EPA 904-R-97-003 THE ANAEROBIC DEGRADATION OF SELECTED CHLORINATED HYDROCARBON PESTICIDES by David W. Hill and Perry L. McCarty 9/27/1966 reprinted by: U. S. Environmental Protection Agency Region 4, Water Division,'Wetlands Section 61 Forsyth Street, S. W. Atlanta, Georgia, U. S. A. 30303-3415 (404) 562-9900 voice (404) 562-8339 TTY/TDD (800) 241-1754 voice WEB site: http://www.epa.gov/docs/Region4Wet/wetlands.html e-mail: burnett.thomasSepamail.epa.gov ------- THE ANAEROBIC DEGRADATION OF SELECTED CHLORINATED HYDROCARBON PESTICIDES by David W. Hill* and Perry L. McCarty Respectively, Doctoral Candidate and Associate Professor of Sanitary Engineering Department of Civil Engineering Stanford University Stanford, California *Now a Research Sanitary Engineer with the Federal Water Pollution Control Administration, Southeast Water Laboratory, Athens, Georgia. Presented on September 27, 1966 at the Water'Pollution Control Federation Meeting, Kansas City, Kansas. ------- THE ANAEROBIC DEGRADATION OF SELECTED CHLORINATED HYDROCARBON PESTICIDES 'by David W. Hill and Perry L. McCarty The persistence of many of the modern organic pesticides in the environment has led to a great deal of concern over their possible harm- ful effects upon both wildlife and human beings. The most persistent of the organic pesticides are the chlorinated hydrocarbon insecticides and certain associated epoxides, commonly referred to as a group simply as the chlorinated hydrocarbons. Residues of these pesticides have been measured in soils more than a decade after application and traces have been found in almost all major U. S. rivers as well as in fish in the open sea. Chlorinated hydrocarbon residues in natural waters are of particular concern because of their great toxicity to many aquatic organisms, because of their possible adverse effect on man through his drinking water sup- plies, and because of their tendency to concentrate in living matter which sometimes results in the death of higher species such as fish and birds. The extremely hydrophobic nature of most of the chlorinated hydrocarbons allows them to concentrate on particulate matter which may settle to form the bottom muds of streams and lakes. There, the pesticides may poison bottom feeding species, they may be churned up and carried along with sedi- ment during periods of turbulent flow, or they may be desorbed to maintain a continuous feed of pesticides to the overlying water. It is important in estimating the quantities of pesticides trans- ported by a river, or in estimating the build-up of pesticides in a reservoir, lake, or bay, to have some indication of rates of degradation ------- -2- or of rates of conversion to other forms. Most transport studies conducted to date have assumed indefinite persistence of all measurable pesticides from which it could only be concluded that there was a continuing increase in the level of pesticide residues, while it may actually be that pesticides slowly degrade so that an equilibrium state is eventually reached. The assumption has frequently been made in the past that if a substance is very resistant to aerobic degradation, then it will probably not degrade at all under anaerobic conditions. This assumption has been applied to the persistence of chlorinated hydrocarbons in the water environment; yet the results of this study have shown that many of these pesticides degrade more quickly under biologically active anaerobic conditions than under correspond- ing aerobic conditions. The term "degradation" is used here in a broad sense to refer to any measurable chemical change in a pesticide under natural environmental con- ditions. This degradation may be "complete degradation" to inorganic end products or "partial degradation" to other organic products. With this def- inition it can be stated that chlorinated hydrocarbons degrade without in- ferring that they are converted completely to inorganic end products. PESTICIDES STUDIED The structures of the eight pesticides studied in this investigation are shown in Figure 1. DDT and DDD are indicated by schematic structures; while the remaining pesticides are illustrated by three-dimensional draw- ings as their schematic structures do not differentiate between possible isomers. Consumption of these pesticides represents more than 60% of the total U. S. consumption of chlorinated hydrocarbon compounds. In chemical ------- -3- structure they represent a range of different types of chlorinated hydrocarbons. DDT and ODD represent chlorinated diphenyl compounds with the basic "DDT carbon skeleton"; lindane represents the benzene hexachloride isomers; aldrin and heptachlor are unsaturated cyclodienes; and heptachlor epoxide, dieldrin, and endrin are epoxidized cyclodienes. Contrasting studies of anaerobic versus aerobic degradation were restricted to DDT, lindane, aldrin, and heptachlor epoxide to obtain a representative of each structure, which was well separated from the others on a gas chromatogram. PREVIOUS STUDIES The persistence of various chlorinated hydrocarbons in different parts of the environment has been extensively documented. Soil pesticide residuals have received the greatest study, and Wheatley (33) lists half lives ranging from 25-40 years for DDT down to 15-20 weeks for lindane. Pesticide residues in water have been receiving increasing attention in recent years, and measurable concentrations of chlorinated hydrocarbons have been found in