7f-8^0 CIVIL ENGINEERING STUDIES SANITARY ENGINEERING SERIES NO. 18 FACTORS INFLUENCING FREE-LIVING NEMATODES IN WATER SUPPLIES ^tOAN COPY feOU*G tu UNiVE 15 1301 WEST SPFUNGF UBBANA,i^ N0SS PROGRESS REPORT: September 1, 1961 Through August 31, 1963 By R. S. ENGELBRECHT R. I. DICK M. R. MATTESON Property of COLLEGE OF ENGINEERING DOCUMENTS CENTER UNIVERSITY OF ILLINOIS 157 GRAINGER LIBRARY 1301 WEST SP D AVEHUE URBANAf'?£B?ltS& B J.a01 USA DIVISION OF WATER SUPPLY AND POLLUTION CONTROL U. S. PUBLIC HEALTH SERVICE RESEARCH PROJECT WP-00047 JWSENTS CENTER SlOlS RRY AVENUE ebi USA DEPARTMENT OF CIVIL ENGINEERING UNIVERSITY OF ILLINOIS URBANA, ILLINOIS SEPTEMBER, 1963 PROGRESS REPORT September I, 1 96 1 through August 31, 1 963 FACTORS INFLUENCING FREE-LIVING NEMATODES IN WATER SUPPLIES University of Illinois Urbana, I 1 1 i noi s Department of Civil Engineering R„ So Engelbrecht R. I. Dick N. Chaudhuri R. H. Siddiqi K. Y. Bal iga Department of Zoology Mo R„ Matteson Co Eo Robbins D. L, Batch September 10, 1963 Research Grant WP-OOO^y Division of Water Supply and Pollution Control Public Health Service Department of Health, Education and Welfare Summary Statement! Major accomplishments to date have been made in the development of techniques to collect representative water and benthos samples, to extract nematodes from water and mud, and to count and identify nematodes. Surveys of the sources of nematodes in surface waters have shown waste treatment plants to be the origin of high nematode populations, while lower concentrations are contributed by surface and subsurface drainage. Extensive studies have been made of the factors governing the number of nematodes carried by a stream as a result of land drainage and of the relative effect of urban and rural drainage. Waste treatment plants have been thoroughly studied because of their importance as sources of nematodes. Effluents from trickling filters have been found to con- tribute a significantly greater number of nematodes per unit volume than activated sludge effluents. The organisms apparently do not survive in anaerobic environments. Nematodes have been found to persist for con- siderable distances downstream from waste treatment plants. Their per- sistence has been found to be related to the stream flow rate, temperature, changes in temperature, and to the heterogeneity of the nematode population. In order of decreasing frequency of occurrence, D? ploqaster , Pi plogaster - oides , and Rhabdi 1 1 s have been found to be the predominant nematode genera associated with surface waters. Laboratory studies have been conducted to determine the effect of environmental conditions upon the survival and growth of pure axenic cultures of Diplogaster nudicapi tatus and Pi ploqasteroides sp. The effect of agitation, pH , temperature, and crowding have been observed. INTRODUCTION The absence of appreciable work on free-living nematodes in the past has made it necessary to conduct surveys on the occurrence of the organisms, as well as develop techniques for collecting, separating, counting and identifying them. As a result of extensive studies on waste treatment plants, urban and rural surface runoff and streams, the relative nematode contribution from each was determined. In addition, the persist- ence of nematodes in streams was investigated. Attempts to use field data to determine the relationships between basic chemical, physical and biological conditions and the occurrence and persistence of nematodes were generally not successful. This necessitated the institution of closely controlled laboratory studies to learn the general characteristics of the organisms and to investigate the influence of environmental con- ditions on their survival and growth. FIELD AND LABORATORY PROCEDURES The majority of the previous studies on nematodes has been with parasitic forms. Study of the work of these researchers has been helpful, but their techniques were often not suitable for use with free-living nematodes obtained from waste treatment plants, streams and bottom muds. Thus, it has been necessary to devise new techniques, to collect, to separate, to count, and to identify nematodes from such environments. Sampling and Extraction Techniques Grab sampling techniques were employed in early stream and waste treatment plant studies. Data on nematode populations obtained from these samples were extremely erratic. These data gave an order of magnitude, but no true quantitative measure of nematode populations was possible. A study of composite sampling was undertaken to overcome this apparent tendency of nematodes to be very irregularly dispersed in flowing water. Four 1-liter samples were collected from the final settling tank of an activated sludge plant. Three of the samples were placed in three individual carboys, and one was retained in a 1-liter bottle. This procedure was then repeated 10 times to give three 10-liter composites and ten 1-liter grab samples. The sampling was accomplished over a period of 10 minutes. The resulting nematode concentrations, as shown in Table I, illustrated the wide variation found between individual grab samples, and demonstrated the advantage to be realized by collecting large composite samples. Although the counts ranged from 1500 to 4780 nematodes per gallon in the individual grab samples, the average count agreed reasonably well with that observed in the three composite samples. The variation between the three composite samples was comparatively slight. From a statistical analysis of these and other data, it was determined that the most practical method of obtaining reliable nematode counts would be by collecting 5 gallon composites consisting of 26 indi- vidual grab samples over a period of 20 to 30 minutes. Several methods for extracting nematodes from water, including plankton nets, sieves, and membrane filters, were studied. The