DDT metabolism in microbial systems.

DDT metabolism in microbial systems By RICHARD

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JoHNSEN°

Contents I. Introduction -----------------------------------------------------II. Nonbiological degradation and other considerations -----------------III. Undefined microbial populations ----------------------------------a) Soils --------------------------------------------------------b) Se\Vage -----------------------------------------------------c) Sediment ----------------------------------------------------d) Silage -------------------------------------------------------e) VVater -------------------------------------------------------f) Digestive systems --------------------------------------------IV. Mixed microbial populations --------------------------------------V. Defined microbial populations -------------------------------------a) Bacteria (including actinomycetes) ----------------------------b) Fungi (including yeasts) -------------------------------------c) Algae -------------------------------------------------------VI. Conclusions -----------------------------------------------------Summary ------------------------------------------------------------Glossary -------------------------------------------------------------References ------------------------------------------------------------

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I. Introduction The metabolic fate of DDT1 is of interest not only because of widespread concern for environmental pollution but also because it affords an opportunity to study the complex metabolic reactions carried out in different organisms and various other ecosystems. Although DDT has been used for more than three decades, much of the knowledge of its metabolism in different systems is incomplete, misleading, or fraught with " Department of Zoology and Entomology, Colorado State University, Fort Collins 80523. Published \Vith the approval of the Director of the Colorado Agricultural Experimental Station as Scientific Series Paper No. 1979, Contribution of VVestem Regional Research Project W-45. 1 Throughout this paper, DDT refers to p,p'-DDT. The chemical names and structures of this and other abbreviations used are listed in the Glossary.

© 1976 by Springer-Verlag Ne\V York Inc. F. A. Gunther et al. (eds.), Residue Reviews © Springer Science+Business Media New York 1976

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inconsistencies. Only with the advent of gas-liquid ( GLC) and thin-layer chromatography ( TLC) and mass spectrometry ( MS) coupled with more extensive use of radiolabeled compounds has real progress been made. For many years, DDE and DDA were considered to be the only major metabolites from biological systems until microbial systems were studied (MATSUMURA and BousH 1971). Part of the reason for this was that the standard Schechter-Haller colorimetric method of analysis did not distinguish DDD ( TDE) from DDT so it is not surprising that it was not until1963 that TDE was shown to be formed from DDT in animal tissues (FINLEY and FILLMORE 1963). Until recent years, the persistence of DDT in natural ecosystems was attributed to microbial inability to degrade it and its related metabolites. Although countless research papers and numerous reviews have been published on the metabolism of DDT and related compounds, no specific review on its metabolism in microbial systems has been published. In the past few years, however, a number of general reviews have appeared which include microbial metabolism of DDT to varying degrees of thoroughness (ALEXANDER 1972, BoLLAG 1972, FRIES 1972, MATSUMURA and BousH 1971, MEIKLE 1972, MENZIE 1969, PFISTER 1972). Microorganisms have assumed an ever increasing importance in the study of pesticide degradation. Various environments or ecosystems supporting or capable of supporting large microbial populations (soil, water, sewage, etc.) have long been thought to be ideal sites for degradation but supporting data were meager. The finding that anaerobic conditions were necessary for noticeable degradation caused an upsurge in research efforts. Natural anaerobic environments are found in river and lake bottoms, in soils, and in the rumen and intestinal systems of many animals. In sludge digestion of municipal and industrial wastes, and in ensilage, similar anaerobic environments exist. Since these systems have undefined and usually very diverse microbial populations, much research effort has been directed toward using various isolates in attempts to find organisms capable of degrading DDT. For this reason, studies with undefined microbial populations will be reviewed separately from those involving defined populations. I have attempted to review the literature in this broad, multidisciplinary field, much of which has appeared since 1970, and to evaluate the work done by reference to the original literature. Every effort has been made to include the more pertinent and recent information to June 1974. II. Nonbiological degradation and other considerations There are numerous examples of pesticides, including DDT, undergoing transformations by nonmetabolic processes ( CROSBY 1969 and 1970, KEARNEY and HELLING 1969). These processes include those initiated by light, water, pH, heat, free radicals, and the complex mixtures of organic and inorganic chemicals in soils. CASTRO ( 1964) has shown that reduced

DDT metabolism in microbial systems

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iron porphyrin complexes are oxidized by DDT and TDE is formed. Similarly, MrsKus et al. ( 1965) observed the same dechlorination in aqueous solutions of reduced porphyrins under anaerobic conditions. GLASS (1972) reported that the iron redox system in water-saturated soil was capable of degrading DDT to TDE and the rate of TDE formation was related to the rate of ferrous iron formation and that this occurred also in a soil-free iron redox system. EcHOBICHON and SAsCHENBRECKER ( 1967) questioned the concept of enzymatic dechlorination of DDT when they found DDT to be converted to TDE and DDE plus unknown metabolites in frozen heparinized blood that was repeatedly thawed. This suggested the involvement of iron porphyrins. However, the fact that they thawed the blood repeatedly would not only release enzymes for activity, but others may have been activated in the process. Overall, many of the papers reviewed herein present strong evidence that the observed degradations are enzymic. OTT and GuNTHER ( 1965) pointed out that various "metabolites" can be formed during analytical procedures, especially those involving GLC. These findings indicate the importance and difficulty in ascertaining whether transformations are biological or are chemical. The work cited regarding porphyrins is especially difficult since reduced iron porphyrins are present in all aerobic organisms and are long-lived in the environment. In addition, KLEIN et al. ( 1964 and 1965) reported the isomeric conversion of o,p'-DDT to p,p'-DDT in the rat. Since these initial reports, it has been shown that this conversion does not take place (CRANMER 1972, BITMAN et al. 1971). The p,p'-DDT was shown to be an impurity in the o,p'-DDT which was detected due to the more rapid metabolism of o,p'-DDT which resulted in an elimination rate differential (BrTMAN et al. 1971 ). Although the simplest procedure for distinguishing between biological and nonmetabolic pesticide breakdown is in comparing the rate of decomposition in sterile and nonsterile systems, care must be exercised in the method of sterilization to prevent untoward changes (ALEXANDER 1965). Perhaps, as stated by CROSBY ( 1969), many of the in vitro studies of pesticide degradation involve both biological and nonbiological processes. III. Undefined microbial populations

a) Soil Reviews by ALEXANDER ( 1965) and EDwARDS ( 1966) point out that DDT is very stable in soils. Even though one of the primary functions of soil microorganisms is the decomposition of a wide array of organic compounds in soil, there are numerous examples of materials, both biological and synthetic, which persist from years to millenia (ALEXANDER 1972). The most abundant soil microorganisms are bacteria but, because of their small cell size, the fungi actually account for the greater portion of micro-

