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The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments


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The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments

  1. 1. Environmental Pollution 106 (1999) 13±21 The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments Turlough F. Guerin* 1691 E Green Briars Drive, Suite 3821, Schaumburg, IL 60173, USA Received 27 July 1998; accepted 24 February 1999 Abstract Indigenous mixed populations of anaerobic microorganisms from an irrigation tailwater drain and submerged agricultural chemical waste pit readily biodegraded the major isomer of endosulfan (endosulfan I). Endosulfan I was biodegraded to endosulfan diol, a low toxicity degradation product, in the presence of organic carbon sources under anaerobic, methanogenic conditions. While there was extensive degradation (>85%) over the 30 days, there was no signi®cant enhancement of degradation from enriched inocula. This study demonstrates that endosulfan I has the potential to be biodegraded in sediments, in the absence of enriched microorganisms. This is of particular importance since such sediments are prevalent in cotton-growing areas and are typically contaminated with endosulfan residues. The importance of minimizing non-biological losses has also been highlighted as a critical issue in determining anaerobic biodegradation potential. Seals for such incubation vessels must be both oxygen and pollutant impermeable. Te¯on-lined butyl rubber provides such a seal because of its resistance to the absorption of volatiles and in preventing volatilization. Moreover, including a 100 mM phosphate bu€er in the anaerobic media has reduced non-biological losses from chemical hydrolysis, allowing biodegradation to be assessed. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Endosulfan; Biodegradation; Anaerobic; Sediment; Cotton; Natural attenuation; Thiodan1 1. Introduction Endosulfan is a chlorinated cyclodiene insecticide currently used throughout the world for the control of numerous insects in a wide variety of food and nonfood crops. Endosulfan (CAS, 115-29-7) comprises two parent isomers, the a-, and b-endosulfan, or endosulfan I and II, respectively. These isomers have a wide distribution in the environment and have been detected in soil and sediments at considerable distances from the point of their original application (Mansingh and Wilson, 1995; Miles and Pfeu€er, 1997). There is limited information on the mechanisms of loss of endosulfan in low-oxygen and anaerobic environments (Cotham and Bidleman, 1989; Peterson and Batley, 1993; Guerin, 1993) and few studies have demonstrated that related compounds can be anaerobically biodegraded. Liquid culture studies with related * Tel.: +1-847-240-4247; fax: +1-847-619-9905. 248 Gladstone Avenue, Coniston, New South Wales, 2500, Australia. E-mail address: (T.F. Guerin) cyclodienes have demonstrated that microorganisms may monodechlorinate the hexachloronorbornene ring. Schuphan and Ballschmitter (1972) ®rst reported the anaerobic dechlorination of hexachloronorbornene and its dechlorinated derivatives by pure cultures of anaerobic soil bacteria. Since then, the dechlorination of the hexachloronorbornene moiety of the cyclodienes aldrin, dieldrin, and endrin, has been demonstrated (Maule et al., 1987), and it has been shown that a consortium of anaerobic microorganisms create conditions for the monodechlorination. Pure cultures isolated from the consortium, including those from the genus Clostridium, were less e€ective in bringing about monodechlorination (Maule et al., 1987). Recent liquid culture studies have indicated that anaerobic soil microorganisms can metabolize selected degradation products of endosulfan (Schneider and Ballschmiter, 1995). There is, however, only limited information on the anaerobic degradation of the major parent isomer (Guerin, 1993, 1995). The current paper describes the biodegradation of endosulfan I in mixed anaerobic cultures by inocula from low-oxygen soils and freshwater sediments. Speci®cally, these environments 0269-7491/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(99)00067-6