nets and sieves gave incomplete or no recovery of nematodes, while complete removal of nematodes was realized using five micron membrane filters. This work substantiates that of Chang and his coworkers (1). The customary bottom samplers such as the Ekman Dredge and Petersen Dredge together with coring devices and a special sampler devised by the Illinois State Water Survey were used during the first phases of the bottom sampling program. It was felt that none of these devices ob- tained a suitably undisturbed sample from the stream bottom. The bottom sampler which was final devised for use in these studies was constructed by merely cutting a one gallon oil can in half longitudinally. The re- sulting semicircular piece was pushed into the stream bottom by a rotating movement with the hands to obtain an undisturbed portion of the stream bottom. The limitation of this technique to shallow streams was recog- nized, but this has not detracted from its usefulness in the present study. Considerable work has been necessary to develop a technique to extract nematodes from bottom samples for counting and identification. The technique which was ultimately developed is an adaptation of the centrifugal flotation technique reported by Caveness and Jensen (2). A five gram bottom sample was placed in a centrifuge tube, and the tube was filled with a sugar solution with a specific gravity of 1.1. The tube was then centri- fuged for three minutes at 4800 rpm. The supernatant sugar solution con- taining the nematodes was then carefully poured into a liter of water. Digitized by the Internet Archive in 2013 http://archive.org/details/factorsinfluenci18enge TABLE I NEMATODE COUNTS IN TEN INDIVIDUAL 1 -LITER GRAB SAMPLES SAMPLE NO. 1 2 3 4 5 NEMATODES/GAL. 4560 2480 4380 3980 4780 AVERAGE 3380 SAMPLE NO. 6 7 8 9 10 NEMATODES/GAL. 3640 1500 2340 1800 4300 NEMATODE COUNTS IN THREE SEPARATE 10-LITER COMPOSITES SAMPLE NO. 12 3 NEMATODES/GAL. 3740 3540 3300 This procedure was repeated three times. Nematodes were then separated from the diluted sugar solution by the same technique described above for water samples. Counting Techniques The procedure used in couting nematodes has been to pass an aliquot through a membrane filter, wash the nematodes from the filter into a counting dish, and examine the contents under a dissecting micro- scope. Difficulty was encountered with the Syracuse and Petri Dishes conventionally used as counting dishes because the sloped sides compli- cated counting at the perifery. More suitable counting dishes were devised by attaching Plexiglas plates, scribed with grids, to k.S inch diameter Plexiglas cylinders. It is often necessary to make separate counts of living and dead nematodes. It is not possible to make this distinction on the basis of movement, because nematodes which have been subjected to adverse con- ditions may remain immotile for long periods of time even after being removed from the harsh environment. An original staining procedure was developed to give rapid and positive differentiation between living and dead nematodes. The procedure involves the use of Eosin-Y dye which was found to readily stain dead nematodes while leaving live nematodes un- stained. Prolonged exposure to dye and/or high dye concentrations results in the staining of live nematodes and in the death of nematodes due to the toxicity of the dye. The effect of contact time and dye concentration upon the survival of nematodes is illustrated in Figure 1. It was necessary to develop a technique using a dye concentration and staining time which would assure that no nematodes were killed by the dye and that only dead nematodes became stained. It was found that 30 minutes contact with 0.67 percent Eosin-Y dye stained 99-5 percent or more of the dead nematodes, did not kill nematodes, and did not stain live nematodes. This work was done with two pure species of nematodes, Pi plo - gasteroides , and Diploqaster nudi capi tatus which were naturally or heat killed. The technique was found to work equally well using mixed popula- tions of nematodes such as obtained from waste treatment plants, surface streams or the benthos. Investigators, using stains other than Eosin-Y (3) (*0 (5) , have often reported inconsistent results when attempting to use a variety of death inducing agents. A variety of lethal agents including sulfuric \ a UJ Z 3 (0 cc o Q UJ o UJ Of - Q < z < z < // \ HI 1- y- o (0 (0 v 1 z \l 3 5 / o *- / < <* / UJ < / Q 0. / Q UJ Z < »- (0 z 3 UJ > o J 1 1 L 1 1 1 , - o CD CO o o lO LU UJ O ? UJ — » h- cr 3 C0 O I 6 0~ U- CO UJ ro o Q UJ O 2 H- fee H o UJ UJ O U- u_ z CM UJ 2 (D o ■z. O _J UJ >- o * Q u_ O O O 00 O CO O O CM !N30U3d acid, sodium hydroxide, ethyl alcohol, freezing, desiccation, and hypertonic saline and sugar solutions were studied in conjunction with the Eosin-Y staining technique. With proper selection of killing agent concentrations and killing and staining times, the method could be successfully employed for any of the chemical or physical killing agents studied. Recommended killing and staining times to obtain 98 percent or more staining of dead nematodes with 0.67 percent Eosin-Y dye are shown in Table II. It was found that sodium hydroxide concentrations in excess of 1/8 N had to be avoided to prevent destruction of nematodes. In the case of sodium hydroxide and hypertonic sugar or salt solutions, staining performance improved when the death inducing agent was removed before the dye was introduced. Because it is easier to count stained dead nematodes than unstained live nematodes, the staining technique is also applicable when a total nematode count is desired rather than a differentiation between dead and live organisms. In this case, total death is induced by one of the methods considered above, dye is introduced, and a total count is made. A more convenient method is to merely apply