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bial mass in most soils (ALEXANDER 1961). Culture studies of microorganisms isolated from soil will be reviewed in later sections under their taxonomic grouping. A number of studies of DDT degradation in soil have shown that under anaerobic conditions reductive dechlorination predominates, whereas under aerobic conditions dehydrochlorination is the dominant reaction. GUENZI and BEARD ( 1967) found 14 C-DDT (ring-labeled) converted directly to TDE when incubated in a moist soil in a C0 2-N 2 atmosphere. After four weeks, TDE accounted for 62% of the recovered radioactivity, 34% as DDT and 4% as other products. However, 43% of the radioactivity was not recovered and they found no activity in either a hexane or NaOH trap designed to trap volatilized materials. Only part of the missing radioactivity was in the water layer of the partitioned hexane extract and they did not try to isolate and identify water-soluble compounds. They indicated finding small amounts of DDA, DDE, BA (p-chlorobenzoic acid), dicofol, DBP, and DDM. Autoclaving of replicate soil samples for 1 hr prevented DDT degradation and the authors concluded that the degradation processes were of microbial origin. Shortly thereafter, GuENZI and BEARD ( 1968) compared DDT degradation under aerobic and similar anaerobic conditions with and without a 1% alfalfa amendment. They found that alfalfa enhanced the degradation of DDT under anaerobic, but not aerobic conditions, finding less than 1% as DDT after 12 weeks. Recovery decreased with time, even in sterile soil, and only 64% of the radioactivity added was recovered after 12 weeks of anaerobic incubation and only 46% was in identifiable compounds. Even combustion of residual carbon after solvent extraction and trapping of the C0 2 brought the total recovery to only 79%. Since in both anaerobic studies they flushed the incubation chambers with nitrogen only at the completion of incubation, the low recoveries, even with combustion, may indicate inadequate recoveries of residues residing in the chambers. This aspect was not described adequately; however, this latter study pointed out that alfalfa, as an added energy source, stimulates microbial activity which hastens the rate of DDT degradation. As GRAY ( 1970) pointed out, soil micro-organisms live under starvation conditions and are largely inactive. Therefore, the addition of an organic energy source should stimulate microbial growth. Ko and LOCKWOOD ( 1968) confirmed the work of GUENZI and BEARD but used water-logged soil. Using several amendments added at the 1% level, they found the conversion of DDT to TOE to be most effective with alfalfa, slightly less so with a peptone-glucose mixture, and least with barley straw. The amendments did not enhance degradation in aerobic soils. They found no conversion of DDT in sterile amended soil for up to five weeks indicating microbial involvement in degradation. They reported TDE to be more stable in soil than DDT and that TDE had a broader antimicrobial spectrum than DDT which may account for its longer soil persistence.


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PARR et al. ( 1970 a) pointed out that laboratory studies using different means of obtaining anaerobic conditions may not be comparable because of varying effects on microorganisms. In a subsequent paper ( 1970 b), they confirmed the work cited above of enhanced DDT degradation to TDE in anaerobic soils amended with energy sources. They found DDT degradation after four weeks in moist soil to approach 100% in soil amended with alfalfa meal, rice straw, or hulls and 85% with glucose. Whereas recoveries of DDT and metabolites exceeded 95% after four weeks in moist aerobic soil, where degradation was minimal, recoveries were often only 60 to 80% in soils where extensive metabolism occurred. This was thought to be due to degradation of DDT to polar metabolites not extracted or detected. JOHNSEN et al. ( 1971), in a related study with cattle manure as the amendment, showed that even after one week most of the DDT had disappeared from flooded soil. TDE was the major metabolite found with only traces of DDE found. Recoveries were uniformly low. Yet incubation in unamended moist soil resulted in recoveries exceeding 96% after one week. To check for other metabolites, 2 mg of DDT in 50 g of flooded soil amended with manure were incubated for one month resulting in a recovery of 71 percent. Only 63 p,g of DDT was found, but 1,322 p,g of TDE, 21 p,g of DDMS, 12 p,g of DDMU, 4 p,g of DBP, and 3 p,g of DDE were identified indicating a slow conversion of TDE to other products. An 18-day time-course study showed recoveries exceeding 90% through the first six days and thereafter decreasing. TDE increased in concentration through 12 days after which it decreased, indicative of the breakdown of TDE. No breakdown products of DDE or TDE were detected when these two compounds were incubated in flooded soil amended with manure. Since autoclaving can cause undesirable soil changes, they incubated 2 mg of DDT in manure-amended soil treated with 40 mg of HgC1 2 • After one week they found no evidence of conversion to TDE, DDE, or other metabolites indicating that the metabolites found were of biological origin. BURGE ( 1971) showed that the dechlorination of DDT to TDE was of biological origin by adding a small amount of viable soil to sterile soil which restored its ability to degrade DDT. He also found that oxygen atmospheres as low as 2% inhibited the dechlorination of DDT and recovery was quantitative. However, with nitrogen only 59% was recovered as DDT and TDE and 41% was unaccounted for after 64 days' incubation. No other metabolites, including DDA, were found. BURGE found both alfalfa and alfalfa-steam distillate to accelerate the anaerobic but not aerobic disappearance of DDT. With alfalfa distillate amendment, he found both TDE and DDE to be stable both aerobically and anaerobically in soil incubated for 31 days. He concluded that unrecovered DDT had not been converted to unidentified compounds through DDE or TDE as intermediates. However, the answer may not be that straightforward. Although direct information is lacking, DDT presumably is being

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dechlorinated after it enters a microbial cell and the resulting TDE may then be further metabolized. According to MEIKLE ( 1972), the major barrier between a foreign organic compound and the metabolic machinery of a microorganism is the cytoplasmic membrane. Perhaps there is a membrane transport differential which DDT can cross to the exclusion of TDE. MEIKLE went into considerable detail on various aspects of membrane structures and membrane penetration rates which are germaine to these considerations. BuRGE ( 1971), in other experiments, could not account for up to 74% of added DDT. CASTRO and YosHIDA ( 1971) reported that TDE accumulated in four flooded, DDT-treated soils and fastest in the one with the highest organic matter content. They also showed that TDE was more persistent in these soils than DDT but residues diminished; they did not indicate finding any TDE metabolites. In all these studies in which DDT disappeared in biologically active anaerobic soils with the accumulation of TDE, recoveries were less than ideal and the various authors speculated as to the cause. KEARNEY et al. (1966), using 14 C-DDT, found that up to 20% of the 14 C could not be extracted from flooded soils after a four-week incubation. They concluded that the 14 C-activity is tightly bound to soil particles in some form and that this loss was real and reproducible, although the mechanism was not known. These missing residues, however, may be locked into microbial matrices rather than soil particles. b) Sewage

HILL and McCARTY ( 1967) were the first to report the breakdown of DDT in sewage sludge. They incorporated DDT into a thick, biologically active, anaerobic, digested wastewater sludge and found DDT to be converted almost immediately into TDE. TDE in turn gradually was degraded with a half-life of about four days. Under aerobic conditions, with several milligrams of dissolved oxygen/L, DDT remained unchanged. They determined the degradation of TDE to follow first-order kinetics, but were unable to classify DDT due to its rapid conversion to TDE. Further work with sewage sludge did not appear until late 1972. ALBONE et al. ( 1972 a), using DDT -treated anaerobic sewage sludge incubated under hydrogen, found by GLC analysis three peaks coinjecting with TDE, DDMS, and DBP. TDE was confirmed by GLC-MS and the peak corresponding to DBP was shown not to have arisen from dicofol. Papers by ALBONE et al. ( 1972 b) and JENSEN et al. ( 1972), appearing back to back in the same journal, reported a new metabolite of DDT from anaerobic sewage sludge abbreviated DDCN [his ( p-chlorophenyl) acetonitrile]. These are the first reports of a nitrogen-containing DDT metabolite. ALBONE and coworkers incubated both enriched and unenriched anaerobic sewage sludge with DDT for periods up to 88 days. Only a trace of DDT remained while TDE predominated and three


