficiency compared to fossil fuel heaters as also % - % reduced co emissions in the cultivation area in comparison with the conventional. there are also examples of greenhouses that use several solar systems that store energy or other photovoltaic systems (pv) that undertake the conversion of solar energy to electricity that meets the heating and cooling needs of greenhouses. based on research conducted by ntinas et al. ( ) , greenhouses that utilize renewable biofuel (wood pellet) present - times lower global warming potential in comparison with a greenhouse that use fossil fuels for heating purposes ( . - . kg of co per kg of harvested tomatoes), even when the required energy is the same for both cases. greenhouses in the netherlands use complex technology for production of various cultivars that gather multiple operation during the production such are nurseries, growing bedding plants and transplants. these systems are highly automated and occupy land approximately ha or more (kozai et al., ) . even if these machineries occupy a lot of potential cultivated space, they reduce the labor cost and therefore the production cost. without the use of highly automated technology, the average work force required in greenhouses for cultivating purposes is estimated at approximately workers per a m production area. according to penissi et al. ( ) greenhouses produce g of fresh weight of romaine lettuce per m daily while traditional farming produce g of fresh weight of romaine lettuce per m per day. as it can be retrieved from table , the required land use for obtaining kg of fresh romaine lettuce daily is m presenting almost % of decreased land usage in comparison to traditional farming. in greenhouses there are different variables that based on their priority can offer different benefits to the farmers. these could be the location of the greenhouse, the product type, the access to capital, the required work force and other requirements. high significance in the cost efficiency is also the upfront cost and the ongoing growing cost of the greenhouse that can also lead to higher cost depreciation and development rates of the production unit. based on a comparative study conducted by , a greenhouse farm consisting of a semi-closed system of m of growing space in denmark, the opex and capex related with the farm were analyzed. their results showed that by assuming that the wholesale price of greenhouse produced greeneries reached at . €/kg, the annual yield production of harvested products reaches at , kg/year. it is also presented that the capital expenses for the installation of the greenhouses was calculated at , € including the hydroponic system and grow unit racks, natural gas, heating and ventilation system, light connection (for supplementary radiation), and electricity distribution. additionally, for the operational expenses the total amount of expenses rises to an annual cost of , €, including the leasing costs, the electricity demand costs (lighting, ventilation), the natural gas heating cost, the water demand, the labor requirements, the packaging expenses and finally the use of organic material (seeds and nutrients). different greenhouse scenarios were presented and a cash flow analysis in a -year projection, indicated that the cumulative gross profit increased in parallel with the increasing wholesale price of greeneries. more specifically, the payback period was calculated much longer than the operational period of the years resulting in negative prices of the net present value (npv), unless the wholesale price of greens increases to . €/kg or more. indoor vertical farming is an innovative type of closed plant production system that provides the opportunity of a controlled-environment agriculture, which can be controlled according to the crop regardless of the weather conditions. indoor vertical farms use artificial lighting as radiation source in order to cover the demand of plants for growth and development via photosynthesis. vertical farms are based in soilless cultivation techniques such as hydroponics, aeroponics or aquaponics. in addition to the hydroponic systems that recirculate the nutrient solution and benefit greenhouse cultivations, vertical farms use systems that condense and collect the water that is transpired by plants at the cooling panel of the air conditioners and continuously recycle and reuse it for irrigation. some principles concerning the structure elements permeate closedsystems of vertical farms. more specifically, vertical farms are thermally well-insulated and nearly airtight structures that are covered with opaque walls. this characteristic makes the farms capable to totally protect the inside crops from the outdoor climatic conditions and make them able to maintain the indoor conditions to the desired levels without having thermal losses. another characteristic that differentiate vertical farms from greenhouses is the multiple layers of stacked plants in the vertical racks or horizontal columns. this way, the construction provides maximization on the possible yield per unit of land in comparison to both greenhouses and outdoor farming. more specifically, vertical farms, according to the size on the installation, have a multilayer system mostly between and rows or columns with approximately cm of distance between the layers (can slightly vary according to the selected cultivated crop). inside vertical farms airconditioners or heat pumps, which principally are used to reduce the heat generated from the lamps and provide cooling and dehumidification for the crop are installed. furthermore, air-conditioners help to eliminate the water vapor that plants transpire in the cultivation area. fans are installed in order to circulate the air in the culture room; at first to achieve a constant and stable spatial air distribution and secondly to improve the photosynthesis and transpiration status of the plants. key factor in the optimal operation of vertical farms is the co delivery units that stabilize the co levels in the cultivation area at around ppm during photoperiod (when lamps are on) in order to increase the level that plants photosynthesize. an important characteristic of vertical farms is the nutrient solution unit that distributes the nutrients to the crops, the electrical conductivity control unit (ec) and the ph controller that monitors the level of the nutrient solution. last, it is very important to analyze the radiation systems inside vertical farms as part of the total structure essentials. as mentioned above, vertical farms are equipped with artificial lighting due to absolute lack of solar radiation. lighting is a key factor in plants development and depending on the selected lighting solution, plants can present differentiations in morphology, flowering and biomass production. light is electromagnetic energy that includes visible as also invisible wavelengths. sunlight is a free resource input that provides plants the whole spectrum of several wavelengths, % of it is within the range of - nm (kozai et al., ) . however, according to a number of researchers over the last decades (hogewoning et al., ; kim et al., ; lin et al., ; liu et al., ) , it is reported that the most important wavelengths for photosynthesis, morphology of plants and flowering are the wavelengths in the visible ( - nm) and the infrared ( - nm) spectrum. lighting emitting diodes (leds) offer advantages in comparison with other types of lamps such as fluorescent, incandescent, high-pressure sodium or high-intensity discharge (hid) lamps. these advantages are the robustness, they produce, a stable output that is immediately activated after the electric current flow, have long life (approximately , h), the opportunity of controlling the light output etc. for this reason, vertical farms focus on applying lighting recipes that combine different nanometers and can promote plants' growth. apart from the spectrum selection of the lamps crucial factors for plants are the dimensions of light, meaning the intensity of light during photoperiod and the duration that lights operate. what has literally been neglected is the potential of indoor vertical farms to act as a demand response provider (aggregators). it may sound weird, but indoor vertical farms could under a multi-value business models create the opportunity to the vertical farm owners to focus on their crop production and at the same time absorb inexpensive electricity offered. usually plants require some hours daylight and fewer darkness. it has been proven that by selecting the hours throughout the day that are not expensive to give the required light, and "give darkness" when electricity price is expensive, has not a significant impact on plants' growth and development. under a mass deployment scenario of such units in major urban environments (xydis, ) , the owners and operators of the indoor vertical farms could create an additional profit under such an approach by entering into contracts with companies in a utility electric region. the opportunity to earn (or at least save) significant amounts will or course be related to the size of the indoor farms and create multiple revenue streams. indoor vertical farms have thermally insulated walls and high level of airtightness that allows a better cooling by air-conditioners during the time that lights operate. this process is functioning even during cold winter nights, as the interior temperature can be increased due to the operating lamps that constantly generate heat in the cultivation rooms. the ultimate goal of air-conditioners is to maintain the indoor temperature at the desired levels. however, during the cooling process, a lot of the water portion is lost due to evaporation of plants or evapotranspiration. indoor vertical farms have heat pumps with cooling panels, which can condense and collect this water, recycle it and via the close irrigation loop, reuse it for watering the plants. according to kozai et al. ( ) , only a small part of the irrigated mass water is getting lost to the outside because of the high level of airtightness inside the vertical farm. it is also pointed out in this research, that the airtightness level of vertical farms should not exceed the . h À . this is suggested because this level of airtightness helps to reduce the co losses to the outside environment and at the same time to maintain the sanitize level inside the farm by preventing pathogens, bacteria, dust or insects to enter the area of cultivation. greenhouses compared to indoor vertical farms, do not provide the opportunity of collection, reuse and recycle of the water masses that evapotranspired from plants, because the majority of the water is lost via the ventilation process to the outside area and furthermore most of the water vapor of greenhouses is mainly condensed at the inner walls, making impossible its collection process. another remarkable point that influences the resulted transpiration in indoor vertical farms is the operation of the artificial lighting. more specifically, when lamps do not function, the relative humidity of the room can reach up to % (little transpiration in the culture room), and cause physiological and morphological disorders to the plants. in order to solve this issue, farmers operate the lamps in rotation after dividing them in groups (two or three) and each group operates for - h per day. with this action, a constant heat generation during the day from the lamps that aligns with the -h function of the heat pumps that dehumidificate and cool the air in the culture room can be achieved. in order to calculate the water use efficiency in indoor vertical farms the following equation is used: where -wc is the water mass (or weight) that is collected in the cooling panel of the air conditioners for recycling purposes (kg*m À *h À ), -wp is the alteration in the water mass that is detained by plants and hydroponic substrates (kg*m À *h À ) and -ws is the irrigated (or supplied) water mass to the indoor vertical farm. in general, co use efficiency in indoor vertical farms is around . - . (when the level of airtightness is between . and . h À ) and the concertation is around ppm-unlike greenhouses which achieve approximately a . cue with closed ventilation system and airtightness level of . h À and co concentration level at ppm (yoshinaga et al., ) . based on these data we can estimate that the cue of indoor vertical farms is . / . ¼ . times higher compared to the greenhouses that do not operate the ventilators and provide co enrichment in the culture room. this phenomenon can be explained because of the amount of co that is released to the outside area from the culture room and keeps increasing with the level of airtightness but also with the difference between the co levels inside and outside. the fact that the co concentration for enrichment in an indoor vertical farm is usually around - ppm in comparison to the greenhouses that have around - ppm can be explained based on that. in order to calculate the cue the following equation is used: where -cp is the net photosynthetic rate (μmol m- h À ), -cs is the enrichment rate of co (μmol m À h À ) and -cr is the rate of respiration of the workers (if there are) in the culture room (μmol m À h À ) the light energy of the lamps that is send in the canopy aims to provide the necessary energy that plants need to grow and photosynthesize. however, the salable part of plants can only fix maximum - % of the electrical energy as chemical energy. the remaining - % of the electrical energy that is not absorbed by plants is converted to heat energy into the culture room and the remaining is removed by air-conditioners to the outside area (avgoustaki, ). the above-described effect can also explain the negligible heating costs in well thermally insulated indoor vertical farms even in the winter cold nights. nevertheless, indoor vertical farms are based in automations and precision agriculture and all the input resources are measured and validated in order to provide the optimal results in the cultivated crop. for this reason, all farms focus on measurements and optimization of the light energy use efficiency both of the lamps and the plant community. what is important for these measurements in the definition and estimation of the par, which in other words, is the wavelengths of light that are in the visible spectrum of the - nm and are the ones that drive photosynthesis. par is not a measurement of light; rather it defines the type of light that is necessary for plants to photosynthesize. apart from the type of light, farmers need to know and further metrics of light such as the amount and the spectral quality of par. in order to estimate the light energy use efficiency of lamps (lue l ) we use the following equation: where f is the convention factor from dry mass to chemical energy that is fixed in dry mass (around mj kg À ) -d is the increase rate of dry mass of the whole unit of plants or only the salable part of plants in the indoor vertical farm (kg m À h À ) and -par l is the photosynthetic active radiation emitted by the lamps (mj m À h À ) respectively, in order to estimate the light energy use efficiency of the plant community (lue p ) is provided by the following equation: where: -par p is the photosynthetic active radiation that is received at the surface area of the cultivation. based on the calculations and experiments conducted by yokoi et al., , it is shown that indoor vertical farms have . to . times higher lue p in comparison to the greenhouses. only % of the light energy is actually converted into salable portion of plants. nevertheless, there are different techniques which can be applied and can improve the conversion factor to % or a little higher. a simple technique that can be followed is the application of interplant lighting, upward lighting, and use of reflectors (fig. ) . traditional lighting that is located only on top of the crop can cause undesirable shading in dense crops by uneven light distribution and lead to senescence of the leaves that are in lower levels. on the contrary, the application of interplant lighting can provide access of light also in the lower levels of the plants, improve the distribution of light and therefore improve the photosynthetic rate of the crop. according to dueck et al. ( ) , the photosynthetic rate of leaves in low levels is usually negative or nearly zero, but the application of interplant light can increase it in positive values. welldesigned reflectors can significantly enhance the lue l as they can reduce the vertical distance between the canopy and the lamps and increase the distance between the plants or the density, since plants constantly grow. same positive results