  2. 2. 14 T.F. Guerin / Environmental Pollution 106 (1999) 13±21 were irrigation tailwater drains and agricultural chemical evaporation (sullage) pits, where there was only limited water perturbation and aeration. Such conditions are prevalent in the cotton-growing areas of northern New South Wales, Australia, but low-oxygen environments in general (such as sediments), are widely distributed throughout the environment. The hypothesis tested in the current research was that endosulfan I can be biodegraded by indigenous microorganisms from low-oxygen soils and sediments under strictly anaerobic conditions. 2. Materials and methods 2.1. Collection of sediment and sullage pit samples and preliminary anaerobic studies on endosulfan I biodegradation A ®eld trip was made to Narrabri, New South Wales, Australia, to obtain samples from agricultural chemical sullage pits and tailwater drains near cotton-farming ®elds. These samples were collected during winter when there was considerable water ponding. Seawater sediment was collected 200 m o€ the shore of Botany Bay, New South Wales, Australia, by dredging a sample from the surface of the seabed (approximately 10±20 cm depth). Six subsamples were carefully removed from the centre of the total mass of sediment that was collected (0.5±1 kg), and these were pooled to give composite samples in duplicate. Tailwater drain sediment samples were collected from the top 10±15 cm of the submerged sediment pro®le and sullage pit samples were collected from 10±15 cm below the surface of the bulk of the sludge. From each of the locations, six subsamples were pooled to give composite samples in duplicate. The duplicate samples from each of the locations were kept in airtight glass jars at 4 C (Shelton and Tiedje, 1984) and were then used as separate sour- ces of inocula for the anaerobic incubation assays. The samples collected were from anaerobic environments and this was evident by: (1) their submerged locations, (2) their colour, and (3) distinct odour of H2S. All the samples had become black and evolved an H2S odour. Table 1 describes key properties of the sediments and soils used. 2.2. Preparation of soil inocula Soil and sediment samples were stored at 4 C (1±3 weeks) in the absence of oxygen prior to their extraction. The samples were then brie¯y shaken for 15 min with sterile and degassed minimal salts medium in a ratio of 1:10 (w/v) (soil:minimal salts medium) in a sealed ¯ask. A 10À1 dilution of this extract was added to the anaerobic media to give a ®nal concentration of 0.4% (v/v). 2.3. Preparation of anaerobic enrichment cultures using technical grade endosulfan and Tween 801 An anacrobic enrichment was prepared using endosulfan (a mix of isomers I and II in a ratio 70:30, respectively, at 98% purity) and the surfactant, Tween 801 (CAS, 9005-65-6, 99% purity, Sigma Chemical Co., Sydney, Australia). The rationale for preparing the enrichment was to develop a culture potentially capable of degrading endosulfan I at high rates and to compare this with an indigenous population (with little or no previous exposure to endosulfan). The reason for using Tween 801 was to increase the aqueous solubility (and bioavailability) of endosulfan so that it may be more accessible to the growing microorganisms for biodegradation (Rouse et al., 1994). Inocula were introduced into minimal salts medium containing (in g/l) K2HPO4 (anhydrous) 6.8, KH2PO4 8.3, NH4NO3 1.0, MgSO4 0.05, CaCl2 (anhydrous) 0.02, and FeSO4 0.01. This medium gave an a€ective bu€ering capacity (100 mM) at Table 1 A description of selected properties of the samples used in the trial Sample Tailwater drain sediment (TD) Sullage pit (new) (NSP) Sullage pit (old) Freshwater sediment Seabed sediment a b c d yga (g/g) 0.460 0.213 0.347 0.497 0.325 OC (%) [Organic Carbon] OM (%) [Organic Matter] Particle size fraction (w/w%) Cb Sc FSd CSe 1.62 1.30 1.20 2.03 0.95 2.28 2.85 2.11 3.56 1.67 53.7 18.3 51.1 16.5 15.9 24.2 7.5 15.9 8.8 2.6 19.7 49.5 13.0 42.9 12.5 2.4 24.7 16.0 31.8 69.0 yg is the gravimetric moisture content of the samples in their ®eld condition. Clay fraction. Silt fraction. Fine sand fraction.
