Environmental Pollution 115 (2001) 219–230
www.elsevier.com/locate/envpol

Abiological loss of endosulfan and related chlo...
220

T.F. Guerin / Environmental Pollution 115 (2001) 219–230

Table 1
Overview of acute toxicity of key endosulfan compou...
T.F. Guerin / Environmental Pollution 115 (2001) 219–230

2.3. Incubation conditions in oxygen-limited study

3. Results a...
222

T.F. Guerin / Environmental Pollution 115 (2001) 219–230

butyl rubber sealed and PTFE-sealed flasks, and the
unsealed...
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T.F. Guerin / Environmental Pollution 115 (2001) 219–230
Table 3
Half-lives (r2 values) in days for pesticide loss us...
224

T.F. Guerin / Environmental Pollution 115 (2001) 219–230

Fig. 3. Experimental vessel and graphical presentation of b...
T.F. Guerin / Environmental Pollution 115 (2001) 219–230

Fig. 4. Dissipation of chlorinated compounds under oxygen-limite...
226

T.F. Guerin / Environmental Pollution 115 (2001) 219–230

Table 4
Summary of bi-phasic loss data from trials with uns...
227

T.F. Guerin / Environmental Pollution 115 (2001) 219–230
Table 5
Half-lives for pesticide loss under oxygen-limited c...
228

T.F. Guerin / Environmental Pollution 115 (2001) 219–230

if there was complete transformation. Some of the differenc...
T.F. Guerin / Environmental Pollution 115 (2001) 219–230

less persistent than the related cyclodienes, aldrin and
dieldri...
230

T.F. Guerin / Environmental Pollution 115 (2001) 219–230

Guerin, T.F., Kennedy, I.R., 1992. Distribution and dissipa...
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Abiological loss of endosulfan and related chlorinated organic compounds from aqueous systems in the presence and absence of oxygen

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Abiological loss of endosulfan and related chlorinated organic compounds from aqueous systems in the presence and absence of oxygen