by interplant lighting have been reported also in greenhouse canopies. the most suitable lamp selection for interplant lighting technique is leds as they have small volume and they perform lower surface temperatures in comparison to fluorescent and other types of light sources. leds have been proven beneficial for reducing the eue l also due to the higher conversion coefficient from electrical energy ( . ) compared to the fluorescent lamps ( . ). although the capital cost of leds is generally higher than the cost of fluorescent lamps, leds have longer operational life and the prices have considerably decreased over the last couple of decades and is expected to continue decreasing. apart from the lighting adjustments, other modifications can improve the lue l such as the control of the environmental conditions. the environment of plants and the ecophysiological status of plants can be enhanced by the optimal selection of air temperature, co concentration, water vapor pressure deficit (vpd), air current speed as well as the combination of ph, electric conductivity (ec) of nutrient solution. these parameters have to be set according to the selected cultivated species. another way to improve the lue l as well as the eue l of the salable part of plants, is to reduce the dry mass of the nonsalable parts of the plants. in indoor vertical farms, the most frequently selected crops for cultivation are leafy vegetables such as lettuce, small fruits and herbs and it is important to limit the percentage of the root mass into less than % of the total mass of the plant (kozai et al., ) . due to of the cultivation technologies used in indoor vertical farms this is an achievable measure only by minimizing the water stress of plants by controlling the water vapor pressure deficit of the room. if the selected crop is root species, then we can significantly increase the salable portion by harvesting earlier than usual in order to have an edible aerial part. finally, other factors that can also help in increasing the relative annual production capacity (per unit land area) of indoor vertical farms are: • limitation of the culture period between transplanting and harvesting by optimal monitoring and controlling of the environmental conditions • increase of the ratio of cultivation area under each farming type (field, tier, floor, culture bed) • increase of the salable part of plants as also the percentage of salable plants. according to kozai et al. ( ) , it is stated that by applying the abovedescribed techniques, the relative production capacity per land area unit in an indoor vertical farm of layers can rise up to - times higher compared to outdoor farming, considering that indoor vertical farms already produce - times more yield than traditional farming (table ). in practice, those techniques could double the efficiency of the whole system. indoor vertical farms use culture beds that are isolated from soil usage and the nutrient solution that enriches the irrigation water is distributed through pumping to the plants. because of the high-automated process of irrigation, the nutrient solution is drained from the culture beds that plants are growing and it follows a close loop by returning to the central nutrient solution tank for recycle and reuse. in order this process to be achieved, nutrient solution is rarely removed to the outside area. this process usually takes place once or twice per year when the level of certain ions such as na + and cl-are not well absorbed by plants and the percentage in the culture beds exceeds the normal levels, requiring discharge. in order this measure to be implemented, the supply of fertilization closes for some days and plants already planted can absorb the nutrient elements existing in the culture beds (kozai et al., ) . on the contrary, the fertilizer use efficiency of greenhouses and of fields in traditional farming is relatively low and occasionally can cause on the soil, surface salt accumulation. in order to calculate the fertilizer use efficiency (fue) the following equation is followed: where: -i u is the absorption rate of plants of ion element i that are in the organic fertilizer and -i s is the supply rate of ion element i into the indoor vertical farm. it is worth to be mentioned that the ion element includes the basic elements of fertilization solutions such as nitrogen (no À and no + ), phosphorus (po À ) and potassium (k + ). artificial lighting apart from a key element in the growth of plants indoor, it does increase the energy consumption of vertical farms. shamshiri et al. ( ) , noted that three major operational expenses in a vertical farm are the electricity cost with - % of the total cost, the operational costs (opex) with % of the total cost and the capital expenditures (capex) with - % of the total cost. indeed, energy consumption is a significant cost of indoor vertical farms and can be used as an measure for their sustainability levels. many research groups and institutes focus on developing innovative technologies and optimizing the lighting recipes in order to reduce the energy footprint of vertical farms and create a more sustainable and cost efficient type of farming. even if the demand for purchased energy is much higher in indoor vertical farms than in greenhouses, the energy efficiency of the former is significantly higher (graamans et al., ) . indoor vertical farms, since are in absolute controlled systems face high efficiency when operating with renewable energy . there are multiple examples of vertical farms that are operating under smart grid systems that generate energy for the demands of the farm via wind turbines or solar panels or even geothermal energy. additional roles in the vertical farm systems towards increasing their efficiency have the connectivity with resourceful batteries that provide the opportunity for smart use of cheap stored electricity from the hours that the electricity prices are lower. an approach gaining constantly more and more attention also under the dynamic pricing concept, where also accurate forecasting plays a crucial role (karabiber and xydis, ) . in order to calculate the energy use efficiency for the lamps (eue l ) is followed the below equation where: h is the conversion coefficient of electrical energy to energy of photosynthetic active radiation that is emitted by lamps. for the latest technology of leds this number reaches the . - . (kozai et al., ) . apart from the energy that is consumed in order to meet the lighting demands, the energy demand of the heat pumps for the cooling (or heating) processes in the indoor vertical farms should be added to the equation. this type of efficiency is often referred in literature as coefficient of performance of heat pumps for cooling purposes. the coefficient of performance of the heat pumps, in a specific room, increases when the outside temperature decreases. the electrical energy use efficiency for cooling by heat pumps (eue c ) is calculated by the following type: where: -h is the heat energy that the heat pumps remove from the cultivation area (mj*m À *h À ) and -a is the consumption of electrical energy by the heat pumps (air conditioners) (mj*m À *h À ). it is worth to mention that the total energy consumption of indoor vertical farms is defined by the sum of the energy consumption of the lamps, the heat pumps/air-conditioners and the electricity demand of other equipment used for the optimal function of the farms such as nutrient solution pumps and air circulation fans. if we focus only in the electricity cost demand of indoor vertical farms, lighting accounts to approx. % of the annual electricity energy use (assuming fluorescent lamps of w), while the electricity cost demand for air conditioning is around % and % the electricity demand of the auxiliary electrical equipment (kozai et al., ) . table presents the estimated representative values of resource use efficiencies in an indoor vertical farm that use artificial lighting. it could be concluded (from table ) in comparison to table , that the relative production capacity per land area unit in an indoor vertical farm of layers is to times higher compared to traditional farming and to times higher compared to greenhouse production. indoor vertical farming is a type of farming which by definition is developed to provide enough production in order to meet the local demand in urban areas with continuous increased demand for fresh and nutritious fruits, vegetables and herbs. in general, the most frequently cultivated species are plants that have higher profitability and have a relatively high price. a significant factor on crop selection is the crop to have a short production cycle in order to reduce the required electricity costs for lighting, heating and cooling of the crop and therefore can be harvested as early as possible. additionally, growers prefer plants that have high harvested yield, meaning a high portion of the crop that can be harvested and sold. for example in crops like lettuce and herbs, growers can harvest and sell the whole unit of the plant, while in tomatoes or peppers they can sell only the harvested fruit but at the same time. therefore the electricity used for the rest of the plant, could be considered as a product waste. another key issue in crop selection is the height of the plants, meaning that it is way more preferable the crop to have a compact status in order to be able to reduce the growing distance between multiple plants and grow more at the same available area. plants are also selected according to the perishability level that they present after harvesting and reaching the market. since indoor vertical farms are mainly located in urban or suburban areas, their goal is to produce crops that can increase their self-life (even of perishable crops), by shortening the harvesting and delivery time to the market. another parameter taken into account when selecting crops is the situation in the local market. if, for instance, tomatoes are missing for some reason from the market, then depending on the price they can get, they could be preferred against of another fruit or herb that is in abundance and its price cannot climb up. finally, most suitable crops are those that have year-round productivity in order to be affordable for the farmers to have a year-round market demand that can be profitable despite the continuous operational expenses. the constant production in a yearly basis of the same crop selection, allows also maintenance of the same, specific engineering settings of the crop, avoiding the modifications in the automations' selection that could cause abnormalities from a horticultural perspective. due to the concept of indoor vertical farming and the technology used in the cultivation areas, growing in an urban environment do not advantage the crops due to possible shading of the building, non-fertile soils or dormant soils. this fact can also be considered as one of the major drawbacks as the land price in urban areas is relatively high. concerning this approach, indoor vertical farms are often installed in large warehouses, industrial factories or even abandoned buildings, where the prices are low. according to kozai et al. ( ) , it is stated that indoor vertical farms can produce the same yield of lettuce heads and other leafy greens in only % of the land required by traditional farming and % compared to a greenhouse construction. based on tables - , it can be retrieved that the land use efficiency of indoor vertical farms ( . m ) required for obtaining kg of fresh romaine lettuce per day is almost % reduced compared to greenhouses and % compared to traditional farming. an indoor vertical farm of layers can produce g of fresh weight of romaine lettuce per m per day ( g fw/m /d for greenhouses and g fw/m /day for traditional farming). adenaeuer ( ) mentions that the increase in yield between indoor vertical farms and traditional farming can be increased by . due to the technology and by due to the technology combined with the stacking ability of the plants. depending on the stacking area and the volume of harvest, cultivation care and crop preparation techniques, the work force can highly vary. , propose that . workers are necessary per , kg of yield, resulting in % of the annual operational expenses of the farm (depending on the labor cost in each country). the same work force is required for a greenhouse production and approximately half of it for an open field farm. more analytically, according to savvas et al. ( ) , in soil-based crops the labor numbers , €/ha while a hydroponic greenhouse or indoor vertical farm requires around , €/ha as production cost. this demand is met by both permanent and by seasonal workers that will be hired for specific labor-intensive operations of the farm (like pruning and harvesting) throughout the year. indoor vertical farms have the advantage that allows them to generate bio-waste as bio-product during the process of edible biomass production. according to the cultivation system that plants grow in (hydroponic, aeroponic or aquaponic), the opportunity to farmers to collect easily all the by-products after the harvest period such as leaves, roots with fibers, stems, or even damaged vegetable and fruits and use it as well waste is offered. based on adenaeuer ( ) , the bio-waste that is collected and used in indoor vertical farms can be metric tons per year and with daily plant wastes that are collected for the indoor farms of roughly . tons. since indoor vertical farms use advanced close loop systems, present also the possibility to convert the daily amounts of biowaste and after careful processing to useful resources material for the crop as liquid fertilizer or biofuel (nikas et al., ) . the are several cases of installation of indoor vertical farms that have designed specific lines of biowaste management in their production line that only serve this specific purpose. it should be stressed that indoor vertical farms have the option to implement high tech equipment for conversion of food waste into energy production via anaerobic digestion. more specifically, this technology is a biogas recovery system that captures methane from food waste and convert it to heat, steam and electricity to meet the energy demands of the farm. this process requires a close-loop system, which creates biogas from organic material by piping it into the turbine generator. the electricity that is finally produced meets the high-energy demand of indoor vertical farms such as the operation of the lamps. anaerobic digestions is also compatible with aquaponic systems by receiving the organic waste of both fish and plants to produce electricity (agstar, ; united states environmental protection agency, ). one of the key factors that influences the selection of the farm system is the selling price of the products. according to tasgal ( ) , traditional farming products are to times cheaper in comparison to greenhouse an indoor vertical farming products. more specifically, traditional farming lettuce price usually costs less than €/head, while greenhouses lettuce and indoor vertical farm lettuce cost - €/head. additionally, based on the same study, the significant upfront capital requirements of indoor vertical farms can highly limit the pool of market participants. this happens because both the land prices, rents and acquisition of high-technology equipment are significantly higher in comparison with the leasing cost of farmland. on the other hand, , by conducting a comparative analysis between indoor urban farms and greenhouses presented slightly different results. in more detail, they assumed an indoor vertical farm with the same growing space and wholesale price of the greeneries as in the greenhouse facility, of m and . €/kg respectively. an interesting point is the massively increased production yield that can be achieved in an indoor vertical farm compared to greenhouses, reaching at , kg of fresh greeneries being annually harvested. the operational expenses of indoor vertical farms according to the examined case reached at , €/year resulting in almost similar numbers with the greenhouse facility. however, the biggest cost of indoor vertical farms noticed were the capital expenditures reaching at , € per grow unit, with the most costly equipment the lamps and integral connection of lamps, installation of growing unit racks and the electric distribution of electricity. subsequently, based on their model and the different cash flow analysis, indoor vertical farms present profitable investment opportunities with a high internal rate of return (irr) and a payback period between and years with a wholesale price equal or more than . €/kg. another research