to & ' CIVIL ENGINEERING STUDIES c.^o SANITARY ENGINEERING SERIES NO. 10 EFFECT OF BIOLOGICAL SLIME ON THE RETENTION OF ALKYL BENZENE SULFONATE ON GRANULAR MEDIA VLetz F ace Room Civil -artmcni BIO ' Unj inois Urbana, Illinois 61801 By SHANKHA K. BANERJI * £ C£/Veo OCT 8l3 76 1 1 Hit Supported By NATIONAL INSTITUTES OF HEALTH U. S. PUBLIC HEALTH SERVICE RESEARCH PROJECT WP-18 DEPARTMENT OF CIVIL ENGINEERING UNIVERSITY OF ILLINOIS URBANA, ILLINOIS JANUARY, 1962 THE EFFECT OF BIOLOGICAL SLIME ON THE RETENTION OF ALKYL BENZENE SULFONATE ON GRANULAR MEDIA by SHANKHA K. BANERJI Supported by National Institutes of Health U. S. Public Health Service Research Project WP-18 9 4 7 b* \ \\ '» 5 >j^ Department of Civil Engineering The University of Illinois Urbana, I 1 1 i noi s January, 1962 Digitized by the Internet Archive in 2013 http://archive.org/details/effectofbiologic10bane iii ACKNOWLEDGMENTS The radioactive alkyl benzene sulfonate used in this study was furnished free of cost by the California Research Corporation. The research was supported by a research grant WP-18 (CI) from the National Institute of Health, Public Health Service. The research was carried out under the direction of Dr. Benjamin B. Ewing, Professor of Sanitary Engineering, University of Illinois. The author is grateful to Dr. Ewing for the assistance and guidance in performing the work. He is particularly grateful for the suggestions and advice In writing out this thesis. Author is also indebted for the valuable guidance of Dr. Richard S. Engelbrecht, Professor of Sanitary Engineering, Univer- sity of Illinois in certain aspects of the research. Thanks are also due to Mr. Louis W. Lefke for his cooperation and help during some of the experimental procedures. Members of Sanitary Engineering Laboratory who helped the author some time or other in connection with this work are also thanked This report was submitted as a thesis in partial fulfillment of the requirements for the degree of Master of Science in Sanitary Engineering under the direction of Ben B. Ewing, Professor of Sanitary Engi neer i ng. iv ABSTRACT This research was conducted in order to determine the factors important in retarding the movement of alkyl benzene sulfonate in ground waters. A method has been developed to study the retention of ABS on soils and biological slimes by modification of existing procedures using a radioactive isotope of sulfur. The presence of active biological slime on granular media like Ottawa sand would increase the ABS retention depending on the concentration of ABS solution and rate of flow. The ABS retention on sand having active biological slime at 10 mg/l ABS concentration was more than twice that obtained for clean sand under similar conditions of saturated flow. The ABS retention on clean sand was of the order of 5.0 micrograms per gram of dry sand, and ABS retention decreased in presence of other ions by 20$. The effect of sterilisation of biologically active slime on sand produced ABS retention equal to that obtained for clean sand. The ABS retention on sand having active slime in a system with unsaturated flow conditions was higher than in saturated conditions. The retention was of the order of 22 micrograms per gram of sand at 10 mg/l ABS concentration. The ABS retention on sand or slime could be reversed into liquid phase by suitable solvent extraction procedure. It is concluded, therefore, that presence of biologically active slime would retard the movement of ABS through the zone of soil containing the slime, but if by any chance the microorganisms die the ABS adsorbed would be eluted with percolating water and would pollute the ground water, as the retention of ABS in soil alone is low compared to biological slime I I I I TABLE OP CONTENTS ACKNOWLEDGEMENTS iii ABSTRACT iv LIST OP TABLES vii LIST OP FIGURES Vlii 1. INTRODUCTION 1 2. UNDERGROUND MOVEMENT OP SYNTHETIC DETERGENTS 5 3. THEORY 8 Ground Water Contamination Theory 8 Probable Mechanism of Retardation of ABS in Ground Water 9 4. APPROACH TO THE ASPECT OP ABS RETENTION 15 5. PROCEDURE 17 Analytical Techniques 17 A. ABS Determination 17 B. Extraction of ABS from Solid Phase 19, Preparation of Columns 19 A. Closed Columns 19 B. Open Column 27 Feeding of Columns for Growth of Slime 30 A. Closed Columns 30 B. For Open Column 31 Performance of Biological Columns 33 A, Closed Columns 33 B. Open Column 35 Application of ABS Solution 35 I I I I I vi TABLE OF CONTENTS (Continued) 6. RESULTS 44 A. Closed Columns A and B 44 B. Closed Columns C and D 45 C. Results of Columns E and P 52 D. Results of Open Column 54 7. DISCUSSION OP RESULTS 59 8. CONCLUSIONS 67 9. REFERENCES 69 10. APPENDIX 72 I, Modified Procedure for Radiochemical Determination of ABS and Degradation Products Using s35 72 II, Revised Procedure for Radiochemical Determination of ABS and Degradation Product Using S35 76 III, Solvent Extraction of ABS from Solid Phase 78 vii LIST OP TABLES Table No. Page 1 Characteristics of Closed Columns 27 2 Characteristics of Open Column 30 3 Peed and Seeding Rates for Column A, C and D 32 4 Open Column Peed Rates and Performance 33 5 Measurement of Biological Activity in Terms of BOD or COD Removal of Closed Column A, C and D 34 6 ABS Uptake on Solid Phase in Closed Columns A, B, C, D, E and P 46 7 Evaluation of the Slime on Closed Columns A, C and D 49 8 Relative Activity of s35 in Various Fractions of Closed Column Effluent 53 9 Open Column Biological Study Results 55 10 Relative Activity of S35 i n Various Fraction of Open Column Effluent 57 ) viii LIST OF FIGURES Figure No. Page 1 Photograph of Columns A and B Showing Eiological Growth in Column A 21 2 Photograph of Columns C through F Before Seeding 22 3 Schematic Drawing Showing Construction of Closed Columns 23 4 Ottawa Sand Size Distribution 24 5 Chloride Breakthrough Curves for Closed Columns 26 6 Line Diagram of Open Column 29 7 Breakthrough Curves for Column A 37 8 Breakthrough Curves for Column B 38 9 Breakthrough Curves for Column C 39 10 Breakthrough Curves for Column D 40 11 Breakthrough Curves for Column E 41 12 Breakthrough Curves for Column F 42 13 ABS Breakthrough Curve for Open Column 43 14 Relationship between Volatile Solids and ABS Uptake for Open Column 56 Metz Reference RoWl JUniv T of Illinol* - S it r "iL 208 '• . ; Si -'sat Urbana, I .s 61001 A3: ■- Jtebana n Illinois fiiSQl 1. INTRODUCTION Synthetic detergents, the most common household cleanser at the present time, Is manufactured from the by-products of the petro- chemical and chemical industries. Thus their ingredients are syn- thetic in nature as opposed to the age-old soap which is made from natural oils. Use of these synthetic detergents since their advent in 1948 has Increased tremendously because of the superior cleansing power, better sudsing action, no reaction with hardness of water and relatively lower cost as compared to soap. Now about 90 percent of household cleansers used in United States are synthetic (1). The average per capita consumption of synthetic detergent is about 100 pounds annually based on the study at Suffolk County, N. Y. (2). Synthetic detergents consists of, (a) a surface active agent or surfactant, (b) phosphate builder compounds, and (c) miscellaneous builder compounds, perfume, etc. The surface active agent is the vital cleansing reagent in the synthetic detergent which amounts to about 30^ of the package product. The surfactant may be anionic, cationic or nonionic, but the most commonly used surfactant in the United States today is the anionic sodium salt of sulfonated alkyl benzene, or alkyl benzene sulfonate (ABS) as it is usually called. The other components of synthetic detergent are not as troublesome to sanitary engineers as this surfactant. The increased use of synthetic detergent in the last decade produced some awkward effects on the sewage treatment plants and the water pollution problems as a whole were affected to some extent. The most common effect noted was that of frothing believed to be due to ABS not degraded by biological processes sometimes called, biologically "hard" ABS (3). Further it was reported that primary- treatment was not at all effective in removing ABS and secondary- treatment could remove only about 50% of the incoming ABS (4) (5). Thus the effluent from such a plant would contain the remaining undecoraposed ABS which would pollute the aqueous environment. It was also reported that ABS reduced the surface tension of the waste and interfered with oxygen transfer (6) affecting adversely the efficiency of activated sludge plants. The physiological effects of ABS on human beings if present in drinking water are reported to be insignificant (7) within the concentrations 10-50 milligram per liter encountered in rivers and other water sources. But off-tastes have been reported (2) when the ABS concentration of water is above 1«5 mg/l. Taste may be due to ABS itself, or to the builder compounds associated with the package products or to ABS in conjunction with the build- ers. Moreover taste may also be due to the sewage associated with ABS contamination. Foaming has been reported in waters having 0.5 milligram ABS per liter (8). As a matter of fact these unaesthetic effects, viz foaming and off-taste, are the reason for limiting the permissible ABS levels in the present Drinking Water Standards (9). The discharge of synthetic detergents in the environment was initiated after their advent in 1948, which subsequently increased in magnitude due to rise in popularity, but curiously enough the first report of ground water contamination by the synthetic detergents was not reported until 1958 (2). Of course when one takes into consideration the slow movement of ground water this lag is easily explained. After this first report there came several other reports of such pollution of ground water by syn- thetic detergents (7) (8) (10) (15). Walton (11) brought to notice instances of such ground water pollution in 9 states. Recently some more cases of ground water pollution in Michigan were reported (12) where all the cases reported were private wells 12 to 30 ft deep and not one was a municipal well. Nichols (13) reported incidents of ABS in privately owned shallow wells in Wisconsin. It is believed there are many other cases of ground water polution by ABS still unreported. In almost all these cases there were some singular simi- larities of environmental conditions. All these cases reported are suburban areas on the fringe of big cities, Suffolk County, N, Y. (2), Copiague, N. Y. (15), Portsmouth, R. I., (8) (16), Minneapolis-St. Paul (17), etc. Here each house lot had a shallow well for water source and a septic tank or cesspool as a means of waste disposal. The waste containing undecomposed ABS percolated through the ground to reach the ground water table and subsequently found its way to the wells which were shallow and quite often downstream from the septic tank as regard ground water flow. The problem was further aggravated because of the unusual stability of ABS under biodegradation conditions. Such a pollution would be of long duration even after the removal of the source of pollu- tion, not only because of the fact that ABS is relatively undegrad- able but also due to slow movement of ground water to flush out the pollution. Walton (14) suggested the use of ABS as an indi- cator of ground water pollution. Nichols (13) considered ABS as a good indicator of virus contamination, since virus might not be removed from waste during percolation in soil like bacteria. 