Bioremediation of DDT-Contaminated Soils: A Review

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Bioremediation of DDT-Contaminated Soils: A ReviewJulia Foght a , Trevor April b , Kevin Biggar c & Jackie Aislabie da Biological Sciences, University of Alberta, Edmonton Alberta Canada T6G 2E9b Biological Sciences, Northern Alberta Institute of Technology, Edmonton Alberta CanadaT5G 2R1c Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta, CanadaT6G2G7d Landcare Research, Private Bag 3127, Hamilton New ZealandVersion of record first published: 03 Jun 2010.

To cite this article: Julia Foght , Trevor April , Kevin Biggar & Jackie Aislabie (2001): Bioremediation of DDT-ContaminatedSoils: A Review, Bioremediation Journal, 5:3, 225-246

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Bioremediation of DDT-Contaminated Soils: A Review 225

1058-8337/001/$.50© 2001 by Battelle Memorial InstituteBioremediation Journal 5(3):225–246 (2001)

Bioremediation of DDT-Contaminated Soils: AReview

Julia Foght,1* Trevor April,2 Kevin Biggar,3 and Jackie Aislabie4

1Biological Sciences, University of Alberta, Edmonton Alberta CanadaT6G 2E9; 2Biological Sciences, Northern Alberta Institute of Technology,Edmonton Alberta Canada T5G 2R1; 3Civil and EnvironmentalEngineering, University of Alberta, Edmonton, Alberta, CanadaT6G 2G7;4Landcare Research, Private Bag 3127, Hamilton New Zealand.

Abstract: The insecticide 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT) has been used extensively sincethe 1940s for control of agricultural pests, and is still used in many tropical countries for mosquito control. Despitea ban on DDT use in most industrialized countries since 1972, DDT and its related residues (DDTr) persist in theenvironment and pose animal and human health risks. Abiotic processes such as volatilization, adsorption, andphotolysis contribute to the dissipation of DDTr in soils, often without substantial alteration of the chemicalstructure. In contrast, biodegradation has the potential to degrade DDTr significantly and reduce soil concentra-tions in a cost-effective manner. Many bacteria and some fungi transform DDT, forming products with varyingrecalcitrance to further degradation. DDT biodegradation is typically co-metabolic and includes dechlorination andring cleavage mechanisms. Factors that influence DDTr biodegradation in soil include the composition andenzymatic activity of the soil microflora, DDTr bioavailability, the presence of soil organic matter as a co-metabolic substrate and (or) inducer, and prevailing soil conditions, including aeration, pH, and temperature.Understanding how these factors affect DDTr biodegradation permits rational design of treatments and amend-ments to stimulate biodegradation in soils. The DDTr-degrading organisms, processes and approaches that maybe useful for bioremediation of DDTr-contaminated soils are discussed, including in situ amendments, ex situbioreactors and sequential anaerobic and aerobic treatments.

* Corresponding author. Tel: (780) 492-3279; E-mail: [email protected]

IntroductionDDT (1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane)was introduced as an insecticide during World War II(Busvine, 1989) and continues to be used successfullyin many tropical countries to reduce the incidence ofmalaria through the control of mosquitoes. Before itsban in most industrialized nations in the early 1970s,DDT was also widely used in North America andEurope for the control of agricultural pests. However,it has been suggested that as little as 1% of the pesti-cide reaches the target pest, with the ultimate sinkbeing soil and water (Sommerville and Greaves, 1987).

Ideally, pesticides should persist long enough tocontrol target organisms, then degrade to inert or in-nocuous products. However, DDT and its residues, col-lectively referred to as DDTr (Figure 1), persist in theenvironment. Two isomers of DDT exist: 4,4′-DDT(predominant) and 2,4′-DDT, and their transformationyields the corresponding residue isomers. The majorresidues detected in the environment include DDT, DDD(1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane, formerlyabbreviated TDE), DDE (1,1-dichloro-2,2-bis-(4-chlorophenyl)ethylene) and DDA (bis(4-chlorophenyl)-acetic acid). DDD and DDE are common co-contami-nants with DDT in soils, arising both as impurities

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during the manufacture of DDT and also as stable trans-formation products from biotic and abiotic processes.DDD is produced by reductive dechlorination of DDTthrough biotic (Table 1) and abiotic processes (e.g.,Sayles et al., 1997), some of which are mediated bybiological molecules (Baxter, 1990). DDE is formed bydehydrochlorination reactions, including photolysis(Maugh, 1973) and aerobic microbial transformation(Table 1). Only recently has the DDT polar metaboliteDDA been detected in the environment in waters down-stream from a DDT manufacturing plant (Heberer andDünnbier, 1999).

Little is known about the toxicity of DDTr, andassessment may be complicated by the synergistic effectsof the original pesticide and its metabolites. Their lipo-philic nature results in bioaccumulation in tissues in non-target organisms, raising concerns about potential long-term health and environmental effects, and warrantingtheir inclusion on the United States Environmental Pro-tection Agency (USEPA) list of Priority Persistent,Bioaccumulative and Toxic (PBT) Chemicals (USEPA,2000a). One of the earliest publicized side effects wasthinning of eggshells, particularly affecting the bald eagleand peregrine falcon populations. Although the effects of

Figure 1. Chemical structures of the predominant 4,4′- isomer of DDT, its residues DDD, DDE and DDA,and the minor 2,4′-isomer of DDT. 4,4′-DDD: 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane; 4,4′-DDD:1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane; 4,4′-DDE: 1,1-dichloro-2,2-bis-(4-chlorophenyl)ethylene);4,4′-DDA (bis(4-chlorophenyl)-acetic acid; and 2,4′-DDT: 1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane. Isomer designations are omitted from discussion in the text.D

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Table 1. Examples of bacteria and fungi that degrade DDTr in pure culture. Refer also to additional examplesprovided in reviews by Johnsen (1976); Essac and Matsumura (1980); Lal and Saxena (1982); Kuhn and Suflita(1989); and Rochkind-Dubinsky et al. (1987)

Degradation mechanism Microbe Source Ref.

BacteriaReductive dechlorination Proteus vulgaris Mouse Barker et al., 1965of DDT to DDD Intestine

Escherichia coliEnterobacter (formerly Rat feces Mendel and Walton, 1966Aerobacter) aerogenes

Pseudomonas aeruginosa Activated Sharma et al., 1987Bacillus spp. SludgeFlavobacterium sp.

Enterobacter cloacae Sewage Beunink and Rehm, 1988Sludge

Bacillus sp. Soil Katayama et al., 1993Unidentified

Cyanobacteria Soil Megharaj et al., 2000

Conversion of DDT to E. aerogenes Not given Wedemeyer, 1966, 1967various metabolitesincluding DDD, DDMU, Bacillus spp. Not given Langlois et al., 1970DDMS, DDNU, DDOH, E. coliDDA and DBP, or DDT E. aerogenesto DDE

Metabolism of 14C-DDT Pseudomonas sp. Sewage Pfaender and Alexander, 1972to DDD, DDMS, DDNU (isolated asand DBP by cell free Hydrogenomonas sp.)extracts under anaerobicconditions

Reductive dechlorination Pseudomonas aeruginosa Soil Golovleva and Skryabin, 1981of DDT and ring 640Xcleavage of metabolites

Transformation of DDT, Strain B-206 Activated Massé et al., 1989DDE, DDD, DDMU to Sludgehydroxylated metabolites

Meta ring cleavage of Ralstonia eutropha Soil Nadeau et al., 1994, 1998DDT (formerly Alcaligenes

eutrophus) A5

Alcaligenes sp. JB1 Soil Parsons et al., 1995

Meta ring cleavage of Pseudomonas acidovorans Genetically Hay and Focht, 1998DDE M3GY engineered

Terrabacter sp. strain Soil Aislabie et al., 1999DDE 1

Meta ring cleavage of R. eutropha A5 Soil Hay and Focht, 2000DDD

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DDTr on humans have been debated and the acute effectsof environmental concentrations are not known, the pos-sibility of chronic effects exists. For example, DDT andDDE have estrogenic (Hansen and Schaeffer, 1994) andandrogen receptor antagonistic effects (Kelce et al., 1995)and may inhibit or limit complete male development. Theeffects of DDTr are not restricted to animals, and toxiceffects on bacteria (e.g., Donato et al., 1997) and eukary-otic microorganisms (reviewed by Lal and Saxena, 1982)have also been observed.