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other GLC peaks relative to DDT of 0.37, 0.57, and 0.62 were found. The latter peak was identified as DDCN by combined GC-MS with synthetic DDCN exhibiting identical GLC and TLC characteristics. They found DDCN to represent 11.7% of the DDT initially incorporated. They speculated that DDCN could be formed from DDA through amide to nitrile formation or directly from DDT. JENSEN and coworkers found DDT to have a half-life of seven hours in their anaerobically incubated activated sludge. They also found DDCN in a local sewage sludge sample in Sweden, the first report of its occurrence in nature. They confirmed the identity of the DDCN using GLC-MS and chemical degradation. Since no DDCN could be detected after adding TDE or DDE to sludge, they postulated its direct formation from DDT. They found DDCJ\' to represent 9% of the original 11 C-activity with an overall 14 C recovery of 40%. In neither paper did the authors ascertain whether DDCN was formed metabolically or chemically. PFAENDER and ALEXANDER ( 1972) incubated 14 C-DDT in sewage for periods up to 24 weeks. TDE and DBP were the major metabolites accumulating but significant quantities of DDMU, DDMS, DDNU, DDM, and DBH also were found. Volatile organic compounds or 14C02 were not produced in significant amounts indicating little or no ring-cleavage occurred. Recovery increased with time and the authors speculated that this was possibly due to binding of DDT to organic matter and microbial cells which was released as the organic material was decomposed. Incubation of DBP in sewage collected at two different times showed DBP to disappear completely in four weeks from one sample and no degradation after six weeks in the other, indicative of seasonal microbial fluctuations. Similarly PCPA ( p-chlorophenylacetic acid), found by FOCHT and ALEXANDER ( 1971) to be a ring-cleavage product of DDM, was incubated in sewage and found to disappear rapidly after the fourth week and to be essentially gone after six weeks. Sterilization by autoclaving prevented any loss of PCPA. Sterile controls were used throughout and any chemical changes were subtracted from the results reported, which in all cases were less than 5%. Since sterile air was passed over the sewage, strict anaerobiosis was not observed. In a subsequent study, PFAENDER and ALEXANDER ( 1973) incubated DDT in raw sewage with added inorganic salts. Glucose was added to four samples (two sterilized), diphenylmethane to another set, and a third set received no additions; the three sets were incubated for seven weeks. As in their 1972 paper, the same products were formed but the amounts varied with the amendment. TDE, DBP, and DDE represented 95% of the metabolites formed. By interval sampling, they found TDE to be formed at reasonably rapid rates in unamended sewage, markedly enhanced by glucose and reduced by diphenylmethane. DBP was formed slowly in the unamended sewage but both amendments reduced its formation even though both amendments resulted in marked bacterial population buildups with the effect of glucose being more rapid.

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c) Sedinumt In studies related to sediments, MATSUMURA et al. ( 1971) studied the metabolism of DDT by unidentified microbial isolates from top silt and bottom silt from Lake Michigan and its tributaries. Of the isolates tested, 77% from top silt (81 of 104) and bottom silt (72 of 92) formed TDE. For DDNS formation, 57 of 104 isolates from top silt and 41 of 92 from bottom silt were active. About one-third of the isolates also formed DDE. Incubation of 14 C-TDE with active isolates indicated that DDNS was formed by dechlorination of TDE (analogous to TDE formation from DDT). In TLC analyses, spots corresponding to TDE accompanied the DDNS spots. PATIL et al. ( 1972), in similar studies, investigated 14 C-DDT degradation in marine environments using unidentified microbial isolates from bottom sediments from bays, estuaries, and ocean floors. Sea bottom isolates were weak in degradative capacity but others formed TDE with lesser amounts of DDNS and DDOH. Varying amounts of unextracted activity, depending on the isolate, resided in the aqueous phase after extraction, due presumably to their polar nature. Their isolate no. 1708, for example, had 74% in the aqueous phase. Additional work on the identity of these compounds is needed. Very few studies of DDT metabolism in sediments have appeared. ALBONE et al. ( 1972 a) studied DDT degradation in estuary sediments both in situ and in vitro. In vitro incubations produced greater conversion of DDT to TDE than in situ studies and was the only metabolite observed although small amounts of polar materials were evident from TLC plates. The search for 14 CO" in the one experiment conducted was negative. jENSEN et al. ( 1972) found the new metabolite DDCN also in a lake sediment layer in Sweden at 0.6 ppm on a dry weight basis. PFAENDER and ALEXANDER ( 1972) also incubated 14 C-DDT in a fresh water-sediment ecosystem for periods up to 24 weeks. As with sewage, the major metabolites accumulating were TDE and DBP with small but significant amounts of DDE, DDMU, DDMS, DDNU, DDM, and DBH also being found. With both sewage and sediment, they reported 99.9% recovery of radioactivity using a 6 hr continuous extraction with diethyl ether, and also no evidence of ring-cleavage. d) Silage

The first report of silage having an effect on DDT was by THORNBURG (1963) who mentioned briefly that corn silage degraded DDT to TDE. FruEs et al. ( 1969 a) ensiled alfalfa treated with DDE, TDE, and DDT in vitro for periods of 28, 56, and 84 days at room temperature. With recoveries of about 90%, they found only 20% of the DDT converted to TDE after 84 days, which is quite low in comparison to other studies cited. They found no change in DDE and did not report on TDE. HENZELL and LANCASTER ( 1969), in a more extensive study, used a rye grass and a rye grass mixture, which had been sprayed with technical


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DDT, as silage. The technical material contained 75.8% p,p'-DDT, 23.8% o,p'-DDT, 0.4% DDE, and 0.015% DDMU. They incubated the silage for periods up to 90 days at 25 and 38°C and found the formation of p,p'-TDE and o,p'-TDE to parallel the level of their DDT isomers in the silage. The higher temperature resulted in a more rapid TDE formation and about 90% loss of DDT compared to 75% at 25°C. However, the formation of TDE from DDT resulted in a net loss of 50%; the remainder was unaccounted for.

e) Water MisKus et al. ( 1965) studied the conversion of 14 C-labelled DDT to TDE by six lake water samples incubated for seven days. They found that the extent of conversion was greater in samples with large amounts of plankton with a 95% conversion in one sample. No change in DDT was evident in either distilled water or boiled distilled water under vacuum indicating that the reaction was biological. EICHELBERGER and LICHTENBERG ( 1971 ) evaluated the persistance of 28 pesticides in raw river water over a period of eight weeks. They found no measurable changes, either biological or chemical, nor any loss of either DDT, TDE, or DDE. Nothing is mentioned regarding the microbial life in the water but it is expected that there would be numerous bacteria and algae. It is surprising then, in light of work already cited and cited in other sections, that no conversion of DDT to TDE was found. 0LOFFS et al. ( 1972) treated water samples from two rivers and from a subtidal zone of Canada with DDT and incubated them in the laboratory for periods up to 12 weeks. They determined bacterial counts periodically with values generally of 104 bacteria/ml and yet found no evidence of degradation. Although they showed substantial loss of DDT with time, glass wool plugs in the flasks only partly accounted for the loss. It seems that their analytical technique, employing extraction in a separatory funnel, would be at fault since this would not readily remove residues from within bacterial cells. Such cells would likely reside in the aqueous phase which was discarded. The first report of the metabolism of DDT by aquatic microorganisms in pure culture was made by MATSUMURA et al. ( 1971). They tested the metabolic ability of unidentified microbial isolates from the water of Lake Michigan and three tributaries to degrade DDT. They found a large majority of 109 isolates capable of forming TDE ( 90 of 109). A considerable number of the isolates also produced DDNS ( 48). In testing the degradation of TDE the finding of DDNS implies that DDNS formation from TDE was the chief metabolic pathway. They postulated that DDT is dechlorinated to TDE which in turn is dechlorinated to DDNS. They also found that 22% of the isolates also formed DDE. In studies related to that above, PATIL et al. ( 1972) investigated the metabolic transformations of 14 C-DDT by microorganisms in marine waters off Hawaii and off

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Houston, Texas. Of six water samples, generally poor metabolic activity was observed and none was found in samples from the open sea. Of active microbial isolates from waters, such as Kaneohe Bay on Oahu and the Houston ship canal, TDE was readily formed. Many of them also formed DDNS and DDOH as minor metabolites. The fact that radioactivity was present in the extracted aqueous phases to various degrees indicated additional polar metabolite formation.