2. UNDERGROUND MOVEMENT OP SYNTHETIC DETERGENTS The reported movement of syndet in the underground water has usually been small as compared to other chemical pollutants. Plynn, et al, (2) reported that chromium waste from a plating plant which had been in operation for 2 l/2 years was found to have travelled about 1200 feet, which corresponded to 1.3 feet per day. As compared to this the movement of ABS was never beyond 65 feet, though the amount of synthetic detergent in use increased every year. This retarded movement of ABS vras also observed by Deluty (8) in the pollution of wells at Portsmouth, R. I., where the maximum distance from the source of pollution to any polluted well was 150 ft. In Mastic, N. Y. , ABS was found to have travelled 1100 feet (10) downstream from a launderette. The launderette had been in operation for the last 12 years, so the rate of movement of ABS in ground water would correspond to only 0.25 feet per day. It must be remembered here that with succeeding years the usage of synthetic detergent had increased and the pollution hazard of the launderette had greatly increased with the years. Possibly the total amount of ABS discharged in the last five years would be double that in the first seven years. Walton (11) found that the wells contaminated with ABS from household waste disposal systems were within 100 feet from the source of pollution except for five instances where the ABS was reported to have travelled 1000 feet from the source which was a municipal or industrial waste pond or recharge pit. At Peoria, Illinois, (11) ABS has been found to have travelled 1800 feet from a recharge pit utilising Illinois River water, A well 200 feet away contained 0.7 milligram per liter of ABS and a well 1800 feet away had 0.17 milligram per liter ABS. The longest reported distance ABS has travelled in under- ground water so far was 4000 feet from a sewage oxidation pond in Kearny, Kansas (11). Neutsch (18) reported an incidence of ground water pollution in Germany from laundry waste. Waters of nearby wells at 200 meters were polluted and produced foaming. He referred to the pollution as a permanent feature and was doubtful if it would be eliminated with the passage of years. From the above instances of ABS movement in underground water, the following becomes apparent: (a) The movement of ABS in ground water is always much less than the water itself. Obviously some sort of retarding mechanism restricts the movement of ABS molecules. (b) The contamination of ground water by ABS from small household waste disposal system is in a much smaller and more con- fined area as compared to that from a municipal or industrial source. The retardation of movement of ABS in ground water men- tioned above points immediately to the fact that the soil through which the polluted water moves may have a retentive capacity for ABS. Chemical or biological degradation may be ruled out because of lack of opportunity and unfavorable conditions in underground water for such reactions. The retention on the solid phase could be surface adsorption. Ion-exchange can probably be ruled out. 7 The ABS molecule is negatively charged and soils are known to exchange predominantly cations. Though some soils may have some anion exchange capacity due to the presence of metal oxides or hydrous oxides but it is negligible compared to the cation exchange capacity. Study of retention of nonionic surfactant on soils would clarify whether ion exchange is effective or not, but this is beyond the scope of this thesis, as we are more interested in ABS for the present. 8 3. THEORY Ground Water Contamination Theory Let us briefly review the observed facts about ground water pollution by leaching from septic tank, pit privies, cesspools, refuse dumps and the like. The pollutant itself may be either chemical or biological in nature. The movement of radionuclides may be assumed to be chemical in nature. Two distinctly different aspects of pollution travel are involved in ground water contamin- ation; first, the movement of bacteria and chemical compounds downward with percolating water in unsaturated soils and, second, the lateral movement of the pollutant with the ground water usually in saturated soils. Observers are generally in agreement that pollution is not laterally extended to any important degree when the water which transport it percolates downward through soil above the ground water table (19). In case of movement of pollution with ground water it is generally agreed that pollution travels farthest in the direction of ground water flow and chemicals travel much farther than bacteria in a water-bearing stratum. Particulate solids are usually removed from water by straining at the narrow openings near intergrain contacts or by adherance on the pore boundaries. Dissolved solids are attracted to the interface by electrical charge attraction. As has been pointed out earlier, most aquifer materials have a negative surface charge caused by the distribution of atoms within the materials. The large surface area of the material, makes the capacity for such sorption greater than is usually imagined. One cubic foot of fine sand has a surface area of the order of 10,000 square feet. Probable Mechanism of Retardation of ABS in Ground Water (20) Movement of a pollute through ground water formation is a displacement process. The vehicle of transportation of synthetic detergent is the waste-contaminated ground water. The pollute moves into a zone of earth which retains an amount of pollute equal to its capacity. After that any further inflow of pollute displaces the pore-retained pollute, and perhaps that retained en the solid phase, to a new zone. There is always, then, a fringe of earth material which is being loaded with the pollute. The pollute moves as a diffuse front through the formation. The width of the front depends upon the kinetics involved in the mechanism representing the capacity of the soil to retain pollute and the hydraulic dispersion phenomena (21) in ground water flow. The mean velocity of movement of a pollute front may be expressed as S = ° I f Eq. (i) where S is the average velocity of pollute front in feet (or meters) per day, R is the retention capacity per unit gross volume of earth for the pollute in micrograms per cubic centimeter, C is the con- centration of that pollute in the feed solution or waste water at the source in milligram per liter, v is average velocity of the transporting water at the same point in the zone in feet (or meters) per day and f is the porosity of soil zone. Also, vf is the dis- charge per unit cross sectional area at the point. Hence the velocity of the pollute front is inversely proportional to the retentive capacity of the formation for the pollute, directly proportional to the concentration of the pollute in waste water and 10 the percolation rate. The retentive capacity would never be less than the amount of pollute contained in the amount of waste water required to displace the pore volume. This would be the product of the concentration of the pollute in the waste water and the porosity of the earth zone, Cf. Substituting this value for R in equation (i), yields, S = v; under these conditions the pollute moves with the percolating water and it is not retarded. Such a pollute would be an ideal tracer of ground water if it met other conditions of detectabllity and stability. In actual practice there are some phenomena by which the retentive capacity of the soil formation is increased over that represented by the pore volume alone. The most probable mechanism responsible for retention of anionic surfactant is physical adsorption on the soil. While soils, particularly the clay fractions , exhibit some cation exchange capac- ity, the anion exchange capacity is insignificant. Concentration of ABS in waste water is generally low, of the order of 5-10 mg/l for household waste (5) and 50-150 milligram per liter for launder- ette waste (10), which is further reduced when diluted by ground water. Renn and Barada (22) investigated the use of various common adsorbants for removal of ABS from water supplies. The mineral adsorbants, being the type which prefer the hydrophilic end of the ABS molecule, are less effective than hydro-carbon-adsorbing surfaces; nevertheless, they do adsorb measurable amount of ABS. Their studies showed suspended silt could adsorb 20-50 mg of ABS per gram of silt. For clay, talc, diatomite, silica and calcium 11 carbonate, the adsorption was less and was of the order of 1 milli- gram of ABS per gram of material. Higher adsorption than the above mentioned materials except silt was obtained with mineral oil and activated carbon. An activated carbon filter has been developed for removal of ABS from household water supplies (23), and the author indicated that removal of ABS on activated carbon conformed to Preudlich equation of adsorption. Eckenf elder and Barnhart (24) have pro- posed the use of a similar device for treating laundry waste on Long Island, N. Y. The retentive capacity of the solid phase for adsorption of a pollute is generally expressed as the weight of adsorbate, X, per unit weight of adsorbent, M. The solid phase retentive capacity would be X/M times p t the bulk density of earth material. The total retentive capacity R would be the sum of the pollute stored on the solid phase and that stored in the liquid phase, r - cf + -i- F M 2q. (ii) substituting this value in equation (i), yields an expression for the relative velocity of the pollute front, S/v = ~! XA P Ec l- (Hi) + Cf or S/v = j[ \ D Eq- (illA) where D is the distribution factor equal to the ratio of pollute on the solid phase to that in the liquid phase. Thus the rate of movement of a pollute is dependent on the distribution factor and the percolation rate of the transporting water. 