  1. 1. Environmental Pollution 115 (2001) 219–230 www.elsevier.com/locate/envpol Abiological loss of endosulfan and related chlorinated organic compounds from aqueous systems in the presence and absence of oxygen T.F. Guerin * 3/32 Wolli Creek Road, Banksia, Sydney, New South Wales 2216 Australia Received 24 July 2000; accepted 25 January 2001 ‘‘Capsule’’: Endosulfan, related OC pesticides, and major degradation products are studied in aquatic systems and factors influencing persistence, and implications to biodegradation studies are explored. Abstract Endosulfan is a cyclodiene organochlorine currently widely used as an insecticide throughout the world. This study reports that the endosulfan isomers can be readily dissipated from aqueous systems at neutral pH in the absence of biological material or chemical catalysts, in the presence or absence of oxygen. The study showed that aldrin, dieldrin, and endosulfan exhibit bi-phasic loss from water in unsealed and butyl rubber sealed vessels. Half-lives are substantially increased for endosulfan I when oxygen is removed from the incubation vessel. The study conditions, where PTFE was used, were such that loss due to volatilization and alkaline chemical hydrolysis was eliminated. Half-lives determined from these data indicate that the parent isomers are much less persistent than the related cyclodienes, aldrin and dieldrin, confirming the findings of previous studies. The major oxidation product of endosulfans I and II, endosulfan sulfate, is less volatile and can persist longer than either of the parent isomers. Endosulfan sulfate was not formed in any of the treatments suggesting that it would not be formed in aerated waters in the absence of microbial activity or strong chemical oxidants. Since endosulfan sulfate is formed in many environments through biological oxidation, and is only slowly degraded (both chemically in sterile media and biologically), it represents a predominant residue of technical grade endosulfan, which finds its way into aerobic and anaerobic aquatic environments. The data obtained contributes to and confirms the existing body of half-life data on endosulfan I and II and its major oxidation product, endosulfan sulfate. The half-life data generated from the current study can be used in models for predicting the loss of chlorinated cyclodiene compounds from aqueous systems. The findings also highlight the importance of critically reviewing half-life data, to determine what the predominant processes are that are acting on the compounds under study. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Endosulfan; Degradation; Pesticide; Half-lives; Hydrolysis; Non-biological degradation; Bi-phasic loss; Persistence; Abiotic loss; Biodegradation; Endosulfan sulfate; Toxicity; Physico-chemical; Risk 1. Introduction Endosulfan is a cyclodiene organochlorine possessing a labile, cyclic sulfite diester group. Endosulfan is a widely used agricultural chemical and it has been detected in an increasing number of environmental samples in recent years (Guerin and Kennedy, 1991; Guerin, 1993; Mansingh and Wilson, 1995; Mansingh et al., 1997; Miles and Pfeuffer, 1997; Guerin, 1999b). Of key * Present address: Shell Engineering Ltd, NSW State Office, PO Box 26, Granville 2142 NSW, Australia. E-mail address: turlough. guerin@shell.com.au. E-mail addresses: turloughg@hotmail.com, turlough.guerin@ bigpond.com (T.F. Guerin). concern regarding its widespread distribution, particularly in water environments, is its high acute toxicity to fish (Table 1). There are, however, relatively few studies describing the fate of endosulfan in aquatic systems (Greve, 1971; Walker et al., 1988; Peterson and Batley, 1991; Singh et al., 1991; Guerin and Kennedy, 1992; Guerin, 1993; Peterson and Batley, 1993; Mansingh and Wilson, 1995; Mansingh et al., 1997; Miles and Pfeuffer, 1997). In addition, it has not been extensively determined to what extent losses of the endosulfan isomers result from chemical degradation as opposed to dissipation by other means, such as volatilization and adsorption, in aqueous systems (Guerin and Kennedy, 1992; Guerin, 1993). Although there are a number of reports describing biological oxidation of endosulfan to 0269-7491/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(01)00112-9
  2. 2. 220 T.F. Guerin / Environmental Pollution 115 (2001) 219–230 Table 1 Overview of acute toxicity of key endosulfan compoundsa Compound Table 2 Liquid-phase physico-chemical properties of major endosulfan compounds Toxicity LD50 (mg kgÀ1) Insects Endosulfan I Endosulfan II Endosulfan sulfate Endosulfan diol Fish Birds Mammals 5.5 9.0 9.5 >500 0.001–0.01b 0.001–0.01 0.001–0.01 1–10 26–1000 26–1000 –c – 9.4–40 177 8–76 >1500 a Summarized from the literature (Guerin, 1993; Anonymous, 1998). b The lower the lethal dose, i.e. LD50 value, the higher the toxicity. c –, indicates that there was no data available. the sulfate, there is no clear evidence for its formation in sterile soils or water (Guerin, 1993). This indicates that living organisms may be necessary to bring about the oxidation of endosulfan to form endosulfan sulfate in the environment. Previous studies have also reported that isomerization can occur between the parent isomers in aqueous systems, with the reaction favouring formation of endosulfan I (a-endosulfan; Schmidt et al., 1997). The primary aim of the research described in this paper was to determine the half-life of the parent endosulfan compounds in sterile aqueous solutions with and without the presence of oxygen. A further aim was to determine whether endosulfan sulfate could be formed under these conditions. 