Removal of DDTr from contaminated soils hasbecome an environmental priority, and both physico-chemical and biological remediation processes havebeen examined. Conventional nonbiological treatmentsfor organochlorine-contaminated soils include excava-tion and incineration, thermal desorption (Norris et al.,1997), microwave-enhanced thermal treatment (Kawalaand Atamanczuk, 1998), soil washing with surfactants(Kile and Chiou, 1989; Parfitt et al., 1995), supercriticalfluid extraction (Sahle-Demessie and Richardson,2000), and sulfuric acid treatment (Singh and Agarwal,1992; Hussain et al., 1994a). Although these physicaland chemical treatments may be more rapid than bio-logical treatment, they are generally intrusive and de-structive to affected soils, energy intensive, and maybe less cost-effective than bioremediation.

DDTr persistence in the environment is a result ofthe recalcitrance of these xenobiotics to biodegrada-tion. This is due to at least three factors: the presenceof chlorine substituents on the molecule (Focht andAlexander, 1971), as discussed below; low water solu-bility leading to poor bioavailability in soil (Alexander,2000); and the fact that, despite its widespread use in

the environment for more than 50 years, no knownmicrobes have yet evolved to use DDTr as a solecarbon and energy source (e.g., Golovleva andSkryabin, 1981). Microbial transformations of DDTrare co-metabolic (i.e., occur fortuitously as the organ-ism uses an alternative carbon and energy source), andtherefore there is no evident energetic or evolutionaryadvantage to the ability to metabolize DDTr. Conse-quently it is difficult to enrich DDTr-degrading organ-isms in contaminated environments or in the labora-tory. Despite these limitations, biodegradation is amajor factor affecting the persistence of DDTr in soils.

Before bioremediation can be considered a viablesoil treatment option, it is important to understand thetransformations of DDT and its residues. In particular,because DDD and DDE are also persistent and toxic,it is important that remediation processes also addressthe fate and potential accumulation of these products.Here we discuss the potential of microorganisms toreduce the toxicity and recalcitrance of DDTr in soils,by reviewing the current literature pertaining to DDTrdegradation by pure cultures, presenting the elucidatedpathways of DDTr transformation, discussing factorsthat influence DDTr bioremediation in soils, and sum-marizing methods most suitable to enhancingbioremediation of DDTr in soils.

Degradation of DDTr byMicroorganisms in Pure Culture

The term “biodegradation” has been used inthe literature with different meanings. In its most

FungiReductive dechlorination Saccharomyces cerevisiae Bakers yeast Kallman and Andrews, 1963of DDT to DDD

Transformation of DDT Aspergillus flavus Not given Subba-Rao and Alexander, 1985to various metabolites Thanatephorus cucumerisincluding DDD, DBP, false smut of wheatand DDE

Involvement of Phanerochaete Not given Bumpus and Aust, 1987lignin degrading system chrysosporiumin mineralization of Pleurotus ostreatus14C DDT via DDD, Phellinus weiriidicofol and FW-152 Polyporus versicolor

DDT conversion to Phlebia strigoso-zonata Rotting wood Katayama et al., 1992dicofol Basidiomycete

Mineralization of P. chrysosporium Not given Bumpus et al., 199314C DDE via DPB

Table 1. (continued)

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restrictive sense, it implies that the compound of inter-est (e.g., DDT) has undergone extensive chemicalchanges, including significant dechlorination and ringcleavage, or mineralization (conversion to CO2 andH2O). In the loosest sense, it includes less extensivechanges to the molecule (i.e., “transformation” or“metabolism” of the compound, including limited de-hydrochlorination and reductive dechlorination) toproducts that themselves may or may not be subject tofurther biodegradation. In the following discussion,DDTr degradative pathways are presented, whenknown, so that the degree of “biodegradation” achievedby the organisms can be assessed unequivocably.

The observed persistence of DDTr in the environ-ment has been attributed to the presence of chlorine onthe molecule hindering biodegradation reactions. Forexample, while DDT is recalcitrant, its nonchlorinatedanalogue diphenylmethane is readily biodegradable(Focht and Alexander, 1971; Subba-Rao and Alexander,1985; Hay and Focht, 1998). Therefore, organismsthat significantly degrade DDT and its residues musteither possess enzymes that are active against chlori-nated substrates, or have the ability first to dechlori-nate DDT then to degrade the products.

Microbes that metabolize DDTr have been iso-lated from a range of habitats, including animals, soil,rotting wood, sewage, and activated sludge (Table 1).Biodegradation of DDTr primarily is cometabolic, asthe microbes involved do not derive any nutrient orenergy for growth from the process, and require analternate carbon source for growth (Bollag and Liu,1990). In most described pathways of bacterial andfungal attack, DDT is reductively dechlorinated toDDD under reducing conditions (reviewed by Johnsen,1976; Essac and Matsumura, 1980; Lal and Saxena,1982; Kuhn and Suflita, 1989; Rochkind-Dubinsky etal., 1987; Aislabie et al., 1997). However, aerobicDDTr degradation has recently been described (Nadeauet al., 1994; Hay and Focht, 1998, 2000; Aislabie et al.,1999) involving aromatic-degrading bacteria. In thefollowing sections, these differing degradative path-ways are described and the mechanisms discussed.

Metabolism via ReductiveDechlorinationProteus vulgaris, isolated from the intestinal micro-flora of a mouse, was one of the first pure culturesobserved to reduce DDT to DDD (Barker et al., 1965).Since then various bacteria and fungi with this abilityhave been described (Table 1). The reductive dechlo-rination of DDT to DDD involves substitution of analiphatic chlorine for a hydrogen atom (Figure 2), andrequires transition metals and metal complexes actingas reductants (reviewed by Holliger and Schraa, 1994).

In most cases, the process involves single electrontransfer, removal of a chlorine ion, and formation of analkyl radical. This not an energy-yielding process forthe organism, unlike other reductive reactions (e.g.,sulfate reduction), as the DDT is not serving as theterminal electron acceptor in a bioenergetic pathway.As mentioned previously, reductive dechlorination ofDDT can also occur in the absence of viable organ-isms, catalyzed by biomolecules like hematins; DDDcan also be reductively dechlorinated by this mecha-nism, but DDE cannot (Baxter, 1990).

Under anaerobic conditions, DDD may be furthermetabolized. For example, pure cultures of Escherichiacoli and Enterobacter aerogenes (formerly Aerobacteraerogenes) incubated with DDT produced trace amountsof 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU),1-chloro-2,2-bis(4-chlorophenyl)ethane (DDMS), 2,2-bis(4-chlorophenyl)ethylene (DDNU), 2,2-bis(4-chlorophenyl)ethanol (DDOH), bis(4-chlorophenyl)-acetic acid (DDA), and 4,4'-dichlorobenzophenone(DBP) (Langlois et al., 1970; Wedemeyer, 1967;Pfaender and Alexander, 1972), in addition to the majormetabolite DDD. The proposed pathway for the anaero-bic transformation of DDT by bacteria (Figure 2) showsthat DDD is reductively dechlorinated to DDNU, whichis successively oxidized to DDOH and DDA, the latterof which in turn is decarboxylated to bis(4-chloro-phenyl)methane (DDM). DDM is oxidized to DBP,which is not further metabolized under anaerobic con-ditions (Pfaender and Alexander, 1972).