an excessive dose of dye, allow the sample to stand overnight, and obtain a total count after the dye has killed and stained all of the organisms. It has been found that the Eosin-Y dye preserves nematodes, and that a sample treated with dye may be held as long as two weeks with no degeneration of nematodes. Nematodes stained with Eosin-Y dye to facilitate counting may later be identified. The Eosin-Y staining procedure does not interfere with the genus identification technique. Identification - Slide Preparation Whenever time permitted, permanent slides were made using specimens from as many samples from the field as possible. The slides usually proved to be excellent for detailed study. The living nematodes were removed from the counting dishes and placed in watch glasses. As little water as possible was carried over during the transfer. They were then fixed by pouring a solution of FAAGO heated to about 50° C over them. FAAGO solution is composed of 90 ml of 50 percent alcohol (methyl), 3 ml of pure glycerin, 2 ml of acetic acid, 5 ml of formalin, and 1-2 drops of osmic acid. The watch glasses were then placed in dust-free containers and evaporation was allowed to proceed until the animals were in a thin layer of glycerin. An additional layer of pure glycerin was then added. They could be stored at this point indefinitely. TABLE II STAINING TIME, KILLING TIME, AND CONCENTRATION OF VARIOUS KILLING AGENTS TO OBTAIN 98% KILL WITH 0.67% DYE Ki 1 1 i ng Agent Concentra - t ion Sulfuric Acid 0.33 N Sodium Hydroxide 0.125 N Absolute 2 ml per ml ethyl alcohol of nematode suspension Freez ing -10° C Desiccation -- Sa 1 ine Solution 3 M (NaCl) Sugar 1 .5 M Solution 1 .0 M Necessity Killing Staining Time of Washing Time to Obtain More off Killing (Hrs.) Than 98% Sta i ning Agent (Hrs.) yes no no 7.00 1.33 0,25 0.50 1 .00 0.33 10.00 0.33 4.00 0.33 yes 10.00 1.00 yes 14.00 0.50 yes 34.00 0.50 Although the worms could be stained following fixation, it was found that their structures were more easily examined in unstained specimens. After placing a small drop of pure glycerin on a slide, one of the fixed nematodes was placed in the drop by means of a very fine glass needle. A drop of glycerin jelly warmed gently until melted was then placed on the warmed slide. After passing a cover slip through steam formed by a container of boiling water, the damp side was placed on the drop on the slide. If the worm was poorly oriented, pressure applied to the cover slip while the slide was still warm would correct the orienta- tion. The formula for making glycerin jelly used for mounting was that of Pennak (Fresh-water Invertebrates of the U. S.) . For a permanent slide, the excess glycerin jelly was scraped away from the sides of the cover slip and a seal of Murrayite was placed around the edge of the slide. Although several thousand slides have been examined, a lmost all have proved to be in three genera: Rhabd i ti s , Pi plogaster , and Pi plo - gasteroides . Occasionally, a slide has appeared on which the nematode has not been placed under any genus. These will be studied further during the coming year. SURVEY OF THE OCCURRENCE OF NEMATOOES Early studies were directed toward an investigation of the major sources of nematodes in streams. The studies served as a background for orientation of the work which has followed, and pointed out the neces- sity of developing the field and laboratory procedures previously discussed The effect of waste treatment plants upon the nematode population of receiving streams was studied by collecting effluent samples and stream samples above, and various distances below, the outfall of five Illinois cities. In each case, the waste treatment plant represented the only source of nematodes in the study section of the stream -- no nematodes were found in the streams above the treatment plants. Significant popula- tions of nematodes were invariably discharged from the plants. Although the concentration of the organisms was observed to decrease as the treat- ment plant discharge proceeded downstream, appreciable nematode populations remained considerable distances below the treatment plants. The occurrence of nematodes in subsurface drainage was investi- gated by studying the discharge from four farm tile drainage areas over a period of ten weeks. Nematode populations were found to vary from zero to about 10 organisms per gallon. Only during periods of high discharge were high concentrations of nematodes found; i.e., 200 organisms per gallon or higher. It was concluded that where appreciable volumes of drainage are involved, subsurface flow can represent a significant source of nematodes. A twenty mile section of the Sangamon River from Mahomet to Monticello, Illinois, was selected to evaluate the contribution of nema- todes from surface drainage. The study section of the river contained no known sources of nematodes other than surface runoff and subsurface seepage The section had a broad, flat flood plain which resulted in extensive flooding. U„S.G„S„ stream gaging stations were located at both the upper and lower ends of the study section. The initial studies indicated that surface drainage maintained the nematode population but did not signifi- cantly increase the nematode concentration in the river. The total number of nematodes at the downstream end of the study section was significantly greater than the population at the upper end because the flow was much higher, but the concentration of nematodes remained nearly constant. A more extensive study of the relationship between runoff and nematode con- centration was later conducted as described below. SANGAMON RIVER STUDY Streamflow and nematode population data collected over an eight- month period from the previously described study section of the Sangamon River were analyzed to determine the relationship between nematode popu- lation and runoff. Nematode population at the upstream and downstream sampling stations are plotted against the corresponding stream flows in Figure 2, and the stage -di scha rge relationships for the two sampling stations are also shown. It was observed that the data could be categor- ized into three different groups depending upon the magnitude of stream-^ flow. Curves have been drawn through the three portions of the nematode data using the method of least squares for best fit. It is seen that the curves are straight lines with different slopes and that they describe a discontinuous function. FIG. 2 VARIATION IN NEMATODE COUNT WITH STREAM FLOW - SANGAMON RIVER 10 o UJ CO CO LJ Q O UJ -J < o 1 1 1 RANGE 2 ■ ■ . t 6 ! i ■ i i /' i - 1 \ s i i i 1 ! 