12 The amount of pollute adsorbed on the solid phase will increase until It equals the specific adsorption capacity in equilibrium with the concentration of the pollute in the liquid phase. For a given weight of adsorbent with a given surface area, the amount of material adsorbed depends on the concentration of the material around the adsorbent. When an adsorbing material is placed in contact with a solution, the amount adsorbed will grad- ually increase, and the concentration of the surrounding molecules will decrease until the rate of desorption becomes equal to the rate of adsorption, and, thus, an equilibrium is established. Several equations have been devised to represent the adsorption data. Freundlich equation represents the equilibrium as: -i- = kon where X, M and C are the same variables as referred in equation (ii). k and n are constant for particular materials and tempera- ture. Langmuir's Isotherm is based on the assumption of mono- molecular layer of adsorbate on the surface area of the adsorbent material. The mathematical expression is: M " Ki + K 2 C where K^ and K 2 are constants. For AES at low concentrations of up to 40 rag/l X/M is linearly related to concentration (20) at a particular temperature. From Equation (iil) it will be apparent that at low concentration up to the linear range any variation in the concentration would not affect the velocity of the pollute front. Thus velocity of pollute front will be independent of the concentration up to a certain maximum value. The limits of the 13 value of S/v, the velocity of pollute front, for finite values of C will be Z < -~ < 1.0 where Z corresponds to the value p — 1 + m — 5— and m is the slope of the adsorption isotherm in the linear range. Whereas the adsorption of ABS on the surface of soil particles may be an important mechanism in retarding the movement of ABS, the presence of zoogleal slime on the soil particle can cause further retention of ABS. The large surface area provided by microbial cells may well offer considerable adsorptive capacity for ABS. In addition it is known that there is some bacterial decomposition of ABS. Many investigators have studied the bio- degradation of ABS in order to determine its fate in biological waste treatment plants (4) (5) (25). They have shown the decompo- sition of ABS is slow; even in the highly active biological systems encountered in trickling filters and activated sludge plants only about 50^ of the ABS is degraded. The proposed reason of the "biological hardness" of the ABS molecule is said to be due to the tertiary ring attachment to the alkyl chain (26). Straight chain alkyl sulphonates of varying chain lengths with a primary ring attachment were readily degraded by activated sludge. Nelson, et al, (27) reported successful degradation of straight chain alkyl benzene sulfonate containing a tertiary attachment of the benzene ring and found that the presence of quaternary carbon near the end of the chain prevented successful degradation of certain branched chain alkyl benzene sulphonate. Nevertheless, the time 14 available for decomposition is much greater in soil and in the first few feet of a soil percolation system it may be that a significant fraction of ABS is actually degraded. This phenomena may be important in septic tank percolation fields, sewage oxida- tion ponds, holding ponds for industrial waste and ground water recharge operations where the water medium contains sufficient organic matter to support the growth of biological slime. It is important, therefore, that the relative magnitude of ABS decompo- sition, adsorption on the surface of microbial cells, adsorption on soil surface and other related phenomena be evaluated. 15 4. APPROACH TO THE ASPECT OP ABS RETENTION Ottawa sand was chosen for all biological column experi- ments because of its uniform size and higher permeability under slimy conditions. The characteristics of Ottawa sand are dis- cussed elsewhere. The approach used to compare the relative effect of biolo- gical slime activity and physical adsorption without slime in re- tarding the movement of ABS through soil was to build identical columns of sand. One of them was a clean sand column to which an aqueous solution of ABS was to be applied and the ABS retention on the solid phase measured by appropriate techniques mentioned later, The other column was to be seeded with settled domestic sewage to get a heterogeneous population of microorganisms on the soil parti- cles. The retained microorganisms were to be developed by feeding synthetic substrate and later fed with an aqueous solution of ABS, the retention on the solid phase being measured once again. The effect of autoclave sterilization of actively meta- bolising microorganism was also to be determined in order to see what effect dead microbial surface had on the ABS uptake. It was felt that the comparison of ABS uptake on dead microbial surface with active onces would clarify the phenomena of adsorption on slime surface. If the retention was a pure surface adsorption and if the total surface provided by microorganisms did not change after sterilization, we would expect to get the same order of ABS retention. If surface adsorption was the phenomena accounting for the retention on the solid phase the presence of other ions (both 16 cations and anions) would reduce the ABS retention because of competition for the available sites on solid phase. There may- be another effect of the presence of other ions on the ABS adsorp- tion. The solubility of ABS would reduce due to salting out effect. In Equation (iiiA) for the amount adsorbed on solid phase X/M would remain unchanged but concentration C would decrease, resulting in a steeper slope of the adsorption isotherm and slower rate of travel of pollute front. This would tend to increase the amount adsorbed on solid phase. This effect of presence of other ion was to be studied by using a feed solution made up of tap water and comparing the ABS retention on sand with a column having ABS feed solution in distilled water. Studies to see if ABS has been degraded to intermediate product or inorganic sulfur during the passage through the biolo- gically active column would also be made. This is necessary because If ABS is degraded and incorporated in the cell the effluent would have less concentration of ABS which would apparently increase the calculation of surface adsorption. If ABS uptake on the solid phase was a mere physical adsorption phenomena it was thought we could reverse the equili- brium by means of solvent extraction. The recovered ABS should compare well with the ABS uptake calculated from breakthrough curve. This solvent extraction technique has also been utilized successfully by University of California research group (28). It was also decided to find ABS adsorption under partially aerobic unsaturated flow conditions and compare it with saturated flow in anaerobic conditions. 17 5. PROCEDURE Analytical Techniques A. ABS Determination It was decided to use a radioassay procedure (30) in conjunction with the Methylene Blue method (29) of ABS determina- tion. Although Infrared spectrometry (29) could be used in place of Methylene Blue method to eliminate some of the interferences caused by alkyl sulfates and other organic compounds, this method is quite time consuming and not suited for routine analyses. The radioassay procedure used for determining daily variation of ABS concentra- tion in column effluent containing organics and biological growths eliminated any interference which would have caused serious error if Methylene Blue method was used only, moreover it was quite fast and fairly accurate method. Methylene Blue method was used to determine the specific activity of radioactive ABS feed solution. Because the feed solution was usually prepared in distilled water, it was felt that any interference of serious magnitude would not result if we used Methylene Blue method. The radioactive ABS was furnished gratis by California Research Corporation, and the sulfur in the sulfonate part of ABS was the radio isotope having atomic weight of 35 atomic mass units. For simplicity the s35-tagged ABS will be designated ABs35. The ABS35 supplied by the manufacturer had an average of twelve carbon atoms in the alkyl chain and an average molecular weight of 246 (31). The radioassay procedure also allows one to determine if the ABS molecule has degraded or not. The proportion of total sulfur in the sample could be determined as well as the fraction of the 18 sulfur which is still ABS, the fraction which has been degraded to inorganic sulfur, and the Intermediate products. The radio- assay procedure and the Methylene Blue method have been described in the Appendix I and II. In certain phases of the research, use was made of direct sampling of the ABS^5 i n the planchets. Two milliliter of sample was transferred to 1 1/8 inch diameter aluminum planchet , which was dried at 103°C and cooled before counting. The procedure was applicable where it was certain the ABS had not degraded and the counts measured the total sulfur associated with the original ABS. For all of the radioassay counting of prepared samples a Nuclear Measurement Corporation Model PC-3A internal proportional counter was used. P-10 gas {90% argon and 10% methane) was used to flush the chamber. All samples were counted for a minimum of two minutes and a maximum of ten. The desirable level of total count was 5000 in order to limit the statistical error to about 1«5^» All samples counted were corrected for background and decay. This enabled the samples to be compared for each individual analysis conducted. Self adsorption correction was not applied since we were primarily interested in relative counts to compare the ABS in the effluent with the feed solution and were not doing absolute radioassays. It may be pointed out, however, that in cases where the sample thickness was great and different compared to feed solution sample thickness, it is essential that self adsorption correction be applied. 19 B. Extraction of ABS from Solid Phase An alkaline 1:1 (by volume) mixture of benzene and methanol (32) was used to extract the ABS from the solid phase in a Soxhlet apparatus. The residual activity of the solvent after extraction gave the amount of ABS present in that particular sample of sand. This solvent is reported (32) to be 90-95/0 efficient in dissolving the ABS from the solid phase. The method has been described in Appendix III. Preparation of Columns A. Closed Columns It was decided to make two columns first and study the effect of biological slime. Later after dismantling these two we rebuilt them and added two more. Therefore column A and column B were the first set of columns; C, D, E and F were the second set of columns built to replicate and expand the results obtained from column A and column B. Columns A, C and D were biologically active columns except D was later autoclaved prior to the ABS application. Columns B, E and F were clean sand columns to study the effect of physical adsorption without any active slime. Columns B and F were fed with ABS solution in distilled water, however, and E was fed ABS solution in tap water, so that the effect of other salts could be studied in relation to ABS retention on sand. Physical Description : The columns were constructed of two-inch diameter Pyrex glass pipe. Each column consisted of two sections of glass pipe with an overall length of 36 inches. Each half was joined with the manufacturer's flange joint as shown in 20 Figure 2. This enabled each half of the column to be sterilized in an autoclave too short to accomodate the entire length. Glass tubings inserted in the rubber stopper permitted entry and exit of liquid to the columns. To prevent loss of earth materials from the column when disassembled at the central joint for steriliza- tion, lucite plugs 1/4 M thick were inserted in both the pipes at the flanged joint. In order to simulate the effect of porous medium through this section of the column, numerous 1/16 " holes were simultaneously drilled through both lucite plugs in concentric circles. The alignment of holes were maintained by an aligning key so that there was hydraulic continuity of flow through the column. A thin mat of glass wool was placed between the sand and the lu- cite plug to prevent loss of material when disassembled. Figures 1 and 2 are the photographs of the columns assembled and Figure 3 is a line diagram. Medium Used : As has been mentioned earlier, the material used in the column was a pure silica sand known as Ottawa sand. This sand had been sieved to produce a very uniform size distribu- tion as shown in Figure 4. The geometric mean size of this sand is O.858 millimeter. The specific gravity was determined to be 2,64. The sand was well washed and dried before packing. Preparation : The sand was packed into the column by allow- ing the sand to fall freely into the glass pipe while the latter was being vibrated with a rubber mallet. This procedure was followed in the preparation of all columns to obtain as uniform and close packing of sand as possible. The amount of sand packed was weighed and the net weight of sand in each part of the column was found. 