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

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               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.

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           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

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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

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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

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                                   -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

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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

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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.

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    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

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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

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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.

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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

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                                    -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.

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     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

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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

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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.

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                                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

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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.

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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.

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                                 -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.

-------
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 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,
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 3.  Allison,  D., Kallman, B. J., Cope, 0.  B., and Van Valin, C. C.,'"Insecti-
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 4.  Barker,  P. S.,  and Morrison, F. 0., "Breakdown  of DDT to DDD  in Mouse
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 5.  Barthel,  W.  F., Parsons, D. A., McDowell, L.  L., and  Grlssinger,  E. H.,
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 6.  Bowman,  M. C.,  Acree, F., Jr., Lofgren, C. S.,  and Beroza, M.,
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 7.  Bridges,  W.  R., Kallman, B. J., and Andrews,  A. K., "Persistence of DDT
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 8.  California,  "Minutes, Conference on Agricultural Drainage Treatment,
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 9.  California,  Department of Water Resources, San Joaquin District,
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10.  Castro,  C. E.,  "The Rapid Oxidation of Iron (II) Porphyrins by
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11.  Cope,  0. B., "Effects of DDT Spraying for Spruce Budworm on Fish
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12.  Croker,  R. A.,  and Wilson, A. J., "Kinetics and Effects.of DDT in
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-------
13.  Dugan, P. R. , Pfister,  R. M. , Sprague, M. L. , "Evaluation of the Extent
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14.  Finley, R. B., Jr.,  and Fillmore, R. E., "Conversion of DDT to DDD in
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15.  Hayes, W. J., "Pharmacology and Toxicology of DDT,"  In DDT, The
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16.  Hoi den, A. V., "Contamination of  Fresh Water by Persistent Insecticides
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17.  Hooper, F. F., and Grzenda, A. R. , "The Use  of Toxaphene as a Fish
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18.  Hunt, E. 6., and Bischoff,  A. I., "Inimical  Effects on Wildlife of
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19.  Kallman, B.  J.,  "Distribution and Detoxication of Toxaphene in Clayton
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20.  Kallman, B.  J.,  and  Andrews, A. K. , "Reductive Dechlorination of DDT
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     Mikroorganismen und Moskitolarven," Justus Liebigs Annalen Per
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23.  Langlois,  B.  E.,  Stemp,  A.  R.,  and Liska,  B.  J.,  "Rapid Cleanup  of
     Dairy Products for Analysis  of  Chlorinated Insecticide Residue by
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24.  Lichtenstein,  E.  P'., Schulz,  K. R., Skrentny, R.  F.,  and  Tsukano, Y.,
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25.  Ludwig,  G» Weis, J., and Korte, F., "Excretion and Distribution of
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     Period ofTCime," Life Sciences. 3. 123-130 (1964).

-------
26.  Miskus, R. P., Blair, D. P., and Casida, J. E., "Conversion of DDT
     to DDD by Bovine Rumen Fluid, Lake Water, and Reduced Porphyrins,"
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27.  Morello, A., "Induction of DDT-Metabolizing Enzymes in Microsomes
     of Rat Liver after Administration of DDT," Canadian Journal of
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28.  Okey, R. W., and Began, R. H., "Apparent Involvement of Electronic
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30.  Standard Methods for the Examination of Water, Sewage and Industrial
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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
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     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
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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).

-------
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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
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^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.

-------