9 ' | t t 1 i X / ; ! i i / i 1 ! j i | i ! y^\ o 5 ... . ,. .] ./ — | W j ■ ; iNGE 3 — H — ! — ^-A — — W R/ — i — \ — J ./ — i 1 / i : 1 : i / \ \ 1 i ; | /♦ i ! 1 ! I 1 1 ' f ! ■ i i i _4 i 1 i ' ! 1 i i i ! 1 i 1 1 y. W i i> i | : 1 / ~1 i /r ' Uf i : ' ! ^ 1 / RANGE 1 ! t • i i o 3 * 1 [ i . 12 i i ! I J k 1 1 ■ j /r^ JMONTICELLO 1 ! i 1 I 1 i 1 . — 1 1 MAHOMET | i ► i Z fee CP UJ I uj 4 < i- (O 1 : i l. . a n - ^ i i I 1 I i i 1 1 10 100 CTDrAU 1000 Fl d\AJ '*' CF 10000 r LUVY m o ** The general equation describing the curves in each of the three ranges is N = k Q. where N = total nematodes per sec. k = intercept on the ordinate when stream flow, Q_, is unity Q. = stream flow in cfs and n = slope of the line The computed values of n and k and the stream flows for the three ranges are shown in Table IN. TABLE II! . VALUES OF CONSTANTS IN THE STREAM FLOW-NEMATODE POPULATION RELATIONSHIP Range Stream Flow n k i n cfs 0.7 1 Q < 100 300 2 1000 > Q > 100 1.1 200 3 Q > 1000 J 40,000 The net increase in nematode concentration (C) for each addi- tional cubic foot of runoff is a'- n It is seen that in Ranges 1 and 3, where n is greater than one, the concentration of nematodes increases with an increase in stream flow, while in Range 2, n is less than one and, thus, the nematode population decreases with an increase in stream flow. It is believed that the population of nematodes in a stream due to seepage and land runoff is related to: (a) The f 1 ushi nq capacity of the seepage flow, overland runoff and the stream itself. Flushing capacity, as defined here, is the capacity of flowing water to scour nematodes from soils. 12 (b) The carrying capacity of the seepage flow, overland runoff and the stream itself. Carrying capacity, as defined here, is the capa- bility of the flowing water to maintain the nematodes in suspension. Study of the shape of the stream channel and the nature of the watershed revealed plausible relationships between flushing and carrying capacity and the nematode population of the Sangamon River. Because nematodes are most abundant in the top few inches of soil, higher nematode populations should be flushed to the river when the scour of the upper soil surface is most efficient. Stream flows less than about 100 cfs are maintained primarily by seepage and thus the nematode concentrations in Range 1 are relatively low. Stream flows greater than 100 cfs are caused by overland runoff, and in Range 2, large numbers of nematodes are flushed to the stream. However, at flows above 1000 cfs, the depth of overland runoff increases causing the efficiency of scour per unit volume of runoff to diminish in Range 3- With regard to carrying capacity, it was observed that changes in slope of the stage-discharge curve corresponded to the points of dis- continuity in the curve describing nematode population as a function of stream flow. Comparison of the stage-discharge relationship with the cross-section of the stream has shown that stream flows of about 100 to 1000 cfs are contained within the stream banks but produce higher velocities than flows below 100 cfs. These high velocities possibly account for the increased nematode populations due to increased carrying capacity and also to increased scour. At flows below 100 cfs or above 1000 cfs stream velocities are reduced and fewer nematodes are carried per unit volume of flow. BONEYARD-SALINE BRANCH DRAINAGE SYSTEM STUDIES The initial studies on the occurrence of nematodes in streams suggested that low concentrations of nematodes in streams are contributed from soil by drainage, that significantly higher numbers are discharged from waste treatment plants, and that nematodes from treatment plants per- sist for considerable distances downstream. The Boneyard-Sa 1 i ne Branch study was undertaken to more closely observe the relative contribution of nematodes from various sources and to study the persistence of the organisms in streams. 13 As indicated in Figure 3» Boneyard Creek flows entirely within the Champa i gn-Urbana , Illinois, urban area. In contrast, the Saline Branch receives entirely rural drainage until it enters Urbana and is joined by the Boneyard. The Urbana -Champa i gn Sanitary District waste treatment plant discharges its effluent below the confluence of the two streams. The Saline Branch has no major tributaries and receives no further discharge of nematodes except surface and subsurface drainage between the treatment plant and its confluence with the Salt Fork Drainage Ditch 15 miles down- stream. This provides an excellent opportunity to study the fate of the nematodes discharged by the treatment plant. Relative Contribution of Various Sources of Nematodes Samples have been collected weekly from the sampling stations indicated on Figure 3 for the past year. Samples were analyzed for various physical, chemical, and biological properties as well as for their nematode content. The average nematode population at each of the stations is indi- cated in Figure k. It is apparent that the Boneyard is relatively free from nematodes in its upper reaches, but that it carries increasingly higher