21 Figure 1. Photograph of Columns A and B Showing Biological Growth in Column A 22 Figure 2. Photograph of Columns C through P Before Seeding rO ^S^^^^vsTw GLASS FEED TUBE J* 2 RUBBER STOPPER ■— *■*— ^~— — ~_ GLASS WOOL MAT EARTH MATERIAL CENTER KEY FLANGE JOINT GLASS WOOL MAT PYREX PIPE ALIGNING KEY TEFLON WASHER ^DRILLED LUCITE PLUGS GLA SS DRAIN TUBE Figure 3* Schematic Drawing Showing Construction of Closed Columns 2h I .9 .8 .7 6. .5 DIAMETER, mm Figure 4. Ottawa Sand Size Distribution Metz Seference Roo.i University of Illinois E10S Nl 208 H sot na, Illinois oiaSSSL 25 Each half was packed separately and assembled for operation with flanged joints. The assembled column was flushed with large amounts of distilled water to wash out the fines still adhering to the coarse sand. To remove all air from the sand pores, a vacuum was applied at the bottom while the inlet for distilled water was closed. When most of the pore was evacuated, suddenly the inlet was opened which allowed all the pores to be filled with distilled water, and there was considerable less air-binding. The pore volume was determined by displacement of the pore fluid by a chloride solution. A dilute calcium chloride solution was allowed to flow through each column with frequent sampling of the effluent for chloride determination. The chloride break- through curve for all the columns are shown in Figure 5» The pore- volume of the column is represented by the area to the left of the chloride breakthrough curve. An attempt was made to show the similarity of physical conditions in the six columns so that their ABS retention could be compared. Table 1 compares the columns on the basis of poro- sity, permeability, and dispersion constant. 26 UJ _l U_ Ll. UJ 3 O o a a H O O •CJ 0) w o H O o U O X! faO o .3 cd u PQ < q: > O z ' T II II ' II 'I M LUCITE DISTRIBUTOR PLATE II WITH 16 No. '/8 HOLES S A N D GRADED 3/8 DIA. GLASS VENT TUBE LUCITE OUTER SHELL EFFLUENT COLLECTOR Figure 6. Line Diagram of Open Column 30 Table 2 Characteristics of Open Column Dimensions : Inside Diameter - cm 14.2 Average Depth - era 30.0 Material : Ottawa sand - pounds 13.07 Calcareous gravel - pounds 4.97 Pore Volume: Prom saturation test - ml 1510 From physical dimensions - ml 1545 Feeding of Columns for Growth of Slime A. Closed Columns To seed the column A, C and D with microorganisms and develop an active slime on the sand, settled fresh domestic sewage which had been filtered through glass wool was applied daily for 4-10 days. A 1:9 mixture of sewage and a synthetic feed described below was also tried for seeding the columns with success. Domes- tic sewage was used in order to have a very heterogeneous popula- tion of microorganisms. The slime developed was initially dark in color which later turned purple at places. In order to maintain the retained microorganisms a syn- thetic feed was used. This permitted not only a close control on the feed biochemical oxygen demand (BOD) but also kept the permea- bility of the column within reasonable limits because it was com- pletely soluble and had no suspended matter. The synthetic feed 31 consisted of 300 milligram per liter of anhydrous dextrose for column A, 225 milligram per liter for columns C and D, Ammonium chloride (50 mg/l for column A, 37.5 mg/l for columns C and D) and 1 milliliter per liter of a buffer solution containing 8.5 grams per liter KH2PO4, 21.75 grams per liter of K2HPO4, 33.4 grams per liter of Na2HP047H20, were added for a nitrogen source and other necessary nutrients. All this was made up and mixed in tap water for a pH 7.20. The tap water used provided the trace requirement of the other growth factors. Table 3 gives the feeding rates of different columns. From clogging experiences of column A (20) we learned to maintain a much better permeability of the column C and D by careful adjustment of feed BOD and by varying the hydraulic head on the column to maintain a constant flow rate of about 10-12 ml per hour or 0.53 cm per hour. B. For Open Column The same procedure was used for feeding this column as described above and a synthetic feed consisting of same ingred- ients was used to culture the microorganisms retained. Clogging of the column was experienced frequently which was remedied by stirring the slimy sand with a sharp edge. The bottom third of the sand turned dark black suggesting anaerobic condition but the top part was clear and suggested aerobic conditions. The pink growth was noticed on the fringes of the dark parts. Table 4 characterises the open column feeding rates, etc. 32 KN. co H a EH •d cd o B o o U o CO w >d a) o ■H -P 2 pl TJ H h> CO O CO CO CO PR o •H M P CO # H CO Cj CO -P«H o EH U O £ •H £ P« CO o p H O O |Sj o s o ri o tj *>i ■a CO cd CO CO 13 CO CO £ CO \ f^ O -P CO CO rH Cd S3. to Ph « o a H £ H CO aJ CO o tJ CO CO CO H iH E ^ ■p 03 -3 CO -p ■P rH •p CO O O •H CO Eh > H d «d • CO -P H O Jh 1 s s P CO • 3 to O bfl o o 03 tO >5 O h aS O cd o 'd E3 -H cd O > ^ >& p! CD ?H t3 ^ «H \ "Ns. E a) co co co • ■^ -t ON • • CM o o o o md CO U LO CO c— p> • •H MO H MD co -3- o\ CM O in o o • H O MD ON O MD • in CM in o o •a cd p. cd •p o ■^■fl CO \ cd to s B o o B 0) « R o o u o p o pq o CO P P) T3 0) $3 EM 03 CO -p H CO > Pi •H a P 3 O H «3* O o H 03 'd o cu •H CO bO o OH HO O H «H P O bO Cfe^ 03 £3 fH CO CD P3 > -33 P o WH 03 0) > bO 0\R oj b Jh CO CO K > «i p c CO H 2P\ H O bD » bO 03 PI T3 •H V. •O CO 03 Pi o /=> o PH. tJ «l H O OP5 O o ■dHfi CO l>-0 T3 •H OJ W CO Pt •h o5 o >>u > P 03 o3 TJ > •CJ 0\ V^ 05 l>- CO HMD oco U cd • p o > «4 o o OJ >A O H C cd 1 > OJ O H B co *d tt CO H n Pi o cd pq > H OJ VO OJ OJ vo in OJ in H H CO CO bO bO cd cd Pi U CO cu > !> < -*l o si" H O H H<1- cd I t><3- O H B CO t* P=S cu H P P) O cd pq > vo OJ ■St -st OJ m in P 35 rate of flow to stabilise the incoming BOD loading. The variation of BOD removal was probably due to fluctuation in the substrate strengths resulting in decrease or increase in the population of the microorganisms. Often chemical oxygen demand (COD) removal was used in conjunction with BOD as a criteria for measuring the biological activity. The average BOD loading was around 1200 pound BOD per acre per day for column A which was much higher than applied to a sand filter in sewage treatment (33). But the loading was around 126 and 155 pound BOD per acre per day for column C and D respec- tively which was quite low loading rate. The wide variation in BOD or COD removals indicated that even with utmost care steady state conditions were not attained. B. Open Column The performance of open column was satisfactorjr, though there was a variation in percent BOD removals from a low value of 39.3/£ to a high value of 98.0^, the average removal was 65*9$ at an average BOD loading of 683 pounds per acre per day. The daily variations of the performance was due to change in feed concentra- tions and stopping of syphon at night because of slime in rubber hose or due to clogging of column and subsequent anaerobic condi- tions. But on the whole the microbial activity was quite signifi- cant after proper adjustment of flow rates and permeability. Table 4 tabulates the BOD results of the column also. Application of ABS Solution The application of ABS was made after an active biological slime had been developed as indicated by satisfactory BOD revmoal. 36 Sufficient ABS35 stock solution was added to the same synthetic feed mentioned earlier consisting of glucose, ammonium chloride and phosphate buffer all in tap water, to give the required con- centration. For most part it was decided to have ABS35 concen- tration of the order of 10 milligram per liter because this is the maximum value commonly encountered in domestic waste. Samples of influent and effluent were analysed for radioactivity by direct sampling in a planchet, drying and counting. The clean sand columns were dealt with similarly except there were no slime growth so ABS solution was fed directly. Column D was sterilised in an autoclave at 120°C at 15 pounds pressure for 30 minutes before feeding ABS. The ABS breakthrough curves for closed columns were plotted as shown in Figures 7, 8, 9, 10, 11 and 12. The same procedure as described for closed biological column was used for open column, the ABS solution was added to the synthetic feed to give a concentration of 10 milligram per liter. Continuous sample of effluent were collected and the activity compared with the feed solution activity. The breakthrough curve was plotted as shown in Figure 13» The ABS application had to be stopped even though the column was not completely saturated because of lack of time and ABs35, 37 1 1 1 1 ' 1 1 1 1 X \ ROUG mg EAKTH N A 5 S BR OLUM ■ CO o <£ - - — — X \ 10 Z> o or X E o lO < CO V UJ or Z \ m 3 _) \ CO CO < O O ^ * — V V. — ~~ — ^A r ^*— LU X o ID o \ O o _i X cc X < o LJ DC CD — 1 1 1 1 , 1 I 1 1 i o o CVJ rO O O CD c\f o O O co (%) O Q O CD 3" CVJ D 0_ ' N011VdlN33NOO lN3mdNl 'N0llVdlN30N0D _LN3m3d3 o o CO < e H O O O o o CVJ — CO E > ^ u O o t- z LU 3 p o _l hO U. . 