Factors in¯uencing the degradation of soil-applied endosulfan
isomers
Niranjan Awasthi, Rajiv Ahuja, Ashwani Kumar*
Environmental Biotechnology Section, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226 001, India
Accepted 21 April 2000
Abstract
The addition of isolated bacterial cells to contaminated soils causes an enhanced degradation of endosulfan isomers. Various factors, including the additional presence of carbon sources, pH, moisture content, concentration of endosulfan, and size of inoculum, in¯uenced the degradation of endosulfan isomers. The degradation was faster in wet soils, as compared with the ¯ooded soils, and was inhibited by the presence of additional carbon sources such as sodium acetate and sodium succinate. The degradation of endosulfan was not detectable at acidic pH and increased gradually to reach an optimal activity at pH 8.5. It chemically converts into endosulfan diol at higher pH values. The rate of biodegradation progressed with the increase in endosulfan concentration up to 5.0 mg gÿ1soil, followed by an inhibitory eect at higher concentrations, reaching a total loss of biodegradative activity at 10 mg gÿ1soil. The addition of 2106bacterial cells gÿ1
soil was optimal for endosulfan degradation and any further increase in inoculum size was of no additional advantage. Initial optimization of these factors is, therefore, essential for successful bioremediation.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Endosulfan; Chlorinated pesticide; Biodegradation; Contaminated sites; Bioremediation
1. Introduction
Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo(e )dioxathiepin-3-oxide, CAS No. 115-29-7) is a chlorinated pesticide of the cyclodiene group. Its technical preparation consists of a- and b-isomers (70:30). It is used extensively, throughout the world, as a broad spectrum insecticide, on cotton crops, ®eld crops such as paddy, sorghum, oil seeds and pulses, as well as vegetables and fruit crops (Goebel et al., 1982). Endosulfan contamination has been detected in soils, water, air and food products because of its abundant usage and potential for en-vironmental transport. It is extremely toxic to ®shes and aquatic invertebrates (Verschueren, 1983; Sun-deram et al., 1992) and is classi®ed as a `priority
pollu-tant' by international environmental agencies (Keith and Telliard, 1979). The persistence of endosulfan in agricultural soils has been studied in many labora-tories. Its life in soils has been estimated to be from 100 to 120 days (Rao and Murty, 1980; Kathpal et al., 1997) to several months (Stewart and Cairns, 1974) and the relative rates of dissipation for a- andb -endo-sulfan have also been shown to be dierent. This dissi-pation depends on a multitude of factors such as its volatilization, alkaline hydrolysis and photodecomposi-tion, besides the presence of fertilizer, crop pattern, at-mospheric temperature, rain and biotic conversions, among others (Goebel et al., 1982).
Bioremediation, which involves degradation of tar-get chemicals by indigenous or added microbial cells, is used to clean up sites contaminated by pollutants such as pentachlorophenol (Miethling and Karlson, 1996; Barbeau et al., 1997), diesel oil (Margesin and Schinner, 1997), herbicides (Kilbane et al., 1983; Kaake et al., 1992), polyaromatic hydrocarbons
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ner et al., 1994; Mueller et al., 1996), petroleum pro-ducts (Balba et al., 1991) and munition compounds (Funk et al., 1993). In many other cases, addition of microorganisms has failed to enhance degradation of polluting chemicals, probably due to any or combi-nation of factors like non-optimal temperature, pH, moisture content, insucient number of added micro-organisms or incompatible soil texture. Besides, the concentration of polluting chemical might either be too high to be toxic to the added microbes or too low to induce the degradative activity (Goldstein et al., 1985; Block et al., 1993; Morra, 1996).
Bacteria and fungi, which degrade endosulfan iso-mers in liquid culture have been isolated and charac-terized in many laboratories (El Zorgani and Omer, 1974; Martens, 1976; Miles and Moy, 1979; Mukherjee and Gopal, 1994; Kullman and Matsumura, 1996; Awasthi et al., 1997). Endosulfan diol, endosulfan sul-fate, endosulfan aldehyde, endosulfan ether and endo-sulfan lactone have been demonstrated as the major metabolites formed during its degradation. The en-vironmental fate of endosulfan in dierent types of contaminated soils has also been studied (Rao and Murty, 1980; Antonious and Byers, 1997; Kathpal et al., 1997; Kaur et al., 1998; Parkpian et al. 1998), but the in¯uence of exogenous bacterial cells on endosul-fan degradation has not been evaluated.