Study of the degradation of DDT by Pseudomo-nas aeruginosa 640× (Golovleva and Skryabin, 1981)has shown that this strain either mineralizes DDT ordegrades it significantly to produce (nonchlorinated)phenylacetic, phenylpropionic, and salicylic acids.However, all steps after the first reductive dechlorina-tion of DDT to DDD until the formation of benzhydrolwere found to require co-metabolic substrates, and theextent of co-metabolism depended on the nature ofthose substrates and on the aeration conditions. Strain640 x was subsequently genetically modified by intro-duction of a plasmid encoding naphthalene and salicy-late degradation, creating P. aeruginosa strain BS827that degraded kelthane (dicofol) to unknown products(Golovleva et al., 1988).

Alternating anaerobic and aerobic incubation con-ditions can enhance DDTr biodegradation by promot-ing reductive dechlorination of DDT to DPB withsubsequent aerobic aromatic ring cleavage. Pfaenderand Alexander (1972) observed that cell-free extractsof a Pseudomonas sp. (Subba-Rao and Alexander,1977; originally classified as Hydrogenomonas sp.)metabolized 14C-DDT to DDD, DDMS, DDNU, andDBP under anaerobic conditions. When these metabo-

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lites were subsequently incubated under aerobic con-ditions with fresh Pseudomonas sp. inoculum,p-chlorophenylacetic acid (PCPA) was identified asthe product (Figure 2). This metabolite was found to besusceptible to further degradation by an Arthrobactersp., producing p-chlorophenylglycoaldehyde. BecausePCPA was not produced under strict anaerobic condi-tions, these in vitro studies implied that the ring cleav-age reactions required molecular oxygen. However,DDT was not metabolized by the Pseudomonas sp.under strictly aerobic conditions either, demonstratingthe advantage of using alternating anaerobic-aerobicconditions to achieve significant DDT degradation inthis system.

Anaerobic bacterial dechlorination of DDE (whichis primarily an aerobic transformation product of DDT)to DDMU was reported in pure culture (Massé et al.,1989) and this transformation has since been describedin soil (Agarwal et al., 1994b) and anaerobic marinesediments under methanogenic and sulfidogenic con-ditions (Quensen et al., 1998).

Aerobic Bacterial Degradation of DDTrvia Ring Cleavage ReactionsMicrobes that transform DDTr co-metabolically can bedifficult to isolate from the environment because thereis no direct means of enriching or selecting for thatproperty. Instead, a technique known as analogue en-richment (Bartha, 1990), in which a structural analogueis substituted for the compound of interest, has provenuseful. Using this method, a “Hydrogenomonas” sp.(Pseudomonas sp.) capable of co-metabolizing DDTwas isolated from sewage by providing diphenylmethane,a structural analogue of DDT, as a growth substrateduring enrichment (Focht and Alexander 1970b). Simi-larly the ability to degrade 4-chlorobiphenyl, anotherstructural analogue of DDT, was used to select bacteriaable to co-metabolize DDTr (Massé et al., 1989; Nadeauet al., 1994; Parsons et al., 1995). Some of the4-chlorobiphenyl-degrading bacteria were subsequentlyshown to degrade DDTr through novel pathways. Cul-tures of Ralstonia eutropha (formerly Alcaligeneseutrophus) A5 slowly metabolized both the 4,4′- and2,4′-DDT isomers (Nadeau et al., 1994), presumablyusing 4-chlorobiphenyl degradation enzymes. Thismechanism was suggested because the initial attack onDDT appeared to involve a dioxygenase. The productwas a dihydrodiol-DDT intermediate that then under-went meta cleavage to produce 4-chlorobenzoic acid(Nadeau et al., 1998), representing extensive aerobicmetabolism of DDT. In contrast, strain B-206, a4-chlorobiphenyl-degrading Gram-negative bacterium,produced phenolic metabolites from DDT, DDE, DDD,

and DDMU (Massé et al., 1989) but no ring cleavageproducts.

Recent studies have shown that degradation ofDDD by R. eutropha A5 (Hay and Focht, 2000), andDDE by Pseudomonas acidovorans M3GY (Hay andFocht, 1998) and Terrabacter sp. DDE-1 (Aislabie etal., 1999) also proceed via meta-ring cleavage (Figure3) under aerobic conditions when the bacterium isinduced with biphenyl. In fact, the ring cleavage path-ways for degradation of DDT (Nadeau et al., 1994,1998), DDE (Hay and Focht, 1998; Aislabie et al.,1999) and DDD (Hay and Focht, 2000) appear to beanalogous based on detection of similar metabolites.Thus, despite reports to the contrary (e.g., Megharaj etal., 1998), DDTr such as DDE are susceptible to aero-bic microbial degradation in vitro. This is a significantobservation, as DDE has previously been considered adead-end metabolite of DDT under aerobic conditions.

Fungal DegradationDegradation of DDTr is not restricted to bacteria. Sev-eral fungal genera (Table 1) have been reported totransform DDT via reductive dechlorination using apathway similar to that described above (Figure 2)(Subba-Rao and Alexander, 1985), whereas others ini-tiate attack through hydroxylation prior to dechlorina-tion (Lal and Saxena, 1982) (Figure 4).

Ligninolytic fungi in particular have been studiedfor their DDTr-degradative abilities, and the majority ofthis work has involved Phanerochate chrysosporium(Bumpus and Aust, 1987; Bumpus et al., 1993; Fernandoet al., 1989; Aust, 1990; Shah et al., 1992; Katayama etal., 1992). P. chrysosporium cultures deficient in nitro-gen mineralized approximately 10% of 14C-labeled DDTto 14CO2 in 30 days, with an additional ca. 50% beingtransformed, and the remainder appearing as metabo-lites, including dicofol, FW-152, and DBP (Bumpusand Aust, 1987). Based on these results a DDT degra-dation pathway was proposed (Figure 4) involving oxi-dation to dicofol, followed by dechlorination to FW-152and DBP with eventual ring cleavage. Because mineral-ization and dicofol production were observed only afteronset of ligninase production by the cultures, biodegra-dation was assumed to be mediated by the fungal lignindegrading system. Interestingly, DDD accumulated dur-ing the initial nonligninolytic lag phase, but was subse-quently degraded. This implied that DDD was producedby a mechanism distinct from the lignin degrading sys-tem, but nevertheless was degraded by it.

Further work by Fernando et al. (1989) showedthat the carbon source greatly influenced the extent ofDDT mineralization by P. chrysosporium. Starch andcellulose were superior to other complex carbohy-

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Figure 3. Proposed meta-ring cleavage pathway for degradation of DDE by Pseudomonas acidovorans M3GY (adapted from Hay andFocht [1998]). Metabolites C, D, and G were also produced by Terrabacter sp. DDE-1 incubated with DDE (Aislabie et al., 1999). Analogouspathways have been proposed for degradation of DDT (Nadeau et al., 1994; 1998) and DDD (Hay and Focht, 2000) by Ralstonia eutropha(formerly Alcaligenes eutrophus) A5. Metabolite A: 1,1-dichloro-2-(dihydroxy-4-chlorophenyl)-2-(4-chlorophenyl)ethylene; B: 6-oxo-2-hy-droxy-7-(4-chlorophenyl)-4,8,8-trichloroocta-2,4-dienoic acid; C: 2-(4-chlorophenyl)-3,3-dichloropropenoic acid; D: 4-chlorophenylaceticacid; E: 4-chloroacetophenone; F: 4-chlorobenzaldehyde; G: 4-chlorobenzoic acid. Dotted lines indicate postulated steps in the pathway.

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Figure 4. Proposed pathway for degradation of DDTr by Phanerochaete chrysosporium(after Bumpus and Aust [1987] and Bumpus et al. [1993]). The dotted line indicatesthat both abiotic and various biotic processes can produce DDE from DDT. DBP: 4,4'-dichlorobenzophenone

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drates or sugars in supporting 14C-DDT mineraliza-tion, and 14CO2 release stopped when available carbo-hydrate was exhausted. Katayama et al. (1992) alsomeasured 14C-DDT degradation by several fungalstrains, including one of Bumpus and Aust’s (1987)strains. They observed 14C-DDT degradation, but withmuch reduced mineralization levels and significantproduction of DDE (1 to 9%) and some dicofol.