3 Lt_ o LU .a o o ID OS CO CVJ _l h O O pq • O *H O 3 00 (sO o o p* o o CD o o PQ (\J 6 3 O O o o Q. o «H E W „ CD 1- > z *H UJ 3 3 o O _l o u. £ CO u_ faO UJ 3 o z u Z o 3 _l o CD o O u t£> PA o o o o c\J CO CD u ps > 3 •H (%) 3>_ t N0llV z CO Q o X LU X CD ^ CD Z> O _) o (T CO Lt X X 1— X z> o or X cr E ■.a cr> CD CO m o o z LU _l U_ u. LU Z X) _J o CJ to OJ o § o NOllVdlN3DNO0 "lNamdNI N0llVdlN3DN03 lN3mjJ3 O o o o u o U £ fi4 40 1 | ■ . | T- 1 ' 1 1 ^ — — X o _ — — 1 > x E V — * o *s) < - nJ V UJ — or ■« CD ~ * V o o \ • • CO — - - — • ^^\^ — - X •N. - o — Z> O • UJ K — LORID EAKTH x a — ■ . U GO - 1 1 1 1 1 , 1 1 o o o CD O ID O O CO o o eg ro o o X CVJ p o O sr 3 CvJ H O o u O o <\J 3 _l O o O H 05 O •H o Pm 00 o o c%) 4- °5_ N0llVaiN33N03 lN3n~)JNI NOUVaiN30NO0 lN3nidd3 41 -| 00 N- W ~ <£> CI O O U o - lO 10 <^ 0) CO 0) — o " £ p o 1- ^ 2 tf III JJ Z> _l Li. o jj u_ ■p 111 ^ Ctf 0) 2 fc 5 w 3 _l • O OJ o H CM o o o 00 o CD o O CM (%) § N0I1V«1N33N0D NOI1VU1N30NO0 lN3mdNI lN3n"1333 o 0) 43 o o CD o f-t o 0) 2 o .a bo o a CD w (%) 4 N0llVdlN33N03 lN3n~ldNI N0llVdlN3DN00 lN3n~l3J3 t 44 6. RESULTS A. Closed Columns A and B The application of ABS for column A was made with an ABS35 concentration 50 milligram per liter in the feed solution. This concentration was chosen in order to hasten the saturation point because the column used to clog and quit discharging any liquid after 2 hours or so. Even with 50 mg/l ABS in feed solution the concentration of effluent was only 50/£ of the influent when the flow was stopped (20) as shown in Figure 7. The calculation of the amount of ABS retained on the solid phase was made on the basis of the breakthrough curve shown in Figure 7. The area between the ABS breakthrough curve and the chloride breakthrough curve repre- sents the amount of ABS on solid phase, which can be converted to milligrams of ABS if the concentration of ABS in the influent is known. For column A the ABS adsorbed per gram of clean sand was 22.8 micrograms at 50 milligram per liter of ABS in the feed solu- tion even though it was only partly saturated. The corresponding figure for the clean sand column B with an ABS concentration of only 10 milligram per liter in the feed solution was 1.01 micro- grams, (Figure 8). In order to have a better comparison, column B was flushed with distilled water till the activity of effluent was practically equal to background indicating complete removal of ABS35. Another application of ABS was made with a feed concen- tration of 50 milligram per liter. From the breakthrough, Figure 8, the resulting uptake was 3.30 microgram per gram of sand. By com- parison with column A, it was apparent that biological slime does help to retain more ABS on the solid phase. How the question arose 45 whether the increased ABS uptake was due to the presence of more surface area provided by the microorganisms or due to the presence of actively metabolising slime. As mentioned earlier, columns C and D were prepared to duplicate the results of column A and to evaluate the ABS retention on active microorganisms on the sand as compared with dead microorganism cell surface, B. Closed Column C and D The conditions of the columns C and D during and prior to ABS application were a bit different than that of Column A. The ABS feed concentration was of the order of 10 milligram per liter instead of 50 milligram per liter because it was felt that the ABS concentration in domestic sewage seldom exceeds this value. Also the flow rate was much slower. In fact the run was continued for 87 days for column C at approximately 0.58 cm per hour whereas the run on column A was hardly 3 hours duration corresponding to 29.5 cm per hour flow rate. The ABS uptake as calculated from the breakthrough curve, Figure 9, are tabulated in Table 6. The column D was sterilised prior to the application of ABS solution by heating in an autoclave for 30 minutes at 120°C and 15 pounds pressure. This would kill the microorganisms without destroying their surface area. The comparison of volatile solids on the sand of column Cand D after dismantling, Table 7, which has been discussed later, did prove that microorganisms may have died but its surface was still present. During sterilization of the column in parts, unfortunately one half of the column broke. The ABS retention was carried out on the other half with sterile feed solution and sterile technique. This prevented actively Table 6 ABS Uptake on Solid Phase In Closed Columns A, B, C, D, E and P Column Type of Concentration ABS Uptake No. Column of ABS35 in in microgram feed solution per gram of mg/l (a) dry sand (b) Benzene Extraction Benzene Extraction of ABS on Sand, of sand from which microgram per slime had been gram of dry sand scrubbed, (c) microgram per gram of dry sand Microbial slime on sand 10 50 22.3 (d) B Clean sand 10 50 1.012 3.304 Microbial slime on sand 10 11.13 (e) 10.75 1.36 D Microbial slime on sand sterili- sed before feeding ABS 10 5.04 3.38 E Clean sand - ABS feed solution in tap water 10 4.04 5.70 Clean sand - ABS feed solution in distilled water 10 5.34 4.12 Note: ® J? a . (a) The concentration of feed solution was obtained by Methylene Blue Method (Appendix II) and have been rounded off to nearest whole number. (b) The ABS uptake was calculated from the ABS breakthrough curve. The milligram of ABS retained la solid phase = concentration mg/l-x (area of the left of the ABS breakthrough curve - Area to the left of chloride breakthrough curve) x Scalef actor. (c) These figures are average over the entire depth. (d) The column was partially saturated, effluent concentration was 50 percent of the influent before it was clogged and no flow was possible. (e) The column was partially saturated, effluent concentration was 84 percent of the influent before feeding was stopped. 47 metabolising microorganisms to grow while the ABS retention on the solid surface was carried on. In order to estimate the altered pore volume of the columns under slime growth conditions a chloride run was made during the period the ABS was being fed. The percent reduction of pore volume for column C was about 17.6$ owing to slime growth. For Column D the altered pore volume for the remaining half was deter- mined. The ABS adsorbed by column C came out to be 11.13 micro- gram per gram of sand which was about half what was obtained for column A where the ABS concentration in the feed solution was five times larger. For column D, under sterile conditions, the ABS uptake came out to be 5.04 microgram per gram of sand, which equaled the amount of ABS adsorbed on sand reported for batch operations at 10 milligram per liter (20). Under batch conditions the opportunity of contact between ABS molecule and sand surface was much more than in the column studies where sand was stationary. Solvent Extraction of ABS on Sand ; When the ABS applica- tion was stopped at the end of the experiment the columns were dismantled and samples of sand were treated with solvent extraction process described in Appendix III in order to extract the ABS into liquid phase. The recovered ABS in the liquid phase was evaporated in a planchet and counted for radioactivity. The quantities of ABS thus recovered has also been tabulated in Table 6 for comparison with results obtained from breakthrough curve. Evaluating the Slime : The amount and nature of slime growth on the sand in column A was reported (20) and has been presented in 48 Table 7. The physical appearance of column C was very similar to column A. The formation of the characteristic pink color developed on the light-exposed part of the column after some days indicated the presence of some photosynthetic chromo-bacterium. The column D was dark all over with suggestion of pink at places. After autoclaving it was greyish in color all over. To evaluate the slime further, samples were removed from the column at different depths. The amount of organic matter on the sand was found by loss of weight of sand on ignition at 600°C. The number of microorganisms in the slime was determined by agi- tating the sand sample in sterile water. The scrubbed slime sus- pension in water was decanted, diluted and plated on sterile nutrient agar. After incubation at room temperature for 48 hours they were counted for total plate counts. The results are also presented in Table 7. For column C, there was a reduction in the number of bacteria as the depth of column increased. For column D, which was supposed to be sterile we did get some counts from bottom samples which possibly was due to access of microorganisms from air at the exposed end of the column. All the top part was sterile, however. For Column C the sand from which the slime had been scrubbed off was examined for any remaining AES associated with the sand surface. The alkaline benzene-methanol solvent extraction procedure was applied to ascertain how much ABS was still retained on the sand. The ABS retained on sand was 1.36 micrograms per gram of sand corresponding to about 10$ of the total retention. 49 » «H 1 W E «H cd -H O Orl CliH n p U Pi fn I A cd • O cd tO m.c O -H bO ! >» p SShhl H In o 13 Pi p< fr M CO 1 B CQ «H H fn Pi E CO •o • O cd +^ to Pi O «H tO fn «H CQ cd JSi S fn 0) r-i O > < s t0 O fn (JOCifi T3 » CO CO O ft cd o >-H •O H ^S O OJ Pi h o CQ cd co E^ E to o-d in *H ■H PJ H » £ 00 fn p J2i O fnQ 5>s >S H fn cd H to f h cd Cd CO H 0\f H CO > > co to H Volatile A Solids V Column Sample mg per No, Depth gram of m T3 H cd H CO E O n ON ho cd "H rH »o P M cd 1 O in^: KN 3 1 • fH vo O Pi F*< M Pi Pi CO i>> 0- S>>0 Cd «H cd «H H »o P iH •CJ P M cd M cd on OJ ^ ON in^c 1 ON p CO 2 • fH • fH rH O Pi H Pi P-4 M P^ M t- O rH M 1 H H • H 1 CO O vo H M H rH & 1 • • H H 0- H OJ O H 0- H 0- O H 0- H X H 1 1 1 1 1 1 t- in CO 0- •st in in O ON -st VO t- t>- ON • • • • • • • • • OJ H O O H co CO CO CO co 00 VO VO vo H H rH H H H O X « « « M W H H H - O OJ m -3- On to H 1 1 t- t— OJ 1 m 1 vo •st C-- • • • • • • • • • • • H CD KN OJ H H OJ H iH H H H in VO in vo m O in in m m • • • • • • • • • • 0- vo OJ - nc; . „ 9-77 gT . ftTn ,»/ ■ ,, * - ■ -* —£ = 56.05 microgram gram of sand 0.1743 cm- per cm 2 . For comparison the ABS adsorption per unit area for sand particles is also calculated. Surface area of a single grain having a diameter of O.838 millimeter would be 4 * ( Q -°Q? 8 ) 2 cm 2 = 220.5 x lCT* cm 2 , and the volume would be _ijL ( 0.0838 )3 om3 „ 307-9 x 10 -6 Cffi3- If the specific gravity is 2.64, then the number of particles in one gram of sand would be 2.64 x 307.9 x 10-<> x graS"^ cm3 = *•&* x lo3 S rara_1 ' 51 total surface area corresponding to this number would be p 220,5 x 10- 4 x 1.238 x 103 g ^ = 27.3 cm 2 per gram. If the total surface area available for adsorption is assumed 75 percent of total surface area, then the ABS adsorbed will be 27.3 x'o?75 gram°x r cm^ ^ gm = 0.24 micrograms per square centimeter, assuming 5 microgram of ABS is retained per gram of sand. It would be interesting to evaluate the amount of ABS adsorbed assuming a monomolecular layer of ABS having an effective diameter of 0.3 millimicrons adsorbed in monomolecular layer in one gram of sand would be ?7t? * °«75.- cm n - 2? 