2. Materials and methods 2.1. Chemicals Endosulfan and its degradation products were a gift from Hoechst, Germany. Aldrin and dieldrin were provided by Shell Chemicals, Australia. cis- and transAldrin diol were kindly provided by Shell Chemicals Research, UK. Hexane (Nanograde) and methanol (ChromAR) were purchased from Mallinckrodt Chemicals. The key physico-chemical properties of the endosulfan compounds are summarized in Table 2. 2.2. Incubation conditions in aerobic study The pesticides, endosulfan I, II, aldrin and dieldrin and the endosulfan degradation product, endosulfan sulfate (100–500 ppm in 10 ml methanol) were added to either Nanopure-filtered, distilled and deionized water, sterilized by autoclaving at 121 C for 20 min, or sterilized 100 mM potassium phosphate-buffered yeast mannitol medium with filtered (45 m) soil extract (10% v/v of a 50 g lÀ1 soil in water extract; (Guerin, 1993) in 4 ml Wheaton vials, to give final amounts of 1–5 mg lÀ1. The headspace volume was 2 ml in the vials. The quantities of 1–5 Compound Log Kow v.p. (Pa)a,b Hc Solubility in water (S) ppma Endosulfan I 0.51 Endosulfan II 0.45 Endosulfan sulfate 0.48 Endosulfan diol 300.0 3.6 3.83 3.66 3.68 0.0004 8.0Â10À5 3.7Â10À5 2.3Â10À6 0.72 0.04 0.03 1.3Â10À4 a Values reported are from the PhysProp and DATALOG Databases from Syracuse Research Corporation where available (Meylan and Howard, 2000). Values for endosulfan diol are from elsewhere (Guerin and Kennedy, 1992; Guerin, 1993). b Vapor pressure in units of Pa. c Henry’s constant (H)=v.p./S in units of Pa m3 molÀ1, calculated from the v.p. and S data reported in this table. mg lÀ1 pesticide used in this study reflects those commonly used in studies of the microbial degradation of pesticides, and in aquatic toxicology, where bioassays are performed. The medium was also sterilized by autoclaving for 20 min at 121 C. It is also noted that the quantities of added pesticide meant that these compounds were close to the limits of the solubility for these compounds in the aqueous phase (Table 2). Duplicate vials were made of unsilanized borosilicate glass and prepared according to the protocol described (Guerin, 1993). The medium contents have previously been described (Guerin, 1993). Vessels were sealed with either Polytetrafluoroethylene (PTFE)-lined butyl rubber seals (Wheaton, Millville NJ, USA supplied by Edwards Instrument, Narellan, Australia) or non-PTFE-lined butyl rubber stoppers. Duplicate vessels were kept at 30 Æ 0.5 C, in an incubator for 30 days. No further attempt was made to artificially aerate the incubation flasks during the course of the experiment, and sterile air at atmospheric pressure from a laminar flow cabinet was used as the gas atmosphere in the headspace of the flasks. The media was kept sterile throughout the experiment, and this was checked by heterotrophic plate counts and microscopic examination (Guerin, 1993). The pH of the media was adjusted to 7 Æ 0.05 with KOH or HCl prior to dispensing into the incubation vessels. Incubation vessels were kept in the dark. Surface microlayer subsamples were also taken, in triplicate, after 4 h (30 C) from vessels set aside especially to determine whether there was any difference in pesticide concentration at the liquid–air interface. This was performed by withdrawing a volume of 100 ml from the surface, an amount equivalent to the top 0.9 mm of the medium, using a pipette. The pipette was rinsed with solvent and the medium extracted as previously described (Guerin and Kennedy, 1992). An equivalent volume removed from the medium bulk was also analyzed for pesticides.
  3. 3. T.F. Guerin / Environmental Pollution 115 (2001) 219–230 2.3. Incubation conditions in oxygen-limited study 3. Results and discussion The incubations conditions were as before except the incubation vessels were evacuated with N2 gas at the commencement of the incubation period. Resazurin was added to the medium and this dye remained in its reduced form throughout the entire incubation period (i.e. clear) in all the incubation vessels. 221 3.1. Analysis using 0–30 day data 2.4. Extraction, recovery and analysis of parent pesticides and degradation products Duplicate incubation vessels containing the aqueous media (2 ml) had their contents quantitatively transferred (i.e. sacrificed) into the reservoir of a 10 ml liquid–liquid partitioning device (Mixxor1 by Genex) and were extracted, recoveries determined, and analyses conducted according to the method previously described (Guerin and Kennedy, 1992). Samples were taken on days 0, 2, 4, 6, 8, 15 and 30. A total solvent volume of 10 ml hexane/acetone/methanol/medium (15:5:2:2) was also added to the Mixxor1 reservoirs. The piston of the Mixxor1 was moved 60 times in its reservoir to partition the pesticides into the solvent phase. After the phases were allowed to separate ($1 min), the solvent layer was