Mineralization of 14C-DDE by P. chrysosporiumwas slower than that observed for DDT, reaching only6% of added label after 60 days (Bumpus et al., 1993),with DBP and unidentified compounds produced asintermediates (Figure 4). Because DDE mineralizationwas enhanced in N-limited cultures compared with N-sufficient cultures, the lignin degrading system wasagain inferred to be involved in DDTr formation.

The role of the lignin-degrading system in DDTrtransformation is an important question, as it suggestsstrategies for selecting competent fungal isolates andsuitable environmental conditions for DDTr degradation.Köhler et al. (1988) have argued against its involvement,based on their observation of DDT “degradation” (mea-sured as disappearance of DDT from cultures) under non-limiting nitrogen conditions where the lignin-degradingsystem would not be induced. However, their observationdoes not actually argue against involvement of the lignindegrading system, because DDT is transformed to DDDunder these conditions (Bumpus and Aust, 1987), ratherthan being mineralized by the lignin degrading system.Furthermore, Aust (1990) has shown that DDT mineral-ization coincides with lignin peroxidase production, andlignin peroxidase inhibitors such as EDTA andtetramethylethylenediamine also inhibit DDT mineral-ization. Although purified lignin peroxidases can oxidizesome xenobiotic compounds, this does not appear tohappen with DDT. The implication then is that DDTdegradation is accomplished through co-oxidation by amediator molecule such as veratryl alcohol (Khindaria etal., 1995), rather than by direct enzymatic attack (Barrand Aust, 1994). It is also possible that lignin peroxidasesmediate oxidation of DDT metabolites rather than ofDDT itself.

In conclusion, multiple mechanisms for degradingDDTr have been demonstrated in a variety of microbes,both bacterial and fungal. In the presence of competentorganisms incubated under suitable conditions, anaero-bic attack primarily produces dechlorinated aromaticresidues, while aerobic attack produces chlorinated aro-matic acids, accompanied by carbon loss.

DDTr Biodegradation in SoilThe DDTr degradation described above was observedin laboratory studies with pure cultures. However, soil

is a complex environment containing mixed popula-tions of microorganisms with synergistic and antago-nistic activities. Also, because soil is not an inert ma-trix, its properties and the prevailing environmentalconditions will influence the behavior of both the mi-croflora and DDTr. It is therefore important to exam-ine DDTr degradation in soils, both in the laboratoryunder controlled conditions, and in the field whereplant exudates may contribute to the activity of the soilmicroflora. The studies discussed below support pureculture observations and, furthermore, allow examina-tion of the environmental factors that influence DDTrin situ.

Early studies (Burge, 1971; Nash et al., 1973;Castro and Yoshida, 1971, 1974) described significantlosses of DDTr from soils incubated in the laboratory.Such studies generally focussed on measuring loss ofthe parent molecule (i.e., “degradation” of DDT) anddetection and identification of its metabolites, withoutdetermining whether DDT had been mineralized toCO2 and Cl–. For example, Guenzi and Beard (1967)reported substantial DDT degradation but recoveredDDD, p,p′-dichlorodiphenylmethane (DBM), 4,4′-dichlorobenzophenone (DBP), 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethanol (kelthane), and DDE from soilsincubated anaerobically with 14C-DDT. More recently,radiolabeled DDT has been used to monitor produc-tion of 14CO2 from soils, enabling mass balance deter-minations and revealing that mineralization of DDT insoils is typically very low.

DDT degradation in soil appears to proceed byone of two main routes, depending on the prevailingenvironmental conditions, and in agreement with pureculture studies in vitro. In anoxic soils, transformationof DDT to DDD by reductive dechlorination is consid-ered to be the dominant reaction (Boul, 1996); minorlevels of DDA, DDM, DDOH, DBP and DDE alsohave been detected (Guenzi and Beard 1967, 1968;Mitra and Raghu, 1988; Xu et al., 1994; Boul, 1996).In contrast, under aerobic soil conditions DDT is dehy-drochlorinated to yield predominantly DDE.

Studies have been conducted comparing soils in-cubated under both aerobic and anaerobic conditions.Aerobic soils were incubated at moisture levels ad-justed to allow aeration (with or without flushing withair) while reducing conditions were generated eitherby flooding the soils with water (Nair et al., 1992;Boul, 1996) or flushing with nitrogen (Scheunert et al.,1987). Mineralization of radiolabeled DDT was lowunder aerobic conditions, with typically ≤ 3% of theadded label recovered as 14CO2 after 42 days incuba-tion (Scheunert et al., 1987; Nair et al., 1992; Boul,1996) or longer (Guenzi and Beard, 1968; Zayed et al.,1994). Anoxic conditions further reduced DDT miner-

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alization (typically <1% of added label), although con-siderable amounts of DDD and low levels of DBPwere detected (Boul, 1996).

Similar low levels of DDE mineralization in soilhave been reported (Zayed et al., 1994; Boul, 1996). Infact, reports of transformation of DDE in soil are few,although Agarwal et al. (1994b) identified DDMU asa minor biotransformation product from 14C-DDE un-der field conditions in an Indian soil. Thus, althoughan isolated soil organism can degrade DDE in vitro(Aislabie et al., 1999), the activity or distribution ofcompetent strains must be limited in situ because ofthe observed persistence of DDE in soils.

Despite the demonstrated existence of DDTr-de-grading microflora in soil, DDTr persist and their min-eralization is limited. Explanation of this phenomenoninvokes the pure culture observations discussed above:anoxic conditions are conducive to dechlorination ofDDT (Figure 2), whereas ring cleavage leading tomineralization requires aerobic conditions (Figure 3).Co-metabolic carbon sources are required to sustainboth these activities. Other soil factors discussed be-low, such as temperature and organic matter content,also affect DDTr degradation. That is, a complex set ofenvironmental conditions is required for complete deg-radation of DDT and its residues, contributing to theirpersistence.

Tropical SoilsDissipation and degradation rates of 14C-p,p′-DDT and14C-p,p′-DDE were studied in tropical soils from Egypt(Zayed et al., 1994), Pakistan (Hussain et al., 1994a),India (Agarwal et al., 1994a; 1994b), China (Xu et al.,1994), and the Philippines (Varca and Magallona, 1994)in trials coordinated by the Food and Agriculture Or-ganization/ International Atomic Energy Agency (FAO/IAEA, 1994). DDT half-lives of 6 weeks to 1.5 yearswere reported, depending on the environmental condi-tions imposed. Flooded soils consistently demonstratedgreater rates of DDT “degradation” than drier uplandsoils, but a large proportion of the DDT initially presentwas subsequently recovered as DDD. Castro andYoshida (1971) also observed greater degradation ofDDT (and concomitant accumulation of DDD) in sub-merged Philippino soils with high organic content,compared with clay soils and soils incubated at 80%field capacity.

Volatilization and microbial transformation areconsidered the main mechanisms of DDTr dissipationin tropical soils (Samuel and Pillai, 1989), while min-eralization and adsorption generally play a lesser role(FAO/IAEA, 1994). Solar irradiation was also shownto be a major factor in tropical soils (Zayed et al.,

1994), presumably through enhanced volatilization.The general consensus of the FAO/IAEA studies wasthat rates of DDT and DDE dissipation and degrada-tion in tropical soils may preclude accumulation ofDDTr, and these rates are significantly greater thanthose measured in temperate soils.

Temperate SoilsIn contrast to tropical conditions, in temperate soilsDDTr persist for long periods of time, primarily asDDE in dry aerobic soils (Boul et al., 1994) and asDDD in anaerobic soils. There is a marked differencein the rate of degradation in temperate soils comparedwith tropical soils (Owen et al., 1977; Boul et al.,1994). Dimond and Owen (1996) have suggested ahalf-life of 20 to 30 years for DDTr in soils in Maine(USA). Generally, the mean half-life of DDT is pre-dicted to be 10.5 years but may be as high as 35 yearsin temperate agricultural soils. In addition to lower soiltemperature and solar irradiation, agricultural prac-tices also contribute to the persistence of DDTr intemperate regions (Boul et al., 1994). Spencer et al.(1996) observed much higher concentrations of DDTpersisting in deep plowed soils; plowing appeared tolimit volatilization and photodegradation of the com-pound.