7 x lO 1 ^ (0.3 x 10-7)2 cm 'd ~ d(i ' 1 x 1U • The average molecular weight of the ABS was reported to be 246 (31) Therefore the weight of ABS adsorbed per gram of sand on the above assumption would be 246 d.03 f x 10^3 =9.24 micrograms per gram which is of the same order of magnitude which has been reported in Table 6. Biodegredation of ABS : In addition to the ABS retained in the column the question was raised as to the possibility that the S^5 counted in the effluent might no longer be ABS, but rather some degradation product or elemental sulfur. To determine the fate of the ABS in the column effluent, an effort was made to evaluate the relative amounts of the sulfur in the column effluent which would still form a chloroform soluble ABS-T-Tethylene Blue 52 complex, the amount -which was degraded to inorganic sulfur, and the amount which could be classed as intermediate products of degradation. This was accomplished by separating the AES fraction in Methylene Blue solution. The extracted aqueous solution after converting any sulfide or sulfite to sulfate was treated with BaCl 2 to precipitate BaSO^. The filtrate would be any intermediate product not completely degraded. Radioactivity of the three frac- tions would indicate the proportion of various fractions. The relative activity of S^5 i n the three fractions of the column effluents are presented in Table 8. C. Results of Column E and F ; It may be recalled these two columns were run to demon- strate the effect of physical adsorption of AES on sand when salts are present in solution. Column E was fed with AES solution of concentration about 10 milligram per liter prepared in tap water containing about 500 milligram per liter total solids whereas column F was fed with AES solution of the same concentration but prepared in distilled water. The feed rates were adjusted by altering the effective head to 10-12 milliliter per hour corres- ponding to 0.5-0.6 centimeter per hour, though later there was considerable less flow because of air binding. Figures 11 and 12 shows the ABS breakthrough curve for columns E and F respectively. The calculated AES uptakes are in Table 6. The ABS uptake of column F gave a value of 5.34- micro- grams per gram of dry sand, which is exactly what was reported (20) for batch studies with 10 milligram per liter ABS solution and n V 53 H H KO co O o3in B » • • PKN\ \>- o ON *— ^ O CO E OJ H H CD >a o EH ft H H H -a CD s — o 03 43 •H H B P CD cm ^S t- On m P •p oukn \ co O H -a -p 03 n bOfn B H O CD • P! 03 H co hD ft H rH CD H aj 43 T3 PS O £ 43 O 4=> CO o P P o o^- P fH Pi H H 0J in VO 03 CD x) o bO T3 pSlO H H o D- •P ft PI 43 PI P s O CD H ^^ •H pq 43 O +3 ■P a +» P O — H 0) CD PS 00 rt B KN o ON H S 43 o fn m \ H H ON ft CD 1 EH o CD P*4 KN E rH H B p$ o o 43 H CO ft aj H fn P P CD JO CO pq c to pq o •H • fH CO 2 «*i H -a £ B CO EH O H «H CO 43 CD fH * «H CD 03 O J3 O -P H H 'd o fn n •p a co pS BE ON ON <+ CD ^H CD cfl o 03 H CO H Pi H \ • • • P o ■P > 4=> -p bO •P >j*H ^H O B KN H K\ O fH 03 0) fH •H H C 43 TJ O ft H H H G3 O 43 a B aj ft fH PS P « HO fn H H n-i pS-P H 3 o •H o o O CD CD B O SiH fn P 43 M O ft H LO O ft •o 0) bO 1 ft O CD O KN > 43 LfN in in a) fH PI CO E - p ft •H o pq o CO CO fn >» * ■< o 03 CD ft PI -3- ^ S > o 03 o g a) fn H o •o S X < P fn43 •P CO H * H CO O » HH" CO Eh O o3 fH H) CO CD H H H S CO H P o > rt pq a> o H E B B B si aJ •H O <* •p o ft 3 O P pq pi > P PI 0$ PS PS P< PS pS O CO U bO •H o o o CO CO ft H fc> CO «H CO P CD H OJ o o o o bOE 43 o o ON O 4=> 13 a) H H ro VO 03 3 bO OJ in co > H CD H ft fnH PS -3- ON t- E * — » ^N co O O in t- 00 CD > a3 o « a} o3 43 o t>> fH H O 03 fH CO ««! 43 Eh CO H ft • E O 03 $25 CO KN. co ON vo o H ftO 43 EH 03 t3 CO O H P PS CD O 43 ft43 Eh PS CO * -P CO H -P H O co a) I Pi= ovo s «a co p \a3 m co Lf\ CD p CD ■P CD 13 O ■P -P P CD B •H Pi CD ft M o a) B •H H CO 13 CD ,£> ^> g o CO «H o bO P •H • ^H CO bO E •H CO sB Pi 13 *- /*! o 0} b p< o p cr o u CO pp cm Q. «i E T3 C7> O C<$ i_ o to E •rt O H o ^. o CO o CO o CD o o CM 5 _i co i D Z < CO UJ < 0. CO CO < CD H +» a aJ 2 HrH O O > o a> ft 5° & o U 6 m cm O CM IT) m Puds jo w6 Jad '6uj ' sailOS 31I1V10A 39VH3AV 57 p CD P o o F3 CD ft O s P P O «H P cd H CD « e H Fl con a PK> ft O CO e-» B ft o CD p Cd p Fl in-Ho i^ ftft • CO iH 03 r-i oftB O CD •H Fl Fl U FIFm CD Q> cd -P ft fcO -^H FlO -H B O CO s CD CD fcsO B cd 3 FiH CD O > > fi H E CD H ft CO o S2i o 0> OJ CO OJ H m o\ ■St r- m m O ON OJ o\ OJ ^t" •st H H H LA I c- vo in oo t- vo vo c- OJ I 0J I ■«t VO 0J 3F vo t— § o u s o cd ^> Js o H CD J=> CO ■p fl p 1 O O CO o •p cd o «H 13 fl *-> a P H cd > a) > «H cd CD £3 CD •P O S2S 58 effluent. Here too there were no counts on BaSO^ precipitate but the counts were lower for the ABS-Methylene Blue Complex in ohloroform. These results were in accord with those obtained for columns A and C. It was also found that the scrubbing of sand to extract the slime out was about 100 percent effective. This was a good method to cross check the volatile solids on sand. 59 7. DISCUSSION OF RESULTS The increased uptake of ABS observed due to the presence of slime on Ottawa sand results undoubtedly from increased surface area provided by the actively metabolising microorganisms. The sterilisation of column D in an autoclave reduced the uptake to the level obtained from physical adsorption on the sand at 10 mg/l ABS concentration as reported (20) and as obtained for column F in Figure 12. This may be due to sterilisation which caused reduction of surface area provided by the microorganisms or the retention of ABS was associated only with growing organisms. There could be some shrinkage of cell volume due to death which reduced the effective area exposed to ABS adsorption. The amount of volatile solids of column D after autoclaving are of same order of magni- tude as of column C which point out that the amount of slime did not reduce much, so the surface area of such dead slime was present but it did not retain any ABS beyond that obtained for clean sand in batch studies. This was quite unexpected. If adsorption had anything to do with the negative charge on the surface of the microorganisms, it was not clear why the anionic ABS would readily adsorb on actively growing cells but would not adsorb so much on surface of the dead cells where the surface charge was probably less if not negligible. Thus it becomes evident that the retention of ABS on biological slime is associated directly with actively growing microorganisms though not necessarily entirely with degra- dation of ABS molecule for energy or growth of the cell. It may be that the ABS is retained on the surface of the extracellular enzymes secreted by the active microorganisms, the size of enzyme 60 may be somewhat larger than ABS molecule which is said to be 1 millimicron in diameter. But the autoclaving would not destroy already formed extracellular enzymes so there should be some increase in ABS retention. The outer surface of an active cell wall may produce some such phenomena which may encourage surface adsorption of the predominant ions. The ions which can penetrate the cytoplasmic membrane and can be degraded by the enzyme systems available are utilised for energy or growth of the cell but ions like ABS which have blocking groups for biodegradatlon in its structure may remain on the surface. Another possible explanation of this paradox could be that the surface area of cells after auto- claving were some how equal to the surface area of the sand on which these cells were growing because of the same order of ABS retention per unit weight of sand, A sand particle having say 5 layers of bacteria over its surface had a tremendous amount of surface area from each cells because of open sponge like character of the layers; but after autoclaving there was a collapse of the form of the sponge resulting in area corresponding to the sand particle itself. The results of benzene-methanol extraction of sand from which slime had been scrubbed we see directly that 90 to 95 per- cent of the ABS adsorbed is associated with the slime rather than the sand around which the slime was developed. The capacity of sand to adsorb ABS at 10 mg/l was found to be around 5«0 microgram per liter as obtained both by batch and column P results. Thus the sand containing slime in Column C having retained 1.36 micro- grams per gram of sand would adsorb more ABS by diffusion if the 61 time of experiment was prolonged due to concentration gradient between the pore liquid and the slime, and between the slime and the sand surface so that the ultimate retention on sand alone would be 5.0 microgram per gram of sand. The experiment to find if sulfonate group of the ABS mole- cule was broken down by the microorganisms did suggest some degra- dation at least for column C, the count of ABS in 20 ml sample extracted and diluted to 100 ml in Methylene Blue -Chloroform were about 50 percent less, though there was no activity indicated in the BaS04 precipitate, but counts on BaS04 precipitate were not corrected for self adsorption. Thus complete break down to ele- mental sulfur was not indicated. As a matter of fact, we could not account for the balance of the radioactivity even in the water phase or in the sulfate precipitate. This could mean that self absorption on the BaSO^. precipitate was accounting for this dis- parity. The self absorption factor for about 35 milligram per ml of solids in the planchet corresponds to about 4.0 for non radio- active ABS as the solid. The effect of flow rate greatly influenced the ABS uptake which was quite understandable. The surface adsorption phenomena is certainly time dependent. The greater opportunity of contact provided by longer time or slower rate of flow will definitely enhance the ABS retention, as much more unexposed surfaces and dead spots would have a better chance of contact with ABS molecules. For column B at 50 milligram per liter AES concentration, the ABS uptake was only 3-03 micrograms per gram of sand whereas columns E and P, both at only 10 milligram per liter of ABS in feed 62 solution but at a much lower rate of flow, retained 4.04 and 5.34 microgram per gram of sand respectively. On a purely linear rela- tionship this is about 6 to 8 times higher retention. This was also observed in the case of column A and C. Column A, partially saturated having effluent concentration 50 percent of influent feed concentration, retained 22.8 micrograms per gram of sand at 50 milligrams per liter of ABS in feed solution, but Column C, at a much slower rate of flow, retained half as much with only 10 milligram per liter. Thus a slow rate allows the unapproached corners and unsatisfied dead areas to have an opportunity to retain ABS which otherwise would have bypassed owing to faster rate of flow. The molecular concentration gradient between the unsatisfied and partially satisfied sites cause a slow diffusion of ABS molecule from a higher to a lower gradient. This process was so slow that in some cases we had to wait 3 to 4 months to get anywhere near saturation. Sand grains have a layer of liquid adhering to the outer surface due to surface tension and the layers of adjoining grains join at the contact boundaries forming a network. The pore water during its passage downwards shears away from the liquid layer on sand without disturbing it. Thus there are many station- ary liquid boundaries in a single pore which do not