  3. 3. T.F. Guerin / Environmental Pollution 106 (1999) 13±21 pH 7.0. Endosulfan I (98% purity) (1000 mg/l in methanol) and Tween 801 (1%) (v/v) were added and in conjunction with the small volume of methanol used for dissolving the endosulfan, were the only sources of carbon in the liquid medium for microbial growth. In other treatments, aldrin (98%) and dieldrin (98%) were added separately at a concentration of 1000 mg/l, instead of endosulfan I. The rationale for this was to compare endosulfan with these related compounds, on which other anaerobic studies have been performed (Maule et al., 1987). Cultures were kept unshaken in the dark for 10 days in an incubator at 30‹1 C before subculturing. The resulting mixed populations of anaerobes grown in this medium containing endosulfan (or aldrin or dieldrin), were subcultured seven times prior to analyzing their degradative capacity in the routine incubations. Methane production was measured on the 10th day of incubation during each of the third to the seventh subcultures. Argon (Ar) was used as the headspace gas in these enrichment cultures and this provided an environment that was selective for the carbon sources in the medium only. 2.4. Incubation conditions for routine anaerobic incubation A medium was prepared for routine anaerobic biodegradation (Maule et al., 1987) and contained (in g/l) K2HPO4 (anhydrous) 6.8, KH2PO4 (anhydrous) 8.3, NH4C1 1.3, MgSO4.7H2O 0.01, FeSO4, yeast extract (Difco) 2.0, Peptone (Difco) 2.0, sodium formate (anhydrous) 2.0, biotin 1Â10À6 and pyridoxal phosphate 2Â10À6. A trace element solution that contained (in g/l) Na2EDTA 15.0, Cu(NO3)2, 1.0, ZnCl2, 2.5, MnCl2, 1.0, CoC12 0.5, H3BO3 1.0, and Na2SO4, 1.0, was added at 1.0 ml/l. The pH of the bu€ered medium was adjusted to 7.0 and then ®ltered with Whatman No. 1 paper prior to autoclaving and adding the heat-sensitive components. The vitamin solution was ®lter sterilized and added when the medium was at room temperature. The medium was reduced by the aseptic addition of cysteine hydrochloride (in sterile distilled water) to give 0.1% (w/v) followed by purging and degassing with Ar for 15 min while the medium was cooled rapidly (Miller and Wolin, 1974). Inocula were then added (as described earlier). All incubations were kept strictly anaerobic during the course of the enrichments and this was checked with resazurin (7-hydroxy3H-phenoxyazin-3-one-10-oxide). In addition to sterilized medium, a second set of controls were included, which was medium with an autoclaved 10-day-old culture. Endosulfan I was added to growth medium in a 1000 mg/l methanol solution so that only a minimal amount of methanol (a potential carbon source) entered the medium. The ¯asks were made anaerobic by evacuation followed by degassing with Ar 6±8Â. Successful 15 reduction was demonstrated by the colour change of resazurin from pink to colourless. 2.5. Incubation conditions and endosulfan I determination 2.5.1. Sealing of the incubation vessels and addition of incubation atmosphere Wheaton serum bottles (125 ml) were used in the non-sterile enrichment and non-enrichment cultures where aliquots of culture were subsampled. Smaller bottles were used for the sterile treatments where it was important that the entire contents (including a solvent rinse of the incubation vessel) be analyzed. In the preliminary experiments, butyl rubber stoppers were used to seal the serum bottles and were the same used in previous anaerobic studies (Miller and Wolin, 1974; Balch et al., 1979; Shelton and Tiedje, 1984; Maule et al., 1987; Holliger et al., 1992; Kohring et al., 1992). In the routine incubations (described later in Materials and Methods), butyl rubber stoppers were replaced with PTFE (Polytetra¯uoroethylene)-lined butyl rubber stoppers, which was reported in the early work by Bouwer and McCarty (1983). This latter sealing system was adopted to overcome diculties in retaining pesticide, due to the low absorptive capacity of PTFE (i.e. Te¯on1) for insoluble and volatile organochlorine compounds such as endosulfan I, while ensuring oxygen impermeability. Subsamples were taken from the serum bottles by removing 1±2 ml from the incubation broth (after homogenizing) using a syringe. Therefore, over the entire period of the trial, there was a decrease in the volume of the broth of 1 ml, and an equivalent increase in headspace. Filter-sterilized gas was introduced to the vessels. Vessels were kept unshaken in the dark for 30 days in an incubator at 30‹1 C. A mixture of CO2:H2 (80:20) or Ar alone was used as headspace. The gas was ®lter sterilized and introduced using 200ml syringes. Prior to sampling when the sampling syringe was used to homogenize the contents of individual ¯asks so that the cell material, and any pesticide residues absorbed into this, would be distributed evenly throughout the liquor. This ensured that subsamples taken from the microbiologically active ¯asks were representative of the total ¯ask contents. In the sterile treatments, subsamples were not taken, but rather the entire ¯ask contents were sacri®ced because of adsorption of endosulfan onto glass. 