conducted by liaros et al. ( ) , a case scenario of a small iuvf of m growing area inside an apartment was presented, showing profitability to smallholders under various scenarios. worth to mention at this point, micro indoor farming in small growing spaces such as containers, garages or even simple rooms can be profitable depending on the demand and the flexibility to rearrange different cultivation parameters aiming for the optimum result. similar findings were also supported by ucal and xydis ( ) . on the other hand, based on a report conducted by agrilyst ( ) indoor micro-farms can be very costly, nevertheless, there are multiple marketing strategies for optimizing the results. according to the united nations (un) projections, the global population will exceed . billion until , all requiring to meet their food demand. additionally, un estimate that % of the global population will be located in urban areas by that time. in order all this increased food demand to be met, it is necessary to produce % more nutritious and fresh food. however, at the same time, land experts such as agronomists and ecologists, already warn of the growing shortages in agricultural land, necessary for sufficient food production (al-kodmary, ). when it comes to high quality food, the fact that already food prices are climbing high also due to limited agricultural resource inputs such as water and energy is a matter of great concern. over the last decade, the increase demand for more farmland in order to meet global food demand it becomes more and more obvious. as an immediate effect a lot forest areas are substituted by new farmlands in order to supply this demand. at the same time, since cities constantly grow in terms of area they occupy, a lot of farmland is lost due to this expansive urban development. it is important to convert the global production line to a greener form for both human beings but also for the planet. this implies that food production will not sacrifice the attention for the human health against the commercial profit. according to world health organization, more than half of the farms globally, still use for fertilization purposes of their crops raw animal waste that can attract insect as flies or contain weed seeds or even diseases which can contaminate the cultivated crops. subsequently, these techniques can highly affect people's health and can cause diseases. nowadays, the majority of the food is produced in large, industrialized farms and is transported, distributed and sold in supermarkets, grocery stores or multinational food outlets. agronomists, engineers and farmers in order to reduce the production cost and resource footprint of food production and at the same time increase the variety of the available food species for the consumers have developed various techniques. the high centralization level of food supply can allow the possibility of infection from foodborne pathogens and toxins that can poison large numbers of consumers. food usually travels thousands of miles every day leaving huge possibilities for contamination threats as it can be infected in one country and develop pollutant populations in another. because of the high logistic complexity of food supply, it is worth to mention the advantages and drawbacks of each farming type during the whole supply chain. what follows is an exploration of the three subjected farming types, including outdoor farming, greenhouses and indoor vertical farming. we will compare and evaluate products and growing process under the scope of food safety practices. outdoor farming is applied for thousands of years, allowing an unprecedented human development. however, over the past years the continuously increasing demand of the population has led farmers to apply chemical inputs for nourishing of plants, fighting pests, insects and improving soil quality. however, because of its nature, crops growing in the open field are facing all the difficulties from severe weather conditions and the danger of infection from various insects and pathogens. traditional farming is a type of agriculture that allows to multiple plant pathogens, bacteria and insect pests to affect crops, causing scalable losses in global crop production. after heavily tilled farming applications, severe irrigations and monocropped selections, soil has been seriously affected causing depletion of its nutrients, highly requiring additional nutrient solutions that can improve its fertile condition, making it appropriate for cultivation. once crops are harvested, a big after-harvest process and logistic supply has to be followed in order food to be transported from the farmer to customers' table. when we are talking about vegetables and greeneries there is a high level of perishability that needs to be confronted. crops have to keep cool in order to maintain the high fresh and nutritious status. in order farmers to retain a high value for their products, after harvesting, food is transported from the field to processing facilities that are responsible for the cutting, washing of plants in cold water applying centrifugation methods in order to remove the excess water from the products. after removing the roots and fulfilling the described procedure, products begin to decompose. an often procedure that farmers follow is to treat their production with chlorine compounds and/or antioxidants that expand preservation during and after washing. continuously, food is usually packaged and stored in refrigerators and very low temperatures in order to remain in inertia status. however, outdoor farmers are not able to perform refrigeration between harvest and transport of the products for water processing, making it more uncertain in pathogens infection. in order groceries to arrive from the processing facilities to the shelves of the markets, they require on average to km, resulting to - days in transportation. according to kublic et al. ( ) , every three days, products lose % of their nutritious value after being harvested and roots' removal, meaning that consumers finally receive severely influenced vegetables in terms of nutritional value. according to the centers for disease control and prevention, each year, "roughly million people ( in )" are food poisoned in the united states. in terms of food safety what products of outdoor farming face is the severe contamination from improper use of manure, either from human fecal that is used as fertilization mainly in developing countries or from contaminated concentrated animal feeding operations (cafos). even if it has been proven by various researchers as a great nutritious source for the crops after proper compostable process, on the other hand the absence of carefulness, targeted application and lack of sanitation can lead to transmission of various types of parasites. a serious parasite that is worth to be mentioned is geohelminths (hookworm, ascaris and whipworm), that can survive their eggs in soil for years when they find the right climate conditions, causing diarrheal diseases as well as permanent learning deficit to children (hotez and pecoul, ) . e.coli was a foodborne illness that took high publicity after infecting approximately , people and causing about deaths, after severe pollution of agricultural water reservoirs in farms of california. to summarize, even if there are multiple technological automations, innovations and outbreaks in outdoor farming over the last decades, the nature of this agricultural type is very open to foodborne illnesses, illnesses extremely difficult to be traced rising the total risks. because of the importance of food safety and in order to avoid further foodborne illnesses, there are several rules that force outdoor farmers to enhance the safety status of their production for the overall benefit of the population. the strictest and most widely recognized organization of food control audits is the global food safety initiatives (gfsi), which was established in to reduce and control the risks associated with food production as also to streamline and improve the overall food safety while reducing the operating costs. various certifications are provided to farmers including the safe quality food (sqf) and hazard analysis critical control points (haccp) that set the necessary rules and prerequisites of a high-level food safety status. this includes some of the following rules: • it is of significant importance the control and validation of the agricultural water. to be more specific, there are rules that prerequisite the testing of the water quality that is applied via irrigation to the crops, but also the water related to the tangential purposes such as hand-washing of the workers during or after harvest, the ice that refrigerates food and the surfaces that food contacts with. • biological adjustments are often applied in soil for or particular nutritional uses that replace chemical fertilization. it is of vital importance that farmers who follow these techniques to follow specific guidelines for the use of raw manure (such as animal and human feces) as also for the use of stabilization compost in order to maintain a high level of sanitation. • there are rules concerning the compliance of domestic and wild animals either they are working in the farm, invade in the farm or graze. • finally, there are high requirements in workers' health and hygiene that need to be followed in order to prevent the contamination that may source by humans. . . food safety status of greenhouses as has already analyzed, there are greenhouses that are soil-based and the more advanced use hydroponic solutions. in hydroponic greenhouses, plants are transported several times according to the growing stage and are monitored throughout the different growing cycles. that give the opportunity to apply the exact resource requirements in every stage, in comparison with soil-based greenhouses and outdoor farms, where the plants remain in the same position until their harvest. another significant advantage of greenhouses in relation with outdoor farming is the high geographical flexibility of installation as it allows a significant reduction of the transit time of the products from the harvesting and processing point to the final consumers. greenhouse plants in an industry that constantly growing, with today's list accounting half of the tomato production and / of the global pepper production that are distributed in the fresh market (brauther, ) . greenhouses are a significant driver of national economies of the agricultural sector because of the high profit margin as also the opportunities for high added-value products. unlike traditional farming products, greenhouse production is highly protected from dangerous elements and various contaminants. however, the technologies that are applied for monitoring and controlling of the environmental conditions do not guarantee crops free of microbes and pathogens. the management practices applied in greenhouses are these that can conduct to growth, survival and spread of foodborne pathogens. a severe contamination thread could be spread by processing equipment since crates and baskets that are used for transportation of products, from propagation tools or even for surfaces that food contacts with. irrigation water is one of the most important food safety risks even in greenhouses as it can be drawn from a wide variety of uncertain sources such as municipality supply, rainwater, underground aquifer, reservoirs or surface water. greenhouses that use untreated surface water as irrigation source face high contamination risks. for example, in , salmonela saintpaul (cdc, found to have infected cucumber greenhouses in us that caused the infection of individuals across the country as they consumed imported vegetables with questionable irrigation water status. because of the high risk of infection of consumers, even from more controlled agricultural systems (compared to outdoor farming), regulations for food safety have become stricter by establishing new standards for food production (produce rules). the four areas that these standards focus on are the followings: • health and hygiene this practice targets in maintaining hygienic conditions of the personnel that is occupied in the greenhouse factories, involving criteria for personnel cleanliness, handwashing and use of appropriate gloves. even if handwashing is considered one of the simplest and cost-efficient practices, it has been reported that only % of greenhouses practice handwashing before the harvesting process. • irrigation water quality and management since water quality is one of the most crucial and contentious factors, it seems absolutely necessary the mandatory establishment of rules that control the water baseline quality profile. greenhouses withdraw water from a bog variety of sources such as municipality supply, wells, reservoirs and surface ponds. by checking and understanding the quality of the quality of various water sources can provide important information and reduce the risk of contamination. by regulation, greenhouses have to determine frequently microbiological testing on the water sources. furthermore, greenhouses that apply hydroponic solutions in semi-close or closed loops that circulate, recycle and reuse water, have to include filtering treatments that remove possible pathogens before re-applying it. methods that are effective and efficient in water recycling is uv light or disinfectants. • animals and waste significant measure for the protection of crops from foodborne pathogens is also to eliminate the restriction of domestic and wild animals at growing activities inside the greenhouses as well as in the outside area of the buildings. practices that contribute in discouraging animal intrusions can be for example the rapid weeding that will minimize rodents attraction and protection. • sanitation of equipment, tools and greenhouse surfaces foodborne pathogens are usually found all over the greenhouse environment such as harvesting bins and boxes and tarp floor covers of the greenhouse (ilic et al., ) . according to produce rule, all the tools and equipment that used in the production line should be inspected, cleaned, sanitized and maintain in this condition throughout the whole production, harvesting and post-harvesting process, in order to prevent contamination. greenhouses in comparison with traditional farming have the advantage of the three-key elements application that can eliminate contamination risk: innovations, automations and control. in specific, innovations provide to greenhouse farms a more secure food safety support such as water filtration systems, integrated pest management and higher quality control systems. automations can reduce the danger of contamination or cross contamination as they minimize or decrease the introduction of foreign specimens. finally, biometric systems provide to growers the ability to detect tracking information concerning the plants. after harvesting, the produce is set up in a traceability system from the greenhouse plant to the customer delivery service. . . food safety status of indoor vertical farms leafy greens, vegetables and herbs are considered of high-risk crops since they are usually not cooked but eaten raw. the usual process of consumers is to rinse their purchased greeneries after purchasing them from their grocery store and then consume them. this is not a particularly effective and protective procedure, since harmful pathogens need interference of chemicals to be detached from plants. outdoor farming and most of the greenhouses perform triple-wash on the harvested plants in order to mitigate the contamination risk, as a post-harvest process. this process consists of the pre-washing, a saline wash and the final bathing of greeneries in sanitizing, choline base solutions. unfortunately, this method cause quality reduction to greeneries, as is observed loss of flavor and texture along with the concurrent risk of contamination existing and spreading under the possibility of incorrect application. greens that grow outdoors follow the triple-wash procedure as a postharvest measure for increasing their health status. harvested crops are transported in the processing facility and sorted, rinsed, put in spinners, apply a second rinse, spinner again, third rinse, sorted (again), packed, and then at the end they get delivered at the grocery market. crops that follow the above washing method bear usually on the packaging labels such as "triple-washed" or "pre-washed". even if this method can provide sufficient results in harvested outdoor crops, if the water used for the triplewashing process is polluted with pathogens, then this can spread rapidly to the rest of the harvested