get displaced by incoming liquid. At the start of the ABS application the con- centration gradient between the pore liquid and the outer liquid layer on the sand was high, resulting in fast transfer of ABS mole- cule to the outer layer. As time passed the layer was eventually saturated by purely molecular diffusion. The concentration gradient between the sand surface ultimately caused the ABS to be retained 63 on sand itself. The surface areas near the contact zone of other sand grain were comparatively far from the high concentration zones, so it would take a long time by molecular diffusion to balance the concentration at those places. The amount of ABS retained on the sand surface would be determined by the sites available to the ABS molecules and also to the size of an ABS molecule. Other salts or ions in water reduced the uptake of ABS on sando Columns E and F were identical in all respects except the solution of ABS in one was made of tap water and the other was distilled water. The ABS uptake was of the same order of magni- tude, 4.04 and 5«34 microgram per gram of sand for columns E and P respectively. There was about 20 percent lowering of uptake in E. The ABS molecule had to compete with other ions in solution for the sites on the sand as such total number of sites being approximately the same, we obtain less retention of ABS in one than the other where there are no competing substances. This explana- tion sounds as if the retention on sand was an ion exchange mechan- ism. The mechanism of adsorption has not yet been fully explained and we cannot rule out a possibility of partial ionic bonds between the adsorbate and the adsorbent. Again the presence of other ions would produce what is known as salting out effect which would de- crease the solubility of ABS. With lower solubility the ratio L would Increase resulting in decrease of pollute front velocity. This would relatively increase the ABS uptake on solid phase. Obviously this effect was not very significant in this case 64 perhaps because there was insignificant reduction of solubility to produce a marked effect on the ABS retention. The reversibility of ABS retention on sand or slime was demonstrated by the extraction of the retained ABS by alkaline benzene -methanol solvent. It was reported by the University of California group (28) that 80-100$ of the adsorbed ABS could be recovered this way. In Table 6 the average values of ABS extracted from sand were lower in all cases except for column E. There was always a higher concentration of ABS associated with the top layer of sand with gradual lowering as the depth increased. The average values noted do not represent the true average which would take into account the amount of sand associated with a particular con- centration of ABS at a particular depth. In fact this may well be the reason why the average value of ABS extracted from sand was in the case of Column E higher than obtained by ABS breakthrough curve. The amount of ABS in the water phase between the pores of the sample of sand being extracted was taken into account although it was only 1-5 percent of the total ABS. The calculation of ABS retention based on surface area of slime produced a very high value of ABS adsorption compared to sur- face area of sand. It was 233 times higher for the slime surface. This may be due to multi -molecular layer adsorption on the bacter- ial cell as compared to mono-molecular layer on sand. Eut this does not explain such great magnitude of variation. Moreover there may be inner surfaces present inside the outer wall of the cell over the cytoplasmic membrane which increased the surface area. The adsorption of ABS on sand was of the order of 9.24 microgram 65 assuming a mono-molecular layer and 0.3 millimicron as the effective diameter of ABS. The 0.3 millimicron was selected as the effective diameter because it was felt that the ABS molecule whose diameter was reported to be 1 millimicron by Nichol (13) was in fact the length of the chain and the side width would be considerable less than this value. For soap molecules in a monomolecular layer the distance between two adjoining molecules was said to be around 0.19 millimicron so with the benzene ring in ABS it was presumed the distance would be higher, around 0.3 millimicrons. The ABS adsorption values for sand from breakthrough curve are less than the value obtained from monomolecular layer calculations. This may be due to the fact that complete saturation was not obtained or the effective area used for calculations was lower than 20.47 cm 2 per gram of sand or the effective diameter of ABS molecule was more than 0.3 millimicron. But in any case the ABS adsorption values obtained from breakthrough curve were of the same order of magnitude of the value obtained from theoretical assumption of a monomole- cular ABS adsorption on sand. The results of the open column showed clearly that under partially aerobic conditions the ABS uptake was very high. It was at least twice that obtained in closed column C although both the columns were not completely saturated. The adsorption of ABS at different depths was proportionate to the volatile solids at that depth. This was significant proof that biological slime are very effective in retaining ABS. The volatile solids and the number of bacteria per gram of sand were significantly higher than column A or C, so it would be expected to get a higher ABS retention if 66 surface adsorption is the phenomena by which retention occurs. The ABS extracted from sand phase by solvent extraction was higher than obtained by breakthrough curve; this may be due to the fact that the value obtained from breakthrough curve was an average of whole column content whereas individual adsorptions values would be higher at top and gradually decrease. The experimental results to find if the ABS was degraded produced results in line with the findings of column A and C. There was no radioactivity associated with the BaSC>4 precipitate, although the counts on ABS-Methylene Blue Com- plex in chloroform were 40-50 percent lower than the direct counts of the sample. The evaluation of weight of slime by scrubbing it off from sand was compared with the volatile solids obtained by igniting at 600°C. There was a very good correlation between the two indicating that the scrubbing of sand for bacterial count and ABS extraction of the scrubbed sand, did succeed in quantitative removal of slime from the sand • The death of microbial cells reduce the retention of ABS so in a practical case of a septic tank tile field slime which had adsorbed ABS, any stopage of growth factor of the microorganisms like shut down or vacation etc. would allow the adsorbed ABS to go into liquid phase and percolate into the groundwater by rain or snowfall. Thus it may be necessary never to stop the septic tank working to eliminate such occurance. This, of course would be very impractical solution, so it would be necessary not to depend on the biological slime at all to retain the ABS but to utilise sound sanitary principles to maintain a proper degree of ABS removal prior to application to ground or surface water. 67 8. CONCLUSIONS The following conclusions are drawn from the above dis- cussion and results. (i) From comparison of ABS retention on a column having biologically active slime and one in which there was no slime, it is concluded that biological microbial growth enhances the retention on Ottawa sand. At 50 milligram per liter in feed solu- tion, 7 times as much ABS was retained on the sand in which slime was present. At 10 milligram per liter ABS in feed solution the retention was only about 3 times as much. (il) Slime developed on Ottawa sand retained 90 to 95 percent of the ABS adsorbed while less than 10 percent of the ABS was associated with the sand particles beneath the slime. It was found that the technique of scrubbing of the slime from the sand by vigorous shaking did remove all the slime. (iii) The effect of ABS adsorption on sterile slime showed that uptake was of the same magnitude as in a column without any slime. Thus the higher uptake due to presence of biological slime is associated with the actively growing organisms rather than with the surface area provided by dead cells. (iv) Amount of ABS adsorbed per square centimeter of slime was about 230 times higher than the value obtained for sand. (v) The retention of ABS on clean Ottawa sand at 10 milli- gram per liter ABS feed solution in column studies checked well with those obtained by batch operations. It was of the order of 5.0 to 5.50 microgram per gram of sand. 68 (vi) The rate of flow or time was an important factor in ABS retention on sand. The overall retention on biologically- active slime grown over sand at 10 milligram per liter of ABS in feed was higher than at 50 milligram per liter of ABS in feed solution because of slow rate of flow. (vii) The retention of AES in open column under partially- aerobic (microbial) conditions was higher than those obtained with closed column under anaerobic conditions. The value of ABS adsorp- tion on sand was 22.47 microgram per gram of sand, which was approximately twice as much obtained by column C. (viii) The retention of ABS on sand was a physical adsorption phenomena easily reversed by suitable solvent extraction. (ix) The variation of ABS adsorption on sand with depth was proportional to the amount of volatile matter present at that depth for open column, which indicated the importance of biologi- cal slime in retention of ABS on sand. (x) The practical significance of these findings mean that a well developed slime in a septic tank drain field or other ground waste disposal system can concentrate a large amount of ABS which otherwise would have gone into the aquifer water by seepage. Death of the microorganisms would elute the adsorbed ABS so it would be necessary to keep the septic tank going on all the time, which is quite impractical. So it would be best not to depend upon the adsorption of ABS on slime but to use better sanitary practice to have a lower concentration of ABS in effluent prior to applica- tion on ground. 69 9. REFERENCES (1) Weaver, P. J., Review of Detergent Research Program, Jr. Water Pollution Control Federation. 52 . 288, (March 19^0). (2) Flynn, J. H., et al, Study of Synthetic Detergents In Ground Water, Jr. AWWA . 50, 1551, (December 1958). (3) Polkowski, L. E., et al, Evaluation of Frothing in Sewage Treatment Plants, Sewage and Industrial Waste. 31 . 1004, (September 1959). (4) Sawyer, C. N., Effect of Synthetic Detergents on Sewage Treatment Processes, Sewage and Industrial Waste. 50 . 757, (June 1958). (5) McGauhey, P. S. and Klein, S. A., Removal of ABS by Sewage Treatment, Sewage and Indistrlal Waste. 51 . 877, (August 1959). (6) Lynch, W. 0., and Swayer, C. N., Effect of Detergent on Oxygen Transfer in Bubble Aeration, Jr. Water Pollution Control Federation. 52 . 25, (January I960) . (7) AAGSP Committee, ABS and the Safety of Water Supplies, Jr. AWWA. 52 . 