2.5.2. Measurement of methane gas production The atmospheres of the incubation vessels were subsampled (500±1000 ml) and analyzed immediately using a Shimadzu gas chromatograph (Model GC-8A) ®tted with a ¯ame ionisation detector (FID) and a 1 m  2.6 mm packed glass column (Poropak T, Waters/Millipore). Temperatures of 130 and 150 C were used for the
  4. 4. 16 T.F. Guerin / Environmental Pollution 106 (1999) 13±21 column and the detector, respectively. The N2 head pressure was 4.0 kg/m2. 2.5.3. Measurement of microbial growth and pH of cultures Microbial growth was measured by determining OD700 nm on a Beckman spectrophotometer (Model DU-64). Controls containing the same media, without added microorganisms, were also analyzed to determine whether the increases in absorbance were due to microbiological activity. 2.5.4. Analysis of endosulfan Endosulfan I was analysed by electron capture detector± gas chromatograph (ECD±GC) and degradation products were analyzed simultaneously with the parent isomers (Guerin et al., 1992). 2.5.5. Microscopic examination of the mixed cultures and enumeration Gram stains of cultures were prepared. Carbol-fuchsin and methylene blue were used as the spore stains. Preparations were examined using a 100 immersion oil objective using light transmission microscopy (Olympus Model BHA). Both non-enriched and routine incubation cultures were examined. Total anaerobic counts were conducted on solidi®ed media (Balch et al., 1979). degradation product, endo (ED), was detected in all the inoculated treatments. No traces of ED were detected in either of the controls. This was consistent with endosulfan I absorption into the butyl rubber stoppers, preventing it from undergoing hydrolytic reaction in the liquor. 3.2. Stability of endosulfan I under routine incubation conditions Butyl rubber seals were replaced with PTFE-lined butyl rubber (with sterilized medium only), to reduce absorption. When sterile controls were prepared using PTFE-lined butyl rubber, the rate of endosulfan I dissipation was relatively slow at applied concentrations of 1, 2, and 10 mg/l after 10 days (Table 3). Thus, provided that incubations in ¯asks were sealed with PTFE, were sterile, and bu€ered at pH 7, relatively little dissipation of endosulfan I occurred. The trend of increasing endosulfan I loss at decreasing spiking concentrations, illustrated a solubility e€ect. At higher concentrations, endosulfan I was adsorbed onto the glass walls and was less available for degradation reactions. There was only very limited endosulfan I degradation in the controls containing sterilized cells, indicating that non-living cell matter was not catalyzing degradation. 3.3. Biodegradation of endosulfan I in routine incubations The inocula used were derived from either sediment or from an irrigation tailwater drain (TD) or from a 3. Results and discussion 3.1. Preliminary studies In all ¯asks (using butyl rubber seals to restrict oxygen penetration) endosulfan I decreased to very low concentrations after 25 days. Controls containing sterile media alone demonstrated a faster initial rate of dissipation than that from inoculated ¯asks, indicating that there were nonbiological losses. It was apparent that the observed losses were due to absorption by the unlined butyl rubber seals (Table 2). These results highlight the importance of using PTFE-lined butyl rubber sealed ¯asks in biodegradation studies of volatile or semivolatile organics to prevent absorption. The hydrolytic Table 3 The stability of endosulfan I in sterilized anaerobic mediaa Days incubation Amount remaining (% of originally applied) 1 mg/l 0 2 4 6 8 100‹3 98‹2 95‹4 90‹2 80‹3 2 mg/l 100‹3 97‹2 94‹2 96‹2 90‹3 10 mg/l 100‹4 100‹1 98‹1 97‹1 98‹2 a Duplicate analyzes conducted and vessels were sealed with PTFElined butyl rubber seals. Table 2 The anaerobic dissipation of endosulfan I using extracts taken from low-oxygen soils and sediments incubated in ¯asks sealed with butyl rubbera Inoculum source Endosulfan 1 (% remaining) ED (mg/l) Biomass (OD700 nm)b 10 days Freshwater sediment Seabed sediment Sterile medium a b c 25 days 10 days 25 days 5 days 10 days 0.04 0.02 0.002c 0.5 0.3 0.002c 70 54 7.3 2.0 0.001c 1.0 0.02 0.05 0.01 0.15 0.20 0.01 1 mg/ml of endosulfan I initially applied and incubated at 30 C. OD, optical density. Not detected.