f) Digestive systems Many earlier studies of the metabolism of DDT by vertebrates and invertebrates, such as insects, can be misleading if the DDT was presented in the feed or otherwise administered orally. With the vast amount of evidence now available on the importance and breadth of microbial degradative capacities, these earlier studies either failed to take into account or to assess properly the role enteric microbial populations had on the fate of the administered DDT. In an early paper, ALLISON et al. (1963) allude to this potential by stating that they found TDE as the only metabolite in microorganisms exposed to DDT and that TDE residues in fish may have been due to metabolic activity of flora prior to the uptake of DDT residues by the fish. Reports using microorganisms isolated from digestive systems will be reviewed under their taxonomic heading. A number of papers report data from monogastric animals. MENDEL and WALTON ( 1966) administered DDT to rats both by stomach tube and intraperitoneally. They found, in extracts of both feces and livers, that TDE was present only with the former route and none by the latter route. They concluded that intestinal microorganisms must be considered the major agents in the formation of TDE in intact rats. WEDEMEYER ( 1968) studied the degradation rates of DDT either injected or ingested in rainbow trout ( Salmo gairdneri). Intestinal microorganisms were implicated further when he showed that after seven days, fish treated with neomycin converted only 20% of the DDT to TDE and DDE, whereas 70% was degraded in fish given only DDT. He showed that neomycin treatment reduced the bacterial count from 106 to 103 I g of intestinal contents. Although he found that DDE and TDE were formed in both injected and fish fed by gavage, degradation was most extensive in the latter. It was shown that liver homogenates do form DDE but intestinal microflora played the major role in DDT degradation. However, he analyzed only the residues in liver and blood and not those in the intestinal system. CHERRINGTON et al. ( 1969) evaluated DDT degradation by intestinal contents of Atlantic salmon ( Salmo salar) . They showed TDE to be formed readily and used streptomycin, as a microbial inhibitor, to lower markedly the rate of degradation which indicated the essential role of the microflora in TDE formation. Since streptomycin is effective primarily against Gram-negative bacteria, this implies the involvement of these bacteria in degradation. Autoclaved samples were essentially in-


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active. They found no conversion of DDT to DDE either under aerobic or anaerobic incubation, indicating that it does not occur in the lumen of the digestive tract and suggesting that it occurs at another site such as the liver. MALONE ( 1970), using the intestinal contents of the northern anchovy ( Engraulis mordax), investigated the role of the microflora and the constituent bacteria and fungi on DDT degradation. He divided cultures of 14 C-DDT-treated intestinal contents into four groups: one with nothing added, one with penicillin and streptomycin to suppress bacteria, one with mycostatin to suppress fungi, and one with all three antibiotics. He had controls of DDT with nutrient broth alone and found no DDE or TDE formed. All the test groups contained high concentrations of TDE but little or no DDE with the uninhibited group forming the highest level of TDE and the lowest level in the group with all three additives. His results indicated that both bacteria and fungi formed TDE. Even after digestion of their cultures with a glacial acetic-perchloric acid mixture, from 18 to 53% of the 14 C-activity was in the particulate fraction after hexane extraction of the first three groups and from 6 to 18% in the group where microflora were most inhibited. Polar phase 14 C-activity was lowest in the latter group and higher in the less inhibited groups which may indicate metabolism of TDE to more water soluble compounds. The following papers describe work done with ruminant animals. MISKUS et al. ( 1965) incubated DDT with cheesecloth-filtered, stagnating bovine rumen fluid (from a fistula ted animal) in stoppered flasks and found 65% of the DDT converted to TDE, the only metabolite found, in 24 hr. A boiled rumen sample similarly incubated was inactive. FRIES (1968) also found only TDE formed from rumen microorganisms and indicated that the disappearance of DDT ( 13% /hr) coincided with an equal formation of TDE. In an expanded study, FRIES et al. ( 1969 b) used "strained" rumen fluid to study the metabolism of both p,p'- and o,p'-DDT. They found both the disappearance of DDT and the formation of TDE to be similar for both isomers, about 12% /hr. About 11% of the 14C-activity resided in the residue after extraction and, even after alcoholic-KOH treatment, was not extracted significantly with petroleum either. Since this activity remained near the origin on TLC plates, it was presumed to be polar. They found neither DDE nor DDMU after this treatment, indicating efficient extraction of DDT and TDE originally, and the authors suggested this fact provides evidence for another microbial route of DDT metabolism. KuTCHEs and CHURCH ( 1971) used cheeseclothfiltered bovine rumen fluid and bacterial and protozoal fractions to determine the contribution of the whole and the two fractions to DDT degradation. They found the cellular uptake of the added 14 C-DDT to exceed 95% in all three systems after 48 hr which the authors suggested indicates that TDE formation is an intracellular process. They analyzed the three systems as cellular and supernatant fractions and found over 95% of the recovered activity in the cellular fraction. Although TDE was the primary metabolite found, along with some DDE, their results are im-

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possible to evaluate since their recoveries of 14 C-activity from rumen fluid were very low ( 3 to 33% ) and varied from 14 to 72% ·for bacterial and protozoal fractions and they did not indicate specific recovery values for each system. Here again, their extraction method, not designed to rupture intact cells, probably minimized their recovery of intracellular residues. Their findings did indicate that protozoa formed little TDE or DDE and generally over 90% of the recovered activity was as DDT even though cellular uptake exceeded 95% of the DDT added to the system. This is indicative of a poor degradative capacity by protozoa. SINK et al. ( 1972) used cheesecloth-filtered ovine rumen fluid from sheep under different nutritional regimes for their DDT degradation study. Boiled rumen fluid served as controls and did not degrade DDT. Rumen fluid from animals on concentrate (com) were more efficient in affecting DDT degradation than that from animals on roughage (alfalfa hay). Microscopic examination of fluid at 0 hr showed the concentrate sample contained two times more bacteria and five times more protozoa than the roughage sample indicating the effect of nutrition on microbial populations. The degradation of DDT did not approach the rapid rate observed by FRIES et al. ( 1969 b). About 20% of the DDT was degraded after an 8-hr incubation. They found considerably more DDE than other workers; usually from two to four times as much DDE as TDE, which may indicate additional metabolism of TDE to DDMU (which they tentatively identified). Nothing is mentioned regarding recovery values for added 14 Cactivity which made evaluation difficult. IV. Mixed microbial populations Very little work has been published on this aspect relating to DDT, especially if identified populations are considered. Its importance is stressed, however, in view of the report by GuNNER and ZuCKERMAN (1968) of a synergistic action by the soil microorganisms Arthrobacter sp. and Streptomyces sp. in degrading the organophosphate insecticide diazinon. It can be assumed readily that actions of this kind probably occur in diverse microbial systems exemplified by soil, sewage, and digestive systems. HALVORSON et al. ( 1971) used a mixed bacterial population, free of algae and protozoa, isolated from a domestic sewage lagoon to study the biodegradation of insecticides including DDT. Recovery of DDT, added to a resting cell suspension and immediately extracted was only 60%. Since their extraction technique, using a Vortex Jr. mixer for 30 sec, would not remove residues from the intact cells this probably accounts for the low recoveries and the authors note they did not attempt to disrupt the cells. They found no aerobic metabolism of DDT whereas anaerobically, 30% conversion of DDT to TDE occurred in two days and was complete in four days. ANDERSON and LICHTENSTEIN ( 1972) studied the effects of adding any


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of eight pure fungal cultures on the capacity of the fungus Mucor alternans to metabolize 11 C-DDT to water-soluble metabolites. In all cases, they found that the added fungus depressed the capacity of M. alternans to degrade DDT. Several fungi, Aspergillus fumigatus, A. niger, Fusarium oxysporum, Rhizopus arrhizus, and Trichoderma viride, depressed the metabolism of DDT to from 0 to less than 10% of the control containing M. alternans alone. This illustrates the negative interactions that may be operational in media such as soils. FOCHT ( 1972), on the other hand, showed a positive interaction whereby the bacterium hydrogenomonas in concert with the fungus Fusarium can metabolize the metabolites DDM and PCP A to C0 2 , HzO, and H Cl (see section V. c). V. Defined Microbial Populations a) Bacteria Literature dealing with the metabolism of DDT by pure bacterial cultures did not begin to appear until 1965. Barker et al. ( 1965) found Proteus vulgaris, isolated from the intestinal flora of a mouse, to dechlorinate DDT to TDE. In a subsequent report, BARKER and MoRRISON (1965) showed that TDE was metabolized itself. by P. vulgaris into DDMU, DDMS, and DDNS, ranked in descending order of abundance. STENERSEN ( 1965) reported that Serratia marcescens and an unidentified bacteria, both isolated from stable fly excreta, and Escherichia coli converted DDT to TDE almost completely (90%) and to DDE (5%) under anaerobic but not aerobic conditions. In the above papers, controls were not reported. WEDEMEYER ( 1966) found the three facultative anaerobes E. coli, Klebsiella pneumoniae, and Aerobacter aerogenes all capable of forming TDE with the latter affecting up to 80% conversion. Further work was done with cell-free systems of A. aerogenes. Small amounts of DDE formed were accounted for by controls. He showed that cyanide inhibited TDE formation and, together with other data, concluded that reduced cytochrome oxidase was the probable cellular agent in the reductive dechlorination of DDT to TDE. The work of MENDEL and WALTON (1966), already cited under section III. f, showed that E. coli and A. aerogenes could affect the conversion of DDT to TDE but low recoveries indicated either undetected metabolites or poor extractability from the cultures. Recovery of DDT from the uninoculated culture medium exceeded 95%, which strengthens this view. WEDEMEYER ( 1967 a) reported that cell-fr.ee extracts of Pseudomonas fluorescens, E. coli, K. pneumoniae, or A. aerogenes, when incubated anaerobically for 24 hr, metabolized DDT to DDE, TDE, DDMU and DDNU. Their identity was confirmed by gas cochromatography on both polar and nonpolar columns and reversed phase paper chromatography. A. aerogenes was used in the remaining reported work. He found that the product pattern was not altered by using cell-free extracts versus