almost all the major U. S. rivers (31). Ordinary background levels of dieldrin, endrin, DDT, DDE, heptachlor, aldrin, DDD and BHC (in order of frequency of occurrence) were found in the parts-per-trillion range in water (1 ppt to 1 ppb) (9, 13, 31) ; and a Mississippi River study showed that many pesticide residues in bottom muds were found at concentrations greater than 0.1 ppm (5). The persistence of chlorinated hydrocarbon pesticides in the aquatic en- vironment is documented mainly in reports of field studies made following in- dividual applications of pesticides. Residues of DDT generally disappear fairly rapidly from water only to reappear in greater concentrations in sedi- ments, vegetation, and aquatic fauna in which they are much more persistent ------- -4- (7, 11, 12). Perhaps the classic example is the persistence of more than 1000 ppm of DDD in fish and birds at Clear Lake, California, more than a year fol- lowing a parts-per-billion application to the lake water (18). A total of 0.05 ppm application of toxaphene to Clayton Lake, New Mexico, persisted in the water at a level of 0.001 ppm for more than one and one-half years (19). EXPERIMENTAL PROCEDURE The conditions that received the greatest amount of study in this inves- tigation were active anaerobic conditions that were idealized or made very favorable for degradation. Here, pesticides were mixed continuously at 35°C with thick, biologically active anaerobic digested sewage sludge obtained from the Menlo Park Sewage Treatment Plant. This environment was chosen to obtain a clear indication of degradation which would probably occur only slowly in less favorable, natural environments. It must be stressed that the anaerobic conditions used in this study were ideal in the sense that an active culture of anaerobic methane producing and sulfate reducing bacteria were present. It is not implied that these particular microorganisms were necessarily involved in the degradation which occurred, but only that their presence in an active state is a useful indi- cator of the conditions under discussion. Significant anaerobic pesticide degradation would probably not occur under conditions which would not be con- ductive to the growth of these particular organisms because under such condi- tions many less resistant materials such as fatty acids are not biologically destroyed. Most of the apparatus was designed with variable volume gas reservoirs as shown in Figure 2. Closed systems were used to minimize pesticide losses by codistillation with water, and the variable volume gas reservoir was essential to avoid excessive pressures in thick-sludge anaerobic units ------- -5- in Which gas production occurred. Sampling was facilitated with this design because application of pressure to the acid-salt confining fluid would force sludge out the withdrawal tube. Pesticide analysis was performed by gas chromatography following organic solvent extraction and clean-up procedures. Samples for analysis were centrifuged and a separate determination was made for pesticides in the supernatant liquor and that sorbed to the suspended solids. The preferred extraction procedure from water or from centrifuged supernatant liquor was to place a small volume of hexane (generally 10 ml) in a separatory funnel on top of the liquid to be extracted (generally about 80 ml). The contents were shaken gently on a horizontal shaker for one hour with the separatory funnel on its side; the water layer was re- moved and discarded; the volume of hexane was measured; and an aliquot was injected directly into a gas chromatograph. (An electron capture gas chromatograph was the most suitable for this procedure as no further pesti- cide concentration step was required). The extraction preferred for centrifuged suspended solids was largely a modification of a procedure designed for pesticide extraction from dairy products reported by Langlois, Stemp, and Liska (23). The centrifuged solids were scraped into a large mortar and ground uniformly into about 25 grams of Florisil. The 60-100 mesh Florisil, obtained from the Floridin Company, Tallahassee, Florida, was previously activated at 600°C for three hours, cooled, mixed thoroughly with 5% water, and stored in an airtight container for at least 48 hours prior to use. Five hundred milliliters of 20% methylene chloride in petroleum ether was mixed in successive increments with the mixture of ground solids and Florisil and then was decanted into ------- -6- the top of a liquid chromatographic column packed with four inches of Florisil. The solvent was eluted through the column into a Kuderna- Danish evaporator and evaporated to about 5 ml. The walls and joints of the evaporator were then rinsed and the solvent was concentrated further by evaporation through a micro-Snyder column. Evaporation was continued to dryness to remove all the methylene chloride using a gentle stream of air directed into the ampule after placement in a flask of hot water to prevent condensation. Finally, the residue was redissolved in 2 ml of hexane, and an aliquot was suitably diluted and injected into a gas chromatbgraph. All solvents were redistilled prior to use, and all glassware was care- fully washed and. given a final rinse with acetone. Teflon stopcocks were soaked in acetone while not in use to help minimize contamination from pesti- cides which strongly adsorbed on Teflon. Pesticide extraction efficiency with both preferred