  5. 5. T.F. Guerin / Environmental Pollution 106 (1999) 13±21 new sullage pit (NSP), and were either non-enriched or enriched. Using NSP inocula, not previously enriched by subculturing, led to an almost complete dissipation of endosulfan I (originally applied at 1 mg/l) after 10 days (Fig. 1). Inoculation with a sample of the TD sediment (that was non-enriched) also led to an almost total loss of applied endosulfan I after approximately 10 days (Fig. 2). The same inocula applied to the medium containing 10 mg/l of endosulfan I, resulted in slower rates of decrease but only slightly higher remaining endosulfan I concentrations (Figs. 1 and 2). In the routine anaerobic incubations containing inocula from enriched NSP and TD sediments, the amount of endosulfan I remaining after 30 days incubation was less than 2% (Figs. 1 and 2). There was no signi®cant di€erence between the extent of degradation of endosulfan I between either the enriched or nonenriched incubations. These results demonstrated that there was no enhancement of degradation in the enriched incubations. An explanation for the extensive degradation observed in both the enriched and nonenriched incubations can be attributed to the primary degradation reaction occurring. The formation of endosulfan diol occurs from opening of the cyclic diester ring Fig. 1. Endosulfan I degradation and endosulfan diol formation in enriched (E) and non-enriched (NE) irrigation tailwater drain (TD) incubations. Fig. 2. Endosulfan I degradation and endosulfan diol formation in enriched (E) and non-enriched (NE) new sullage pit (NSP) incubations. 17 system on endosulfan, and the current ®ndings suggest that this may be catalyzed by a non-speci®c enzyme. In the 10 mg/l incubations, there was an apparent increase in the endosulfan I in the subsampled aliquots. This was observed 2 days after the addition of inoculum (Figs. 1 and 2). This phenomenon was due to the partitioning of adsorbed endosulfan I from the glass surfaces on the inside of the incubation vessels to the more lipophilic microbial cells. Endosulfan I was distributed to the glass surfaces in the ungrown (or very young cultures) because 10 mg/l is 4±5 higher in concentration than its water solubility (Guerin and Kennedy, 1992). 3.4. Formation of endosulfan I degradation products ED, a hydrolysis product of the parent endosulfan isomers, was formed in all the non-sterile treatments. The dissipation of endosulfan I from the non-enriched and enriched cultures correlated with an increase in ED. This occurred in treatments containing either 1 or 10 mg/l of applied endosulfan I. The highest concentrations of ED were present in the incubations containing 10 mg/l of applied endosulfan I (Figs. 1 and 2). The formation of ED was higher in the medium inoculated with microorganisms from the NSP, compared to TD sediment. Only traces of ED were reported in the nonsterile treatments, where concentrations did not exceed 3 detection limit, or 0.006 mg/l. There were signi®cant increases in the biomass of all the inoculated treatments. In the sterile treatments, and in the ®ltrate from biologically active incubations with endosulfan I added, there were no increases in optical density (OD 700 nm). This indicated that the turbidity in the inoculated vessels was due to microbial growth and not a result of chemical processes (e.g. precipitation) and this was con®rmed by microscopic examinations, and there were no metabolites formed during the incubations with absorption at 700 nm. Lower maximum biomass values were recorded in the incubations with 10 mg/l of applied endosulfan I, compared with Fig. 3. E€ect of endosulfan I concentration on growth rates.