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whole cells, using different carbon sources, or incubation temperatures from 10° to 37°C. However, with cell-free extracts that eliminated permeability considerations, he found that varying the pH from 6.0 to 8.0 did alter the relative amounts of metabolites which indicated the involvement of discrete enzymes rather than a single system. To elucidate the sequence of DDT metabolism, each of the metabolites above, as well as DDMS, DDA, and DBP, was incubated similarly. DDE was found not to be degraded further under aerobic or anaerobic conditions, indicating it is not an intermediate in DDA formation. The DDT metabolic pathway was indicated as being DDT ~ TDE ~ DDMS ~ DDOH ~ DDA ~ DBP ~ or DDT ~ DDE, with DDOH postulated as an intermediate. Incubation of each metabolite with a metabolic inhibitor (cyanide, fluoride, iodoacetate, or malonate) gave additional evidence that discrete enzymes were involved in each step. For example, DDA decarboxylation to DBP was not inhibited by any agent, whereas DDMS formation from DDMU was suppressed by all inhibitors. Neither DDT nor any of the metabolites could be metabolized as a sole carbon source and the metabolism of DDT was dependent on exogenous energy sources. In subsequent work, WEDEMEYER ( 1967 b) studied the degradation of DDA in more detail with A. aerogenes and found the sequence to be DDA ~ DPM ~ DPH ~ DBP. The DBP product was the most highly degraded DDT metabolite reported to date. MENDEL et al. ( 1967) also worked with A. aero genes and showed that o,p'-DDT was reductively dechlorinated to o,p'-TDE. They found that this dechlorination did not take place in DDT analogues where the chlorines in the aromatic rings were replaced by either ethyl or methoxy groups. PLIMMER et al. ( 1968) used this same organism to prove that TDE formation proceeds by direct reductive dechlorination of DDT without the formation of DDE by using deuterated DDT labeled in the 2-position. CHAcKo et al. ( 1966), in the first report of soil microorganisms degrading DDT, found six of nine actinomycetes tested (Gram-positive filamentous bacteria) capable of forming TDE from DDT. Those with degrading capability were five species of Steptomyces and a N acardia sp. Because the cultures were shaken during incubation, strict anaerobiosis was not maintained which may have diminished positive results in lieu of the anaerobic requirements cited above. JoHNSON et al. ( 1967) evaluated 27 pathogenic and saprophytic bacterial species associated with plants for their ability to degrade DDT. They found the dechlorination of DDT to TDE to be widespread under anaerobic incubation conditions but essentially absent aerobically. With incubations of one and two weeks, more TDE was formed generally during the second week period. Of these species, 24 formed TDE after two weeks and several species, including A. aerogenes, were found to produce a number of unidentified metabolites. This was implied also in the finding that A. aerogenes had considerably less TDE detected at two


15

weeks than was found after one week. This report indicated a broad range of bacteria capable of reductively dechlorinating DDT to TDE. BRAUNBERG and BECK ( 1968) studied the degradation of DDT in pure cultures of bacteria, typical of the intestinal flora of laboratory rats. Of the six genera and "coliform" group represented, only Micrococcus did not form TDE. The data are difficult to interpret because the results are presented as percentages of recovered residues and not of starting material. The recovery of residues from Micrococcus was below 30% and, since their extraction method did not rupture cells, considerable amounts of intracellular residues may not have been extracted. It is interesting to note that the three genera with the lowest metabolism of DDT (Micrococcus, Lactobacillus, and Streptococcus) also were those in the lowest recovery class (below 30% ) . Since various numbers of isolates were tested for each taxonomic group, it is assumed that the results are average values, but no data on this point are given. The "coliform" groups, representing 47 isolates, were in the high recovery class (more than 70% ) and yet, as a group, showed the highest amount of DDD formation. This seems contradictory if extraction of intracellular residues was a problem with their procedure. This discrepancy may be explained if extracellular enzymes were involved in the formation of TDE. Similarly, an explanation may lie in the inherent differences in cell wall thicknesses. SALTON ( 1964) stated that the wall of the coliform E. coli is 80 A whereas it is as much as 800 A for Lactobacillus acidophilus ( p. 69). These considerations, however, are outside of the scope of this review. In a separate, largerscale experiment, BRAUNBERG and BECK ( 1968) incubated TDE with E. coli and recovered only TDE. Recoveries, however, decreased to only 16% at nine days even though the harvested cells were ground with sand prior to ether extraction. LANGLOIS ( 1967) studied the metabolism of DDT by E. coli grown in various broths or skimmilk and reported over 50% dechlorination to TDE after two days and over 90% after seven days in the broths but very little dechlorination in skimmilk. In later work, LANGLOIS et al. ( 1970) concluded that casein in milk complexed with DDT preventing its degradation. They studied seven bacteria, incubated in tripticase soy broth both aerobically and anaerobically, for their degrading ability. Aerobically, Pseudomonas fluorescens and Staphylococcus aureus did not degrade TDE, whereas Bacillus cereus, B. coagulans, and B. subtilis did degrade DDT. For E. coli and Enterobacter (Aerobacter?) aerogenes, TDE was the major or only product aerobically but anaerobically, trace amounts of DDMU, DDMS, DDNU, DDOH, DDA, and DBP were detected. Aerobic incubation of the three bacilli gave trace levels of the same metabolites. It is probable that their aerobic incubation became anaerobic during the 30 days of incubation since their liquid media were in screwcap flasks. No recovery data were given. KrM and HARMON ( 1970) found that Gram-positive cultures of Lactobacillus casei, Streptococcus lactis,