nematode con- centrations as it proceeds through the cities and receives urban drainage. The Saline Branch normally remains essentially free of nematodes until it enters Urbana and receives the impact of urbanization. Only during periods of high runoff, such as in April, 1 9^3 , does the Saline Branch contain a significant nematode population after flowing through the agricultural area above Urbana. The increase in nematode concentration below Station U/2 reflects the larger nematode population discharged by Boneyard Creek into the larger, less polluted Saline Branch. The contribution of nematodes by rural runoff and even urban runoff in this basin was almost insignificant as compared to the contribu- tion from the waste treatment plant. The relative effect of waste treatment plant effluent upon the nematode population in any other stream would, of course, depend on such factors as the size and type of the treatment plant, the amount of dilution available in the receiving stream, the effect of preditors, and the predominance of nematodes from other sources. Persistence of Nematodes in Receiving Stream The tendency for nematodes to remain in the stream below the waste treatment plant is illustrated in the 7-71 mile section between 14 15 rr ,*. ssoil Q , 1 CO i i o Q H L o ! t_ l*"«l * ! 2 ■s. 1 O 1 LU >- CO ■ UJ o < z < CC iCO la j Itseil Q 2 4 o < , | HI s' \ j t CC (- CO x § ! j ^ UJ !$8S3| o O o z < z *N.j_! CO N'N — li K> 6ddl CD y i^n 1 V 3 Q UJ z V EH z>] ' j _l „ \ ! 4- RDSA / \ CO | Ul ' _J 2 o ? - / \ IT) - g z \ r«S o ' \ CD \ ro I V 3i z o » i ~P3ET CO m ' : 1- 1 < I _l 1 3 j Q. l o 1 0. 1 CO i CO UJ ' 1 y o z Ul Q O 2 * * < or 2 lO < 1 (uU) u CO > _j 2 1 ^ UJ UJ — lj z "1V9/ 621 1 CM -J CC i ° (0 UJ < or HONvaa \ CO J- CC < > oavA3Noa \LZZ\ UJ > < CD UJ z o CO _1_ UJ z I regT _l < CO O o UJ tr (0 UJ Q O 6 UJ I- z UJ O o: uj o. DOWNSTREAM SAMPLING STATIONS rate of change remained constant. The curves in Figure 5 indicate that the rate of disappearance of nematodes is directly related to stream temperature. Curve 6, which illustrates a condition in which no nematodes disappeared, was obtained under conditions of complete ice cover. Curve 11, which shows the highest rate of disappearance of nematodes in Group IA, also is the one with the highest water temperature. Occurrence of Nematodes in the Benthos Bottom samples obtained throughout the Boneyard-Sa 1 ine Branch Basin have been studied in an attempt to determine the relationship between the nematode population of the benthos and that in the stream, to evaluate the possible role of the benthos as a secondary source of nematode infesta- tions, and to study the relationship between environmental conditions and the occurrence of nematodes in the benthos. The waste treatment plant effluent has been found to be the source of nematodes in downstream bottom samples. Comparatively few nema- todes have been found in bottom samples taken from the Boneyard and the Saline Branch above the treatment plant. However, no correlation has been discovered between the nematode population of the stream bottom and distance downstream from the treatment plant. A study of the cross sectional distribution of nematodes indicated extreme variation in the bottom nematode populations at individual sampling stations. The bottom population underlying the main body of flow was found to be relatively uniform while erratic results were typically obtained near the stream banks. For example, at station D/l below the waste treat- ment plant, 5 bottom samples were collected across the 40-foot cross section of the stream. Three of the samples obtained from the main flow region of the stream indicated an average nematode population of 8 nematodes per 5 grams of wet sample with a range of only 6 to 1 1 organisms per 5 grams, while populations of 26 and 119 nematodes per 5 grams were observed at the two banks. The high nematode bottom populations near the banks correspond to regions of sluggish velocities with resulting bottom sludge deposits. In addition to their nematode content, bottom samples tA/ere analyzed for C.O.D., volatile matter, nitrogen content, effective size and uniformity coefficient. To date, no definite correlation has been estab- lished between the bottom nematode population and these environmental con- ditions. However, this phase of study is continuing. 19 Identification of Nematodes Nematodes in stream and bottom samples were identified by the techniques discussed previously. With only a few exceptions, nematodes identified from these sources belong to one of the three genera: Pi plo - qaster , Pi ploqasteroi des , or Rhabdi t i s . With the exception of one appearance of Rhabdi t i s all nematodes identified in the samples from the Boneyard and Saline Branch above the waste treatment plant were members of the genera Pi pi oqaster . Immediately below the waste treatment plant, Pi pi oqaster and Pi pi oqasteroi des predom- inated and were identified with equal frequency. Again, Rhabdi t i s was identified only once. Further from the treatment plant, from station P/l downstream. Pi pi oqaster again predominated. On only one occasion was another genera, Pi pi oqasteroi des , found below station P/l. Nematodes belonging to all three genera were isolated from bottom samples. Again, Pi ploqaster predominated. It is seen that Rhabdi t i s is the least common of the three genera. Pi ploqaster appears to be the best adapted to stream environments as it is the genera most commonly found in streams, and it persists for longer distances below the treatment plant. Possibly Pi ploqasteroi des is more exacting in its food requirements, since it appears in significant numbers only immediately below the waste treatment plant. WASTE TREATMENT PLANT STUPIES The Urbana-Champai gn Sanitary Pistrict Waste Treatment Plant utilizes both trickling filters and activated