decanted off the aqueous phase directly from the Mixxors1 into volumetric flasks and the total volumes were made to either 10 or 25 ml with hexane. Subsamples were dried with anhydrous sodium sulfate prior to analysis by GC–ECD. 2.5. Analysis of data Half-lives for all the compounds were determined. The data analysis was conducted in three stages. During the initial data analysis (incorporating the data collected at all sampling times, i.e. 0–30 days), the square root of the correlation coefficient (r2) was determined from the log exponential decay plots i.e. log 100ÂC/Co vs t plots using the trend line function in Microsoft Excel 2000 (Microsoft). In the second stage of the analyses, data were analyzed, excluding the data at day 15 and 30, to determine whether the pesticide loss was bi-phasic, that is, exponential during the initial phases of the experiment (0–8 days), followed by a later linear phase (8–30 days). Where there was bi-phasic (i.e. non-first-order or non-linear) loss occurring, evident from the 100ÂlogC/Co vs t plots, 8–30 day data was plotted and half-lives were also calculated using this data. Multivariate analysis were conducted to compare sets of data using the regression function in Microsoft Excel 2000 (Microsoft). This generated P-values (5% level of significance) for these comparisons. Univariate analyses were conducted on individual treatments (e.g. aldrin, unsealed, 0–30 days) to determine P values (5% level of significance). For each of the treatments, a line was plotted through the data to determine whether the data collected at all the sampling times fitted an exponential decay curve. Since the concentration data was plotted as log values, then an exponential decay plot would be a straight line on such graphs. The dissipation of aldrin and dieldrin from PTFE sealed vessels observed the first order decay model. This was also the case with the data from dissipation of endosulfan I and II from the unsealed and PTFE sealed flasks, and the dissipation of endosulfan sulfate from unsealed, BRS and PTFE sealed flasks. This is illustrated in Fig. 1A–C. These data indicated, however, that the first order exponential decay model did not adequately describe the losses of pesticide over the entire period of the experiment (0–30 days), particularly for losses of the more volatile pesticides from the unsealed and butyl rubber sealed vessels. The dissipation of endosulfan I and II from water in PTFE-sealed vessels (r2=0.93) and endosulfan sulfate from water in unsealed vessels (r2=0.95) fitted the first order decay model well, and therefore a single half-life value was adequate to describe the loss of these compounds within these treatments. There was a significant difference (P0.05) between the pesticide half-lives in the butyl rubber sealed and PTFE-sealed flasks, and the unsealed and PTFE-sealed vessels, but no differences were measured between the butyl rubber sealed and the unsealed vessels. The half-lives of the compounds in each of the aerobic treatments are recorded in Table 3. 3.2. Analysis using 0–8 day data The data that were obtained earlier in the trial (i.e. up to the 8th day of incubation, only) for the unsealed and BRS sealed treatments, generally showed larger r2 values and an improved fit to the first order exponential decay model (compare columns 2, 3, 5, 6 in Table 3). In the unsealed and butyl rubber sealed treatments, it was found that data from the first 8 days fitted the model of first order exponential decay better than data from over the entire experiment (i.e. 30 days). These findings indicated that the loss of pesticides from the aqueous media, particularly in the butyl rubber-sealed and the unsealed vessels, did display a first order exponential decay, but only over the initial phase of the experiment. Volatilization from the unsealed vessels, and absorption by butyl rubber in the butyl rubber sealed vessels, could explain these losses early in the experiment. As with the 0–30 day data, there was a significant difference (P0.05) between the pesticide half-lives in the
  4. 4. 222 T.F. Guerin / Environmental Pollution 115 (2001) 219–230 butyl rubber sealed and PTFE-sealed flasks, and the unsealed and PTFE-sealed vessels, but no differences were measured between the butyl rubber sealed and the unsealed vessels. The 0–8 day data for aldrin, dieldrin and endosulfan are plotted in Fig. 2A and B. 3.3. Analysis of 8–30 day data When the 8–30 day data from the unsealed treatments was analyzed, increased half-lives were obtained. These half-lives increased from 0.5, 2, and 14 (for the 0–8 day data), to 78, 46, and 29 days for aldrin, dieldrin, and endosulfan I, respectively. These increased half-lives reflected the predominant dissipation processes which were acting on the compounds after the initial fast rate of loss due to volatilization. These longer term rates of loss reflecting the chemical degradation and slow desorption processes, showed that there was an 18–75Â increase in the half-life length when data from 8–30 days was considered. These slower processes were chemical degradation and slow desorption of the compounds from the glass and through the aqueous media, and subsequent volatilization (Fig. 3). 