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S. cremoris, and S. diacetilactis in sterile milk containing DDT did not cause any measurable degradation in DDT after 14 days, supporting the findings of LANGLOIS cited above. LEDFORD and CHEN ( 1969) isolated cheese microorganisms and incubated them in a media containing DDT, DDE, and lindane. Their means of determining degradation was by comparing peak areas on gas chromatograms of uninoculated controls with those of treated media. Using this method, they showed two isolates of Brevibacterium linens, Geotrichum candidum, and G. sp. to degrade DDE and DDT. No metabolites, other than a small amount of TDE, were formed indicating a possible extraction problem. Their chromatogram of the extract from Geotrichum sp. showed an unusually broad peak for lindane, suggesting the existence of a metabolite peak overlapping with that of lindane. They did state that binding of residues by Geotrichum was not involved in apparent low recoveries but no data were given. PATIL et al. ( 1970) compared a number of soil microbial isolates, previously known to degrade dieldrin, for their ability to metabolize DDT. For 18 isolates, including nine Pseudomonas sp., four unidentified (presumably bacteria), three Bacillus sp., a Micrococcus, and an Arthrobacter sp., all but one unidentified species formed at least TDE. None produced DDE. Ten isolates formed a dicofol-like compound (identified later by MATSUMURA et al. 1971 as DDNS) and 12 degraded DDT to DDA, indicating complex metabolic routes. Since their 30-day incubation was in screw-capped tubes using unshakened liquid media, anaerobic conditions must have predominated even though the authors stated that conditions were aerobic. FRENCH and HooPINGARNER ( 1970) studied the capacity of cellular components of E. coli to metabolize 14 C-DDT. They found that TDE formation occurred in the membranous fraction when stimulated by a factor( s) present in the cytoplasmic fraction. Neither fraction alone produced significant amounts of TDE. Boiling of the membrane fraction, followed by addition of the cytoplasmic fraction, produced little TDE indicating TDE formation is enzymic. FocHT and ALEXANDER ( 1970) and ( 1971) studied the degradation of TDE, DBP, DBH, and DDM and their monochloro and/or unsubstituted analogues using cell suspensions of Hydrogenomoruzs sp. grown aerobically on diphenylmethane ( DPM) as sole carbon source. They found that PCPA was formed from DDM, a known metabolite of DDT. This ringcleavage product, the first to be proven, was in turn degraded but the products were not identified. No chloride was found to be released microbially. Hydrogenomonas neither grew on nor cometabolized DDT or DBP but the DDT analogue without phenyl chlorines and the monochloro or unsubstituted analogues of DBP were cometabolized. Cometabolism is a term used by ALEXANDER ( 1967) to mean the phenomenon whereby an organism metabolizes a compound coincidentally without apparent benefit to the organism: it cannot use it as a carbon source for growth. Both DBH and DDM, in contrast, were cometabolized and


17

Hydrogenomonas did grow on their analogues which were tested. This pointed out the strong resistance to degradation which is imparted to the molecules by p-chlorine substitution on the phenyl rings. PFAENDER and ALEXANDER ( 1972), following up the reports just cited, demonstrated extensive enzymatic degradation of DDT by Hydrogenomonas. In addition, they reported for the first time a ring-cleavage product when DDT was the starting substrate. They incubated 14C-DDT anaerobically with cell-free extracts of DPM-grown Hydrogenomonas and found, in decreasing order, TDE, DBP, DDMS, DDMU, and DDE. Much smaller amounts of DDNU, DDA, DDM, and DBH also were found. This reaction mixture then was incubated aerobically, since ring-cleavage reactions usually require oxygen, after fresh DDM-grown cells were added to compensate for any denatured enzymes resulting from anaerobiosis. They found, based on GLC and GLC-MS data, that PCPA formed as a result of cleavage of one or more of the previously formed DDT metabolites. Using Arthrobacter sp. cells incubated with PCPA, they found PCPA in turn to be degraded to p-chlorophenylglycolaldehyde; their identity was based on mass and IR spectra and a positive Tollen's test for aldehydes. In their conclusions, they supported earlier reports in work on soils that DDT metabolism is enhanced when nutrients are added to the soil. However, they pointed out that the extensive degradation observed necessitated high protein concentrations in bacterial extracts or thick cell suspensions. They postulated that since DDT is not used as a sole energy or carbon source by microorganisms, they have no selective advantage in DDT -containing environments. b) Fungi (including yeasts)

KALLMAN and ANDREWS ( 1963), using commercial yeast cake ( Saccharomyces cerevisiae), were the first to demonstrate the microbial conversion of DDT to TDE. Using an autoclaved nutrient medium to which was added the yeast and 14 C-DDT, they incubated the mixture for periods up to 187 hr. Since about 75% of the added radioactivity was not recovered, it is difficult to evaluate their data except that they did ascertain the formation of 14 C-TDE from labelled DDT which did not require the intermediate formation of DDE. They postulated that their low recoveries were due to co-distillation since a similar recovery was obtained using boiled yeast. However, later workers [e.g., ANDERSON et al. ( 1970) and RICE and SIKKA ( 1973)] have shown that dead microorganisms can pick up DDT from solutions or media passively so there may have been an extraction problem. CHIBA and DooRNBOS ( 1971) using Fleishmann's active dry yeast (S. cerevisiae) and an unidentified selected yeast, found 14 C-DDT degraded to TDE during fermentation. However, TDE was found only in the fermenting lees and not in the clear wine. They found also that unfiltered wine (with lees), after a one-month fermentation, that was col-

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Iected and stored at 1 oc for three months, also gave rise to DDA and two unidentified compounds. They also found DDA to be formed from DDT in filtered wines that were bottled after one month and stored at 1 °C for three months. Only DDT was present in the filtered wine and only DDA was found after storage. CHACKO et al. ( 1966) tested eight soil fungi (including Trichoderma viride) cultured separately in a nutrient medium for six days, for their ability to degrade DDT. They found no detectable degradation for any of the fungi although they did for six of nine actinomycetes studied (see section V. a). MATSUMURA and BousH (1968), however, found T. viride able to degrade DDT. They cultured the fungus in screw-capped tubes in liquid media containing 10- 3 M 14 C-DDT for three days. Of the 18 variants tested, eight cultures produced both TDE and dicofol as the major metabolites, three produced only TDE, and one produced TDE and DDE. It is interesting to note that in later work they used "dicofollike" in place of dicofol ( PATIL et al. 1970) and still later MATSUMURA et al. ( 1971) replace "dicofol-like" with DDNS. It is probable that the dicofol, as used here, is really DDi\'S since MATSUMURA et al. ( 1971) showed DDNS to arise from TDE. Six variants, under their conditions, did not metabolize DDT. They also found that DDT degraded slowly in the absence of the fungus. For their culture no. 12, they found 89% of the DDT degraded after three days at 30°C. In later related work, PATIL et al. ( 1970) found two isolates of T. viride capable of degrading DDT to TDE, DDA, and a dicofol-like metabolite (probably DDNS). It is apparent from the studies using T. viride that broad differences must exist in their enzyme systems enabling them to either degrade DDT to various compounds or not at all. ANDERSON et al. ( 1970) studied DDT degradation in shake cultures of a nutrient media containing Mucor alternans. Mycelia killed by autoclaving or HgCl 2 were used in controls. In their experiment with 14 C-DDT using a four-day incubation, they found five metabolites by TLC and radioautography. Three were in the hexane phase after extraction of the mycelia and media and two were in the remaining aqueous phase. None were identified since their R1 values did not coincide with those of either DDA, DBP, TDE, dicofol, or DDNS. This is surprising in light of the work just cited and yet to be reviewed. The major metabolite was one in the aqueous phase. Total recovery was not given but based on GLC data of the hexane phase, about 36% was found in this phase and, of the total activity recovered, it was about equally distributed between the phases; therefore, recovery was about 70%. Degradation was noted only with live mycelia and control recoveries were about 85% for autoclaved and 75% for HgCl 2 -killed mycelia. ANDERSON and LICHTENSTEIN ( 1971) subsequently studied various nutritional factors influencing DDT degradation by M. alternans. They found that DDT was not used by the fungus as a source of carbon and that degradation was not related to the mycelial mass. Any degradation, then, would be considered cometabolism as pre-