In order to develop a suitable technology package for bioremediation of soils contaminated with endosul-fan, we have studied the ecacy of an isolated
bac-terial culture (Awasthi et al., 1997) on the
biodegradation of soil-applied endosulfan isomers. The in¯uence of factors, such as moisture content, pH, pre-sence of additional carbon sources, size of inoculum and initial pesticide load in soils, on endosulfan degra-dation were also evaluated.
2. Materials and methods
2.1. Chemicals
Commercial endosulfan (Parrysulfan, 35% EC, by EID Parry, Chennai, India) was purchased from a local market. Pure a- and b-endosulfan were obtained from Farbwerke-Hoechst AG, Frankfurt, Germany. 2-Phenoxy ethanol was purchased from Sigma, St Louis, MO, USA. All other reagents were of analytical grade.
2.2. Bacterial cells
A bacterial co-culture consisting of two dierent strains of Bacillus sp., isolated earlier from a contami-nated site by selective enrichment, was used (Awasthi et al., 1997). Either of the isolated bacteria can med-iate the degradation of endosulfan but together they
were found to be more eective. For this study, each of the isolated bacteria were grown separately in a rich medium (Peptone 1.0 g; Yeast Extract 0.5 g; NaCl 0.5 g; dissolved in distilled water, pH adjusted to 7.5 and made up to 100 ml) overnight, centrifuged at 10,000 rpm for 10 min, washed and resuspended in a mineral medium (KH2PO4, 170 mg; Na2HPO4, 980 mg; (NH4)2SO4, 100 mg; MgSO4, 4.87 mg; MgO, 100 mg; FeSO4, 50 mg; CaCO3, 20 mg; ZnSO4, 80 mg; CuSO45H2O, 16 mg; CoSO4. 15 mg; H3BO3, 6 mg; dis-solved in 100 ml of distilled water, the pH of the med-ium was 7.0) to the density of 2108 colony forming units (CFU) mlÿ1. Equal volume of both the cultures were mixed to make a stock co-culture. For degra-dation experiments, bacteria from stock co-culture were grown in rich medium overnight, centrifuged, washed twice and suspended in mineral medium to 2
108 CFU mlÿ1
before use.
2.3. Soils studied
Two soils, one (Soil A) from the Banthara ®eld station of the National Botanical Research Institute, Lucknow, containing, clay 60%, silt 30%, sand 10%, organic carbon 0.38% at pH 8.7, and the other (Soil B) from the Gheru Campus of Industrial Toxicology Research Centre, Lucknow, containing clay 39%, silt 12%, sand 49%, organic carbon 0.59%, at pH 9.37, were used. Collected soils were air dried and sieved (2 mm). Four hundred milliliters of distilled water con-taining 6.0 g of commercial endosulfan (35% EC) were added to 1000 g of each soil and mixed thoroughly. The concentration of the active ingredient was esti-mated to be 2.35 mg gÿ1 soil, i.e. 1.64 mg of a -endo-sulfan and 0.71 mg of b-endosulfan. After air drying for 24±48 h, the soils were pulverized and used for degradation studies.
2.4. Degradation of soil applied endosulfan and in¯uence of external factors
Degradation of soil-applied endosulfan in uninocu-lated or inocuuninocu-lated conditions, was studied in parallel sets of three containers each, for each variable and time point of the study.
were taken after 0, 1, 3, 5, 8, 12 and 14 weeks, and the residual endosulfan concentrations were quanti®ed.
For the experiments, where eect of pH was to be evaluated, portions of unspiked soil A (75 g each) were adjusted to pH values of 3.0, 5.0, 7.5, 8.5, 10.0 and 12.0 by addition of either 2 N HCl or 1 N NaOH. Adjustments to desired pH were made at least three times over 10 days, till the pH values of the soils were stabilized. Excess liquid from each soil was drained after pH adjustment and commercial endosulfan was added to yield the concentration of 2.35 mg gÿ1active ingredient. For each pH adjusted soil, two sets were made containing 10 g soil in each beaker. One set (uninoculated) received 3.0 ml of mineral medium, and other (inoculated) received 3.0 ml of mineral medium containing 2108 CFU of bacterial cells. Soils were incubated at 288C for 6 weeks and 1 g samples were taken after 0, 3 and 6 weeks to quantify the residual endosulfan present in each sample.