crop. for this reason, triple washing cannot categorized as the most effective and guaranteed process. indoor vertical farms apply only nutrient elements in the irrigation system and completely avoid the use any chemicals during the growing period of plants, excluding all the types of pesticides, herbicides and chemical spaying for fertilization. the philosophy of indoor farming depends on monitoring and constant controlling of the crops as also of all the resources that come in and out from the farm and they are isolated from mother nature where many threats and contamination sources may appear. for this reason, indoor farmers suggest that their products do not need to be washed before consumption, as they are already clean by a protected and purified growing process and a quick delivery to local grocery stores. hermetically sealed environments, inside highly controlled spaces that are designed to offer the highest possible level of food safety particularly for the growing period, surround the cultivation rooms of indoor vertical farms. since there are no seasons to be followed as in outdoor farming neither humidity, temperature fluctuations nor long gaps on post-harvesting processes and packaging, indoor farmers can dramatically reduce a potential contamination with precise systems. in addition, the hermetically sealed environment protects crops from being exposed to outside elements such as harmful pests, insects, fungi and bacteria. in one of other type of such systems, aquaponics, co-cultivation of fish with plants is done. this method of cultivation uses very innovative water filtration systems, which extract solids from the fish tanks. continuously, solid break down to beneficial bacteria that transforms them into nitrates. then, the nitrate-rich water circulates to the plant culture area where plants absorb the nutrients and purify the water. since the aquaponic system follows a close-loop, the clean water is circulated and reused into the fish tank. plants that grow in soilless systems can travel along their production process giving the opportunity to be inspected for health status. for example, after sowings, seeds are moved to germination rooms with high humidity that boosts their sprouting. then, seedling is moved to propagation room with controlled climatic conditions that promote their development. next, young plants usually located in the main part of the cultivated room in floating rafts, receiving a nutrient-rich water. after finishing their development and reaching their mature stage, they are daily harvested and shipped. between every translocation of plants, there is intensive quality check to prevent crops' contamination. high precision irrigation systems are used in order to monitor the water that travels throughout the crops. innovative hydroponic or aeroponic methods usually draw water from filtered and drinkable sources and distribute it at each crop often without even touching the salable part of the plants. this is achieved either by the use of water in liquid form, mist or fog that sprays it only into the root section of the plants and not in the parts consumed. extensive sterilization and supplier are also applied methodologies of indoor vertical farms that control and assure the input resources of the farms such as seeds, nutrients that need to be absolutely safe and clean. because of control and monitor mechanisms that are carried out indoor, there is clear advantage of indoor farms. they are aware of the cleaning status of plants and maintain it with further regulations during the cultivation period and finally harvest and deliver a healthy and fresh product. even if indoor vertical farms produce food safer to consume than the open field grown products, bottlenecks and hazards can still be introduced during the growing process of crops. such threats can be dirt and bacteria transferred from the workers and dangerous threats in the nutrient medium that include chemical sources, cleanliness and water safety. further risks can also detected at the post-harvest activities such as trimming, sorting and delivery of the products. thus, it is of vital importance even for indoor farmers to perform high status and certified systems for detection, monitoring, testing and evaluation as in outdoor farming and greenhouses. a study conducted by purdue university (wang et al., ) , found that there is also high risk of crops contamination due to pathogen pollution in vegetables grown in hydroponic or aquaponic systems. more specifically, they reported that e.coli o :h was found in fish feces and because of the circulation that close loops systems, it caused water contamination of the plant root surfaces that were in the aquaponic and the hydroponic systems. since fish probably were contaminated by the bacteria, it is important to follow a proper and certified handling, cleaning and sanitizing process in order to reduce the contamination risk in hydroponic and aquaponics. it is a very difficult, time consuming and α costly process to control all the plants even in an indoor vertical farm for having a % safe food product. indoor vertical farms use controlled environment of humidity and temperature in order to provide plants the most suitable conditions. however, in the case that unpredictable production errors occur, e.g., technical malfunctions with the engineering equipment, temperature and humidity can get out bounds to unwanted levels and create fertile environment for bacteria growth. this incident could be possibly avoided in the case of traditional farming, as the constant natural air circulation and the sunlight could smooth out some of these errors. bacteria population are not biased, meaning they do not grow or prefer targeted geographic locations, but they are transported to different locations by human activities as they can be brought by clothes, shoes or skin. furthermore, it should be noted that even if indoor farms consist a safer environment compared to other farming types, if a controlled environment develops for some reason bacterial infection, it will be extremely difficult to eliminate the contamination and protect the rest of the growing crop. for this reason, indoor farms follow high sanity level protocols to avoid the possibility of crops' contamination by human contact that involves all the workers involved with various cultivation processes of the plants. that include strict control by imposing the use of facemasks, hair and beard net, footbaths and clean or single-use suits, which can diminish the risk of contamination. another solution for further risk elimination from potential contamination, is the application of innovative technologies that operate extensive integrated pest monitoring. this can be achieved with the use of ultraviolet light outside of the farms that detect possible threats as also air curtains that are installed in every door and can control air that enters the cultivation room protecting it from the danger of contamination. additional solution that can increase the sanitation levels of indoor crops, is the application of certified hvac filters, in order to perform an extensive pest monitoring. indoor vertical farms belong to a novel type of farming cooperating with innovative technologies in order to provide the safest, higher quality and most fresh and nutritious groceries. both advocates and critics of this technology seem to recognize that indoor vertical farms under suitable circumstances (mainly of the high demand on electricity loads), could offer a solution to the safety and sustainability problems faced in traditional farming. however, consumers seem to be more skeptical and critical on this technology. potential explanation of the consumers' skepticism is the uncertainty and lack of trust in other food innovations such as genetically modified crops, food nanotechnology and artificial irradiation that struggled to find acceptance in the market. nevertheless, the subjective knowledge and awareness level of consumers on the indoor vertical farming is still limited, even with the excessive spread of technology and information globally, it is of vital importance to increase the education of people on this new technology by informing them on the actual growing properties and impugn the unjustified myths and dangers. because of the increasing demand of indoor vertical farms and their establishment in the market, many researchers have focused on designing and addressing customer surveys and other research methodologies in order to define the public opinion on this technology and the status of their trust and preference on already existing agricultural production systems. significant angle on these researches is to explore the existing knowledge and perception of customers between the three different farming systems; traditional farming, greenhouses and indoor vertical farms, in respect of the cultivation techniques, safety, resource sustainability, quality and their willingness to buy products from each category. for this reason, primarily it was of high importance to validate that consumers are able to recognize the different agricultural systems between them in order to provide valid, clarify and comprehensive results. different customer studies that investigated customers' opinion on different agricultural methods show a more skeptical belief concerning novel technologies on food production. more specifically, peoples' perception with technological innovations in agriculture are associated with high risks for food production presenting low expectations on the provided benefits of technology used (sparks et al., ) . in another research (coyle and ellison, ) participants rated higher the greenhouses facilities and the outdoor farms compared to the indoor vertical farms in terms of naturalness in the production process and the final product. concerning the quality status of the final product people also seem to present higher levels of confidence and trust on the greenhouse products and subsequently indoor vertical farms and finally in outdoor farming products. naturalness seems to be a high influencing indicator for consumers' selection globally as also a critical significant factor on the usefulness of the agricultural system. according to j€ urkenbeck et al. ( ) , customers replied that led lighting is not considered a too artificial tool for horticulture and slightly agreed that they do not consider indoor vertical farming too artificial concerning the overall production system. even if consumers in general prefer naturally and traditionally produced food, nevertheless the fact that food of indoor vertical farms grow without chemical additives is highly considered. on the other hand, under a customer research conducted by j€ urkenbeck et al. ( ), it is noticed that consumers seem to present a high acceptance on indoor vertical farming concerning the offering sustainability and the high ecological footprint. people seem to select their purchased food based on their concerns on the naturalness, ethics and environmental status. in more details, % of the respondents in that research declare that they put an extra effort to select and buy locally grown food because of its high level in freshness, nutrition and reduced food mile emissions compared to traditional farming methods. on the other hand, a significant share of the consumers evaluates indoor vertical farming as an artificial agricultural process in order to trust their footprint outcome. for this reason, it is pointed out that knowledge, information and nutritional awareness can become a solid solution for the higher acceptance of indoor farming and irradiated food products. respondents of the specific survey showed a strong willing on buying products that were produced in an indoor vertical farm with . % of the total sample, . % partly agreed on that statement, and finally only . % were not willing to purchase these products. however, it should be noted that the perceived behavioral control does not influence the customers' willingness to buy, but it has some influence on the behavioral intension of willingness to purchase the product. overall, the behavioral intention of customers to purchase products from indoor vertical farms is highly dependent on sustainability. under a different analysis, it has become very clear that perceived sustainability of indoor vertical farming is the main reason of acceptance. it has been observed that the more positive the resulted sustainability status of the system is, the higher and the customers' acceptance and willingness to purchase the product is. furthermore, based on the perceived sustainability level of indoor vertical farms it seems that customers increase and their acceptance of this innovative technological food production system. based on these results, we could indicate that the growing involvement and concern of consumers to select products from agricultural systems that present high environmental performance. indoor vertical farming can be very advantageous in terms of resources sustainability since because of the high technology and the soilless cultivation systems it consumes way less on natural resources (e.g., water and nutrients). additionally, indoor vertical farms significantly decrease the co emissions that are correlated to food transportation from the producers and the processing facilities. in specific, indoor vertical farms can provide times higher productivity per year per unit land area compared to traditional farming due to the zero dependence on weather conditions, seasonality and possible infections from insects, pests and bacteria. due to the evolution of technology it is not anymore a prerequisite holding a large area of land for sufficient fresh food production, but the use of multiple layers, optimally controlled (environmental conditions and physiological parameters of the crops and minimum possible loss from crop threats). significant characteristic of indoor vertical farms in terms of sustainability is the minimization food delivered losses. in addition, significant reductions can be observed in the cooling fuel demand, necessary to cool the production in order to be transported in long distances. this can be achieved since indoor vertical farms are usually installed in the urban or suburban areas in shaded and/or abandoned buildings (or even basements) due to the soilless farming techniques and the artificial lighting, providing access to fresh and nutritious greeneries to citizens. finally, one of the significant benefits that indoor vertical farms provide is the ability after proper processing of the use of waste water, crop wastes and excessive co produced in urban areas, as input resources of water, nutrients and co in the culture area. to summarize some of the basic improvements in resource savings provided by indoor vertical farms compared to the immediately following high technology cultivation system, the greenhouses are the following: • indoor vertical farms save % of the pesticide use in their interior by maintaining the culture area clean and insect-free. • because of the application of close loop irrigation systems and of the collection, recycle and reuse of the water vapor that plant leaves transpire, indoor vertical farms can reduce up to % the water consumption. furthermore, the use of closed loops can decrease up to % the fertilizer usage since it is feasible to recirculate and reuse the nutrient solution. • significant land reduction up to % can be achieved with the application of indoor vertical farming, due to the important increase (more than times) of the annual productivity of crops per unit land area. • yield variation can also reduced by % because of the constant monitoring and control of the crops and the lack of influence from the outdoor environmental conditions. food safety and traceability of products is another important factor highly relevant to indoor vertical farming. even if it does not provide a % safety for consumers, despite the fact that crops grow in a controlled environment protected by wildlife, animals, birds and insects, it upgrades the safety and security feeling of the products than those that grow in open field. the majority of the selected cultivated crops of indoor vertical farms are among the species with the higher contamination risk when they grow outdoors or unprotected, because they grow very close to the ground level. furthermore, one of the most crucial factors that greatly affect the possibility of contamination is the water quality that involves during the whole production process, including the irrigation water as also the washing water at the post-harvest processing techniques. farmers of all categories should follow high standards