methods was approximately 100%. The gas chromatographs used during the course of this investigation were a tnicrocoulometric gas chrotnatograph of the type manufactured by Dohrmann Instruments Company and an electron capture Hy-Fi Gas Chromatograph manufactured by Wilkens Instrument and Research, Inc. (now Varian Aerograph). These instruments are described adequately in the literature. Other analyses were performed using techniques essentially as described in the llth Edition of Standard Methods (30). EXPERIMENTAL RESULTS o Degradation in Thick Anaerobic Sludge at 35 C o Experiments with thick anaerobic digested sewage sludge at 35 C formed the major portion of this investigation as it was with this material that degradation was the most pronounced. ------- -7- All concentrations of pesticide products are reported as equivalent concentrations of the parent pesticide. Calculations were made by comparing peak areas generated by the pesticide products with those generated by the parent pesticide injected as a standard. The one exception was DDD formed from DDT which is reported as DDD as determined from DDD standards. The pH of the sludge varied from one experiment to another but it remained generally in the range of 7 to 8. All pesticides were dissolved in acetone prior to injection in the units. The sludge for the first two studies with lindane was diluted 1:1 with deaerated tap water to produce an average total dry solids concentration of 1.5% of which an average of 56% was volatile solids. The sludge was seived and placed under nitrogen in the apparatus described previously. Units were agitated on horizontal shakers because the sludge was too thick to stir with magnetic stirring bars. In the first study two units were dosed with one and ten ppm lindane, respectively; and a third unit was maintained as a control. [Throughout this discussion pesticide concentrations expressed as parts per million (ppm) refer to micrograms of pesticide per milliliter of sample and parts per billion (ppb) refer to micrograms of pesticide per liter of sample!] The results of this experiment, indicated in Figure 3, show an immediate and sharp decrease in the total concentration of lindane in both units. The second study was performed to determine if this rapid anaerobic degradation of lindane was a biological or a chemical phenomenon. Four units were prepared of which two were heavily poisoned with a total of 4 gm/1 of cobaltous and mercuric salts. One active and one poisoned unit were dosed with 10 ppm lindane each, and the other two units were maintained as controls. The results of this experiment shown in Figure 4 indicate a very significant ------- -8- difference between the rates of degradation in these two units. The half life of lindane in the active unit was in the order of 1 day while that in the poisoned unit was approximately 170 days* The third study was designed to measure the degradation of other chlorinated hydrocarbon pesticides in thick biologically active anaerobic sludge at 35°C. Total solids averaged 4% by weight of sludge and volatile solids averaged 48% of the total solids. Each pesticide was dissolved in 350 jul of acetone and injected into a separate one-gallon bottle containing 3500 ml of undiluted, seived, digested sewage sludge under nitrogen; giving a 1 ppm concentration of pesticide in each bottle which was connected to a gas reservoir as shown in Figure 2. These units were also agitated on hori- zontal shakers. The results of this study are shown in Figure 5. DDT was converted almost immediately to DDD, which was identified by its characteristic re- tention time on two types of chromatographic columns; and the DDD was de- graded gradually but steadily with a half life of less than one week. Aldrin was degraded at approximately the same rate as DDD. Heptachlor was converted to an unidentified early elution product whose retention time on a Dow- Corning 200 Silicone Oil chromatographic column was three-quarters that of heptachlor. This product persisted for at least 42 days but was degraded completely within 266 days. The fourth study with thick anaerobic sludge was conducted to measure the degradation of large doses of pesticides added in combinations to units containing sludge of different characteristics. Two combinations of pesti- cides were each added to a Thick Sludge unit (7.2% total solids) and to three units (4% total solids) used previously in the third study. Of these ------- -9- three units one was lightly poisoned with one-half gram per liter of mercurous chloride as mercury, and two were left unchanged (Active I and Active II). The first combination of pesticides used was lindane, aldrin, heptachlor epoxide, and DDT in concentrations of 40, 40, 100, and 100 ppm, respectively; and the second combination was heptachlor, dieldrin, and endrin; 10, 30, and 150 ppm, respectively. These concentrations were chosen to give approximately the same peak heights on the gas chromato- graphic chart. The results of this study are shown in Figures 6 and 7. Lindane de- graded very rapidly in all units and was the only pesticide to show a sig- nificantly greater persistence in the lightly poisoned unit than in the active units. Other differences between the units were largely masked by the variability of the results. However, slightly faster degradation rates were suggested for lindane and aldrin in the Thick Sludge unit than in other units. DDT and heptachlor were