To study the eect of additional carbon sources, four beakers, each containing 75 g of spiked soil A were used. A 2% aqueous solution of glucose, sodium succinate and sodium acetate were added to beakers 1, 2 and 3, respectively, to yield the ®nal concentration of 10 mg gÿ1of additional carbon source. The fourth beaker received no additional carbon source. Each soil was then apportioned (10 g) into two sets of beakers. One set received 3 ml of mineral medium and served as uninoculated soils. The other set received 3 ml of medium containing 2108 CFU of bacterial cells. All containers were incubated at 288C for 8 weeks. One gram soil samples were taken after 0, 3, 5 and 7 weeks, and residual endosulfan concentrations were quanti®ed.
To evaluate the eect of inoculum size, six sets of beakers containing 10 g of spiked soil B were used. Three milliliters of mineral medium containing 2106, 2107, 2108, 4108 and 109 CFU of bacterial cells was added to sets 1±5, respectively. Three millili-ters of mineral medium without any bacterial cells was added to containers of set 6, which served as uninocu-lated controls. All the containers were incubated for 7 weeks at 288C. One gram soil samples were taken after 0, 1, 3, 5 and 7 weeks, and the residual endosulfan concentrations were quanti®ed.
In the experiments where eect of wet and ¯ooded conditions was to be investigated, 12 sets of beakers containing 10 g of spiked soil A were used. Three milliliters of mineral medium containing 2108 CFU of bacterial cells was added to a group of six sets (inoculated). Three milliliters of mineral medium with-out bacterial cells was added to the other group of six sets (uninoculated). Three sets, from both inoculated and uninoculated groups, received an additional 20 ml of mineral medium to represent ¯ooded samples. All the beakers were incubated at 288C for 6 weeks. One
¯ooded and one wet set from each group were har-vested after 0, 3 and 6 weeks, and residual endosulfan concentrations were quanti®ed.
When the eect of initial endosulfan concentration on its own biodegradation was studied, commercial endosulfan was added to 75 g portions of unspiked soil to arrive at a concentration of 0.05, 0.1, 0.4, 2.0, 5.0 and 10.0 mg gÿ1. Two sets of three replicate bea-kers containing 10 g soil per beaker were made. One set (uninoculated) received 3.0 ml of mineral medium and other set (inoculated) received 3.0 ml of mineral medium containing 2108 CFU of bacterial cells. Soils were incubated at 288C for 7 weeks. One gram soil samples were taken after 0, 1, 2, 3, 5 and 7 weeks, and residual endosulfan concentrations were quanti-®ed.
2.5. Extraction and analysis of residual endosulfan from the soils
Approximately 1.5 g of wet soil was removed from the incubating soil samples at the sampling times men-tioned earlier for dierent treatments, and air dried. One gram of dried soil was transferred to a test tube and extracted with 3 ml of ethyl acetate by vortexing. The ethyl acetate layer was decanted after 5 min. This extraction was repeated two more times. The ethyl acetate fractions were pooled, passed through anhy-drous sodium sulfate and evaporated at room tempera-ture. The eciency of extraction was 8522%: In the
experiment with wet and ¯ooded soils, the volumes in all the containers were raised to 25 ml with mineral medium before harvesting. An equal volume of ethyl acetate was added and samples were shaken on orbital shaker at 220 rpm for 30 min. Contents were trans-ferred to separatory funnel and the organic layer was collected. The aqueous layer was extracted two more times with 20 ml of ethyl acetate. Ethyl acetate frac-tions were pooled, passed through anhydrous sodium sulfate and evaporated at room temperature.
temperature for column injector, and detector were maintained at 220, 250, and 2808C, respectively, and nitrogen (IOLAR grade-1) was used as carrier gas. Retention times for a-endosulfan, b-endosulfan and endosulfan sulfate were 11.6, 20.2 and 23.6 min, re-spectively.
2.6. Statistical analysis
The values of the residual endosulfan, obtained from triplicate samples, were analyzed using analysis of variance (ANOVA), and Fischer's LSD was used to compare means. P values less than 0.05 were con-sidered signi®cant.