In a study of long-term monitored DDT applicationto temperate pasture soils, Boul et al. (1994) found thatintermittent addition of superphosphate fertilizer com-bined with regular irrigation regimes served to enhanceDDTr removal compared with unamended controls. Thelevels of DDTr detected were a function of the totalamount of DDT applied (beginning about 35 years pre-viously) and the mean soil moisture over the summerseason. In non-irrigated soils, DDE was the principalresidue detected, while in irrigated soils, DDD levelswere enhanced at the expense of DDE, without exces-sive accumulation of DDD. This suggested that produc-tion of DDD was followed by further transformation tonondetected products in situ, whereas DDE formationled to persistence of that product. Boul et al. (1994)postulated that several factors contributed to DDTr trans-formation in situ, including creation of anaerobicmicrosites for microbial and abiotic reductive dechlori-nation of DDT, altered binding to soil particles, andlosses of lesser importance such as increased plant oranimal export, leaching and volatilization.

Owen et al. (1977) attributed the slow decay ofDDTr in temperate soils to low pH. Alkaline soil pHpromoted faster dissipation rates of DDTr along withhigher tropical temperatures, compared with acid soilconditions and more temperate climates (Xu et al.,1994).

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236 Foght et al.

Subsurface SoilsSubsurface DDTr contamination has not been ad-dressed to any extent in the literature, probably be-cause leaching via diffusion or by bulk transfer withwater (Khan, 1980) plays a relatively small role in theabiotic dissipation of DDTr (Xu et al., 1994) due tothe hydrophobic nature of the compounds. A 5-yearcomprehensive study of the behavior of DDT in tropi-cal soils found no evidence of DDT leaching down-wards through the upper soil strata (FAO/IAEA,1994). However, subsurface contamination has beenobserved in some instances, especially in the pres-ence of hydrophobic co-contaminants, such as xy-lene, that promote mobility by co-solvation (Morrisonand Newell, 1999). Lindqvist and Enfield (1992) alsodetermined that biosorption of DDT to soil bacteriasignificantly enhanced mobility of the DDT in satu-rated soil columns by co-transport. Results arisingfrom studies conducted with surface soil contamina-tion cannot be extrapolated to subsurface conditions,and therefore this area requires more study (Morganand Watkinson, 1989).

Factors InfluencingBiodegradation of DDTr in SoilsReduction of DDTr concentrations in soils occursthrough physical means such as adsorption, volatiliza-tion and erosion, and through chemical means such asabiotic reduction reactions involving biological reduc-tants (e.g., porphyrins and hematins; Zoro et al., 1974;Baxter, 1990), nonbiological chemical reductants (e.g.,zero-valent iron; Sayles et al., 1997), and photochemi-cal reactions (Miller and Narang, 1970), all of whichare beyond the scope of this review. These physico-chemical processes can contribute significantly to over-all DDTr losses, and in practice it may be difficult toapportion the contributions of abiotic and biotic degra-dation in situ. Biological reduction of DDTr in soils isprimarily influenced by factors that affect bioavailability(e.g., adsorption) and microbial activity (e.g., soil tem-perature, pH, moisture, and organic matter content), asdiscussed below.

BioavailabilityThe term “bioavailability” describes the ease with whicha chemical (nutrient, substrate or toxicant) can be ac-cessed by biological processes. In soils, bioavailabilityis reduced by physical limitations such as adsorptionto mineral surfaces or soil organic matter (to variousdegrees of reversibility), and by limited aqueous solu-bility of the chemical, reducing its diffusion in porewater.

Adsorption to and within soil particles plays amajor role in reducing the bioavailability of DDTr.With time, residues may become bound and resistantto chemical extraction, may be reversibly adsorbed tosoil matter, or may diffuse into pores less than 100 nmin diameter where bacteria, fungi, or root hairs cannotpenetrate (Alexander, 2000). Soil temperature(Cornelissen et al., 1997) affects the kinetics of bind-ing of DDTr to soil particles, as does soil moisture. Forexample, Nair et al. (1992), Xu et al. (1994), and Boul(1996) found that flooding of soils can significantlyincrease the amount of soil-bound DDT residues, likelyby increasing diffusion rates of DDTr into microporesor other sites that make the compounds subsequentlyinaccessible to microbes. Studies (e.g., Peterson et al.,1971; Castro and Yoshida, 1974; Khan, 1980; FAO/IAEA, 1994) have found that soils high in organicmatter, such as peats (Vollner and Klotz, 1994) havesignificantly lower concentrations of bioavailable DDTrthan sandy soils, due to adsorption to soil organicmatter. The results of Khan (1980), Khan and Dupont(1987), and Vollner and Klotz (1994) support thisview. Hydrophobic adsorption onto the surface of hu-mic materials and trapping within a matrix of humicmacromolecules have been proposed as importantmechanisms of DDT binding (Senesi and Miano, 1995).Soil microorganisms themselves can play a role inbinding residues to soil organic matter (Bollag andLiu, 1990; Boul, 1996), mediated through enzymaticor chemical reactions involving microbial products.Mineral surfaces are also involved in adsorption, andin a study with clayey soils as much as 25% of DDTand 65% of DDE contamination may not have beenbioavailable (White and Herndon, 1995).

Bioavailability diminishes with time (Singh andAgarwal, 1992), and although indigenous soil micro-organisms may achieve significant initial rates of deg-radation of DDT freshly added to soil, the rate de-creases substantially with time despite the continuedpresence of extractable concentrations of DDT in thesoil, so that aged DDTr persist (Alexander, 2000). Infact, there is considerable debate about whether slowadsorption processes result in irreversibly adsorbedresidues (Kan et al., 1998). In this way, soil appears toremove or diminish the toxicity of a compound beforethe actual disappearance of the compound, throughreduced bioavailability, sequestration, and adsorption(Alexander, 2000). This is confirmed by Peterson et al.(1971) who proposed that DDTr toxicity decreasedwith the length of time the compounds remained in thesoil. Similarly, Singh and Agarwal (1992) andAlexander (2000) found that organic compounds incu-bated in sterile soils became progressively lessbioavailable to microorganisms with time, and these

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compounds became increasingly difficult to removethrough conventional solvent extraction methods.

Biosorption of DDTr, for example to bacterialcells (Katayama et al., 1993) and fungal mycelia (Juhaszand Naidu, 1999), also occurs. This can be viewed aseither an immobilizing or a mobilizing process:Lindqvist and Enfield (1992) determined thatbiosorption of DDT to soil bacteria significantly en-hanced mobility of the DDT in saturated soil columnsby co-transport, whereas Boul (1996) proposed thatmicrobes contributed to immobilization of DDTr inflooded agricultural soils.

If DDTr were not bioavailable at all (i.e., becameirreversibly adsorbed to soil particles), they would beof less practical concern and could be left in place.However, the chemical nature of bound residues isoften not known (Boul, 1995), and the residues may bereleased slowly over time or bioaccumulated in ani-mals that ingest contaminated soil particles, passingDDTr into their products (e.g., meat and milk).

Soil PropertiesSoil microbial biomass is intimately associated withsoil organic matter, a complex and heterogeneous mix-ture of humic substances, polysaccharides, lignin, car-bohydrates, lipids, proteins, and organic acids (Scow,1993). The presence and quality of soil organic matterpositively influence biodegradation by sustaining anactive microflora and providing a carbon source forco-metabolism of DDTr. Similarly, the presence ofnatural inducers (e.g., lignins, terpenes, discussed be-low) may also stimulate DDTr biodegradation. Con-versely, soil organic matter has an indirect negativeeffect on bioavailability as a matrix for adsorption ofDDTr. Thus, for example, Szeto and Price (1991) re-ported that organochlorine residues persisted longer intemperate soils with high organic matter content (e.g.,27 to 56%) than in silty loams (3.7% to 6.5% organicmatter) or sandy loams with low organic matter (1.0 to1.8%). In addition, free radicals in solution derivedfrom soil humic and fulvic acids may be inhibitory toDDT-degrading microbial populations (Fujimura etal., 1994).