19

viously defined. The quantity of water-soluble metabolites of DDT was largely dependent on the carbon and nitrogen sources in the culture medium, especially glucose and ammonium nitrate. In continuing this approach, ANDERSON and LICHTENSTEIN ( 1972) studied the effects various soil fungi and insecticides had on the capacity of M. alternans to degrade DDT. In most cases, addition of one of the various fungi to 14 C-DDTtreated cultures of M. alternans totally depressed the appearance of watersoluble DDT metabolites in the media. Several insecticides similarly reduced DDT degradation. These results imply that it is difficult to extrapolate laboratory findings using pure cultures with those found in nature when one considers the many possible detrimental interactions involved. In a comprehensive series of papers, ENGST and co-workers reported the enzymic degradation of DDT by molds. Having discerned earlier that Fusarium oxysporum was the most active of five molds degrading DDT, ENGST and KuJAWA ( 1967) found this mold to decompose DDT to DDE, TDE, DDMU, DDOH, and DBP. They reported that TDE was formed directly from DDT and indirectly from DDE. This latter point could be disputed as indicated by the work of PLIMMER et al. ( 1968) and KALLMAN and ANDREWS ( 1963), previously cited. Upon losing HCl, DDMU was formed from TDE which in turn formed DDHO, an aldehyde, with water, followed by rapid dismutation into DDOH and DDA. Although it is not clear, presumably the DDMU was dechlorinated to DDNU before DDHO formation. The existence of the aldehyde was indicated by the reaction sequence being stopped by semicarbazide. DDA was degraded to DBP by oxidative decarboxylation. ENGST and KuJAWA ( 1968) synthesized the aldehyde DDHO which confirmed its role as one of the degradation products of DDT. In subsequent allied work, FRANZKE et al. ( 1970) found that 0.1 ppm of DDT added to F. oxysporum culture inhibited fungal growth and esterase activities whereas TDE stimulated both. In vivo, they found both the inhibitory and stimulatory effects were superimposed due to the enzymic formation of TDE from DDT, which simulated an activating effect by DDT. After 10 to 14 days of incubation, DDT no longer was detectable but esterase activity continued to increase. DDE was found to have no effect on the esterase system. KuJAwA and ENGST (1970) added additional proof to the enzymatic concepts of DDT degradation by fractionating the enzymes of the DDT-treated F. oxysporum culture. Separation of the culture filtrate, lyophilizate, and acetone powder precipitate on Sephadex G-100 and G-200 yielded only one fraction which contained all the DDT-decomposing enzymes. Exchange chromatography on DEAE-cellulose yielded five enzymatic fractions. Fraction 1 was inactive toward DDT and five metabolites. Fractions 2 and 3 were similar in containing enzymes which metabolized only DDMU (to DDA, DDOH, and DBP), DDA (to DDOH and DBP) and DDOH (to DDA and DBP). Fraction 2 was less efficient than fraction 3 since only trace amounts of the metabolites were detected. All of the enzymes concerned with the

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metabolism of DDT occurred in fractions 4 and 5. These two fractions were similar in that they both metabolized DDT to DDE, TDE, and DDMU. Although fraction 4 was the most active, they both also degraded TDE (to DDMU), DDE (to TDE), DDA (to DOOR and DBP), and DDOH (to DBP). From their data it is apparent that differentiation of the responsible enzymes was difficult since gel filtration gave negative results as did sedimentation experiments using an ultracentrifuge. They concluded that more than one enzyme is responsible for DDT degradation, that these enzymes were very similar in their molecular weights, and that their charge behavior agreed extensively with one another. They isolated from the five fractions, two different types of effects which brought about different steps in the degradation of DDT. The only previously published work on DDT degrading enzymes has been that with DDTdehydrochlorinase. This report opens up new areas for further research on the characterization of the enzymatic degradation of DDT. FocHT ( 1972), continuing studies of FOCHT and ALEXANDER ( 1970 and 1971), reviewed in section V. a, used extracted products from bacterial cell suspensions (Hydrogenomonus), incubated with either DDM or PCPA, which were each re-extracted into basal salts media and incubated with Fusarium sp. He found growth to be evident within five days, indicating utilization of these chemicals or metabolites as carbon sources. He also found that both fungal supernatant liquids, from DDM and PCPA bacterial incubations, were positive for chloride whereas uninoculated controls were negative. These facts established that DDM, and its ring-cleavage product PCPA, were further degraded to C0 2 , H20 and HCI. It has thus been shown that, using both a bacteria and a fungus, DDT can be completely metabolized to its basal components through DDM and PCPA. c) Algae

There are only a few papers in the literature dealing with the metabolism of DDT by algae and some of these do so in only a cursory way. MooRE and DoRWARD ( 1968), using axenic cultures of A1UlCystis nidulans in time-course studies, found that DDT metabolism was concomitant with growth. They reported metabolism to occur at or near the cell surface although no mention was made of any metabolites. KEIL and PRIESTER ( 1969) studied the marine diatom Cylindrotheca closterium for its ability to accumulate and metabolize DDT added to culture media at 0.1 ppm. Their results indicated that only DDE was formed. However, the fact that their untreated control series averaged over 8.6 ppm compared to 30 ppm for their DDT-treatment indicates a far more serious contamination problem than that coming from the water or "minute" residuals on the glassware. Since no information was given on the background of the diatom, culture methods, etc., many speculative causes can be made. One wonders why there is over three times more


21

DDT residue in the control series receiving no additives than the control series receiving only the carrier acetone. MIYAZAKI and THORSTEINSON ( 1972) reported on the metabolism of 14 C-DDT by the fresh-water diatoms Nitzschia sp. and an unidentified species. Using an aerobic incubation for two weeks, they found DDE as the only metabolite. However, these conversions were less than 1%. The fact that from 2 to 3% of the activity was found in the aqueous phase indicates that possibly other routes of metabolism were present. Since only about 60% of the activity was recovered from the treatments versus 75% for the controls (without diatoms), it is apparent that some residues were not extracted from the cultures. Their conclusion that diatoms may be significant factors in degrading DDT to DDE hardly seems justifiable on the basis of their data. PATIL et al. ( 1972) studied several field-collected, unidentified marine algal cultures and a laboratory colony of Dunaliella sp. for their 14 C-DDT metabolizing capabilities. Unfortunately, many of their isolates were not identified by organism type and are listed only as "microbial isolates" from various sources. In one algal culture, 74% of the activity resided in the aqueous phase after chloroform extraction. Of that remaining in the solvent phase, about 20% was TDE, with less than 2% as DDT and with lesser amounts of DDE, DDNS, and also DDOH being found. For Dunaliella sp. and another unidentified alga, 11.5 and 1.5% of the activity remained in the water phase after extraction, respectively. With Dunaliella, over twice as much activity ( 33%) in the solvent extract resided at the origin on thin-layer plates than existed as TDE, indicating the formation of polar metabolites such as DDA. For the other alga, 52% was identified as TDE with 37% as unchanged DDT. Possibly because they were handling so much data and included only a small amount in their article, the paper is difficult to follow in spots. They concluded that algal cultures appeared to convert DDT exclusively to a DDOH-like compound, although from their data this conclusion is not justified. It is apparent, though, that TDE is the main metabolite with lesser amounts of DDE, DDNS, and DDOH. It would be interesting to know, for the one algal culture in particular, the identity of the unextractable activity in the aqueous phase after solvent extraction. BowES ( 1972) studied the effect of DDT on seven marine algae representing five algal divisions in unaerated, axenic cultures and found their ability to metabolize DDT varied. The only significant hexane-soluble metabolite found was DDE with a maximum conversion of only 7.4% for Dunaliella tertiolecta. Total recovery of added DDT after at least 14 days' incubation ranged from 63.7 to 90.7%. Hexane rinses of D. tertiolecta yielded only DDT and DDE was found only when the cells were digested. For the three diatoms Skeletonenw costatum, Thalassiosira fluviatilis, and Cyclotella nanna, they converted 4.5, 3.9, and less than 3%, respectively, of the DDT to DDE. For Coccolithus huxleyi, Amphidinium carteri, and Porphyridium sp., no metabolism was apparent. Details of their analyses