  6. 6. 18 T.F. Guerin / Environmental Pollution 106 (1999) 13±21 incubations of 1 mg/l (Fig 3). Growth rates and maximum biomass values were only marginally higher in the incubations inoculated with microorganisms from the enriched treatments (data not shown). The growth rates in the cultures inoculated with soil extracts from the sullage pit (both new and old) and TD were similar. In all the treatments, the maximum growth was reached by the sixth day of incubation. OD700 nm values in control media containing inocula from TD and NSP without endosulfan I (but containing solvent carrier), reached a maximum of 0.45±0.47 at 2 days. This indicated that there was only very minor inhibition of biomass production from added endosulfan I. Increased biomass production did not correspond to higher ED formation as the most ED was produced when biomass production was lowest. In many of the incubations it was noted that there was evidence of ED dissipation, suggesting further degradation. There was evidence that degradation to metabolites, other than ED, was occurring from the detection of traces of endosulfan hydroxy ether (EHE), endosulfan ether (EE), and endosulfan lactone (EL) in selected incubation vessels. In an enriched TD incubation, containing 10 mg/l endosulfan I, in addition to the 4.3 mg/l ED formed, EHE and EE were found at 0.21 and 0.04 mg/l, respectively, at 10 days. In a separate trial, EL (2.6 mg/l) was detected in a NSP enrichment culture, containing 10 mg/l of originally applied endosulfan I. EL was not consistently formed in all the non-sterile treatments, re¯ecting its aqueous instability (Miles and Moy, 1979). The identity of the degradation products was con®rmed by the use of synthesized standards and chromatographic behaviour on two gas chromatography (GC) columns (Guerin et al., 1992). The formation and subsequent detection of degradation products (at the end of the incubation period) could not account for all the originally applied endosulfan I. This lack of stoichiometric mass balance when endosulfan I and the identi®ed degradation product concentrations were considered, indicates that endosulfan I is most likely being converted to anaerobic degradation products which are escaping detection. These could include dechlorinated forms of endosulfan. Endosulfan sulfate (ES), the major oxidation product of both the parent endosulfan isomers, was not detected in any of the anaerobic incubations. This is consistent with the predicted pathway of degradation for this compound where oxygen is limiting. These include tailwater drain and seabed sediments; freshwater muds and soils that remain saturated with water for extended periods of time. Thus from the current study, it appears that the presence of ES in sediments is most likely attributable to the transport and deposition of this degradation product from its site of formation (such as in the cotton ®eld itself), to the waterways and subsequently to sediments. 3.5. Minimizing the non-biological hydrolysis of endosulfan It is generally recommended that the pH of media for biodegradation studies be in the region of pH 6±8 (Painter, 1992). Also, bu€ered media has been recommended for use in biodegradation assays to o€set the e€ects microorganisms can have on broth pH and subsequently on any compounds added to the broth (Painter, 1992). The ®ndings of the current study indicated that using a 100 mM phosphate-bu€ered medium provided pH stability against the growing anaerobic cultures (Fig. 4). The growing cultures did not increase the pH of the medium more than 0.25 pH units. Klein and Alexander (1986) have reported that the survival of microorganisms from lake water was improved if it was bu€ered with 100 mM phosphate bu€er, indicating that such an approach is unlikely to a€ect the selection and growth of microorganisms. The degradation observed for endosulfan I in the current experiments was due therefore to the activity of microorganisms and not a result of nonbiological hydrolysis. 3.6. Analysis of the incubation vessel atmosphere Methane was detected in the headspace of all the enrichment cultures as well as those from the routine, non-enrichment incubations. In the enrichment cultures, methane production was measured in enrichments 3±7 (Table 4). Methane was also produced in cultures enriched with high concentrations of aldrin and dieldrin as well as endosulfan, which indicated that these related organochlorines did not inhibit the growth and proliferation of methanogenic microorganisms. Methane production was at its peak at the sixth subculture for both the enriched sullage pit (new and old) and TD treatments and for each of the cyclodiene enrichments. Methane production was slightly higher in the endosulfan-enriched, TD sediment. In all of the treatments, methane concentrations decreased at the fourth and seventh enrichment indicating that there was a reduction Fig. 4. pH stability at varying endosulfan I concentration in the nonenriched TD incubation.