3. Results
3.1. Degradation of soil bound endosulfan isomers
The degradation of endosulfan in uninoculated soil A and soil B, measured by the disappearance of the parent chemical after 14 weeks of incubation, was 20 and 38%, respectively (Fig. 1). Addition of isolated bacterial cells enhanced this degradation to 90 and 78%, respectively. Degradation rates for a- and b
-iso-mers of endosulfan were comparable in uninoculated or inoculated conditions in both the soils (Fig. 1). In the inoculated soil samples, besides enhanced degra-dation of endosulfan, degradegra-dation of formed endosul-fan diol was also observed (Fig. 2, lane 8.5 I). Accumulation of endosulfan sulfate, a metabolite known to be formed primarily by fungal activity (Mar-tens, 1976; Kullman and Matsumura, 1996) and also in the contaminated soils (Kathpal et al., 1997; Rao and Murty, 1980) was not observed at any time during the degradation of endosulfan.
3.2. Eect of soil pH on endosulfan degradation
In uninoculated soils, after 6 weeks of incubation, no signi®cant degradation of endosulfan isomers at pH 3.0, slight degradation at pH 5.0 and a signi®cant degradation at pH 7.5 and 8.5, was observed (Table 1). In proportion to the degraded endosulfan, small amounts of endosulfan diol were formed at pH 7.5 and 8.5, whereas at higher pH values, i.e. 10.0 and 12.0, almost all the added endosulfan was rapidly con-verted to endosulfan diol (Fig. 2). Inoculated soils pre-sented no enhancement in degradation at pH 3.0, slight at pH 5.0, and good to optimal (>40±50%) enhancement at pH 7.5 and 8.5, respectively (Table 1). At pH 10.0 and 12.0, since all the added endosulfan was converted to endosulfan diol in uninoculated soils, no further change in its degradation due to added cells could be found. Formed endosulfan diol, however, underwent degradation at pH 8.5, 10.0 and 12.0 (Fig. 2), suggesting that the bacterial activity towards the degradation of endosulfan and its metabolites was operating in these alkaline conditions also.
3.3. Eect of additional carbon sources
In uninoculated soils, during 7 weeks of incubation, no signi®cant change in the degradation of endosulfan isomers was observed in the presence of glucose, sodium acetate or sodium succinate (Fig. 3). In inocu-lated soils, where no auxiliary carbon source was added, 75% of the added endosulfan was degraded after 7 weeks of incubation. A signi®cant inhibition was observed in presence of sodium acetate and sodium succinate, while the inhibition in presence of glucose was statistically not signi®cant. The inhibition was highest (100%) in the soils that received sodium succinate, as the amounts of residual endosulfan in uninoculated and inoculated soils, after 7 weeks of in-cubation, were identical.
3.4. Eect of inoculum size
Addition of 2105 CFU gÿ1soil had no signi®cant eect on endosulfan degradation, when compared to Fig. 1. Degradation of endosulfan isomers in two dierent soils:
the uninoculated soil samples (Fig. 4). Addition of 2
106 cells stimulated a rapid degradation, and after 7 weeks of incubation 60% of the added endosulfan was degraded. Addition of higher CFU, i.e. 2107, 4 107 and 108
, resulted in an initial increase in endosul-fan degradation P<0:05which attenuated with time
to show no signi®cant increase in endosulfan degra-dation over a period of 7 weeks (Fig. 4).
3.5. Degradation in ¯ooded and non-¯ooded soils
In uninoculated soils, after an incubation of 6 weeks, the degradation of endosulfan isomers was bet-ter in the non-¯ooded condition (26±29%) than in the ¯ooded condition (15%). Addition of bacterial cells
enhanced the degradation of both isomers of endosul-fan to 68±69% in non-¯ooded and 43±48% in ¯ooded soils, after the same period of incubation (Table 2).
3.6. Eect of initial concentration of endosulfan on its degradation
At low initial concentration of endosulfan i.e. 50 mg gÿ1 soil, there was a rapid degradation of endosulfan isomers in uninoculated soils. Here, addition of bac-terial cells, had no appreciable in¯uence on the degra-dation rates (Fig. 5). The dierence in endosulfan degradation rates, between uninoculated and inocu-lated soils, increased progressively up to 0.4 mg gÿ1 soil. At higher concentrations, endosulfan began to Fig. 2. Thin layer chromatogram depicting the degradation of endosulfan isomers at dierent pH values, after 6 weeks of incubation: aES Ð a-endosulfan, bES Ðb-endosulfan, ED Ð endosulfan diol, UI Ð uninoculated and I Ð inoculated.