Soil pH levels may also affect degradation anddissipation of DDTr. Andrea et al. (1994) reported thatacidic soil (pH 4.5 to 4.8) inhibited volatilization andmineralization of DDT. They also found that low pHgreatly reduced the ability of microorganisms to re-lease bound DDTr from soils. Nash et al. (1973) stud-ied the effect of pH on DDT stability and found thatsoil pH values above pH 7 resulted in significant con-version of 14C-DDT to DDE in both moist and drysoils. This conversion was attributed to microbes in the

moist (but not saturated) soils, and to chemical reac-tions in the dry soils at high pH.

Besides affecting general microbial activity andcontaminant adsorption (Nair et al., 1992), soil mois-ture affects DDTr metabolism by influencing aeration.As discussed, the major residue under aerobic condi-tions is DDE, whereas under anaerobic conditions suchas those produced by flooding (i.e. water-saturatedsoil), DDD is the major product that accumulates (Boul,1996; Xu et al., 1994).

Soil temperature directly affects microbial activ-ity (and hence DDTr biodegradation) and indirectlyaffects soil DDTr concentrations through volatilitylosses. Agarwal et al. (1994b) measured appreciablevolatility of radiolabel added as 14C-DDE to soils ex-posed to solar irradiation and having temperatures of43°C to 60°C. Nair et al. (1992) similarly observedthat up to 30% of radiolabel added as 14C-DDT wasrecovered in the volatile fraction from soils exposed tosolar irradiation for 42 days.

DDTr Concentrations and Presence ofCo-contaminantsDDTr are often present in agricultural soils at rela-tively low levels (e.g., 1 to 5 mg/kg in New Zealandtopsoils; Holland, 1996). Low concentrations, com-bined with poor bioavailability, can contribute to thepersistence of those contaminants that serve as growthsubstrates and require induction of degradative en-zymes (Boethling and Alexander, 1979), but low con-centrations should be immaterial for DDTr as degrada-tion is co-metabolic. Indeed, Katayama et al. (1993)observed degradation by soil bacteria of 14C-DDT inliquid culture at concentrations as low as 160 pg/ml(although parallel studies in soil at these low concen-trations were not conducted).

Conversely, at the higher concentrations encoun-tered in soils at cattle tick dip sites (e.g., 100,000 mg/kg soil; DIPMAC, 1992), pesticide mixing plants (e.g.,1000 mg/kg soil; Morrison and Newell, 1999) andmanufacturing sites (e.g., 10 to 100 mg/kg soil;Richardson, 1995; USEPA, 2000b), DDTr can be toxicto soil microflora (Donato et al., 1997; Megharaj et al.,2000). Most of the experiments in the FAO/ IAEA(1994) study used high acute DDT loading rates of 5g/kg soil (5000 ppm), orders of magnitude greater thanthe typical DDTr levels in temperate agricultural soils.Therefore, the conclusions from laboratory studies usinghigh DDT loading rates may not accurately reflect thedegradation rate potential of authentic contaminatedsoil. Similarly, soils freshly amended with DDT maynot be good predictors of the biodegradation of agedDDTr that have become less bioavailable.

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Soils may be impacted by more than one contami-nant. For example, soil at a pesticide mixing facilitywas contaminated to depth with DDT, toxaphene andxylene (Morrison and Newell, 1999). Such co-con-taminants can complicate biodegradation (1) by pro-viding more readily utilized substrates for the microf-lora, thus diverting enzymatic activity from thecontaminant of concern; (2) by having specific or non-specific toxic effects on the soil microflora; or (3) byaffecting the solubility or adsorption of the contami-nant of concern, decreasing its bioavailability or in-creasing its mobility through co-solvation transport.Conversely, co-contaminants theoretically could en-hance biodegradation by serving as a co-metaboliccarbon and energy source or an inducer of DDTr deg-radation.

Enhancing DDTr Biodegradationin SoilsRemediation of contaminated soils must consider meth-ods to reduce the concentrations of DDT as well as itsresidues. For example, although parent DDT concen-trations may be reduced through biotic and abioticdechlorination, accumulation of the product DDD isproblematic, as DDD is also a pesticide and persistentpollutant with chemical properties similar to its parentcompound. As You et al. (1996) have observed, accu-mulation of DDD under anaerobic conditions and itsstability under aerobic conditions has hampered devel-opment of DDTr bioremediation processes, and well-defined methodologies to minimize DDD accumula-tion during DDT biotransformation are lacking.Similarly, accumulation of DDE under aerobic condi-tions does not lessen the contamination, as DDE iseven more persistent than DDT. Therefore, consider-ation of methods to enhance biodegradation must in-clude approaches that lead to overall reduction of DDTrby stimulating extensive dechlorination and mineral-ization beyond the initial steps that “remove” the par-ent DDT from the soil.

Abiotic processes such as incineration and solventwashing are expensive and intrusive, whereasbioremediation, although slower, has the potential to bemore cost-effective and less damaging. Therefore, know-ing the factors that limit natural DDTr biodegradation insoils allows design of appropriate treatments to enhancebioremediation in situ or ex situ. Table 2 presents asummary of processes with the potential to overcomelimitations inherent to biodegradation of DDTr in soils.Notably, combinations of these treatments can be de-signed to accommodate the specific soil conditions en-countered (Morgan and Watkinson, 1989).

In Situ ApproachesCo-Metabolic Carbon Sources. Because soil microor-ganisms co-metabolize DDTr, successful bioremediationdepends largely on the presence of an alternative carbonsource in situ. In early studies, simple and complexamendments (see Table 2) added to anaerobic soil werefound to increase the rate of DDT degradation. Golovlevaand Skryabin (1981) found that the addition of glycerolor n-alkanes such as octane was effective, and the great-est degradation occurred with the addition of hexadecaneor glycerol. Complex co-substrates that increased ratesof degradation of DDT to DDD under anaerobic condi-tions included alfalfa (Guenzi and Beard, 1968; Burge,1971), “green manure” (Mitra and Raghu, 1988), andrice straw and cellulose (Castro and Yoshida, 1974).Notably, as a result of oxidation of the additional carbonsubstrates by microflora in saturated soils, further re-duction in oxygen levels occurred, thus promoting areducing environment and stimulating transformationof DDT to DDD.

A recently publicized commercial process (Walker,1999) used organic waste amendments such as chickenmanure, old newspapers, straw, and wood chips tostimulate the indigenous soil microflora to degradeDDT and DDE. The amended soil was incubated inbiopiles with intermittent mixing to provide alternat-ing anaerobic and aerobic conditions, reportedly re-sulting in reduction of DDT levels by more than 95%(Gray et al., 1999). However, no degradative mecha-nisms were defined for the process, nor was the poten-tial for volatilization to occur assessed.

Microbial Inoculants. Because biodegradation of DDTrrequires a substantial and active population of soilmicrobes, measures that stimulate the natural flora(such as carbon amendment, above) should enhanceDDTr bioremediation. Bioaugmentation of the naturalflora with specialized DDT-degrading microbes ispossible, but since the initial transformation of DDT toDDD and DDE in soils occurs relatively readily bybiotic and abiotic processes, seeding with DDD- andDDE-producing microbes should not be necessary.Instead, it may be useful to introduce mixed popula-tions of anaerobic bacteria (e.g., enriched from sewagesludge; You et al., 1996) that degrade DDTr by reduc-tive dechlorination, or ligninolytic fungi (Bumpus andAust, 1987; Bumpus et al., 1993) or aerobic bacteriawith ring-cleavage activities (Nadeau et al., 1994;Aislabie et al., 1999; Hay and Focht, 2000). Of course,consideration must be given to the in situ survival andcompetitiveness of introduced strains (Sayler and Ripp,2000), particularly because co-metabolic DDTr degra-dation affords no energetic advantage. For this reason,

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Table 2. Treatments with potential for alleviating limitations to bioremediation of DDTr in soils

Limitation and treatment Ref.