observed only in samples taken initially, 20 minutes after injection of the pesticides. All later analyses of samples for these compounds showed only DDD and "heptachlor early elution," respectively, to be present. Several degradation products of endrin were observed. The first and most clearly defined product had a relative reten- tion time of about 2.34 on a Dow-11 column; three other less distinct products were formed later with relative retention times ranging from 2.12 to 5.55 on a Dow-11 column. These products were not formed from heptachlor or dieldrin, the other pesticides added in the mixture, because the products were observed in greater concentrations than the total added heptachlor and because dieldrin persisted in almost its initially injected concentration. ------- -10- Degradation gollowing Daily Pesticide Injections into Dilute Sludges Daily injections of a pesticide mixture were made into dilute sludges contained in five-gallon units illustrated in Figure 2. The daily injections were made to examine variations in pesticide degradation rates which might be caused fry repeated doses of pesticides; and dilute sludge was used to allow measurement of pesticide degradation at slower rates than observed previously in the thick sludge studies at 35°C. With dilute sludge it was possible to maintain aerobic conditions in some units to allow a comparison of aerobic and anaerobic rates of pesticide degradation. Degradation products were again measured as the parent pesticide with the exception of DDD which was measured as DDD. Lindane, aldrin, heptachlor epoxide, and DDT were chosen as easily separable and as representative pesticides for these studies, and were injected daily for 57 days in quantities calculated to give incre- mental increases of 0.4, 0.4, 1.0, and 1.0 ppm, respectively. Three units were initially set up for comparison of degradation at 20 C: (1) an aerobic unit containing diluted aerated digested sludge mixed under oxygen, (2) an anaerobic unit containing diluted digested sludge mixed under nitrogen, and (3) an autoclaved unit containing a suspension of fine glass beads, 0.0029 centimeters in diameter, mixed in BOD dilution water under nitrogen. The 11 grams of beads added to 18 liters of water was the maximum that could be suspended with a 50 watt magnetic stirrer and had a total surface area equal to that of the walls of the bottle. The beads served as an adsorption surface for the pesticides and were removed along with periodic water samples in order to measure the adsorbed pesticide concentrations. The sludge used was digested sewage sludge diluted 19 to 1 with either ------- -11- deaerated or oxygenated tap water. The sludge was initially the same for both the anaerobic and aerobic units, but that for the aerobic unit was aerated for five days and seeded with one liter of dilute activated sludge before being transferred to the five-gallon test unit. Alkalinity was kept low, initially between 300 to 400 tug/1 as CaCO^, and the pH was kept at approximately 7 by varying the CO content of the confined gas. The confined gases were changed periodically to maintain a high level of dissolved oxygen in the aerobic unit and to avoid a large CO, increase in either sludge unit. Dissolved oxygen in the aerobic unit began at 1 ppm and rose to 26 ppm by day 118; ORF in the anaerobic unit ranged from -453 mv to a high of -100 mv. Total suspended solids averaged 0.2570 in both sludge units and volatile solids were approximately 59% of the suspended solids. The results of the first study are shown in Figure 8. In the anaerobic unit DDT converted to DDD within a period of two days; no further DDT was detected even though daily additions of 1.0 ppm DDT were continued. By day 41 a waxy scum had accumulated in the autoclaved glass bead unit that proved to be fine glass beads coated with a sufficient quantity of pesticides to cause them to float. This scum layer continued to grow and was probably the cause of the apparent decrease in DDT concentration in the autoclaved glass bead unit between day 57 and 118. The anaerobic degradation of lin- dane and aldrin was slightly greater than the corresponding aerobic degra- dation. Lindane appeared to degrade steadily under anaerobic conditions at an approximate rate of 0.1 ppm per day; a continuation of this rate from day 57 to 118 would have left a residue of 11.3 ppm lindane, which checks fairly closely with the 10.4 ppm measured. There was no significant degradation of heptachlor epoxide in any of the units. ------- -12- The second study with daily pesticide injections into dilute sludges was conducted primarily at 35°C. A total of five units were used: three at 35°C were set up similarly to those in the previous experiment at 20°C; an additional anaerobic unit at 20°C was set up, also similar to the pre- vious one except that the sludge was older; and an autoclaved sludge unit at 35°C was set up the same as the anaerobic units except that it was also autoclaved for 12 hours and cooled prior to pesticide injection. The 20°C unit was used as a check on the constancy of the sludge from one experiment to the next, and the 35°C autoclaved sludge u»iL was added to examine the' importance of biological activity on anaerobic pesticide degradation. The measured sludge characteristics were essentially the same as in the previous experiment. The results of this study are shown in Figure 9. Conversion of DDT to DDD was