and criteria for the water sources that channel water into the farms as also frequent control and monitor of the crops for potential threats of contamination. it is now clear, that indoor vertical farms are a high necessity for tackling the challenges concerning the conservation of their resources. nevertheless, in order to enhance the environmental sustainability and improve the efficiency and sufficiency of food production supplies for our society, it is necessary to develop more diverse, effective and ecological agricultural systems including both the traditional farms and the greenhouses. further research and experimentation it is absolutely necessary in order both to improve the efficiency of resources in an indoor vertical farm but also to possibly eliminate the possibilities for contamination threats and constantly provide the outmost safe, fresh and nutritious fresh fruits and vegetables to the human population. notwithstanding the promising benefits that are linked with indoor vertical farming, there are also important challenges in the further implementation of this farming system in the future. it is of vital importance further improvements on the efficiency and effectiveness of the equipment that will lead to a significant decrease of the energy demand of the systems. by achieving the reduction of energy demand, it will add extra value in the environmental sustainability of the system but also it would also make it more appealing for the public, the investors and the industry and will increase the viability and profitability. however, it is pointed out by despommier ( ) that there is the opportunity for energy recovery from the non-salable crops' parts and capture of renewable sources of energy that can create zero energy building for hosting indoor vertical farms. at the same time, the whole system of indoor farming can synchronize and manipulate huge amounts of carbon and simultaneously release into the atmosphere oxygen from plants' respiration. significant is also the start-up costs that are associated with indoor vertical farms as it is clear that it is more expensive to develop a vertical greenhouse than a normal greenhouse (fletcher, ) . as it has been highlighted by many studies one also key barrier that indoor vertical farmers have to confront is the public resistance to these type of products as social masses face difficulty in accepting indoor vertical farms instead if traditional farming ones because of the natural way that food is produced. additionally, as indoor vertical farms serve the concept of local, fresh food production and they are mainly installed in urban or peri urban areas, they have also to salient the issue of affordability because of the expensive land and space use. for this reason, key factor is the productivity rate of indoor vertical farms that can maintain them profitable and keep them prevailed in the future. more specifically, if indoor vertical farms achieve to produce up to times more yield compared to traditional farming, then they can offset the capital expenditures and the expensive land use. previous research conducted by , presented that indoor urban vertical farms regardless the financing scheme, are much more profitable in comparison to greenhouse constructions. in the specific work, different investment scenarios are presented based on the cash flow analyses and show that iuvf can present high irr (investment return rate) as also a payback period between and years. finally, another drawback that is linked with indoor farming production is the limited variety of crops that can be produced with this technology, such as lettuce, herbs, tomatoes and berries. even if theoretically, all types of crops could be cultivated indoors, that would not be economically feasible due to the highly increased energy demand. thus, low-value agricultural crops such as wheat and barley will continue to grow under economically and environmentally unviable conditions. under these circumstances, the indoor vertical farms have to face a limited production compared to the "limitless" hectares of traditional farming and a reconsideration of scaling up would be particularly costly and complicated. the last years that indoor vertical farming gained more recognition and research interest, a plethora of new studies, prototypes and innovation designs have been presented under the academic and industrial scope. indoor vertical farming presents a high interest and potential to play a critical role in the demanded sustainability in food of urban areas. this becomes even more important by the multiple studies that estimate and analyze the significant increased food demand in urban areas. indoor vertical farming presents important advantages compared to traditional farming, concerning the required sustainability in our times by focusing in three main categories: environmental, economic and social. there is a high demand for further development in automation. this will be scaling up the projects in order to create more feasible scenarios both from economic and commercial perspective. future research is necessary towards a holistic approach via the investigation and the analysis of the full life-cycle of indoor vertical farms and the impact to the environment compared to the traditional farms and greenhouses. up, up and away! the economy of vertical farming state of indoor farming agstar data and trends. united states environmental protection agency the vertical farm: a review of developments and implications for the vertical city benefit cost analysis of the maize crop under mechanized and traditional farming systems in the nwfp optimization of photoperiod and quality assessment of basil plantsgrown in a small-scale indoor cultivation system for reduction of energy demand plant factories in the water-food-energy nexus era: a systematic bibliographic review indoor vertical farming in the urban nexus context: business growth and resource savings basil 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and the waste-to-bioproducts approach carbon footprint and cumulative energy demand of greenhouse and open-field tomato cultivation systems under southern and central european climatic conditions resource use efficiency of indoor lettuce (lactuca sativa l.) cultivation as affected by red: blue ratio provided by led lighting environmental, energetic, and economic comparisons of organic and conventional farming systems crop growth and development in closed and semi-closed greenhouses. thesis recharging greek youth to revitalize the agriculture and food sector of the greek economy survey and evaluation of heating technologies for worldwide agricultural greenhouse applications advances in greenhouse automation and controlled environment agriculture: a transition to plant factories and urban agriculture gene technology, food production, and public opinion: a uk study the economics of local vertical and greenhouse farming are getting competitive multidirectional relationship between energy resources, climate changes and sustainable development: technoeconomic analysis agstar: biogas recovery in the agriculture sector the occurrence of shiga toxin-producing e. coli in aquaponic and hydroponic systems watering the farm: comparing organic and conventional irrigation water use in the murray-darling basin development of an integrated methodology for the energy needs of a major urban city. the case study of athens wind energy integration through district heating. a wind resource based approach a wind energy integration analysis using wind resource assessment as a decision tool for promoting sustainable energy utilization in agriculture small scale plant factories with artificial lighting and wind energy microgeneration: a multiple revenue stream approach effects of leaf area index of tomato seedling population on energy utilization efficiencies in a close transplant production system energy and mass balance of a closed-type transplant production system (part ): carbon dioxide balance discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin key: cord- -vyyboega authors: mulvihill, peter r.; ali, s. harris title: disaster incubation, cumulative impacts and the urban/ex-urban/rural dynamic date: - - journal: environ impact assess rev doi: . /j.eiar. . . sha: doc_id: cord_uid: vyyboega this article explores environmental impacts and risks that can accumulate in rural and ex-urban areas and regions and their relation to urban and global development forces. two southern ontario cases are examined: an area level water disaster and cumulative change at the regional level. the role of disaster incubation analysis and advanced environmental assessment tools are discussed in terms of their potential to contribute to more enlightened and effective assessment and planning processes. it is concluded that conventional approaches to ea and planning are characteristically deficient in addressing the full range of impacts and risks, and particularly those originating from pathogens, dispersed and insidious sources. rigorous application of disaster incubation analysis and more advanced forms of ea has considerable potential to influence a different pattern of planning and decision making. in this article we explore the general utility of recent work from disaster research for the enhancement of contemporary methods and approaches to environmental assessment (ea). in particular we discuss the potential disaster research has for assessing slowly evolving and underappraised environmental problems, including environmentally related health impacts and those problems involving the cumulative impacts of the ever changing relations between the urban, exurban and rural regions. to illustrate and discuss this potential of disaster research we give context environmental impact assessment review ( ) - www.elsevier.com/locate/eiar and substance to our analysis by focusing on two case studies, the first involving water contamination/pollution problems in rural ontario and the other focusing on the changing ecological and social landscapes of areas which surround the greater toronto area (gta). these particular cases were selected because frequently ea may neglect or at least minimize the potentially significant impacts of the urban/ex-urban/rural dynamic. this may be perhaps because of the complexities inherently involved in this dynamic, often involving longer time scales and expansive distances, thus making the incorporation and consideration of the factors related to this dynamic difficult. nonetheless, the adoption of certain principles and the general orientation of disaster research, especially if coupled with cumulative impact assessment, may better enable environmental assessors to broaden the conventional lens of ea. in turn, this may facilitate the identification of not only the physical and biological antecedents to many environmental problems, but their social and political economic undercurrents as well, thereby providing a means through which some of the difficulties of incorporating the urban/ex-urban/rural dynamic can be addressed. notably, the analysis of both the physical and social dimensions in a disaster research inspired ea process will enable assessors to more effectively grapple with the vexing issues related to the complex cumulative and latent effects of many understudied and conventionally neglected environmental problems. we begin with an overview of recent approaches in disaster research that are of relevance to ea. generally speaking, disaster research has tended to evolve as an interdisciplinary and applied field, although notable pioneering contributions have been made primarily by geographers and sociologists. traditionally, the field has investigated a large array of issues often related to practical issues such as the role of organizations during emergencies, evacuation behaviours, emergency medical service delivery, the protection of property, the spatial aspects of disasters and so on. recently, there has been increased attention focused on issues related to what turner ( ) has termed "disaster incubation"the period before disaster onset when the causal factors that contribute to, or precipitate, a disaster accumulate and interact in an unnoticed manner. for our discussion in this article, the key feature of the research on disasters is this idea of disaster incubation. the notion of disaster incubation has particular relevance to ea because of the analytical emphasis on the latent character of the causal factors and processes that underlie a given disaster, or more specifically in the case of ea, those latent factors/processes that underlie environmental impacts and problems. over the last several years, there have arisen a number of case studies that illustrate the incubation of various disasters. for example, ali ( ) discusses how the political economy of an industrial city in southern ontario had contributed to a large toxic conflagration at a private recycling firm, while beamish ( beamish ( , relates how one of the largest oil spills in american history occurred on a stretch of land on the guadalupe dunes in california over four decades by chronic leaks that were the result of numerous instances of institutional neglect and corporate malfeasance that accumulated over time. perhaps one of the most notable studies of a the incubation period of a technological disaster was conducted by diane vaughan ( ) who reveals how the space shuttle challenger disaster ( january ) was the outcome of social phenomena involving, for example, among other social factors, the transformation of deviant practices into acceptable behaviours at nasa. specifically, this transformation process is referred to as the "normalization of deviance" wherein danger signals occurring during the pre-disaster period gradually become accepted as normal by those in the organization. as vaughan shows through her institutional ethnography, it was just such a process that ultimately and tragically led to the decision that the space shuttle could be safely launched despite certain technical concerns on launch day. in addition to technological disasters, the analysis of disaster incubation has also been applied to natural disasters, as for example in the case of hurricane katrina in (bakker, rydin, ; seager, ) and the chicago heatwave (klinenberg, ) , in which analysts have noted that particular social groupings, such as poor african-americans and women, were particularly vulnerable to the impacts of the "natural" agents because of land use and political decisions which put the particular groups in harms way. the latter two cases point to the important issue of disaster vulnerability in the urban context, but, as will be discussed, disaster vulnerability is also a particularly relevant consideration for ea in the rural context. at this point in time, research progress in the area of disaster incubation has proceeded on a somewhat incremental and ad hoc basis with particular insights gained through case study analyses. as such, the work on disaster incubation has not yet crystallized into a set of propositions in such a way as to define a formal theory. although it is perhaps true that the study of disaster incubation has not yet achieved paradigmatic status, we contend that there now exists sufficient theoretical and empirical development in this area to define a coherent approach which for the purposes of this article we will refer to as disaster incubation analysis (dia). largely informed through research in organizational sociology, dia has tended to focus on the organizational foundation of disasters. in this vein, turner's pioneering work in the area has been based on the analysis of the inquiry proceedings that followed three high-profile disasters in the united kingdom. notably, his analyses examined how regulatory agencies and firms were implicated in the disaster incubation process. in this connection, turner identified several important underlying organizational factors, including: rigidities in institutional beliefs; the presence of distracting decoy phenomena; the neglect of outside complaints; multiple information-handling difficulties; the exacerbation of the hazards by strangers; the failure to comply with regulations; and the psychological tendency to minimize emergent danger. more recent efforts have built on the work of turner, both by expanding his focus on the organizational foundation of disasters (see for example : clarke, : clarke, , kroll-smith, , ; dynes, ; erikson, ; freudenburg, freudenburg, , freudenburg and gramling, ) as well as through expansion of the analytical scope of dia to now consider the role of biophysical and environmental factors alongside the social factors emphasized by