3.4. Losses of aldrin and dieldrin There were high rates of loss of aldrin and dieldrin from both unsealed incubation vessels and flasks sealed with butyl rubber when the 0–30 day data was considered. In the butyl rubber sealed vessels, aldrin and dieldrin had largely disappeared at the 30th day, with half-lives of 2.9 and 3.7 days, respectively. These results were similar to the values of 0.5 and 2 days for half-lives in the open flasks. The only difference in the pattern of loss between these two treatments was that the initial rate of dieldrin disappearance in the unsealed vessels was slightly higher than in the butyl rubber sealed vessels. In contrast, when aldrin and dieldrin were incubated in similar sterile media or in water alone with PTFE-lined butyl rubber seals, there was a considerably slower rate of loss. The half-lives under these conditions were 58 and 22 days, respectively. Losses of aldrin and dieldrin were very low under conditions of limited oxygen with half-life values of 134 and 46 days, respectively (Fig. 4A and Table 7). The bi-phasic pattern of loss was particularly pronounced for aldrin, dieldrin and endosulfan I loss from unsealed flasks (Table 4). A possible reason for this non-first order loss could have been that a large proportion of pesticide was concentrated at the liquid–air interface (surface microlayer) as previously reported (Guerin and Kennedy, 1992). As a consequence, these pesticides would be expected to volatilize early in the experiment because of their close proximity to the liquid– air interface. Peterson and Batley (1991) have also suggested this mechanism as a reason for the loss of Fig. 1. Dissipation of chlorinated compounds under oxygenated conditions (0–30 day data) (A) aldrin and dieldrin, (B) endosulfan I and II, and (C) endosulfan sulfate. endosulfan from aqueous media in laboratory experiments. After this surface quantity had entirely volatilized, further losses must have come from the solution bulk through the process of diffusion. Therefore another possible reason for this bi-phasic loss may be due to a rim effect, where the more rapid diffusion occurs at high adsorptions (i.e. at concentrations higher than the compound’s solubility in the aqueous medium
  5. 5. 223 T.F. Guerin / Environmental Pollution 115 (2001) 219–230 Table 3 Half-lives (r2 values) in days for pesticide loss using the first order exponential decay model on data from various timesa Compound Unsealed (days) 0–30 Aldrin Dieldrin Endosulfan I Endosulfan II Endosulfan sulfate a b 13.6 2.8 25.8 18.0 37.3 BRS-sealed (days) 0–8 (0.37) (0.65) (0.83) (0.90) (0.95) 0.52 2.01 13.8 )200b 51.8 8–30 (0.73) (0.98) (0.89) (0.001) (0.48) 77.9 46.4 29.0 18.2 35.3 0–30 (0.55) (0.90) (0.93) (0.91) (1.0) 1.1 3.4 7.9 12.7 34.0 PTFE-sealed (days) 0–8 (0.79) (0.94) (0.83) (0.83) (0.5) 2.9 3.7 6.6 9.6 12.4 8–30 (0.98) (0.98) (0.96) (0.96) (0.78) 21.6 2.3 3.0 5.7 68.9 0–30 (0.71) (0.99) (0.66) (0.63) (0.18) 200b 75 21.7 24.2 102.5 0–8 (0.22) (0.54) (0.93) (0.93) (0.56) 57.8 21.8 13.8 36.2 65.1 (0.34) (0.82) (0.89) (0.36) (0.37) Unsealed=cotton wool was used to stopper the flasks, BRS=butyl rubber sealed vessels, and PTFE=Teflon-lined butyl rubber seals. Values of 200 and )200 days were given because of the relatively short time of the trials (30 days). Fig. 2. Dissipation of chlorinated compounds under oxygenated conditions (0–8 day data) (A) aldrin and dieldrin, and (B) endosulfan I and II. early in the experiment) across the glass to the surface. As the pesticide levels decrease in the vessels, the amounts remaining after the initial high losses will more closely approximate the upper limits of the compound’s solubility. This may explain, at least in part, why the pesticide loss is slower after 8–30 days incubation as the forces of solubilization would tend to override those exerted by volatilization (Guerin and Kennedy, 1992; Guerin, 1993). In the current study, approximately 40–50% of the originally applied pesticides recovered from the vessels containing aqueous medium (1–5 mg mlÀ1 originally added), was found to be concentrated at the interfaces of the system. Also, an attempt was made to subsample the liquid–air interface and measure the pesticide concentration. However, there was no detectable difference between the surface sample and that in the bulk of the medium. Therefore, the pesticides must have accumulated at the liquid–glass interface only under these conditions. Although it is possible that a slower rate of hydrolysis could occur at this interface, insulated from the effect of the hydroxyl ion, this is unlikely to explain the increased half-lives for the endosulfan isomers reported in this study with effective sealing. A similar increase in half-life was also observed for the more stable compounds such as dieldrin, when the flasks were sealed with PTFE. This distribution to the glass-medium interface was observed when the compounds under study were added to either water or microbial growth media. An homogenous distribution of pesticide throughout the entire system was achieved by adding 0.1% Tween 80. All of the compounds studied were distributed throughout the system in a similar fashion, and all responded similarly to the detergent treatment in both the sterile distilled water and growth medium. There was a greater distribution of