again noted only under anaerobic conditions. The conversion was almost immediate in the biologically active anaerobic units at both 20°C and 35°C, and it was peculiarly delayed in the autoclaved anaerobic unit. Apparently this unit became contaminated and the conversion to DDD began sometime between day 14 and 41. By day 57 the conversion was complete and the resultant DDD concentration was approximately one-half that in the active anaerobic unit at 35°C. Aerobic degradation of DDT was apparent at 35°C, although it had not been clearly defined previously at 20°C. Anaerobic lindane degradation appeared to be very temperature sensi- tive because at 35°C it proceeded at a rate of approximately 0.3 ppm per day which was contrasted with very little degradation at 20°C in this experiment. The contrast between these and the previous 20°C results suggest that the biological activity of the sludge might have decreased during the time inter- val of 1 1/2 months between the two experiments while the undiluted sludge ------- -13- was stored at 20°C. A slight amount of aldrin degradation was noted in all three anaerobic units, the active units at 20°C and 35°C and the autoclaved sludge unit at 35°C. These results suggest that neither temperature nor biological activity are significant in influencing the rate of anaerobic aldrin degradation. No significant degradation of heptachlor epoxide was noted in any of the units. Sorption and Desorption £f Pesticides in Water Sorption and desorption tests were conducted to determine the relative uptake of pesticides by suspended particulate matter. Strong sorption of pesticides on particulate matter in streams and subsequent settling in still water would allow the pesticides to be carried to the bottom where anaerobic conditions often prevail. Adsorption from phosphate buffered water onto bentonite clay was explored as an index to adsorption on inor- ganic particulate matter; and sorption on algae was investigated as these organisms frequently occur in significant concentrations in lakes and streams. Algae eventually die and settle to the bottom where they may decay anaerobically. The importance of the anaerobic degradation of pesti- cides is thus highlighted when algae are present. The algae were a mixed culture consisting primarily of Vaucheria. possibly V. sessilis or a similar filamentous species. The algae were homogenized in a blender before being dosed with pesticides to facilitate the taking of representative samples. For comparison, a single test of adsorption and desorption for some of the pesticides was also made with fine sand (115-250 mesh). The adsorption isotherms obtained for'the pesticides tested with ------- -14- bentonite clay are presented in Figure 10, and detailed sorption data for all three sorbing media are illustrated for DDT in Figure 11. Sorption was fairly reversible for all media tested, and the extent of sorption was in the inverse order of pesticide solubility. Sorption on algae was approximately one to two orders of magnitude greater than adsorption from the same water concentration of pesticide on bentonite, and adsorption on sand was about one order of magnitude less than on bentonite. The isotherms show that sorption on particulate matter was significant for all the pesticides tested. For example, a turbid river con- taining 0.1 ppb DDT in solution and carrying a suspended load of 100 ppm bentonite clay would be carrying slightly more DDT on the clay than in solution. Similar relationships can be obtained with these isotherms for other pesticides. ------- -15- DISCUSSION As a group the chlorinated hydrocarbon pesticides are resistant to any change. They have been found throughout the environment and are con- tained even in rain water (34). On the basis of the assumption that per- sistent compounds do not degrade under anaerobic conditions, they would be expected to persist indefinitely in anaerobic muds. Comparison of Anaerobic and Aerobic Degradation of Chlorinated Hydrocarbon Pesticides The results of this study show, however, that such a sweeping generality cannot be applied to the anaerobic environment. All of the chlorinated hydro- carbons tested showed some degradation under anaerobic conditions; and all of them, with the exception of dieldrin and heptachlor epoxide, consistently showed more degradation under anaerobic conditions than under corresponding aerobic conditions. DDT showed the most striking qualitative difference between anaerobic and aerobic degradation. Under anaerobic conditions it converted very rapidly to DDD, but under aerobic conditions with several mg/1 of dissolved oxygen DDT remained unchanged as DDT. The identification of DDD as a degradation product of DDT was supported by reports in the literature by other investigators who also identified DDD as a common product of DDT (3, 4, 10, 14, 15, 20, 26, 27). Castro (10) demonstrated a chemical conversion of DDT to DDD under nitrogen using ferrous deuteroporphyrin as a reducing cofactor; and Miskus, Blair, and Casida (26) extended these findings to aqueous systems using hematin and hemo- globin as reducing porphyrins. In both studies anaerobic conditions were essential to preserve the reduced state of the porphyrins needed for the re- action. Miskus, Blair, and Casida .also showed that DDD was the only ------- -16- signlficant metabolic product of DDT produced by unsterllized, stagnating bovine rumen fluid. On the basis of these