  7. 7. T.F. Guerin / Environmental Pollution 106 (1999) 13±21 19 Table 4 A comparison of methane production from mixed anaerobic cultures enriched with high concentrations of endosulfan, aldrin and dieldrina Compound Source Methane produced at enrichment No. (nmol/25 ml culture) 3 b 4 5 6 7 Endosulfan NSP TDc 59.4‹1.6 50.7‹0.4 18.95‹2.3 20.9‹0.5 65.4‹13.9 101.4‹64.8 70.9‹8.0 138.9‹14.2 17.3‹3.5 76.6‹21.0 Aldrin NSP TD 74.5‹15.0 63.9‹4.7 26.5‹15.0 15.8‹3.1 44.2‹16.2 36.0‹4.6 52.3‹1.6 79.9‹19.4 28.4‹1.7 27.8‹6.1 Dieldrin NSP TD 68.1‹8.2 56.6‹11.9 15.2‹3.0 19.4‹2.9 85.0‹64.8 34.4‹20.9 98.5‹29.6 90.8‹16.5 48.8‹4.4 15.5‹0.9 a Endosulfan I:II (70:30) parent isomers (98%), aldrin (98%) and dieldrin (85%) were added to anaerobic medium at 1000 mg/l with Tween 801 (1%). Incubations were subcultured every 10 days and kept under Ar. b Soil from a new agricultural chemical sullage pit (10±15 cm below surface). c Sediment from an irrigation tailwater drain. in the activity of the methanogenic bacteria at these times. This suppression of activity, however, was not maintained. No methanogenesis was detected in the sterile controls. In the routine non-enriched incubations, increases in endosulfan I caused a signi®cant reduction in methane production. At 20 and 50 days of incubation in the treatments containing 1 mg/l applied endosulfan I, the methane production was 136‹12 and 119‹10 nmol/25 ml of growing culture, respectively. The values for the 10 mg/l treatments were 27‹7 and 47‹9, respectively. These results indicated that endosulfan I may be inhibitory to methanogenic organisms in non-enriched cultures. The cells observed in both the enrichment cultures and routine cultures were predominantly Gram-negative anaerobic rods and methane-producing bacteria. The relatively high concentrations of methane produced in the enrichment cultures indicated that methanogenic bacteria were not inhibited to any great extent by high concentrations of endosulfan, aldrin or dieldrin. This is consistent with the ®ndings of Mancinelli (1982) and Thomann (1976) who have shown that cyclodiene pesticides a€ected the activities of Gram-negative soil bacteria to a much lesser extent than Gram-positive isolates. The current ®ndings indicate that methanogens were active in the endosulfan I degrading cultures. Maule et al. (1987) similarly found that methane was produced by enrichment cultures growing in the presence of dieldrin (10 mg/l). These researchers, however, found that methane production ceased in routine anaerobic cultures that were inoculated with microorganisms grown initially in these enrichment cultures (containing dieldrin non-enriched at 10 mg/l). In both these enriched and non-enriched cultures, dieldrin was monodechlorinated. However, in the current study, there was no evidence for the signi®cant formation of dechlorinated endosulfan I compounds. Any dechlorinated forms of endosulfan I would have been extracted, analyzed and detected by the chromatographic procedure used (Guerin et al., 1992). In previous studies it has been found that dechlorination was greatest with the populations of anaerobes that did not produce methane (Maule et al., 1987). This may indicate the reason why endosulfan I dechlorination was not observed in the current study, where all the incubations contained methanogens. 3.7. E€ect of headspace atmosphere composition on endosulfan I biodegradation Endosulfan I was biodegraded in incubations containing TD inoculum and a headspace that was ¯ushed with Ar instead of the H2:CO2 (80:20) atmosphere that was routinely used (Fig. 5). Endosulfan I degradation, was not signi®cantly di€erent between these two treatments. As described in the previous section, the treatments without any added H2:CO2, i.e. with Ar headspace, still produced methane. This indicated that these cultures were not forming methane from CO2, but from one of the carbon sources in the medium. These ®ndings indicate that the endosulfan-degrading populations studied were not H2, CO2, or H2:CO2 dependent. 