Low total microbial numbers in soil or low carbon source as co-metabolic substrateSimple Amendments:

Glucose, yeast extract, peptone Chacko et al., 1966Glucose plus diphenylmethane Pfaender and Alexander, 1973Octane, hexadecane or glycerol Golovleva and Skryabin, 1981Cellulose Castro and Yoshida, 1974

Complex Amendments:Alfalfa Guenzi and Beard, 1968; Burge, 1971Rice straw Castro and Yoshida, 1974; Mitra and Raghu, 1986“Green manure” Mitra and Raghu, 1988

Low numbers of DDT-degrading microorganismsInoculation with competent microbes:

Phanaerochaete chrysosporium Fernando et al., 1989; Lestan et al., 1996Aromatic ring cleavage organisms Nadeau et al., 1994; Aislabie et al., 1999; Hay and Focht, 1998, 2000Aerobacter (Enterobacter) aerogenes Kearney et al., 1969

Low DDTr-degradative activity of soil microfloraAddition of inducers or analogues

Diphenylmethane Pfaender and Alexander, 1973Biphenyl Hay and Focht, 1998; Aislabie et al., 1999Terpenes Hernandez et al., 1997

Poor DDTr bioavailability in soilSoil treatment

Surfactant addition Kile and Chiou, 1989; You et al., 1996Cyclodextrins Fava et al., 1998pH adjustment Nash et al., 1973; Xu et al., 1994Addition of fresh soil Hussain et al., 1994a; Varca and Magallona, 1994

Sub-optimal conditions in soilIn situ treatment

Aeration (by soil moisture adjustment) Spencer et al., 1996; Boul, 1996pH adjustment Andrea et al., 1994, Owen et al., 1977Phytoremediation Garrison et al., 2000; Fletcher and Hegde, 1995

Ex situ treatmentWindrows Gray et al., 1999Bioreactors (with or without alternating Beunink and Rehm, 1988; White and Herndon, 1995; Fiedeiker etanaerobic and aerobic conditions) al., 1995; Sharma et al., 1987; Corona-Cruz et al., 1999Redox potential adjustment You et al., 1996UV Irradiation Katayama and Matsumura, 1991

multiple inoculation is likely required (Morgan andWatkinson, 1989).

Selection of potential inoculants from the envi-ronment has been achieved using DDTr analogues.For example, DDT-degrading strains of Pseudomonassp. (isolated as Hydrogenomonas sp.; Subba-Rao andAlexander, 1977) and Enterobacter cloacae were iso-lated from sewage using PCPA (Pfaender andAlexander, 1972) and diphenylmethane (Beunink andRehm, 1988) as the co-metabolic substrates, respec-tively. In an interesting variation on selective tech-niques, a DDT-degrading Cladosporium sp. was iso-

lated from DDT-contaminated forest soil (Juhasz andNaidu, 1999) by screening for the ability to decolorizethe polymeric dye Poly R-478, which is a useful indi-cator of ligninolytic activity by microorganisms.

Genetically modified bacteria with DDTr-degrad-ing abilities such as P. acidovorans M3GY (Hay andFocht, 1998) may prove useful in bioremediation ofDDTr (Golovleva et al., 1988), as has been proposedfor other persistent pollutants (Chen and Mulchandani,1998).

Methods have been developed for encapsulatingbacterial biocatalysts for pesticide detoxification (re-

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240 Foght et al.

viewed by Chen and Mulchandani, 1998) and forligninolytic fungi such as P. chrysosporium to inocu-late soils contaminated with organochlorines (Lestanet al., 1996). However, another consideration raised byLinqvist and Enfield (1992) is that adsorption of DDTrto introduced microbes could increase the mobility ofDDTr into groundwater.

Regardless of the proposed inoculant, the limita-tions to biodegradation discussed above that are im-posed by the in situ conditions must still be consideredand ameliorated if possible.

Inducers and Analogous Substrates for Enrichmentof Microflora. Although a variety of carbon substratesappear to enhance DDTr degradation, an energy sourcethat most closely resembles that chemical structure(i.e., DDTr analogues) may be the most successfuladditive. For example, Pfaender and Alexander (1973)observed an increase in the metabolism of DDT insewage with the addition of diphenylmethane, andBeunink and Rehm (1988) used diphenylmethane toenrich sewage sludge cultures for DDM-degradingbacteria. More recently, biphenyl (Aislabie et al., 1999;Hay and Focht, 1998) and chlorobiphenyl (Nadeau etal., 1994) have been found in laboratory studies toinduce production of enzymes that degrade DDTr.These successes may in part be a result of enrichmentfor certain bacteria having greater enzymatic capabili-ties to degrade DDTr than nonenriched populations,and in part due to induction of the DDTr-degradingenzymes in those organisms. Although these reportswere laboratory studies, their findings may be appli-cable to the field. However, as cautioned by Aislabieet al. (1997), care must be taken that the substrateanalogues do not contribute to the toxicity of the soilor pose a threat to the environment. Natural, non-toxicinducers such as terpenes in orange peels, pine needles,etc., may serve as natural substrates for biphenyl-de-grading bacteria (Hernandez et al., 1997) and may besuitable analogues for stimulation of DDTr-degradingmicrobes. Combination of microbial seeding with ana-logue enrichment, such as that demonstrated with PCB-containing soils by Brunner et al. (1985), may bebeneficial.

Surfactants to Enhance Bioavailability. As previouslydiscussed, limited bioavailability can impede biodeg-radation of aged DDTr in soils. This limitation can berelaxed to some degree by the addition of surfaceactive agents (Keller and Rickabaugh, 1992) to en-hance desorption of residues from soil particles. Parfittet al. (1995) demonstrated that DDT residues adsorbedto soil contaminated 30 to 40 years previously weresolubilized and desorbed by treatment with Triton X-

100™ and polypropylene glycolethoxylate. However,subsequent biodegradability of residues in the leachatewas not determined. Surfactant soil washings can beused as a substrate for microbial growth. Such anapproach has been tested with a surfactant/polychlori-nated biphenyl (PCB) mixture inoculated with a sur-factant-degrading bacterium that had been geneticallyengineered to constitutively express a bph operon fordegradation of PCBs (LaJoie et al., 1997). In a soilslurry reactor, You et al. (1996) combined Triton X-114™ or Brij 35™ treatment with addition of chemi-cal reducing agents to achieve greater DDTr dechlori-nation than either treatment alone. However, the highconcentration of DDT used in that series of experi-ments (initially 2500 mg/kg soil) precludes extrapola-tion of the data to authentic soils with low-level con-tamination. A related approach is to stimulate the natu-ral or introduced microflora to produce biosurfactantsin situ.

Another method that may be employed to releaseadsorbed residues is the use of cyclodextrins, cyclicsugar polymers that enhance the availability of water-insoluble compounds in the water phase. This approachhas been used with PCB-contaminated soils (Fava etal., 1998) in soil-slurry and fixed-phase reactors.

Other Amendments. The addition of reducing agentssuch as zero-valent iron to accelerate reductive dechlo-rination of DDT to DDD has been suggested (Sayles etal., 1997) as an adjunct to nonbiological treatment ofDDTr-contaminated soils. You et al. (1996) found thatthe addition of reducing agents (cysteine or sodiumsulfide) to anaerobic microcosm soil slurries acceler-ated DDT degradation. Whether this approach wouldbe compatible with enhanced bioremediation practicesor be cost-effective has yet to be determined in situ orex situ.

Hussain et al. (1994b), Varca and Magallona(1994), and Xu et al. (1994) added fresh soil to DDTr-contaminated soil, resulting in the release of DDTr.They concluded that this release was the result ofmicrobial activities.