sludge as secondary treat- ment processes. This provides a unique opportunity to study the behavior of nematodes in the two different waste treatment processes utilizing the same raw waste. This ideal arrangement is somewhat impaired by the fact that a portion of the effluent from the trickling filter plant is recycled to the activated sludge process. Samples have been collected weekly during the past year from the raw sewage and from the final effluent of the activated sludge and trickling filter portions of the plant. In addition to determining the nematode population, various physical, chemical and bacteriological analyses have been conducted on the samples. The treatment plant has been found to be a consistent source of high concentrations of nematodes. Analysis of data 20 from September, 1962, through the early part of September, 1963, indicated that the nematode population of the combined effluent from the entire plant exceeded 5000 organisms per gallon 50 percent of the time. The 10 and 90 percentile values were 21,000 and 1000 nematodes per gallon, respectively Some of the extreme nematode populations could be associated with plant operational procedures. The data also showed that nematodes were always present in the raw sewage. The nematode population in the raw sewage ex- ceeded 1700 organisms per gallon 50 percent of the time. The 10 and 90 percentile values were 4700 and 400 nematodes per gallon, respectively. Considerable nematode propogation was found to occur in the biological filters. Figure 6 shows the actual nematode population in the settled waste and the final effluent of the trickling filter process. It can be seen that the nematode output from the trickling filter process in general exceeded the input, normally by a factor of five to ten. Less propogation occurred in the activated sludge process than in the trickling filters. The nematode population of the activated sludge influent and effluent are shown in Figure 7. The high influent concentra- tions reflect the return of final effluent from the trickling filter process to the aeration tanks. While the overall average population of nematodes in the effluent of the activated sludge process exceeded that of the influent, nematode reproduction in the process was not consistent and was much less than in the trickling filter process. Two possible explana- tions for the smaller contribution of nematodes from the activated sludge process may be that nematodes have difficulty propogating in the suspended state, and that nematodes are more efficiently removed in the final settling tank of the activated sludge process than in the final tank of a trickling f i 1 ter plant . The data shown in Figures 6 and 7 indicate lower nematode popula- tion during periods when the waste temperature was below l4 a C and above 20 9 C. A complete analysis of variation in nematode population relative to temperature, waste flow, and other physical, as well as chemical, charac- teristics is being made. While large numbers of nematodes are included with the primary and secondary sludge pumped to the digesters at the plant, the anaerobic environment is not suitable for their growth. Samples obtained from the supernatant of the secondary digesters occasionally contained a few dead nematodes, but live ones were never present. 40,000 21 3 2,000 SEP- 2 4.000 < 6.000 Id Q O H < UJ 8,000 / \ ODE POPULATION I SYSTEM OCT — JUL V NAL EFFLUENT ER D SEWAGE AUG SEP ii M /> / I 40,000 3 2,000 -SEP-- —OCT NOV DEC JAN 2 4.000 » I' H l\ I I / ' / I < I I / ' / I 6,000 Q O < Ld 8.000 i \ L'-N I / \ / \ ' \ I \ ' FIG. 6 VARIATION OF NEMATODE POPULATION IN TRICKLING FILTER SYSTEM FEB MAR -APR MAY JUN JUL i I ' I ' I I I I I I I NEMATODE IN THE FINAL EFfLUENT OF TRICKLING FILTER NEMATODE IN SETTLED SEWAGE AUG 21 SEP 50,000 22 SEPi OCT JUL- - AUG _ SEP J 4 0.000 30,000 2 0,000 < o Id Q O < UJ I 0,000 POPULATION IN M 50.000 4 0,000 30,000 0.000 10,000- 22 FEB - \- . MAR APR NEMATODE IN THE FINAL EFFLUENT OF ACTIVATED SLUDGE NEMATODE IN ACTIVATED SLUDGE INFLUENT MAY JUN JUL- AUG FIG.7 VARIATION OF NEMATODE POPULATION IN ACTIVATED SLUDGE SYSTEM - SEP 23 EFFECT OF ENVIRONMENTAL CONDITIONS UPON SURVIVAL OF NEMATODES In each of the studies discussed above, samples were analyzed for their chemical, physical, and bacteriological properties as well as for their nematode content. Because of the many environmental variables involved, it has not been possible to determine the effect of individual parameters from these data. Thus, it has been necessary to utilize closely controlled laboratory experiments to study the effect of individual envi- ronmental conditions upon nematodes. Pure cultures of two nematode species commonly found in surface waters, Diploqaster nudicapi tatus and Dlplo - gasteroides sp. have been used in these studies. Effect of Temperature Studies were conducted to determine the effect of temperature upon the motility of the nematodes and to observe the lethal effect of extreme temperatures. Effect of Temperature on Motility Cultures of D i plogasteroides sp. and Diploqaster nudicapi tatus were suspended separately in a phosphate buffer solution at pH 7> and exposed to temperatures varying from 5 s to 40" C. At the end of a 30- minute contact period, the numbers of active nematodes were determined. Pi plogasteroides sp. was most active at 20 s C, while Diplogaster nudicapi - tatus preferred a temperature of 25° C. Both species remained active within a temperature range of 1 5° C - 30° C. At temperatures above or below this range, the nematodes became sluggish or their movement ceased alto- gether. Since immobile nematodes would be unable to copulate, it might be expected that reproduction would cease at adversely high or low tempera- tures. This supposition was verified in later growth studies. Thermal Death Kinetics Nematodes suspended in phosphate buffer solution