turner (ali, ; klinenberg, ; murphy, ) . it is perhaps this focus on both the biophysical and the socio-organizational factors that holds the most promise for the formal impact assessment and management of natural resource use, such as in the case of ground and surface water impacts we will discuss shortly. in this light, a critical feature of adopting dia for environmental assessment is to address the latency dimension of the problem incubation; in particular, the question as to why the conditions that fostered a particular disaster went unnoticed. as much of the research in dia has been based on post-hoc analyses, the challenge for the environmental assessor is to bring together the relevant insights from these retrospective case studies in order to apply them in a prospective manner to future scenarios in the planning process. this leads to a second point. as foresight is an essential foundation for effective planning, the need to identify the reasons for the failure of foresight is critical and dia facilitates this identification by focusing explicit attention on these very reasons. third, dia is especially well-designed to address those situations in which largely overlooked cumulative impacts are critical. this is the case for example in relation to such processes as: those related to development pressures (such as incrementally increasing urban sprawl), globalized economic activities (such as intensification of livestock operations (i.e. "factory farms)", as well as creeping "natural" disasters such as desertification, global climate change, and the pathogenic contamination of groundwater, all of which may develop gradually over time in an unnoticed manner until the catastrophic impacts can no longer be ignored. let us now consider empirically how such latent biophysical and social processes have environmental impacts and how they can be analyzed using dia by considering the following two cases involving rural ground water and water use within the context of toronto and its ex-urban and rural regions. while public concern about southern ontario's water future is substantial, it is usually focused on more immediate and higher profile issues, at the expense of some of the more insidious and cumulative risks. the broader surroundingsthree of the great lakes are in close proximityhave long ensured public consciousness of water pollution, but also a complacent if misplaced sense of infinite supply and regeneration. surface water issues have commanded most of the attention, while the magnitude of groundwater problems and risks remains largely unknown and neglected. this changed to a significant extent in with the walkerton drinking water contamination disaster. situated within the agricultural heartland of ontario, the town of walkerton is located on the saugeen river about km northwest of toronto and km east of the shores of lake huron. although relatively close to the great lakes, the town is entirely dependent upon groundwater that is drawn from wells. during the third week of may , walkerton (population: ) became the site of the largest outbreak of a waterborne infectious disease in canadian history. after a public outcry, an independent inquiry was commissioned a month later to determine the causes of the outbreak as well as to fulfill the extended mandate of making recommendations about how best to manage the water supplies in the province. the inquiry concluded that the contamination of the municipal water supply occurred after a period of very intense rainfall whereupon cattle manure containing the pathogen e. coli o :h spread on the fields of a nearby farm was washed into a particular well (walkerton inquiry, ) . as this pathogen can only survive in groundwater for very short periods of time, it was suspected that rapid flow of water contaminated with the pathogen was able to reach the municipal water supply because of the presence of fractures in the carbonate substrata structure of the region. aside from identifying the source of contamination, the inquiry commission also identified a list of other factors that contributed to the outbreak, including malfeasance on the part of the local water operator and serious deficiencies in the provincial regulatory framework that governs water management. the case of walkerton immediately raised the question of the magnitude of the groundwater contamination problem within the national and regional context. one of the difficulties faced in identifying the particular pathogen involved in the walkerton case was that e. coli o :h was normally associated with food-borne, and not waterborne, outbreaks. sometimes referred to as "hamburger disease", illness due to e. coli o :h first began to be recognized as a newly emerging public health problem in the s with the onset of several sporadic outbreaks throughout north america (nadakavukaren, ) . from a dia perspective, the association of the disease with food served as distracting decoy phenomenon. as dia casts a wider net in its analysis, it often considers the wider biophysical and social context of a disaster. such an orientation has practical implications for ea, which may benefit from such an expanded perspective by employing a wider scan that would be alert to broader social and ecological trends (for example, see krieg, krieg, , for trends regarding the distribution of hazardous waste in north america). in this spirit, let us consider some of the relevant trends in terms of the walkerton disaster, starting with that related to increased incidence of water contamination in the north american context. by being knowledgeable about such trends, ea processes may be redirected to focus on the more likely scenarios in environmental and land use planning. one relevant trend that ea professionals may want to consider is the increase in new and emerging diseases more generally, as it is likely that the protection of groundwater from such threats will become an issue of growing importance in the future. in recent times, western societies have witnessed the rise of a new class of emerging (and re-emerging) pathogens that are extremely insidious and virulent, including, to name just a few: the severe acute respiratory syndrome (sars) coronavirus, hiv/aids, west nile, huntavirus, ebola, the lyme disease virus, the avian flu virus, the monkey pox virus, and the hoof-and-mouth pathogen (drexler, ; levy and fischetti, ; garrett, , institute of medicine, . the bacteria e. coli o :h involved in the contamination of the walkerton groundwater is another example from this new group of pathogenic threats. the emergence of these new pathogens has several important implications for planning in general and rural planning in particular. first, it needs to be recognized that a wide range of social and environmental factors are involved in the evolution of these pathogens as well as in the onset of the respective disease epidemics themselves. such factors include: ecological, human demographic and behavioural changes; travel and commerce; technology and industry; microbial adaptation and change; and the breakdown of public health measures. for this reason, whether contamination occurs within the groundwater source, through the food distribution system, or through the air and soils, the wider contextual factors that could amplify or mitigate the spread of the pathogen need to be considered as part of the assessment and planning process. in particular, the possible interactions of such factors need to be studied. for example, as is argued below, the need to monitor groundwater for pathogens may not be possible under the tight fiscal circumstances that many resource-based communities face. the rural geographic dimension of the new and emerging pathogenic threats is often neglected in groundwater management. part of the latency of the disaster incubation involved in disease outbreaks is a function of the fact that most of the relevant incubating processes and factors are occurring in remote locationsthey are "under the radar" of most observers. such tendencies are especially worth noting in light of such disaster incubation elements as rigidities in institutional beliefs (as for example with respect to rural versus urban, as well as municipal versus provincial versus federal government agencies) and neglect of outside complaints (as for example, the neglect of the input and concerns of farmers by government agencies). furthermore, most of the new and emerging pathogens are based on animals raised or otherwise inhabiting relatively remote areas. for example, it is thought that the human immunodeficiency virus (hiv) originates from the simian reservoir in the rural jungles of east africa (institute of medicine, ); the sars coronavirus in the palm civet cat reservoir of rural southern china (guan et al., ; martina et al., ) and the avian flu virus in the wild duck and fowl reservoir of the same region (institute of medicine, ) . the crossover of the pathogen from the animal to the human reservoir is influenced by distinctly social processes; thus, for example, it has been noted that the paving of the kinshasa highway through the forests of the rift valley led to increased traffic thereby facilitating the spread of disease from remote posts to urban areas as prostitutes working the stretch of road plied their trade. local doctors estimate that as many as % of those living in this area are infected with hiv (preston, : ) , while strong evidence indicates that the sars coronavirus crossover occurred in live animal markets in urban centers where animals were brought in from rural areas to be sold. the significance of the interrelationship between humans, animals and the environment highlights the importance of the interface of urban-rural areas and particularly the encroachment of humans into formerly untouched environs, as seen by the rise in cases of hantavirus pulmonary syndrome (hps) (buck and aron, : ) and lyme disease. incidence rates increased as exposure to the vectors transmitting the viral agent increased as development into forested areas brought humans into closer contact with deer mice carrying the sin nombre virus in the case of hps and wilderness ticks hosting the bacterium borrelia burgdorferi and carried in songbirds and mice in the case of lyme disease (bourette, ) . a second general trend refers to increased incidence of contamination within rural areas themselves. although drinking water contamination problems tend to be associated with developing nationsthe world health organization notes that the leading cause of illness and deaths in most developing countries is attributable to gastrointestinal infections from the ingestion of water containing pathogens (nadakavukaren, ) the walkerton outbreak dramatically illustrates that the waterborne route of the disease may pose an equal, if not greater, threat to public health in the north american, and particularly, canadian context. the potential for groundwater contamination is particularly noteworthy in light of the finding that approximately % of canada's drinking water is stored as groundwater (howard, ) . at the same time, almost % of those reliant on groundwater as a source of drinking water live in rural areas (howard, ) . the center for disease control and prevention data indicates that between - there were on average outbreaks of waterborne diseases per year in the united states (nadakavukaren, ) while there were published reports of waterborne outbreaks/ incidents associated with drinking water in ontario alone from to (edge et al., ) . in many cases, waterborne disease afflictions run their course in - days and the victim recovers completely without treatment (nadakavukaren, : ) . as a result, it should be noted that the u.s. environmental protection agency (nadakavukaren, ) estimates that for every case of waterborne disease identified, more never appear in the statistics because of haphazard reporting. edge et al. ( ) estimate that only one-tenth of waterborne outbreaks are reported. thus the problem of pathogenic contamination of drinking water may be much more pervasive than what is currently realized. in this way, the neglect of the issue of pathogenic contamination of groundwater may in fact illustrate turner's observation that in disaster incubation, there is a psychological tendency to minimize an emergent threat. again, ea would clearly benefit by being cognizant and vigilant of such tendencies. rural areas tend to face especially challenging water quality problems due to high levels of dissolved organic material and predominantly agricultural drainage basins (peterson, ) . in this connection, the epa has found that the leading source of pollution of contaminated rivers in north america is agricultural runoff (mallory, ; nadakavukaren, ) . in the case of ontario, agricultural runoff in the form of manure (i.e. nutrients), as opposed to herbicides or pesticides, is particularly problematic because of the nature and type of farming practiced in this region. reflecting a more general trend throughout north america, from the second world war onward, agriculture in the province has become more industrialized, with fewer farms that are larger in size and growing many more animals per farm. thus, for example, although today agriculture and food is ontario's second largest industry, employing some , people (omafra, b) , the number of farmers has dropped dramatically over timebetween and the number of dairy farmers in ontario went from approximately , to ; while the number of pork producers dropped from , to (omafra, b) . notably, walkerton itself is situated within one of the rural ontario counties with the highest number of cattle per unit area grown in what are referred to as "factory farms" (nikiforuk, ) . typically, factory farms (also known as feedlots) may contain as many as , cattle feeding in crowded areas the size of a city block (other regions of such intensified farming include specialized regions in alberta, south carolina and iowa). the animal waste generated by these intensive livestock operations is enormous. for example, a , head feedlot will produce , tonnes of waste annuallymore fecal matter than that produced by a city of , humans in the same period (nikiforuk, ) . the animal waste is stored in manure lagoons before being applied to farm fields. contamination problems often arise because these lagoons tend to leak into groundwater and streams, while sometimes the lagoon walls may even collapse, resulting in massive manure spills. further problems can also arise because the manure is frequently applied at rates far above what the crop and soil base can absorbthe resultant excess finding its way into surface and groundwater. the entry of manure into ground and surface water pathways becomes a public health threat if the manure contains pathogens. elevated levels of bacteria in ontario's rural drinking water sources have been detected for at least a decade. for example, a survey of groundwater quality in farm wells across ontario found that % of the wells had bacteria levels above the safety guidelines (michael goss cited by rankine and boyd, ) . second, a health canada study published in found that those counties with the highest cattle density (including walkerton) routinely registered the highest rates of e. coli o :h infection in the province between and (nikiforuk, ) . further still, for several years prior to the outbreak, ontario ministry of environment inspectors were aware that the well contaminated in walkerton was vulnerable to contamination by surface water (referred to as gudigroundwater under the direct influence of surface water) and that the ontario drinking water objectives of required that such sources be monitored continuously for chlorine residuals and turbidity, which they were not in the case of walkerton (walkerton inquiry, ) . if such knowledge existed, the pertinent question that arises is why were preventative planning measures not taken and how could such information be incorporated into effective drinking water management and planning practice? in other words, to use the lexicon of dia, why were danger signals missed? as we shall see, by addressing such questions through a dia-inspired ea process, the knowledge attained from the past experiences of a disaster can be used to more effectively identify and better plan for the impacts of the situation or undertaking currently under consideration. the walkerton case suggests a number of significant implications for water management and planning. first, it suggests that other similar disasters may be incubating, since waterrelated incidents tend to be underreported and since monitoring has been shown to be frequently inadequate. ex-urban areas appear to be at