studies it appears that the conversion of DDT to DDD requires a reducing cofactor. If conditions are not sufficiently aerobic to oxidize all such cofactors, it seems probable that some conver- sion to DDD may occur even in the presence of a slight amount of dissolved oxygen. In the study cited above, Miskus, Blair, and Casida demonstrated a variable amount of this conversion in surface water from Clear Lake, California; and in preliminary experiments for this investigation the authors observed a variable amount of the DDT Co DDD conversion in dilute sludges containing traces of dissolved oxygen. DDT also degraded aerobically, but in sludges containing several mg/1 of dissolved oxygen the extractable residual remained as DDT in greater con- centrations than the corresponding anaerobic DDD residuals (all concentrations measured in ppm of the identified pesticide). Lindane degraded much more rapidly under anaerobic conditions than under aerobic conditions at 35°C, but at 20°C this difference was much less pronounced. Aldrin also degraded more rapidly under anaerobic conditions, but the differences between anaerobic and aerobic rates were not as great as for lindane. Heptachlor epoxide showed varying results, most of which indicated very little degradation under either anaerobic or aerobic con- ditions; although some anaerobic degradation was noted in thick sludge. Many extractable degradation products were observed under anaerobic conditions, but very few were found in the aerobic sludges. This obser- vation suggests that small changes in pesticide molecules may be more common under anaerobic conditions. ------- -17- Importance of Biological Activity 'It has been fairly well established that none of the chlorinated hydrocarbon pesticides are used by any organism as a carbon source for metabolism. Okey and Bogan (28) arrived at this conclusion after a series of Warburg experiments; and Alexander (1, 2) has justified this conclusion at some length. However, in this study and in other studies (6, 16, 17, 24, 32), pesticide degradation or change has been found to occur most readily under conditions of significant biological activity. It seems likely that tuese changes are enzymatic in nature and related more to detoxification mechanisms than to metabolism. Radioactive tracer studies with the cyclodiene insecticides (21, 22, 25, 29) have suggested that the final products formed by biological processes are frequently water-soluble compounds that are easily excreted by organisms. It also seems possible that the degradation noted in microbial sludges may be caused by extracellular enzymes, which would cause a desorption of the more soluble degradation products. Of the pesticides studied lindane was by far the most sensitive to biological activity, as it was also to temperature. Although the anaerobic conversion of DDT to DDD was rapid, the "delayed reaction" conversion in the anaerobic, autoclaved sludge suggested that at least a minimum of biological activity was required for this conversion. Anaerobic aldrin degradation was also increased by biological activity but only slightly compared to that of lindane. Reaction Kinetics Although this investigation was not a study of the kinetics of pesti- cide degradation some comments can be made about the rates of degradation observed. ------- -18- It appears that anaerobic lindane degradation was a function -of the concentrations of lindane and of the sludge. In the thick sludge experi- ments lindane degradation appeared to follow first order kinetics as it was proportionally faster for larger concentrations of lindane. In dilute sludge, however, degradation appeared to proceed at a constant rate, zero order kinetics, probably because the degrading capacity of the sludge was saturated. Aerobic lindane degradation was too slight to classify. The anaerobic conversion of DDT to DDD was too rapid to classify, but the subsequent degradation of DDD in thick sludge following a 1 ppm dose of DDT appeared to follow a fairly smooth curve characteristic of first order kinetics with a half life of about 4 days. Slower degradations of DDD in both thick and dilute anaerobic sludges following larger doses of DDT suggested that the degrading capacity of the sludge became exhausted, possibly because a complexing capacity of the sludge for DDD became satu- rated or because degrading organisms became poisoned. Aerobic degradation of DDT in dilute sludge was too variable to classify at 20°C but at 35°C zero order kinetics were suggested. The anaerobic degradation ofaldrin in both thick and dilute sludges suggested first order kinetics. Aerobic degradation of aldrin and degra- dations of other pesticides were not classified. Relative Rates of the Anaerobic Degradation £f_ Chlorinated Hydrocarbon Pesticides The results of the thick sludge experiments showed that all the chlorinated hydrocarbon pesticides studied degraded under suitable an- aerobic conditions. Dieldrin was almost an exception, but it degraded eventually in the third thick sludge experiment as shown by a final ex- traction using the methylene chloride technique. ------- -19- An approximate ranking of the pesticides studied and their extractable degradation products according to increasing persistence under anaerobic con- ditions is: lindane, heptachlor, DDT, ODD, aldrin, endrin, heptachlor epoxide, and dieldrin. Reservations are made concerning the relative order of DDT, DDD, aldrin, and