Table 1
Eect of soil-pH on the degradation of endosulfan (ES) isomersa
Incubation (week) pH 3.0 pH 5.0 pH 7.5 pH 8.5
UI I UI I UI I UI I
show an inhibitory eect on the degradative activity and at the concentration of 10 mg gÿ1 soil, in both uninoculated and inoculated soils, nearly no degra-dation of endosulfan was observed, up to 7 weeks of incubation (Fig. 5).
4. Discussion
The environmental fate of organic pollutants in soils is in¯uenced signi®cantly by the pH and texture of the soil, and also by the presence of organic matter and
copollutants. Accordingly, biodegradation of endosul-fan isomers was studied in two dierent soils that have contrasting properties in terms of their texture, pH, or-ganic content, etc. When inoculated the degradation of endosulfan was faster in soil A than in soil B, prob-ably because of their dierent physico-chemical prop-erties. During the degradation, the formation of endosulfan sulfate, a metabolite known to accumulate Fig. 3. Eect of additional carbon sources on the degradation of
endosulfan isomers: unamended (-w-w-), and amended with glucose (-q-q-), sodium acetate (-r-r-) or sodium succinate (-t-t-), UI Ð uninoculated and I Ð inoculated. Amounts of endosulfan recov-ered at 0 time were taken as 100%. Vertical bars represent2 stan-dard deviation of the mean of three replicates and where no bar is shown it is less than the size of the symbol.
Fig. 4. Eect of inoculum size on the degradation of endosulfan iso-mer: uninoculated (-w-w-), and inoculated with 2105 (-q-q-),
2106(-r-r-), 2107(-t-t-), 4107 (-r-r-) and 108 (-D-D-) CFU gÿ1 soil. Amounts of endosulfan recovered at 0 time were taken as 100%. Vertical bars represent2standard deviation of the mean of three replicates and where no bar is shown it is less than the size of the symbol.
Table 2
Degradation of endosulfan isomers in non-¯ooded and ¯ooded soilsa
Time (week)
Non-¯ooded Flooded
a-Endosulfan b-Endosulfan a-Endosulfan b-Endosulfan
UI I UI I UI I UI I
0 1.4020.04
(100)
1.4120.04 (100)
0.6120.05 (100)
0.6020.04 (100)
1.4020.05 (100)
1.3920.04 (100)
0.6020.04 (100)
0.5920.05 (100)
3 1.1220.04
(80)
0.6320.05 (45)
0.4820.04 (80)
0.2720.06 (46)
1.2220.05 (87)
0.8420.05 (60)
0.5520.04 (92)
0.4120.06 (68)
6 1.0020.05
(71)
0.4520.06 (32)
0.4520.06 (74)
0.1820.07 (31)
1.1920.06 (85)
0.7220.05 (52)
0.5120.05 (85)
0.3420.06 (57)
a
during incubation with certain fungi (El Zorgani and Omer, 1974; Martens, 1976; Kullman and Matsumura, 1996) and also in contaminated soils (Rao and Murty, 1980; Kathpal et al., 1997; Lee et al., 1997; Kaur et al., 1998) was not observed at any time. This could be due to lack or non-functioning of fungi in the soils stu-died here. These results are in accordance with our earlier studies, where, in culture conditions, endosulfan sulfate was not formed (Awasthi et al., 1997).
Under uninoculated conditions, increased degra-dation of endosulfan isomers was observed at higher pH values and is primarily due to its alkaline hydroly-sis into endosulfan diol. This probably is the reason for the increased degradation of endosulfan isomers in soil B (pH 9.37) as against soil A (pH 8.5) at the ear-lier time points (Fig. 1). Under inoculated conditions, however, the degradation of endosulfan isomers was
optimal at pH 8.5. This could be due to better bio-availability of endosulfan, and optimal biotic activity of cells at this pH value. Better degradation of con-taminating diesel oil (Margesin and Schinner, 1997) as well as herbicide atrazine (Hance, 1979) has also been reported, under alkaline conditions.