at pH 7 were subjected to constant temperatures in the range between 40 e C and 50 s C. The rate of death as a function of time was observed by means of the Eosin-Y dye staining technique previously discussed. It was observed that thermal death kinetics could be described by a first order reaction; i.e., the death rate was directly proportional to the number of live nematodes remaining. Diplogaster nudicapi tatus was 2k observed to be more resistant to extreme temperature than was Pi plogaster - oides sp . At a given temperature, it took a greater period of time to kill Diplogaster nud icapi ta tus . Effect of pH Nematodes were suspended in solutions buffered to various pH values from 2 to 12 and the time required for complete immobilization, 90 percent kill, and 100 percent kill was observed. The motility and reproduction of D i ploqasteroi des sp. was impaired at hydrogen ion concen- trations above pH 9 and below pH 5- The pH range in which propogation was possible with Diplogaster nud icapi tatus was from 5 to 8. With both species, immobilization was more rapid at alkaline pH values than under acidic con- ditions. Pi ploqasteroi des sp. was found to be more resistant to pH variations than Diplogaster nudicapi tatus . Similar studies were conducted utilizing synthetic culture media buffered to various pH values. The synthetic medium employed was that developed by Chang (6) which consists of antibiotics, nutrients, and a non-specific assortment of organic materials. The desirable pH ranges for growth of Piploqaster nudicapi tatus and Pi plogasteroides sp. were again found to be 5 to 8 and 5 to 9, respectively. Immobilization by adverse pH values outside of these ranges was produced more slowly in the buffered synthetic medium than in the plain buffer solutions. GROWTH STUOIES It has been necessary to investigate the life history and growth characteristics of nematodes to permit interpretation of the field surveys. Studies have been made of the growth characteristics under ideal condi- tions and the effect of crowding, shaking, temperature and pH have been considered . All of the growth studies were conducted in T-type culture flasks as manufactured by E. H. Sargent & Company. T - 1 5 and T-30 flasks with areas of 15 and 30 sq cm, respectively, were used. "Chang's medium" was used throughout the study and Sorensen's buffer was used to adjust and maintain pH . In each study 12 to 15 identical flasks were prepared by adding the desired amount of medium and a few adult nematodes from pure cultures of Pi plogasteroides sp. and Diplogaster nudi capi tatus . Nematode 25 populations were then monitored by periodic counting. Nematode populations up to 600 in the T-15 flasks or 1000 to 1200 in the T-30 flasks could be counted directly in the flasks without difficulty. Higher populations had to be removed from the flasks for dilution before counting. Typical Growth Pattern D i plogasteroides sp. has demonstrated an egg-to-egg period of 7 to 8 days, while for Diplogaster nudi cap! tatus this period has been k to 5 days. Average initial rate of reproduction has been found to be three nematodes per adult per day, with the bulk of reproduction occurring over a period of k days. A modified technique has been developed for studying the typical growth pattern. The above results of the original growth study are to be verified by this technique before they are released. Effect of Area Upon Growth Pi plogasteroides sp. was observed to have a higher growth rate when grown in containers with greater surface area. When 1.1 ml of medium was placed in T-15 flasks, and 1.1 ml of medium and 1.1 ml of distilled water was placed in T-30 flasks, the larger flasks gave growth rates 1.2 times those of the smaller flasks. These results were verified by using Petri dishes with an area of 57 sq cm as culture dishes. The Petri dishes gave growth rates 1.6 times those of the T-15 flasks. Area was not found to influence the growth rate of D? plogaster nud i capl tatus . Effect of Agitation Upon Growth Agitation of nematode cultures was accomplished by shaking a raft holding T-15 culture flasks. The raft moved through k5 three-inch strokes per minute. Agitated cultures of D i ploga steroids sp. have demonstrated growth rates about 90 percent of undisturbed culture growth rates. Agi- tation studies using the other specie are in progress. Effect of Temperature Upon Growth Experiments conducted at 1 0" C have verified the environmental studies by showing that no growth takes place. Cultures of Pi plogaster - oides sp. resumed their normal growth rate when returned to room 26 temperature after six and nine days' exposure to 1 0" C . The same phenomena occurred when Diploqaster nud i capi ta tus cultures, refrigerated for \S and 30 days, were returned to a favorable environment. Effect of pH Upon Growth At a pH value of 5, Diploqaster nud icapi tatus cultures experience an initial lag period before starting to reproduce at their normal growth rate. No lag period was observed with Diploqaste roides sp. cultures. Diploqaster nudi cap? tatus cultures reproduce at their normal rate at pH 8. Growth studies of Pi ploqastero? des sp. under alkaline con- ditions have shown normal growth up to pH 9. CONCLUSIONS 1. The practice of compositing a large sample from many smaller grab samples is an effective means of compensating for the irregular dis- tributions of the organisms. 2. Sieves and Plankton nets are not suitable for extraction of nematodes from water samples. Five-micron membrane filters give complete re- covery of the organisms. 3. Conventional bottom sampling devices are not suitable for collecting undisturbed samples for nematologica 1 analysis. Satisfactory bottom samples may be obtained from shallow streams by using a semicircular scoop. k. Satisfactory extraction of nematodes from bottom samples can be accomplished by sugar flotation and centri fugation . 