endrin because of small variations in relative rates of degradation from one experiment to another. Possible Degradations in the Natural Environment The anaerobic environment can be very biologically active and can cause significant degradation of the. notoriously persistent chlorinated hydrocarbon pesticides. This fact must be recognized and evaluated in making long term predictions of aquatic pesticide residue levels. Greater degradation of resistant chlorinated hydrocarbon pesticides should be expected in lakes, streams, reservoirs, or other bodies of water in which anaerobic bottoms are prevalent than in those that are aerobic throughout. The amount of degradation would depend on temperature, bio- logical activity in the anaerobic bottoms, and probably on many other factors not evaluated here. If anaerobic conditions are widespread such as in Clear Lake, California, with its bottom of soft black ooze (18), residues of DDT in particular would be expected to be very small because of its very rapid anaerobic conversion to DDD. Studies by other investigators have indicated that virtually no deg- radation occurs at freezing temperatures. However, when anaerobic muds in algae ponds or in natural waterways reach temperatures of 20°C or warmer, a significant amount of degradation can be expected for lindane, aldrin, DDT, DDD, heptachlor, and possibly endrin. Heptachlor epoxide and dieldrin would persist longer. The restrictions currently being ------- -20- placed on the sale and use of dieldrin would help reduce some of the problems caused by this most persistent pesticide; and if the presence of heptachlor epoxide could somehow be limited, a fairly effective an- aerobic treatment pond could possibly be built to remove the other pesti- cides. ------- -21- qONCLUSIONS The following conclusions can be drawn from this study: 1. Many chlorinated hydrocarbon pesticides were degraded under suitable, biologically active, anaerobic conditions. 2. Degradation of most of the chlorinated hydrocarbon pesticides studied was more rapid under anaerobic than under corresponding aerobic conditions; exceptions were heptachlor epoxide and probably dieldrin, which were very persistent in both environments. 3. Extractable degradation products were more common under anaerobic than under corresponding aerobic conditions. 4. The pesticides studied and their extractable degradation products can be ranked in the following approximate order of increasing persistence under anaerobic conditions: lindane, heptachlor, DDT, DDD, aldrin, endrin, heptachlor epoxide, and dieldrin. 5. The anaerobic degradation of lindane was greatly increased with increased biological activity and was generally more rapid in anaerobic than in corresponding aerobic environments. 6. DDT converted very rapidly to DDD under anaerobic conditions, but under aerobic conditions of several milligrams per liter of dissolved oxygen it persisted as DDT. 7. Under anaerobic conditions heptachlor converted quickly to extract- able degradation products which in turn were more persistent than the initial heptachlor. 8. Endrin also formed extractable degradation products under anaerobic conditions. 9. The increase of temperature from 20 C to 35°C produced no signifi- cant increases in pesticide degradation rates except for the anaerobic ------- -22- degradation of lindane and the aerobic degradation of DDT. 10. The chlorinated hydrocarbon pesticides were more strongly sorbed by algae than by bentonite clay. 11. The adsorption of the chlorinated hydrocarbon pesticides was inversely related to their solubilities. ACKNOWLEDGMENTS This investigation was supported by Research Grant WP-715 from the Federal Water Pollution Control Administration. The generosity of Dr. Dale M. Coulson of Stanford Research Institute in granting the use of his instruments, particularly at the beginning of this study, is gratefully acknowledged. ------- REFERENCES 1. Alexander, M., "Persistence and Biological Reactions of Pesticides in Soils," Proceedings of the Soil Science Society of America, 29, 1-7 (1965). 2. Alexander, M., "Biodegradation of Pesticides," Presented before the 57th Annual Meeting of the American Society of Agronomy held in Columbus, Ohio, Oct. 31 - Nov. 5, 1965. 3. Allison, D., Kallman, B. J., Cope, 0. B., and Van Valin, C. C.,'"Insecti- cides: Effects on Cutthroat Trout of Repeated Exposure to DDT," Science, 142. 958-961 (1963) 4. Barker, P. S., and Morrison, F. 0., "Breakdown of DDT to DDD in Mouse Tissue," Canadian Journal of Zoology, 42. 324-325 (1964). 5. Barthel, W. F., Parsons, D. A., McDowell, L. L., and Grlssinger, E. H., "Surface Hydrology and Pesticides - A Preliminary Report on the Analysis of Sediments of the Lower Mississippi River," Presented before the 57th Annual Meeting of the American Society of Agronomy held in Columbus, Ohio, Oct. 31 - Nov. 5, 1965. 6. Bowman, M. C., Acree, F., Jr., Lofgren, C. S., and Beroza, M., "Chlorinated Insecticides, Fate in Aqueous Suspensions Containing Mosquito Larvae, + Anopheles quadrimaculatus - Say," Science, 146, 1480-1481 (1964). 7. Bridges, W. R., Kallman, B. J., and Andrews, A. K., "Persistence of DDT and Its Metabolites in a Farm Pond," Transactions of the American Fisheries Society. 92, 421-427 (1963). 8. California, "Minutes, Conference on Agricultural Drainage Treatment, April 11, 1963," 11 participants from Departments of Fish and Game, Public Health, and Water Resources, Sacramento (1963). 