Carbon sources, other than the target chemical, are present in natural soils and may in¯uence degradation rates. Accordingly, the eect of some of the common carbon sources was evaluated on the biodegradation of endosulfan isomers in soil. In inoculated soils, the pre-sence of sodium acetate or sodium succinate inhibited the degradation of endosulfan to dierent extents. The inhibition of degradation in presence of additional car-bon sources can also be among the reasons for less degradation of endosulfan isomers in soil B (organic content 0.59%) than in soil A (organic content 0.38%). In many other studies also, the presence of more favorable carbon sources have been shown to impede the degradation of less favorable chemicals, e.g. xenobiotics (Hartline and Gunsalus, 1971; Sahu et al., 1993). This could be due either to the mechanism of catabolite repression (Hartline and Gunsalus, 1971; Botsford and Harman, 1992) or decrease in the rates of transcription either due to supercoiling of promoter DNA (Assinder and Williams, 1990) or by decreased binding of transcription factors (Holtel et al., 1994).
Exogenous microorganisms, when added to the soils, run the risk of getting out-competed by native microbial communities and also of predation by proto-zoans, etc. To arrive at the optimal number of bac-terial cells for eective degradation of endosulfan, the in¯uence of dierent inoculum sizes ranging from 2
105 to 108
CFU gÿ1 soil was studied. While the ad-dition of 2105 CFU caused no enhancement in the degradation rates, addition of 2106 or more cells caused a substantial enhancement in the degradation of endosulfan. The enhancement in the degradation over 7 weeks was comparable to the inoculation with 2106, 2107, 4107 or 108 CFU gÿ1 soil. It is possible that during incubation in the soils, when the added bacterial cells were 2105 or below they were out-competed or preyed upon by the indigenous micro-¯ora. At higher inoculum densities i.e. 2106, 2107, 4107, 108
CFU gÿ1, the bacterial popu-lation might have equilibrated to a common eective level. In the experiments, where in¯uence of dierent water regimes on endosulfan biodegradation was eval-uated, the higher degradation of endosulfan in non-¯ooded conditions, in uninoculated and inoculated soils, was probably due to better oxygen availability, as against ¯ooded conditions.
Concentrations of target pollutant can vary con-siderably in dierent contaminated soils and exert a signi®cant in¯uence on the degradative activity of microorganisms. While very high amounts of the pol-Fig. 5. Eect of initial endosulfan concentration on its degradation
lutant can be toxic to the microorganisms, their low concentrations might fail to induce degradative ac-tivity. At low initial concentrations of endosulfan, i.e. 50 and 100mg gÿ1soil, the degradation was very rapid in both uninoculated and inoculated soils. At higher concentrations, however, the degradation rates were slower, leading to a total inhibition of the degradative activity at the initial concentration of 10 mg gÿ1 soil. The inhibition of degradation was probably due to the cytotoxicity of endosulfan at this concentration.
In this study, we have used bacterial cells that had been isolated and characterized for the degradation of endosulfan isomers in liquid-culture conditions (Awasthi et al., 1997), and have presented evidence for their viability as well as capability to enhance the degradation of the chlorinated pesticide, endosulfan, in contaminated soils. The enhancement is in¯uenced greatly by factors such as soil-pH, presence of carbon-aceous material, water content, size of bacterial inocu-lum and concentration of the pesticide present in the soils. Initial optimisation of these factors is therefore essential before undertaking any bioremediation ac-tivity. Further, in view of the in¯uence of external fac-tors on the degradation activity, half-lives of chemicals in the soils reported in literature should be considered with caution.
Acknowledgements
We thank Dr P.K. Seth, Director, Industrial Toxi-cology Research Centre, Lucknow for his constant support to this work, Mr Neeraj Mathur of this insti-tute for statistical analysis, Dr S. K.Tiwari of National Botanical Research Institute, Lucknow, for analysis of the soils and Dr Rakesh Jain of Institute of Microbial Technology, Chandigarh, for identi®cation of the bac-terial strains. Generous gift of endosulfan isomers and metabolites by Hoechst, AgrEvo, Germany, and ®nan-cial assistance from Department of Biotechnology, New Delhi is gratefully acknowledged.
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