particular risk as the hazards associated with factory farming accumulate and converge. the relatively brief history of farming on such a scale makes it difficult to understand its implications fully and in historical context. second, the case points to the risks of incomplete or partial approaches to monitoring in which some of the risk factors may be watched, but less so in relation to other factors. over time, key risk interrelationships may thus unfold but be missed until disaster strikes. third, the case casts doubt on the reliability or effectiveness of environmental assessment processes to identify the probable cumulative effects of an activity like factory farmingor, more precisely, the recurring reality that many significant development activities are not subject to rigorous or formal assessment at all. fourth, the case confirms the paradox of groundwater resources: they are relied on heavily but taken for granted, literally out of sight and mind most of the time. rural-urban interfaces therefore provide excellent opportunities for such crossovers to occur and need to be incorporated into assessment and planning considerations. the outbreak in walkerton is another case in point; it involves the relationship between the bacteria e. coli o : h , the surface and groundwater environment, livestock and humans. it was clear that the humanmade water distribution system, the geophysical characteristics of the land in the region, and agricultural practices in the area were instrumental in the spread of the disease. in light of the above, it is also apparent that rural and ex-urban areas may be highly vulnerable to waterborne disease outbreaksa situation which could be described in terms of a "rural health penalty" (ali, ) in which such areas are prone to confluence of circumstances that make residents vulnerable to certain health problems. this includes health-threatening conditions due to the rise of factory farming and the use of agricultural pesticides and herbicides that may contaminate drinking water. this increased vulnerability to health threats may also be due to broader social and political circumstances, as an analysis based on an expanded scope of dia reveals. under ontario's climate of cost-cutting that began in earnest in the 's, activities related to the prevention of health threats, such as the protection of drinking water, became particularly difficult for smaller rural communities. not only did such communities have to deal with reductions in the transfer payment funds available to them, but, at the same time, they were forced to absorb the costs of new responsibilities and practices that were formerly conducted by higher levels of government (i.e. the downloading of costs and services from federal and provincial levels to the municipal level). the smaller tax base of rural communities meant that these municipalities did not have an abundance of public money available to them to invest in infrastructure and public projects. consequently, they lacked the sophisticated equipment and facilities as well as the highly trained personnel generally found in larger cities. under such circumstances, it is not surprising that most of the recent outbreaks of e. coli o :h have occurred in municipalities with small water systems. (nadakavukaren, : ) . walkerton was further prone to e. coli o :h contamination because of the costs it had to assume in relation to privatized laboratory testing of water (previously done by the provincial ministry of the environment). the need to pay for private water testing was especially difficult for the town of walkerton. to compete with other small towns in luring new residents, business and industry, inexpensive water is a major selling point, and walkerton offered the cheapest water rates in the area even though its utility company was losing money (perkel, : ) . therefore, any additional financial costs associated with the water monitoring functions (such as privatized water testing) could not be easily assumed by the walkerton public utilities company (puc). furthermore, as part of ontario's neo-liberal initiative to cut public spending, the number of inspections conducted by the ministry of the environment, the number of site visits and other contacts between moe officials and municipal water officials, all decreased dramatically (walkerton inquiry, ) . for example, from to , annual planned inspections for the walkerton region decreased from to , the number of actual annual site inspections decreased from to , while the amount of employee resources expended on municipal water requirements decreased from . % to . % (on a province-wide scale, the number of inspections fell by %)(walkerton inquiry, ). a dynamic and diverse place, southern ontario defies simple description, but a number of aspects are worth noting. in socioeconomic terms, it is an industrial center, dominated by automobile and related industries. it is home to more than a quarter of canada's population, clustered in cities like hamilton, london, windsor, kitchener and, to a rapidly increasing extent, the greater toronto area (gta). the sprawling gta exerts a shadow effect over much of southern ontario, driving development patterns and real estate speculation hundreds of kilometers away. in turn, its economy is shaped by close proximity to some of the most populous regions of the united states. the wide ranging environmental impacts of southern ontario's development have been well documented; loss of high quality farmland in the niagara peninsula, contamination of the great lakes and other waterways, increasing incidence of smog, habitat fragmentation and loss of biodiversity are a few of the many problems. (u.s. epa and environment canada, ) . in recent years, some of southern ontario's most bitterly contested development battles have centered around land uses on urban-rural fringes, pitting forces of sprawl against conservation values. diminishing physical and psychological boundaries remain between the highly urbanized areas and the prized rural landscapes and cottage country. in between, the ex-urban areas are in transition, in some instances clinging to rural character and in others imminently suburban. on a typical journey across this increasingly ex-urban landscape, one would see aggregate pits, horse farms, touristy villages, densely developed subdivisions, factories, brownfields, "estate" properties, woodlots, wetlands, moraines, watercourses and lakes. the geologically and ecologically significant niagara escarpment, some million years old and more than km long, crosses the length of southern ontario, defining and highlighting much of the landscape. (niagara escarpment commission, ) . although development-driven politics have tended to dominate land use planning processes in southern ontario, a mix of environmental laws, policies and practices have ensured a measure of protection. a system of conservation authorities, historically created to address flooding risks, manage many of the river areas. the niagara escarpment commission is responsible for planning and development control along the areas close to the escarpment. a variety of local, national and binational initiatives seek to protect and remediate great lakes waters. growing realization about moraine destruction has led to new greenbelts and other policies limiting development in areas of prime concern. the strategic location of the moraines, just north of toronto's city limits, and their hydrological importance as the "rain barrel" for millions of people helped make one initiativeprotection of the oak ridges morainea galvanizing and consciousness raising ecological issue. while not unequivocal environmental victories, the oak ridges battles have helped foster a broader and more regional view of water resources (oak ridges moraine, ; oak ridges moraine foundation, ) . the ongoing environmental challenges in southern ontario continue to exceed the capability of current management and planning practices. even when the more conventional planning practices such as official community planning processes, land use planning regimes and environmental assessment are augmented by additional protection regimes (such as greenbelts, the niagara escarpment commission, moraine protection measures, conservation areas or trusts) the southern ontario landscape continues to be transformed and urbanized at a rapid and largely uninterrupted pace. the change is particularly evident when considered from a cumulative perspective and the landscape is compared to even the fairly recent past. valuable natural features, resources and systems remain at risk as the transformation unfolds. canada's largest city extends over a vast area, but for most of toronto's history it was known primarily for its setting on lake ontario. many of its current ecological issues continue to concern, directly or indirectly, the lake and its associated waterways. toronto's immediate waters are now synonymous with pollution; its beach waters are hazardous and its surviving rivers and creeks are long-term restoration projects. toronto's drinking water comes from lake ontario via elaborate water treatment systems. water supplies for its suburban and ex-urban areas, however, are more likely to come from groundwater, unless costly and ecologically risky "big pipes" draw the water from the great lakes and transport it to considerable distances. with lake ontario deeply embedded in toronto's identity, it is not surprising that its headwaters tend to be less well known. toronto's waterfront has been the subject of numerous campaigns and planning exercises, most notably a royal commission chaired by former mayor david crombie, which culminated in the early 's (government of canada, ). the crombie commission received national attention and reflected at the time some leading edge thinking about ecologically enlightened planning for water. it advocated a watershed-based approach and went beyond a toronto-centric perspective. as an educational initiative, it made an important contribution in encouraging a more regional-based dialogue on development and restoration, situating the challenges facing toronto's waterfront in a wider perspective. the still largely rural region known as headwaters country lies directly north of the greater toronto area (fig. ) . the contrast in landscape between the two regions is striking, although it is diminishing through sprawl and the expansion of transitional ex-urban areas. the relationship between the two regions raises many questions about urban/rural relationships and sustainability at local, regional and trans-regional scales. headwaters country, comprised of two counties -dufferin and caledonfeatures the origins of four substantial waterwaysthe grand, humber, nottawasaga and credit rivers (fig. ) . it lies just north of the gta, but its ecological significance for canada's most populated region is generally under-appreciated. despite its ecological aptness, the name "headwaters country" is unofficial and used mainly for tourism purposes. in physical fig. . rivers of headwaters country. terms, the region still presents a striking contrast to the highly developed gta, with extensive rural areas, small towns and villages. its inhabitants include many prosperous retirees and urban commuters, but the economy of the region is still based largely on agriculture and related industries. important secondary industries in headwaters country include tourism, recreation and crafts. agriculture remains the largest industry and employer in dufferin county and is equally important in the rest of headwaters country. some of the most contentious environmental issues revolve around the tension between the traditional agricultural economy and newer development trends and impacts. global market forces are pressuring the region's farmers to find new efficiencies, economies of scale, technologies and vertically integrated operations (omafra, a) . the trend to larger, factory farms is strong, conflicting with ideals about traditional farming. factory farms typically demand a large and continuous supply of water to maintain their operationsmuch of it drawn from aquifers. meanwhile, large agribusiness can reduce local autonomy and bring increased vulnerability to globally determined commodity prices. in ontario the percentage of rural non-farm inhabitants continues to grow; one estimate claims that it grew by nearly % in the to period (omafra, a) . rural newcomers often have limited understanding of modern agriculture, and their lifestyles and expectations may clash with those of neighboring farmers. many parts of southern ontario that were rural until recently now feature an uneasy mix of livelihoods, lifestyles and expectations. municipalities are taking planning measures to address impacts of farming that were more acceptable in a rural context but less so in an ex-urban one. these include, for example, nutrient management plans (including adequate manure storage facilities), moratoria or limits on the construction of new livestock barns, and minimum distances between land uses (omafra, ) . meanwhile, farm lot severances are one of the most contentious issues, since they can bring much-needed income to farmers but also tend to fragment landscapes and facilitate sprawl. as agriculture intensifies in scale and is combined with other activities and hazards, groundwater risks can increase. contamination can result from point sources such as landfills, distributed sources such as fertilizers, or line sources such as road salt. hazards may be surface (e.g. direct runoff into watercourses or spills infiltrating into the ground) or sub-surface (e.g. septic tanks, underground storage tanks, improperly sealed abandoned wells or buried waste producing leachate) (howard, ) . sinton et al. ( ) showed that viruses are much more readily transported through sub-surface substrata than are bacteria. in the united states, the epa estimates that agricultural runoff from animal factories and traditional farms are the leading source of water pollution in that country. the practice of "sludging" is increasingly prevalent in headwaters country. it involves the spreading of partly treated human waste on farmers' fields as a form of fertilizer. most of the sludge is trucked in from the gta and applied to fields at little or no direct cost to local farmers. sludging may be a quick fix both from the perspective of urban waste managers and farmers, but it can entail significant ecological and human health risks. the longer-term effects of the pharmaceutical content of the sludge are worrisome and may pose another hazard to groundwater systems, but the full effects may not be apparent for many years. finally, although the region is largely defined by water, the full hydrological picture is not well known, either in terms of quantity or quality. the groundwater resources are typically surveyed only when required and paid for as a direct cost of development processes. with water extraction and export on the increase, and not tightly regulated, groundwater levels may be lower than believed, making assumptions about regeneration speculative at best. compared to the gta, headwaters country is popularly perceived as a quiet, rural hinterland. the reality is quite different and the speed of cumulative change is dramatic, with impacts that may or may not be perceived, assessed or managed. sprawl continues despite greenbelts and moraine protection, leapfrogging over urban/rural boundaries as new subdivisions appear. farmland is lost through severances or conversion, in some cases replaced by large residential estates and in many instances further fragmenting habitats and ecological corridors. moraines are depleted or damaged by development. groundwater resources are depleted or neglected. big pipes traverse regions, distorting regional hydrological systems. rivers and sub-watersheds are stressed. factory farming intensifies contamination risks. overall, cumulative impacts at the regional level are insidious, obscured by the urban/ex-urban/rural dynamic. urban/rural boundaries have significance at administrative, geographic and psychological levels, but in an ecological sense these boundaries exist to a diminishing extent. what is assumed to be an urban shadow, with incremental influence over the hinterland, is in practice a more rapid, synergistic process of regional transformation. in this regard, the rural image that is cultivated in places like headwaters country can backfire, reinforcing visions of an undisturbed and salubrious setting even as irreversible change occurs. with the gta as the main driver of change in headwaters country, and with global economic forces also exerting a strong influence, a