Nash et al. (1973) found that high soil pH (achievedby adding various forms of lime) enhanced conversionof DDT to DDE at the expense of DDD in both moistand dry soils. They postulated that such conversionwas predominantly microbial in moist soils and abioticin dry soils. They did not recommend liming as apractical means of enhancing DDT conversion, how-ever, as the total production of DDTr (DDD + DDE)was comparable regardless of soil pH.

Boul et al. (1994) observed that the common ag-ricultural practices of irrigation and superphosphatefertilizer amendment enhanced DDTr losses in a tem-

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perate soil, and significantly diverted DDTr transfor-mation from production of persistent DDE to produc-tion of DDD, which apparently was further transformed.In this study, tillage was limited to that required forcrop management, and did not have a measurable ef-fect on DDTr losses, although intensive tilling mayhave benefits in tropical soils due to increased pho-tolysis and volatilization (Samuel and Pillai, 1989).

Although technically not an amendment, photo-chemical treatment has been used to enhance DDTdegradation by ligninolytic fungi (Katayama andMatsumura, 1991). Combination of short wavelengthUV irradiation (254 nm) and incubation with the UV-resistant P. chrysosporium strain BU-1 enhanced DDTdegradation significantly, and no residues were re-ported to accumulate.

Phytoremediation Potential. The role of plants androot exudates in DDTr biodegradation has yet to bestudied extensively, but Garrison et al. (2000) havereported that two plants, and extracts from those plants,transformed a proportion of DDT to DDD in labora-tory studies. Furthermore, the plant components boundup to 22% of the 14C-DDTr generated. The dechlorina-tion activity did not require either a surface microbialflora or living plant tissue, and was assumed to becatalyzed nonenzymatically by a biological reductant.Phenolic root exudates (Fletcher and Hegde, 1995)and terpenes (Hernandez et al., 1997) may also fosterthe activity of rhizosphere bacteria that degrade DDTrby inducing enzymes or providing co-metabolic growthsubstrates, although the available concentrations ofsuch exudates may be low outside localized pockets.Whether phytoremediation is appropriate for DDTrcontamination remains to be demonstrated.

Ex Situ Approaches and BioreactorsWhile the treatments described above are suitable forin situ bioremediation, ex situ approaches such as theimplementation of bioreactors and biopiles permit thecontrol and monitoring of variables such as pH, tem-perature, water content, aeration, nutrient amendment,etc., that cannot be readily achieved in situ. Feidiekeret al. (1995) have proposed the use of bioreactors forremediation of surface soils contaminated with chlori-nated aromatics where the prognosis for in situ treat-ment was poor. White and Herndon (1995) observedrapid and efficient DDT degradation in anaerobic soilslurry reactors at 25°C, although residual concentra-tions of DDE were removed at a slow rate, if at all, forthe duration of their tests. Sharma et al. (1987) used anactivated sludge reactor inoculated with cow dung andsoil from a DDT manufacturing site to degrade DDTat concentrations of up to 500 mg/L of sludge.

The benefits of using an alternating anaerobic-aerobic treatment in a bioreactor have recently beendemonstrated by Corona-Cruz et al. (1999). Using amixed microbial inoculum derived from industrial sew-age effluent for the initial anaerobic phase, and subse-quent aerobic incubation with either another mixedmicrobial population or with a pure culture of P.chrysosporium, they measured significant reductionsin DDT and DDE concentrations in an authentic DDTr-contaminated agricultural soil. In contrast, Strömpland Thiele (1997) reported recalcitrance of DDE andits dehalogenated derivative 1,1-diphenylethylene(DPE) under alternating aerobic and anaerobic condi-tions in batch reactor systems.

The bioreactor constructed by Beunink and Rehm(1988) achieved synchronous reductive dechlorinationof DDT to DDD and oxidation of the metabolite DDMusing a mixed culture of Alcaligenes sp. andEnterobacter cloacae co-immobilized in calcium algi-nate. Diphenylmethane and lactose, respectively, weresupplied as co-metabolic growth substrates for the twoorganisms. Under low oxygen flow rates the periph-eral region of the alginate beads remained aerobic,permitting oxidative degradation of DDM by the Al-caligenes sp. The redox potential of the center of thebeads decreased due to the metabolic activity, encour-aging reductive dechlorination of DDT to DDD by E.cloacae. Although both processes occurred more slowlyin mixed than in pure culture, nearly 40% of the initialDDT was degraded, and almost all the DDM wasremoved in a 12-day period at 30oC. Although thisstudy points out the potential benefits of a designedbioreactor, it should be noted that mineralization ofDDT was not achieved in this system; only the firststep (DDT to DDD) and the last steps (DDM to uni-dentified products plus chloride ion) were documented.

A recent encouraging study investigating the po-tential of on-site ex situ bioremediation of DDT-con-taminated soils has been reported by Gray et al. (1999).They successfully remediated a contaminated site con-taining several pesticide pollutants including toxaphene,DDT, DDD, DDE, dieldrin, and chlordane. This full-scale ex situ study treated 1000 m3 of contaminatedsoil from a pesticide formulation site using a compostwindrow amended with organic material, mixed usingmechanical methods to help alternate aerobic andanaerobic conditions. However, little detail was pro-vided in the report about the type of organic amend-ments, the temperatures, redox, oxygen or moisturelevels of the soils. Initial concentrations of DDT (88.4ppm) were reduced by 98% in 14 weeks, while DDDand DDE were degraded from initial concentrations of242 and 11.3 ppm by 90% and 40%, respectively.After this demonstration of the potential for clean-up,

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242 Foght et al.

the USEPA recommended bioremediation as the pre-ferred technology for treatment of the soil at that site(Gray et al., 1999).

The use of flow-through biofilters, based on mi-crobial adsorption of chlorinated pesticide residuesfrom water and their subsequent degradation, has beentested (Torres and Bandala, 1999). Given the low watersolubility of DDT and its metabolites (except DDA),however, the practical application of this technologyto treatment of soil or leachates remains to be proven.

ConclusionsNumerous soil microorganisms exist with the ability toco-metabolically transform DDTr into products hav-ing varying susceptibility to further degradation. Thepredominant persistent residues detected in soils areDDD and DDE, formed under anaerobic and aerobicconditions, respectively. Despite the apparent recalci-trance of DDTr in the environment, certain soil bacte-ria and fungi have the potential to mineralize or sig-nificantly degrade DDTr, given suitable conditions.

Addition of organic amendments (e.g., glucose,plant matter, or DDT analogues) permits the micro-flora to proliferate, and under properly designed con-ditions oxidation of the amendment generates a reduc-ing environment. Under such conditions, DDT andDDD can be transformed anaerobically by reductivedechlorination, thus limiting the formation of recalci-trant DDE. Alternation of anaerobic and aerobic con-ditions has the potential to stimulate mineralization ofDDTr by mixed populations. Using these principles,Gray et al. (1999) have accomplished significant re-duction in soil DDTr concentrations. Alternatively, insoils where DDE is persistent, inoculation or enrich-ment with competent microflora and concomitant pro-vision of inducers or analogous substrates may proveeffective. Successful bioremediation processes willexploit these findings to enhance DDTr degradation.

Persistent residues may be treated in situ by ma-nipulation of soil conditions (e.g., adjustment of soilmoisture, addition of co-metabolic substrates or en-zyme inducers), or ex situ by constructing bioreactorsor biopiles in which incubation conditions can moreeasily be adjusted to enhance degradation. After suc-cessful treatment ex situ, the remediated soil can bereturned to its place of origin, thus reducing the overallenvironmental impact on that site. Combinations ofnonbiological and biological approaches can be envi-sioned to make treatment regimes site-specific.

Bioremediation, although influenced by envi-ronmental conditions in a complex manner, can be aneffective and efficient remedial method that not only

reduces or eliminates contamination, but more impor-tantly has little adverse impact on the physical andchemical properties of the soil. Further research andespecially controlled in situ studies are needed to pro-mote DDTr bioremediation as a viable treatment op-tion.

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Bioremediation of DDT-Contaminated Soils: A Review

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