9. California, Department of Water Resources, San Joaquin District, "Summary of Water-Borne Chlorinated Hydrocarbon Pesticide Program, September 1963 through December 1964," Sacramento (1965). 10. Castro, C. E., "The Rapid Oxidation of Iron (II) Porphyrins by Alkyl Halides. A Possible Mode of Intoxication of Organisms by Alkyl Halides," Journal of the American Chemical Society. 86, 2310-2311 (1964), 11. Cope, 0. 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Morello, A., "Induction of DDT-Metabolizing Enzymes in Microsomes of Rat Liver after Administration of DDT," Canadian Journal of Biochemistry. 43. 1289-1293 (1965). 28. Okey, R. W., and Began, R. H., "Apparent Involvement of Electronic Mechanisms in Limiting the Microbial Metabolism of Pesticides," Journal Water Pollution Control Federation. 37, 692-712 (1965). 29. Poonawalla, N. H., Korte, P., "Metabolism of Insecticides. Excretion, Distribution and Metabolism of Alpha Chlordane- Life Sciences. 3. 1497-1500 (1*64). 30. Standard Methods for the Examination of Water, Sewage and Industrial Hastes, llth Edition, American Public Health Association, New York (I960)'. 31. Weaver, L., Gunnerson, C. G., Breidenbach, A. W., and Lichtenberg, J. J., "Chlorinated Hydrocarbon Pesticides in Major U. S. River Basins," Public Health Reports. 80. 481-493 (1965). 32. Werner, A. E., and Waldichuk, M., "Decay of Hexachlorocyclohexane in Sea Water," Journal of the Fisheries Research Board of Canada. 18, 287-289 (1961). 33. Wheatley, G. A., "The Assessment and Persistence of Residues of Organo- chlorine Insecticides in Soils and Their Uptake by Crops," Annals of Applied Biology. 55. 325-329 (1965). 34. Wheatley, G. A., and Hardtnan, J. A., "Indications of the Presence of Organochlorine Insecticides in Rainwater in Central England," Nature. 207, 486-487 (1965). ------- Cl Cl Cl- C-CI I Cl DDT Cl Cl I Cl -C - Cl I H ODD ALDRIN LINDANE H DIELDRIN Cl HEPTACHLOR Cl ,H HEPTACHLOR EPOXIDC FIGURE 1. Structures of Pesticides Studied. ------- T 7-—OVERFLOW V J RESERVOIR GAS VENT TO LOW PRESSURE AIR VALVE WITHDRAWAL TUBE — TEST UNIT INJECTION PORT (ON FIVE-GALLON UNITS ONLY) MAGNETIC STIRRER 8 SUPPORT BLOCKS PIEZOMETER TUBE (PRESSURE INDICATOR) GAS RESERVOIR (PRIMARILY N2 OR 02) TO OTHER UNITS CONFINING FLUID RESERVOIR FIGURE 2. Schematic Diagram of Closed System with Variable Volume Gas Reservoir Used for Pesticide Degradation Studies. ------- 100 * I o UJ cr t 4 • • 10 DAYS AFTER PESTICIDE INJECTION FIGURE 3. Anaerobic Degradation of Lindane in Sludge at 35 C. ------- 10 Q. Q. O < 2 UJ tr 2 6 UJ z ACTIVE POIS( NED 4O 80 120 I6O DAYS AFTER PESTICIDE INJECTION 200 .0 FIGURE 4 .Anaerobic Degradation of Lindane in Active and Poisoned Sludge at 35 C ------- I ppm DDT ADDED E a a. 1.0 1 0 0.8 Z z — 0.6 Z ( LJ Q» O 4 LJ O — 0.2 — i H - CO n i i i r> 1 ppm HEPTACHLOR ADDED i — — L /— HEPTACHLOR /—"HEPTACHLOR EARLY ELUTION" _ m A A J .20 40 60 200 300 DAYS AFTER PESTICIDE INJECTION 20 40" 60 " 200 300 DAYS AFTER PESTICIDE INJECTION ppm ALDRIN ADDED 20 40 60 200 DAYS AFTER PESTICIDE INJECTION LEGEND O MEASURED CONCENTRATION OF INJECTED PESTICIDE A MEASURED CONCENTRATION OF APPARENT DEGRADATION PRODUCT FIGURE 5. Anaerobic Degradation of Single Pesticides in Thick Sludge at 35 C. ------- 50 40 ppm LIN DANE ADDED POISONED ACTIVE I • THICK SLUOOC 10 20 30 40 50 DAYS AFTER PESTICIDE INJECTION 10 20 30 40 SO DAYS AFTER PESTICIDE INJECTION 60 100 ppm H E PTACHLOR EPOXIDE ADDED 40 ppm ALDRIN ADDED 40 •2 0. 10 20 JO 40 SO DAYS AFTER PESTICIDE INJECTION 30 ppm DIELDRIN ADDED ACTIVE t THICK SLUOOE POISONED- 10 20 30 40 SO DAYS AFTER PESTICIDE INJECTION 60 FIGURE 6. Anaerobic Degradation of Pesticide Mixtures in Thick Sludge at 35 C (Pesticides without Extractable Degradation Products). ------- *\ £ 80 a O 4 z z 60< 5 1 u ac g « | B a. 20 100 E _" U Z K 2 u K 60 Vt H 0 O £ « hi ft ESTICII 8 0. n iii.. 100 ppm DDT A DDEO ' 1 . ALL UNIT* (OOTI W / ^^ i x--» r^ J^ ""'lO 20 v JO 40 50 6 DAYS AFTER PESTICIDE INJECTION ' ' DDT PRODUCT ( DDD) , — THICK ILUDtE / •*•», / J ACTIVE I f^^^T^ «"- \ ^POUONEO ' ~ — ^_ ' ACTIVE H ,11,1 K) I 8I a o * 6 Ul • c 0 * 0 K m.n c 200 E 160 a a 0 z [ Z 120 z u K O O Lo n 0 °< 2OO E a i 0 z 160 z J •u B 120 M y o 0 £ BO " S u H 4O Iff Ul a. o 1 150 ppm ENDRI N ADDED K o \ • \ /— ACTIVC H V \ \ , THICK SLUDtE ^v ^v \ \*V\ / ACTIVf I -D V\Xv 10 20 JO 40 50 61 DAYS AFTER PESTICIDE INJECTION E N D Rl N PRODUCTS ACTIVE II v \ THICK ILUDCE — i \ \ POItONID \ \ "^-^^^^^^ • /'^^-'^ o ri in 90 \n Ait «vn 10 20 30 40 50 60 DAYS AFTER PESTICIDE INJECTION DAYS AFTER PESTICIDE INJECTION DAYS AFTER PESTICIDE INJECTION (J FIGURE 7. Anaerobic Degradation of Pesticide Mixtures in Thick Sludge at 35 C (Pesticides with Extractable Degradation Products). ------- 40 SO 60 70 »0 DAYS AFTCftlNITIA. PESTICIDE INJECTION 20 30 40 SO tO '0 60 DAYS AFTER INI TIAL PESTICIDE It.'JECTION 40 50 (0 70 60 DAYS AFTER INITIAL CCSTICIDE IUECTION 40 SO «0 TO DAYS AFTERINITIM. PESTKIDC IIUECTIOI/ FIGURE 8. Pesticide Degradation in Dilute Sludge at 20 C (Daily Injection Units). ------- O 20 JO 40 60 OATS AFTCH INITIAL PESTICIDE INJECTION D 00 I PRODUCED FROM DDT) 10 10 >o «o so DAT* AFTCK INITIAL PESTICIDE INJECTION FIGURE 9. Pesticide Degradation in Dilute Sludge Primarily at 35 C (Daily Injection Units). ------- 10,000 aoi 0.01 I IJO WATER CONCENTRATION (ppb) 100 IOOO FIGURE 11. DDT Adsorption and Desorption from Several Materials in Buffered Water, 20° C. ------- |