number of unsustainable patterns are unfolding upstream and downstream, and the ability of ea and planning processes to intervene and influence is limited. this is due partly to the legal planning arrangements in headwaters country (dufferin county, for example, has official community planning at the municipal level, but no official planning at the regional or county level). it is also due partly to the general under-use of regional level tools such as cumulative effect assessment or strategic environmental assessment. the recent history of southern ontario demonstrates that virtually no region, area or water resource is fully protected from sprawl. history also reveals misplaced confidence in conventional approaches to planning, with the cumulative picture consistently ignored or underestimated. there are strong trends but also key uncertainties driving southern ontario's future; continued strong population growth and sprawl appear to be inevitable for the foreseeable future. the full impacts of sprawl can be unpredictable, cumulative and insidious. vulnerability to pathogens adds further complexity and unpredictability. the ex-urban/rural/urban relationship, along with global forces, thus creates a dynamic that enables a variety of impact scenarios to unfold quietly and cumulatively. greener futures are also possible for headwaters country, but would require a shift to more explicitly ecological forms of planning and development. in support of such a shift, the use of advanced forms of ea would need to be normalized. this might include rigorous approaches to cumulative assessment, use of scenarios and regional visioning exercises, disaster incubation analysis and strategic, regional level ea. more routine approaches to ea, combined with controversial exemptions of undertakings from rigorous ea, tend to merely reinforce the current path of development. the experience of environmental assessment (ea) has provided abundant evidence that impacts frequently originate from sources and pathways that are difficult to detect, assess and manage. even as the art and science of ea continue to be refined, impacts are to a great extent becoming more difficult to detect and studyoriginating, accumulating and playing out in ways that undermine common assumptions. at the regional scale, the urban/ex-urban/rural dynamic exemplifies the complicated challenge of perceiving impacts early enough to influence their timely management. in this dynamic, when rapidly growing urban centers exert inordinate development pressure, sprawl transforms rural places to ex-urban, and less visible but insidious impacts unfold in remaining rural areas. in a context of increasingly globalized economic activity, this unfolding dynamic increases the challenges of cumulative impact assessment and management at the regional scale. the sustainability challenges facing rural and ex-urban places have been well documented in a variety of literature (for example, audirac, ; daniels, ; duany et al., ; nelson, ; storper, ) . in this article we have examined two types of changean area level disaster and the specter of transformative regional changein rural and ex-urban settings in southern ontario. in both cases, water systems, one underground and the other watershed-level, figure prominently. both cases underline the need for more enlightened and sophisticated approaches to assessment and planning. at present, there is relatively little use of longer-term, wider scale and preventive approaches to planning for water in southern ontario, but accumulating experience with water crises of various scales should stimulate increased experimentation. we have argued here that the foundations of improved practice include: ) use of disaster incubation analysis to consider a fuller range of present and historical risk factors; ) increased use of advanced ea tools such as cumulative effects assessment (cea) and strategic environmental assessment (sea), and use of scenario planning to detect weak signals of problematic development trends, cumulative effects and future risk factors. in sum, both the walkerton and headwaters country cases reflect the shift from the standard perspective that impacts arise primarily from primarily local human activities and development processes to more of a pathogen-driven view of impacts in which they originate from dispersed and insidious sources, not only accumulating but also incubating in ways that are difficult to perceive and monitor. they reflect the increasing vulnerability of rural and ex-urban places to an intensifying urban shadow along with global forces. the resulting challenges are complex, transjurisdictional and multi-scalar. the lurking risks and cumulative changes are compounded by incomplete approaches to assessment, underreporting, latency of risk phenomena and decoy issues. in the particular case of southern ontario, persistent urban sprawl ensures continued challenges. ea, in its advanced forms, when applied in a consistent and rigorous fashion and supplemented with innovative approaches such as those informed by disaster incubation analysis, could make a substantial difference and nudge the urban/ex-urban/rural dynamic toward a different pattern of planning, decisions and outcomes. disaster and the political economy of recycling: toxic fire in an industrial city a socio-ecological autopsy of the e. coli o :h outbreak in walkerton rural sustainable development in america katrina: the public transcript of 'disaster' waiting for crisis: regulatory inaction and ineptitude and the guadalupe dunes oil spill ticking bomb? toronto star epidemiological study designs context dependency and risk decision making explaining choices among technological risks the chronic technical disaster: toward a social scientific perspective communities at risk: collective responses to technological hazards when city and country collide: managing growth in the metropolitan fringe secret agents: the menace of emerging infections suburban nation: the rise of sprawl and the decline of the american dream disaster reduction: the importance of adequate assumptions about social organization communities at risk: collective responses to technological hazards contamination, corrosion and the social order: an overview research in social problems and public policy bureaucratic slippage and failures of agency vigilance: the case of the environmental studies program the coming plague: newly emerging diseases in a world out of balance. n.y.: penguin; . government of canada. regeneration: toronto's waterfront and the sustainable city. final report of the royal commission on toronto's waterfront isolation and characterization of viruses related to the sars coronavirus from animals in southern china expert testimony at the walkerton inquiry. walkerton ontario: publications ontario: ministry of the attorney general emerging infections: microbial threats to health in the united states. washington: national academy of science denaturalizing disaster: a social autopsy of the chicago heat wave a socio-historical interpretation of toxic waste sites: the case of greater boston the two faces of toxic waste: trends in the spread of environmental hazards the new killer diseases: how the alarming evolution of germs threatens us all factory farming: an issue affecting human health, the environment, workers, and animals sars virus infection of cats and ferrets nature's temporalities and the manufacture of vulnerability our global environment: a health perspective characterizing exurbia niagara escarpment commission. niagara escarpment plan when water kills: the dangerous consequences of factory farming are being felt all across the country farming and rural affairs). task force on intensive agricultural operations in rural ontario consultation. government of ontario discussion paper on the intensive agricultural operations in rural ontario. government of ontario well of lies: the walkerton water tragedy maintaining drinking water quality, lessons from the prairies and beyond anchor books doubleday when water's not well: contaminated well water is linked to gastrointestinal problems justice and the geography of hurricane katrina noticing gender (or not) in disasters the regional world: territorial development in a global economy the organizational and interorganizational development of disasters implementing indicators : a technical report the challenger launch decision: risky technology, culture and deviance at nasa brief of part one report of the walkerton inquiry he has years of experience with ea as a researcher, educator, practitioner and consultant. his previous articles in eia review appeared in his research interests include environmental disasters, environment and health, environmental sociology and preventive engineering the authors wish to thank the social sciences and humanities research council of canada for its financial support. thanks also to an anonymous reviewer for a number of very helpful comments and suggestions. key: cord- - zg q b authors: weng, chih-huang title: water pollution prevention and state of the art treatment technologies date: - - journal: environ sci pollut res int doi: . /s - - - sha: doc_id: cord_uid: zg q b nan this special issue (si) of environmental science and pollution research (espr) includes a collection of peer-reviewed articles relating to water quality and toxicity risk assessments, ecosystem protection, groundwater contamination assessment, soil and sediment remediation technologies, water treatment technologies, climate change adaptation and mitigation strategies, and control of carbon intensity that were formally presented at the th international conference on water resources and environment (wre ) , the th international conference on water resources and environment (wre ), and the st international conference on advances in civil and ecological engineering research (aceer ). wre was held in i-shou university, kaohsiung, taiwan, from july th to st, . wre was held in macau university of science and technology, macao, china, from june th to th, . wre conference started in , when the first wre was held in beijing. as an annually held conference, the upcoming th wre conference is prescheduled held in tokyo, from rd to th, ; however, due to the outbreak of covid- and for the safety of participants, wre will be held online via virtual presentation (http://www.wreconf.org/index.html). the wre conference highlighted the needs to maintain the sustainability of indispensable water resources under increasing uncertainty and to protect the fragile water environments under the growing concern of intensive use of water we are facing today. while civil engineering provides us a better living environment, there is a need not only to protect our fragile environment but also to ensure it is sustainable for future generations. thus, the initiation of aceer is to emphasize links between civil engineering research and knowledge of ecological/environmental issues. the nd aceer will be held on october th- rd, , in beijing, china (http://www.aceerconf.org/index.html). the present si of espr was guest-edited by professor chih-huang weng (i-shou university, kaohsiung, taiwan). the papers selected in this si were based on the ones originally presented at the conference; however, having gone through the regular peerreview process of espr, the contents of those papers have been changed. a brief highlight of the papers included in this si is as follows: & constantly consumption of caffeine around the world has led to increase in caffeine concentration in water bodies and agricultural soil, and thus the impact of caffeine on the environment should not be ignored. korekar & yeh et al. ( ) demonstrate that the synergistic heavy metal contamination degree of an industrially affected river and its adverse biological effects can be assessed by employing the heavy metal pollution index, the degree of contamination index, the contamination factor, the index of geo-accumulation, and hazard quotients. this assessment practice can be quite useful to provide a reference for establishing river pollution control and management strategies. & with the help of ionic tracers and employing the spiraling curve characterization approach and the michaelis-menten equation, song and song ( ) revealed that the dynamic absorption characteristics of no -n in a rural-urban ecotone channel is slightly influenced by the hydrological factors, but significantly influenced by the geomorphic features of the channel. & on the basis of a -year dataset of eleven cities in the songhua river basin, wanhong et al. ( ) ultimately developed two econometric models to quantify the indust r i a l w a s t e w a t e r d i s c h a r g e a l l o w a n c e a n d ecocompensation of the investigated cities, respectively. they suggested that the ecosystem can be protected via an emissions trading program, which permits the wastewater discharge allowance to trading market. & peng et al. ( ) found that poly-γ-glutamic acid (γ-pga), an environmentally friendly washing reagent, is effective in removing heavy metals (cu, zn, ni, and cr) from metal-contaminated farmland soil. the efficacy of metal removal in the field pilot-scale tests is mainly governed by the order of γ-pga concentration, washing time, liquid/soil ratio, and rotational speed. & lee and lin ( ) provide relationships between integrated vulnerability (biophysical and social) and personal ecological footprint of taipei (urban) and yunlin county (rural), taiwan, for governments and communities to establish implementation strategies in risk areas to adapt and mitigate to climate change. & the carbon intensity of a country is closely related to its economic system. based on a selection of countries and application of endogenous growth model, wang et al. ( ) revealed that countries with high or medium high income show convergence of carbon intensity; however, countries with medium or low income exhibit insignificant convergence tendency in carbon intensity. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. he serves as the editor, editorial board member, and guest editor of several academic journals, such as agricultural water management, coloration technology, environmental geochemistry and health, and environmental science and pollution research. he has also organized and chaired several international conferences. his main research interests focus on using advanced oxidation processes and adsorption for the treatment of wastewater and bacteria inactivation, ground water modeling, and application of electrokinetic technologies to soil remediation/sludge treatment/activated carbon regeneration. biosynthesized fe-and ag-doped zno nanoparticles using aqueous extract of clitoria ternatea linn for enhancement of sonocatalytic degradation of congo red comparative toxicity of five dispersants to test organisms at different trophic levels: platymonas helgolandica removal of polycyclic aromatic hydrocarbon (pah)-contaminated sediments by persulfate oxidation and determination of degradation product cytotoxicity based on hepg and zf cell lines assessment of background levels and pollution sources for arsenic and fluoride in the phreatic and confined groundwater of xi'an city occurrence, fate, persistence and remediation of caffeine: a review application of progressive freezing on forward osmosis draw solute recovery vulnerability and ecological footprint: a comparison between urban taipei and rural yunlin impact of carrier on ammonia and organics removal from zero-discharge marine recirculating aquaculture system with sequencing batch biofilm reactor (sbbr) remote sensing-based water quality assessment for urban rivers: a study in linyi development area enhancement of sonocatalytic degradation of organic dye by using titanium dioxide (tio )/activated carbon (ac) derived from oil palm empty fruit bunch a field pilot-scale study on heavy metal-contaminated soil washing by using an environmentally friendly agent-poly-γ-glutamic acid graywater treatment technologies and reuse of reclaimed water for toilet flushing fish habitat evaluation based on width-to-depth ratio and eco-environmental diversity index in small rivers spiralling curve characterization of nitratenitrogen absorption in a channel at a rural-urban ecotone in northeast china convergence of carbon intensity: a test on developed and developing countries industrial water pollution and transboundary eco-compensation: analyzing the case of songhua river basin assessment of heavy metal contamination and adverse biological effects of an industrially affected river hydrogeochemical and isotopic characteristics of groundwater in xinchang preselected site and their implications