all the pesticides to the liquid–glass interface in the vessels containing pure water. A similar effect was observed by Peterson and Batley (1991) when they subsampled from a larger total volume of water containing both endosulfan isomers at lower concentrations (0.1 ppm) in polycarbonate vessels. These findings and the results from the current study therefore illustrate the importance of avoiding subsampling when analyzing aqueous extracts containing relatively high concentrations of endosulfan and related cyclodienes. The distribution of pesticides in aqueous systems is of particular importance in microbial degradation studies where the availability of the compound is likely to affect its degradation. Thus, when the pesticide is added in small amounts of solvent to the aqueous phase (as has generally been reported in microbial pesticide
  6. 6. 224 T.F. Guerin / Environmental Pollution 115 (2001) 219–230 Fig. 3. Experimental vessel and graphical presentation of bi-phasic loss. degradation studies, often followed by evaporation of solvents with N2 gas), its distribution in the incubation vessel will tend to be associated with the interfaces. The geometry of the incubation vessel as well as the constituents of the medium will effect the pesticide distribution. In microbial degradation experiments, where insoluble compounds are added in methanol or a similar solvent, an apparent increase in pesticide concentration with time will be observed in the bulk of the medium, once exponential growth commences and lipids increase in quantity. This effect may be overcome by completely sacrificing the entire treatment incubation flasks at each sampling time. The inclusion of the very chemically stable cyclodienes, aldrin and dieldrin, in PTFE-sealed vessels in this study provided internal controls that indicated disappearance predominantly from physical losses, thereby providing the maximum limits of these processes in the system studied. The experimental conditions were too mild and the incubation period was too short to allow substantial chemical degradation of these compounds. The persistence of aldrin and dieldrin in these incubations therefore represents the maximum limits for either slow volatilization or other possible processes such as irreversible binding to container surfaces. Thus, any differences between the persistence of these internal controls and that of the endosulfan compounds represents the actual disappearance by chemical reaction. The very slow rate of disappearance of aldrin and dieldrin in the PTFEsealed vessels in both water and growth medium confirmed that the system was well sealed. 3.5. Dissipation of endosulfan isomers In both the butyl rubber sealed and the unsealed vessels, the endosulfan isomers were lost at fast rates. After 8 days of incubation, the calculated half-lives of endosulfans I and II in the butyl rubber sealed flasks were 7.9 and 12.7 days, respectively. When endosulfans I and II were incubated in unsealed vessels, the half-lives varied from 13.8 to 29 and 18 to )200 days, respectively. In both the butyl rubber sealed and the unsealed flasks, comparing 0–8 day data, endosulfan I loss was higher than that of endosulfan II. This result reinforces previous findings that endosulfan I is more volatile than endosulfan II (Goebel et al., 1982; Worthing and Walker, 1987; Singh et al., 1991; Guerin and Kennedy, 1992; Guerin, 1993). When the vessels were sealed with PTFE, rates of disappearance for both isomers in both water and
  7. 7. T.F. Guerin / Environmental Pollution 115 (2001) 219–230 Fig. 4. Dissipation of chlorinated compounds under oxygen-limited conditions (0–30 day data) (A) aldrin, dieldrin, endosulfan I and II (1 ppm), and (B) endosulfan I and II (10 ppm), and (C) endosulfan sulfate (5 and 50 ppm). microbial growth medium were considerably lower comparing 0–30 day data. The half-lives were 21.7 and 24.2 days for endosulfans I and II, respectively. Under oxygen-limited conditions, the half-lives of endosulfan I and II, were )200 and 58 days, respectively (Fig. 4A, B and Table 7). The effect of PTFE sealing was to substantially reduce the volatilization of the parent compounds from the flasks. 225 It was clear from the butyl rubber sealed and unsealed treatments that endosulfan I is more volatile than endosulfan II. Given the relative chemical inertness of the PTFE-sealed systems and that traces of endosulfan diol were detected in the same system, it is reasonable to conclude that both endosulfan isomers were chemically degraded in the aqueous incubations. Based on this data, endosulfan II may be more chemically labile than endosulfan I. In previous studies, it has been observed that endosulfan II also disappeared at a faster rate than endosulfan I. Under aerobic conditions at a lower temperature of 22 C, the half-lives of endosulfans I and II in a potassium phosphate buffered, minimal salts medium (pH 6.5), were 88 and 40 days, respectively (Miles and Moy, 1979). In their paper no mention was made on how the vessels were sealed. The half-lives of endosulfans I and II in non-sterile seawater (pH 8.0) were 4.9 and 2.2 days, respectively (Cotham and Bidleman, 1989). These incubations were carried out aerobically and at 20oC under laboratory lighting. In another