3.8. Enumeration and microscopic examination of the cultures Microscopic examination of the inoculated anaerobic medium con®rmed that cell matter increased with time, indicating that the increase in turbidity was due to microbial growth. The number of microorganisms able to grow on Medium No. 1 of Balch et al. (1979) solidi®ed with 1% agar (Oxoid), under strictly anaerobic conditions, were 105±106 cfu/ml. These were enumerated in the routine incubations at 10 days. The numbers of microorganisms in cultures after the seventh enrichment were 108±109 cfu/ml. Within the non-enriched cultures
  8. 8. 20 T.F. Guerin / Environmental Pollution 106 (1999) 13±21 Acknowledgements Fig. 5. E€ect of incubation atmosphere on endosulfan I degradation. and the enriched cultures there was no signi®cant di€erence in the numbers of microorganisms (at 5% signi®cance level). No growth was observed in the sterile incubations and no growth was evident when the same inocula were incubated under aerobic conditions. A small proportion of the cells in the enrichment cultures were endospore-forming bacteria (approximately 5% of all ®elds viewed). The majority of the routine cultures possessed cells that were either free or in small chains of two to three cells in length. The enriched cultures contained clusters of Gram-negative rods, often very large in size (50±100 cells each), but were not present in the routine incubations. The predominant Gramnegative rod identi®ed was of the genus Desulfotomaculum (Allen et al., 1985; Dindal, 1991). This genus reduces sulfates to sul®des, and may have been responsible for the black colour of the media. The methanogenic bacteria identi®ed in the cultures were those from the genus Methanobacterium (Allen et al., 1985; Dindal, 1991). This genus is Gram negative which is commonly rod shaped and in chains, and it is mainly found in anaerobic muds. Clostridium and Desulfotomaculum were identi®ed as the genera of the endospore-forming bacteria present in the enrichment cultures (Allen et al., 1985). All the incubations, including those containing 1, 10 and 1000 mg/l endosulfan, possessed predominantly Gram-negative cells. This was also observed when enrichments were made with aldrin and dieldrin (1000 mg/l). These ®ndings can be explained from previous research that has shown that Gram-positive bacteria are more sensitive to the e€ects of cyclodienes (Thomann, 1976; Mancinelli, 1982). Mancinelli (1982) has suggested that the reason Gram-negative bacteria tend to predominate in the presence of organochlorine pesticides, is due to the presence of an outer lipopolysaccharide on their cell walls. These compounds have been postulated to protect the cell from high concentrations of pesticides (Mancinelli, 1982) and this is likely to have been the case with the anaerobic cultures in the current study. To I.R. Kennedy, Department of Agricultural Chemistry and Soil Science, University of Sydney, Australia, for helpful feedback on early drafts. The ideas of Andrew Maule, PHLS Laboratories (Salisbury), are also appreciated, as were the helpful suggestions of Ron Harris (University of Guelph) and Graeme Batley (CSIRO). Funding from the Cotton Research Development Corporation, Australia, and support from Hoechst (Melbourne and Frankfurt) is gratefully acknowledged. References Allen, S.D., Siders, J.A., Marler, L.M., 1985. Isolation and examination of anaerobic bacteria. 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