786, (June I960). (8) Deluty, J., Synthetic Detergents in Well Waters, Public Health Reports. 75 . 75, (January, I960). (9) Drinking Water Standards, 1961, U. S. Public Health Service, Jr. AWWA. 53 . 8, 935, (August, 1961). (10) Lauman, C. W., Co., Effect of Synthetic Detergents on the Ground Waters of Long Island, N. Y. , IT. Y. State Water Pollution Control Board Research Report No. 6 (I960). (11) Walton G., ABS Contamination, Jr. AWWA . 52, 1354, (November I960) . (12) Oliver, G. E., ABS in Michigan Supplies, Jr. AWWA. 53 . 301, (March 1961). (13) Nichols, S. M. and Koepp, E., Synthetic Detergents as a Criterion of Wisconsin Ground Water Pollution, Jr. AWWA . 53, 303, (March 1961). (14) Walton G., Chemical Indicators of Sewage Contamination of Ground Water, Technical Division Activities. National Water Well Association . 11. (1960). (15) Schmidt, 0. J., Significance of Detergents in Water Pollu- tion Control, Public Works. 12 . 93, (December 1961). 70 Corapenni, L. G., Synthetic Detergents In Ground Water - Part 2, Water and Sewage Works , 210, (June 1961). Woodward, F. I., et al, Experiences with Ground Water Con- tamination in Unsewered Areas in Minnesota, Presented before conference of State Sanitary Engineers and Conference of Municipal Public Health Engineers, APHA Annual meeting, San Francisco, California, (November I960). Neutsh, F., Pollution of Ground Water by Detergent Residues from Laundry; Eull. of Hygiene. 55 . 102 , (February I960). Gotaas, H. E., et al, Studies in Water Reclamation, Sanitary Engineering Research Laboratory, University of California, Technical Bulletin No. 13 . I.E.R. Series 37. Ewing, B. E., et al, Synthetic Detergent and Ground Waters, 1st Progress Report, Department of Civil Engineering, University of Illinois, 3, (January 1961). Rifai, M. N., et al, Dispersion Phenomena in Laminar Flow Through Porous Media, Sanitary Engineering Research Labora- tory, University of California, Report No. 3. I.E.R . Series 90 . Renn, C. E. and Barada, M. F., Adsorption of ABS on parti- culate Material in Water, Sewage and Industrial Waste. 31 * 850, (July 1959). Lieber, M., Syndet Removal from Drinking Water Using Activated Carbon, Water and Sewage Works. 107 . 299, (August, I960). Eckenfelder, W. W. , and Barnhart, E., Removal of Synthetic Detergents from Laundry and Laundromat Wastes, New York State Water Pollution Control Board. Research Report No. 5 . — £— McKinney, R. E. and Symons , J. E., Bacterial Degradation of ABS-1. Fundamental Biochemestry, Sewage and Industrial Waste . 21, 549, (May 1959). Ryckman, D. W. and Sawyer, C. N., Chemical Structure and Biological Oxidizability of Surfactants, Proceedings 12th Industrial Waste Conference. Purdue University. 1957 . 270. Nelson, J« F. t et al, The Biodegradability of Alkyl Eenzene Sulfonates, presented at the American Chemical Society meeting Cleveland. Ohio . April I960. Studies of Synthetic Detergents, News Quarterly. Sanitary Engineering Research Laboratory . University of California, Volume XI - July 1961. 71 (29) APHA, Standard Methods for Examination of Water, Sewage and Industrial Waste, 11th Edition (I960), 246. (30) Pinal Report on the Fate of ABS in Sewage Treatment, Sanitary Engineering Research Laboratory , University of California, (July, 1957). (31) Rutherford, J. T., California Research Corporations, personal communications, (32) Cohen, J., personal communications. (33) Steel, E. W. , Water Supply and Sewerage . Fourth Edition, p. 519, KcGraw Hill Book Company, IT. Y. , (I960). 72 10. APPENDIX Appendix I Modified Procedure for Radiochemical Determination of ABS and Degradation Products Using S35 A. Determination of ABS Step 1. A 50 -ml aliquot of the ABS^5 solution is placed in a 250-ml separating funnel and acidified with 1 ml of concentrated HC1. Step 2. The 50-ml aliquots are extracted using 25 ml of ether. The extraction is performed by shaking vigorously for exactly 2 min. After the ether and ABS solution has separated, the ABS solution is drawn off into a second separatory funnel. The extraction is repeated three more times. The four ether portions are accumulated in the second separatory funnel. Step 3. The collected ether is then washed twice with 20 ml of 211 HC1 for exactly 2 min. each time. Again the ether and 211 HC1 wash solution are separated between washings.. The wash solution is collected in a third separating funnel. Step 4. 1.000 gm of activated carbon (Nuchar) is placed in a 300 ml Erlenmeyer flask to which the ether is added. This mixture of carbon and ether is then evaporated to dryness on a steam bath. 73 Step 5. The dried carbon is further treated by adding 50 ml of acetone and 50 ml of distilled water. The Erlenmeyer flask is then stoppered until samples are taken in planchet. Step 6. Two ml of the carbon-acetone-water suspension is pipetted and transferred to a planchet. After drying in an oven at 103°C, the samples are cooled in a dessicator. Counting may be performed after drying. Step 7. The planchets are counted in an internal propor- tional counter and the count is corrected for background, self-absorption and decay. The net corrected count per ml of solution found. Step 8. The concentration of ABS is Corrected Net Counts per mln. 100 x 1000 Specific Activity CIICPK/mg AES x ' 50 in mg ABS/l. Sample volume is 50 ml and after extraction was diluted to 100 ml in acetone and water, 1000 is conversion from mg/ml to mg/l. B. Determination of Inorganic Sulfur Degradation from ABS Step 1. To the original water sample after extraction, together with the wash water, 0.5 gm of sodium sulfate are added. Step 2. The solution is heated to boiling and 15 ml of concentrated Bromine water were added. The bro- mine water will oxidize the inorganic sulfur to sulfate. 74 Step 3. After the bromine color is gone, 10 ml of 10 percent barium chloride is added and a white precipitate of BaS04 is formed. Step 4. The solution is then vacuum filtered through Whatman No. 2 filter paper to remove the BaS04 precipitate, using a Tracerlab Model E8B preci- pitation apparatus. Hot distilled water is used to wash the precipitate from the Erlenmeyer flask. Step 5. The precipitate on the vacuum filter paper is placed in a 103° C oven for drying and then counted. The count is corrected for background, self -absorption and decay. The corrected net counts per minute per ml found. Step 6. The concentration of inorganic sulfur is Corrected Net Counts -per mln per ml on „/•, Sp# Activity CITCPK/mg ABS x dU mg/x as ABS. Specific activity must be determined under similar counting condition. C. Determination of Intermediate Products Step 1. Two ml aliquots of the filtrate from Step 5 are transferred to a planchet by pipette and dried in an oven at 103° C. Step 2. The planchets is counted in the internal propor- tional counter and the count rate is corrected for background, self-adsorption and decay. The corrected net counts per ml found. 75 Step 3. The concentration of intermediate products in the degradation of the original ABS-55 equals Corrected net counts per rain per ml Specific Activity CNCPl-I/mg ABS _ ml. of filterate _ on „„/, __ A -n C x in step 5 Above x 20 m sA as ABS. 76 Appendix II Revised Procedure for Radiochemical Determination of ABS and Degradation Product Using S35 A. Determination of ABS (29) Step 1. 20 to 50 milliliter aliquots of the ABS35 solution are placed in a 250 ml separatory funnel and using phenolphthalein indicator it is just made basic with IN HaOH and acidified with HI HC1. Step 2, 25 ml of Methylene Blue solution are added. Step 3. 10 ml of chloroform are then added and the ex- traction of ABS-Methylene-Blue complex in chloro- form is performed by vigorous shaking for 30 seconds. After the layer of chloroform and water are separated, the chloroform is drawn off into a second separatory funnel. The first funnel liquid is washed with 3 ml of chloroform and chloroform is drawn into the second separatory funnel. The extraction and washing with chloro- form is repeated two more times. All the chloro- form from the three extractions are accumulated in the second separatory funnel. Step 4. The chloroform ABS-Methylene Blue complex in the second separatory funnel is washed by shaking for 30 sees with 50 ml of wash solution containing Na2HP04 and H2SO4. The separated chloroform layer is transferred through glass wool to 100 ml. volu- metric flask. The separatory funnel content is rinsed with 3 ml chloroform which is also drawn 77 Into the volumetric flask. The washing with vigorous shaking of the remaining liquid in the separator^ funnel with 10 ml chloroform is repeated twice, each time after a wash the funnel is rinsed with 3 ml chloroform. Step 5« The collected chloroform solution of Methylene Blue-ABS complex is diluted to 100 ml mark with chloroform. Step 6. Two ml samples of the chloroform solution of Methylene Blue-ABS complex is transferred to a planchet and dried in the oven at 103°C. Step 7« The dried planchets are counted in an internal proportional counter at 1800 V and the counts are corrected for background, and decay. The cor- rected net counts per minute per milliliter is found. Step 8. The concentration of ABS in the sample . Corrected ITet Counts per minute per milliliter Specific Activity CNCFM per milligram ABS x 100 x 103 / x Volume of sample II1 S/- L The determination of inorganic sulfur degraded from ABS and intermediate products are the same as in Appendix I para B and C respectively. 78 Appendix III Solvent Extraction of ABS from Solid Phase Step 1. A known amount of sand whose moisture content was also known is placed in the porous container and inserted in the syphon tube of the Soxhlet apparatus. Step 2. 100 milliliter of Methanol is mixed with 100 milliliter of Benzene and 4 milliliter of 2N NH4OH is added so that the final normality of the mixture is about 0.04. The corresponding pH will be of the order of 7.5« This solvent is placed in the distilling flask of the Soxhlet apparatus. Step 3. The distillation is continued in a water bath with temperature controlled around 65°C + 5°C. A condenser at the outlet of the Soxhlet appara- tus returns any solvent vapours condensed to the syphon tube. The extraction is continued for 8 hours . Step 4. All the extracted solvent is collected and its volume measured. 2 ml. is transferred to a planchet and dried at 103°C. The radioactivity is counted in an internal proportional counter at 1800 V. The counts are corrected for back- ground and decay, and the corrected net counts per minute per milliliter is found. 79 The milligram of ABS per gram of sand - Corrected net counts -per min per ml x ml solvent Specific Activity CNCPM per mg ABS x M where M = weight of dry sand as sample in gms. Step 5« A correction may be applied for the ABS^5 activity of the pore liquid of sand. The amount of moisture being known, the concentration of ABS is assumed to be equal to the column influent after saturation conditions have been attained. Total ABS associated with pore liquid is sub- tracted from the gross total obtained above. «p^\* ' &