study, incubations in lake water showed that the half-life of endosulfan I was 35 days at pH 7 and 105 days at pH 5.5 (Greve, 1971). It was shown in the same study that when iron hydroxide gel is mixed with water, the rate of hydrolysis is considerably accelerated. Other researchers have reported half-lives of 10–43 days under controlled laboratory conditions, at pH values of 8.5 (Southan and Kennedy, 1995), and values of 3 days for both isomers in laboratory water columns of unreported pH (Logan and Barry, 1996). Guerin (1999a) has reported losses of endosulfan I under sterile anaerobic conditions, as part of a 30-day anaerobic biodegradation study, with losses of endosulfan I at 20, 10 and 2% (of that originally applied) when this compound was added at 1, 2, and 10 ppm, respectively, indicating rates of loss are dependent on the mass of added pesticide. The latter findings are consistent with water insoluble pesticides desorbing from the glass surface into the medium. Also, in the Guerin (1999a) study, when microorganisms were present, half-lives of endosulfan I varied between 5 and 15 days, substantially increasing its loss. It has also been shown that endosulfan losses can be significantly minimized from water solutions if the incubation vessels are sealed to prevent volatilization (Guerin and Kennedy, 1992; Guerin, 1993). Several biodegradation studies in liquid culture have demonstrated the importance of sealing incubation vessels with Teflon or PTFE as previously discussed (Guerin, 1995, and references cited therein). However, not all biodegradation studies have employed PTFE and this should be considered as a critical criterion when reviewing and evaluating degradation data reported in the literature. It was noted that there was no interconversion between isomers under the conditions described in the current study.
  8. 8. 226 T.F. Guerin / Environmental Pollution 115 (2001) 219–230 Table 4 Summary of bi-phasic loss data from trials with unsealed vessels Phase of trial (days) Main loss mechanisms Relative rate of loss Calculated half-lives (days) Aldrin Initial (0–8) Latter (8–30) Volatilization Desorption, chemical degradation The role of pH is important, particularly when the rates of endosulfan loss are compared across different studies. This is because the endosulfan isomers are susceptible to alkaline hydrolysis (Goebel et al., 1982). Thus, rates of hydrolysis at pH 8 will be a $10 times faster than the rates at pH 7. Some differences in the half-lives previously reported may be due to differences in temperature, which may also affect the hydrolysis rates of pesticides. Since endosulfans I and II are volatile, the temperature at which the experiments are carried out is also very important. In the current study there was no significant difference (P0.05) in the degradation rates of either endosulfan isomer comparing incubation in water and in the microbial growth medium. This indicates that the soil, peptone, or yeast extract and inorganic minerals had no measurable effect on the persistence of the isomers. 3.6. Losses of endosulfan sulfate In all of the experiments conducted, endosulfan sulfate was relatively stable and considerably more persistent than the parent isomers. The half-life of endosulfan sulfate in the sterile water was calculated at 103 days when sealed with PTFE compared with 30 days in the unsealed vessels. Its persistence in the vessels sealed with butyl rubber (10 day half life), compared with that in the unsealed vessels (30 day half-life), was not significant, and this was likely to be due to the wide variation in the analysis of endosulfan sulfate data as previously reported by (Guerin et al., 1992). The data on the dissipation of endosulfan sulfate from the PTFE-sealed vessels fitted the model of first order exponential decay poorly, when all the sampling times were analyzed. The very low r2 values obtained with endosulfan sulfate in the PTFE-sealed vessels, suggests little or no relationship between endosulfan sulfate concentrations and time. However, from the extraction and analysis of endosulfan sulfate previously reported (Guerin and Kennedy, 1992), it is likely that analytical error was also important and contributed to the very low r2 values. Endosulfan sulfate was even more stable under conditions of limited oxygen, with half-lives typically )200 days (Fig. 4C and Table 5). The results of the dissipation of endosulfan sulfate therefore indicate a limitation of calculating the half-life of this compound using the approach described here. Fast Slow Dieldrin Endosulfan I 0.52 77.9 2.01 46.4 13.8 29 This approach is more appropriate for determining the half-lives of the parent isomers, where the differences between the half-lives are not as great, and where analytical variation is low. Miles and Moy (1979) have also reported on the persistence of endosulfan sulfate in aqueous media and have given a value for its half-life, under the previously described conditions, as 140 days. The reported persistence of endosulfan sulfate in the aqueous systems studied in the current work, and from this report in the literature, indicates that this endosulfan transformation product is likely to remain in water environments much longer than the parent isomers. It has previously been shown not to be readily biodegradable. However, in real environments, there may be other processes of endosulfan sulfate removal such as strong adsorption to soil and sediment particles. It should be recognized that because of the relatively short time frame of the trials, the r2 values and corresponding half-life data for endosulfan sulfate, has been included in the data set for the sake of completeness and these do not represent definitive values. Further research would be needed to determine definitive values for the persistence of this compound in aqueous systems. 