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were not given in this paper and no indication was given as to possible aqueous metabolites. RicE and SIKKA ( 1973), in work similar to BoWEs ( 1972), studied 14 C-DDT metabolism using six marine algae, representing four algal divisions. Their cultures were grown axenically and agitated on a reciprocating shaker in stoppered flasks. The only metabolite found was DDE and the % conversion, after 24 days' incubation, varied from less than one for Olisthodiscus luteus and Isochrysis galbana, to 1.2 for C. nana, 2.81 for A. carteri, 4.9 for S. costatum, to a high of 11.5 for Tetraselmis chuii. Since they only analyzed cell extracts, activity in the medium and metabolites not soluble in petroleum ether were missed using GLC. However, their TLC method used acetone extracts but they reported DDE as the only metabolite. These latter two papers indicate much less metabolic activity of marine algae toward DDT than found by PATIL et al. ( 1972). VI. Conclusions Although DDT has been used extensively throughout the world for three decades, it is only in recent years that it has been shown to be metabolized by microorganisms. Considerable evidence now exists that many diverse microbial organisms, including bacteria, fungi and algae, are capable of degrading DDT and some of its metabolites. Sufficient evidence has shown that most of the observed changes in DDT, when incubated under various conditions and with different organisms or groups of organisms, are biologically rather than nonbiologically induced. Numerous authors have shown, using controls which are sterile, employing dead organisms, or using microbial inhibitors, that these degradative reactions are of enzymatic and not chemical origin. It is apparent that the major route of DDT metabolism by microorganisms is through TDE formation by reductive dechlorination under anaerobic conditions. A considerable number of reports show that DDT is degraded, through various dechlorination steps and a decarboxylation step to DBP or to the more reduced form DDM. A few reports indicate that ring-cleavage of DDM can occur to form PCPA which requires aerobic conditions, which in turn was shown to be degraded to p-chlorophenylglycolaldehyde. Aerobically, DDE is the prime DDT metabolite and, despite some evidence to the contrary, appears to be a stable end product incapable of being further degraded. It can be seen from various experiments that p-chlorine ring substitution makes DDT and its metabolites much more resistant to microbial degradation than their unsubstituted analogues. It appears that no organisms can use DDT, or its main metabolites, as sole carbon sources for growth but rather that any degradation is coincidental with the organisms own metabolic processes ( cometabolism). Two recent papers reported a new metabolite, DDCN, to occur in


23

DDT-treated sewage sludge. These were the first reports of a nitrogencontaining DDT metabolite. How this metabolite fits into the scheme of degradation and whether it is widespread in the environment is 110t known at this time. Recent work on fractionating the proteins in culture filtrates of a DDT -treated fungus indicated that at least several enzymes are involved in DDT metabolism and that they are very similar in molecular weight. Other work indicates that discrete enzymes are involved in the various degradative stages. Various authors have shown that often there were rather extensive amounts of aqueous metabolites formed which were not extracted by the usual nonpolar solvents and which remain unidentified. Identification of these products is essential for a more complete understanding of DDT metabolism. That there are complex interactions at work with microbial mixtures is evident from reports which show that the presence of a second microorganism inhibits DDT degradation and in others where additional microbial species enhance metabolism. In the environment, then, it is apparent that both negative interactions and a type of synergistic interaction can occur and that extrapolation of laboratory findings to field situations can be difficult at best. The metabolic pathway of DDT by microorganisms appears to be similar to that proposed by MENZIE ( 1969) ( p. 131) whereby DDT ~ DDE and DDT ~ TDE ~ DDMU ~ DDMS ~ DDNU ~ DDNS ~ DDOH ~ DDHO ~ DDA ~ DDM ~ DDBH ~ DBP. Variations on this path would include DDA ~ conjugates with amino acids, etc., DDM ~ PCPA ~ p-chlorophenylglycolaldehyde, DBP ~ C0 2 , H 2 0, and HCL These steps are a synthesis of the data and no single paper reports all these stages. Only a single paper reports complete degradation. However, the stage is now set for additional research to resolve the discrepancies which remain. Summary The metabolism of DDT by microorganisms has been reviewed and is shown to be a major factor in its environmental degradation. For extensive degradation to occur, anaerobic conditions must prevail and microbial populations must be high. It is clear that the preponderance of observed changes in DDT induced by microorganisms are of biological origin and are mediated by enzymes. DDE apparently does not undergo further biological alteration and is formed aerobically. The major route of DDT metabolism is through TDE which can be degraded to DBP or DDM, the latter being subject to ring cleavage (aerobically) and may be degraded completely to C0 2 , H 2 0, and HCl. A new nitrogen-containing metabolite, DDCN, was noted but its significance is not known. Un-

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identified aqueous metabolites, noted in many reports, may indicate conjugates of DDA or other ring-cleavage products. There are still many gaps in the knowledge of microbial degradation of DDT that remain. Microorganisms, however, are very involved in DDT degradation but none have been shown to utilize DDT or its main metabolites for growth, and metabolism of DDT is considered a coincidental process. Glossary Abbreviations, chemical formulas and names of DDT and its degradation products Abbreviation•

Formu[ab

p,p'-DDT

(R) 2-CH-CCL

p,p'-DDD (TDE) p,p'-DDE

( R) 2-CH-CHCL

p,p'-DDMU

(R) 2 -C=CHCI

p,p'-DDMS

( R) 2-CH-CH2Cl

p,p'-DDNU

(R)z-C=CHz

p,p'-DDNS p,p'-DDOH

( R) 2-CH-CH3 ( R) z-CH-CHzOH

p,p'-DDA p,p'-DBH p,p'-DBP p,p'-DDM p,p'-dicofol

( R) 2-CH-COOH (R) 2-CHOH (R) 2-C=0 (R)2-CHz (R) 2-COH-CCI3

o,p'-DDT

(R) 2-C=CCI 2

R, Rl/

CH-CCI, .

Name

1,1'-bis( p-chlorophenyl) -2,2,2trichloroethane 1,1'-his ( p-chlorophenyl) -2,2dichloroethane 1,1'-bis( p-chlorophenyl) -2,2dichloroethylene 1,1'-bis ( p-chlorophenyl )-2chloroethylene 1,1'-bis ( p-chlorophenyl) -2chloroethane 1,1'-his ( p-chlorophenyl ) ethylene 1,1'-bis( p-chlorophenyl) ethane 1,1'-his ( p-chlorophenyl) -2hydroxyethane his ( p-chlorophenyl) acetic acid 4, 4'-dichloro henzhydrol 4,4'-dichlorobenzophenone his( p-chlorophenyl )methane 1,1'-his ( p-chlorophenyl) -2,2,2trichloroethanol 1- ( o-chlorophenyl) -1- ( pchlorophenyl) -2,2,2trichloroethanec

• Abbreviation of compounds principally after C. M. Menzie ( 1969): Metabolism of Pesticides, Bur. Sport Fish. Wildlife, Spec. Sci. Rept.-Wildlife No. 127, USDI, Washington, D. C. • R p-chlorophenyl and R1 o-chlorophenyl. c Other o,p'-isomers would be similarly described.

=

=


25

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Metabolism of 14C-DDT in Pheretima posthuma and effect of pretreatment with DDT, lindane and dieldrin.

Antimutagenesis in microbial systems.

Metabolism of 14C-DDT in the mouse and hamster.

DDT uptake and metabolism by a marine diatom.

Carbon metabolism in model microbial systems from a temperate salt marsh.

Metabolism of DDT analogues by a Pseudomonas sp.

Microbial lipid metabolism.

Systems biology approaches for inflammatory bowel disease: emphasis on gut microbial metabolism.

The role of microbial amino acid metabolism in host metabolism.

Recombinant protein expression in microbial systems.

Posttranslational regulation of microbial metabolism.

Spatiotemporal modeling of microbial metabolism.

Microbial models of mammalian metabolism.

Microbial metabolism of aliphatic glycols. Bacterial metabolism of ethylene glycol.

Systems Biology of Microbial Exopolysaccharides Production.

Induction of abnormal fatty acid metabolism and essential fatty acid deficiency in rats by dietary DDT.

Systems strategies for developing industrial microbial strains.

Pathways of microbial metabolism of parathion.

Microbial metabolism of monoterpenes--recent developments.

Microbial acetyl-CoA metabolism and metabolic engineering.

Human intestinal microbial metabolism of naringin.

Chlorine stress mediates microbial surface attachment in drinking water systems.

Heterogeneity in Pure Microbial Systems: Experimental Measurements and Modeling.

Changes in Microbial Biofilm Communities during Colonization of Sewer Systems.