5. Nematodes can be more readily counted in counting chambers with vertical walls than in the conventional Syracuse or Petri dishes. 6. Use of Eosin-Y dye is an effective method of distinguishing between dead and live nematodes. With proper stain concentration and contact time, dead nematodes become stained, while live nematodes remain unsta i ned . 7. The Eosin-Y staining technique may be used in conjunction with any of the lethal agents investigated. It is necessary to alter the staining time to accomplish effective staining with some of the lethal agents. 27 A 30-minute contact period with 0.67 percent Eosin-Y dye is recommended for naturally or heat killed nematodes. 8. The Eosin-Y staining technique is also convenient to use when total dead and live nematode counts are desired. The dye preserves the organisms and does not interfere with subsequent identification pro- cedures . 9. Subsurface drainage, urban and rural runoff, and waste treatment plants are sources of nematodes in surface waters. 10. The nematode population of a stream receiving rural drainage is related to the flushing capacity and the carrying capacity of the stream. The flushing and carrying capacities of the Sangamon River are related to stream discharge. 11. Drainage normally contributes "background" concentrations of nematodes to streams. High concentrations may be expected to be caused by waste treatment plant effluents or similar discharges. 12. Drainage from urban areas produces higher nematode concentrations in streams than does rural drainage. 13. The persistence of nematodes in streams is related to the average stream temperature, changes in stream temperature, rate of stream flow, and the heterogenous nature of the nematode population. \k. Comparatively few nematodes are normally found in the benthos except below waste treatment plants or similar discharges. 15. The distribution of nematodes in the benthos is extremely variable. While nematodes may be fairly uniformly distributed in the bottom underlying the main flow region of a stream, extreme variation occurs in other portions of the stream's cross section. Particularly high nematode concentrations are found in sludge deposits in areas of slow stream velocities. 16. Rhabd? t is , Pi ploqaster , and D iplogasteroides are the three genera of nematodes found in streams to the exclusion of all others. 17. Rhabdi t i s is the least common of the three genera, while Pi plogaster appears most frequently. Diplogasteroides has been found in signifi- cant numbers only in the area immediately below the waste treatment plant. 28 18. Raw sewage continually supplies nematodes to waste treatment plants, and considerable multiplication occurs within the plants. 19. Greater reproduction of nematodes occurs in trickling filters than in the activated sludge process. Nematodes are unable to survive in anaerobic processes. 20. Nematodes remain active within a temperature of 15 9 - 30 a C. Optimum temperatures for Diplogaster nudi capi tatus and D? plogasteroides sp. are 25 e and 20 s C, respect ively. 21. Diplogaster nudicapi tatus is more resistant to high temperatures than is Pi plogasteroi des sp . Thermal death kinetics of both species are described by a first order reaction. 22. Immobilization of nematodes occurs more rapidly in alkaline than in acidic conditions. Pi plogasteroi des sp. can survive and grow within a pH range of 5 to 9- With Piplogaster nudi cap i tatus , the favorable pH range is 5 to 8. 23. The rate of growth of D iplogasteroides sp. is diminished by a reduction in the size of the growth container. Similar growth curtailment is not observed with Piplogaster nudicapi tatus . 2k. Agitation reduces the growth rate of Pi plogasteroi des sp . 25. Although no growth occurs when nematodes are subjected to 1 0° C temperatures, normal growth resumes when the organisms are returned to a favorable environment. 26. Piplogaster nudicapi tatus reproduces at its normal rate when exposed to pH values of 5 or 8, although an initial lag period occurs at the low pH . P i plogasteroi des sp. cultures reproduce normally with no lag at a pH of 5 and 9- 29 REFERENCES 1. Chang, S. L., Austin, J. H., Poston, H. W., and Woodward, R. L. "Occurrence of a Nematode Worm in a City Water Supply." Jour. AWWA , 51. 6 ?1> Ma y» 1959- 2. Caveness, F. E. and Jensen, H. J. "Modification of the Centrifugal Flotation Technique for the Isolation and Concentration of Nematodes and their Eggs from Soil and Plant Tissue," Proceedings of the Helminthologica 1 Society of Washington, July, 1955. 3. Boyd, A. E. W. "Determination of Death in the Larvae of the Potato Root Eelworm." Nature , 1 48 , 782, December, 19^1. h. Fenwick, D. W. and Franklin, M. T. "Identification of Heterodera Species by Larval Length. Technique for Estimating the Constants Determining the Length Variations Within a Given Species." Jour. Helminthology , 2£, 67, December, 19^2. 5. Peters, B. G. "Toxicity Tests with Vinegar Eelworm. I. Counting and Culturing." Jour. Helminthology , 26 , 97, 1952. 6. Chang, S. L. Private Communication. 30 (2) Publications Supported by Grant "Source and Persistence of Nematodes in Surface Waters," N. Chaudhuri , R. Siddiqi and R. S. Engelbrecht. Accepted for publication, Journal American Water Works Assn . (3) Staffing Name Title Period Percent Time R. S. Engelbrecht Prof, of San. Engr. 1 Sept 61 - 31 Aug 63 10 M. R. Matteson Asso. Prof, of Zoo. 1 Sept 61 - 31 Aug 63 20 R. H. Siddiqi Res. Asst. 15 Sept 6l - 31 Aug 63 60 K. Y. Baliga Res. Asst. 15 Sept 62 - 31 Aug 63 60 D. I. Batch Res. Asst. 15 Sept 62 - 15 June 63 50 C. E. Robbins Res. Asst. 15 Sept 61 - 15 June 62 50 R. I. Dick Instructor 15 Sept 62 - 15 June 63 50 Additional Staffing N. Chaudhuri - Supported through AID Fellowship - Completing Ph.D. Thesis; 15 September 1 96 1 - 31 August 1963 (k) Foreign Travel - None