3.7. The role of volatilization in pesticide disappearance From the increased losses in the open vessel incubations, it is evident that the endosulfan dissipation in these treatments was primarily owing to volatilization. The rates of volatilization of the endosulfan isomers in the open-vessel experiments were similar to those from the butyl rubber sealed experiments. These results indicated that volatilization of these compounds from the aqueous media has a similar effect as that of the butyl rubber seals. Aldrin and dieldrin were also lost at fast rates in similar incubations, confirming that absorption into the butyl rubber seals was the major cause of loss (Guerin and Kennedy, 1992). Extraction and analysis of the butyl rubber seals after the incubation indicated that all the cyclodienes had become absorbed into this sealing material. No hydrolysis products of endosulfan, endosulfan sulfate, aldrin or dieldrin were detected in hexane– acetone extracts from the open or butyl rubber sealed treatments. Thus, it is evident that the major cause of dissipation of all compounds in the unsealed flasks was
  9. 9. 227 T.F. Guerin / Environmental Pollution 115 (2001) 219–230 Table 5 Half-lives for pesticide loss under oxygen-limited conditions using the first order exponential decay model (0–30 days)a Compound 1 ppmb 10 ppmc Half-life (days) Aldrin Dieldrin Endosulfan I Endosulfan II Endosulfan sulfate r2 Half-life (days) r2 134 46 )200 58 )200 0.003 0.14 0.001 0.24 0.43 – – 200 97 )200 – – 0.001 0.71 0.01 a PTFE-lined butyl rubber sealed vessel, evacuated 7Â prior to incubation (see methods); values 200 days were given because of the relatively short time of the trials (30 days). Values of )200 indicated that the calculated half-lives were greater than 1000 days. b 5 ppm of endosulfan sulfate was used because of its higher analytical detection limits. c 50 ppm of endosulfan sulfate was used because of its higher analytical detection limits. volatilization, and absorption in the butyl rubber sealed flasks, rather than chemical degradation. Volatilization from uninoculated controls in aerobic microbial degradation studies is likely to be a significant factor in overall pesticide disappearance in unsealed systems, particularly with organochlorine pesticides such as endosulfan. In one study, 30% of applied endosulfan I was reported to have volatilized from a seawater/sediment microcosm (sealed with polyurethane) during the first 4 days of the experiment (Cotham and Bidleman, 1989). Others demonstrated that polystyrene absorbed both endosulfan isomers strongly, compared with glass (Peterson and Batley, 1991). The current findings therefore confirm these findings, and illustrate the importance of sealing aqueous systems containing these compounds, with an inert material such as PTFE. The high volatilization rate of endosulfan I, is due to its low water solubility and relatively high vapor pressure, or its high Henry’s constant. The ratio of liquidphase vapor pressure and solubility, or solid-phase vapor pressure and solubility, provides a value for the Henry’s constant. This relationship may be used to show the difference in the relative rates of volatilization of the parent endosulfan isomers and of the recalcitrant cyclodienes, aldrin and dieldrin. In illustrating the importance of the Henry’s constant of a compound, it is convenient to introduce the concept of fugacity. The fugacity is the escaping tendency of a compound from a particular phase. This can be expressed mathematically as f=C/Z. In this expression, f is the fugacity (units of pressure Pa), C is the concentration (units of mol mÀ3) and Z is the fugacity capacity (units of mol mÀ3 PaÀ1). Each compound has its own fugacity and at equilibrium, compounds will accumulate in phases with the lowest fugacity, or highest Z values. So in water, the fugacity capacity is the inverse of the compound’s Henry’s constant (H) (Guerin and Kennedy, 1992). This is described by the equation, Z=Zwater=1/H. The calculated fugacities of the cyclodiene compounds under study are equivalent to their vapor pressure values in the same phase because the concentration (C) is equal to their water solubilities for the solid compounds. In calculating the Henry’s constant, values for water solubility and vapour pressure must be for the same phase, that is, both for the liquid-phase or both for the solid-phase. The values presented in Table 2 are for the solid-phase for each of the pesticides. Some of the behavior observed in the butyl rubbersealed and unsealed vessels can be accounted for by differences in their calculated fugacities. The fastest rates of disappearance from both of these treatments were that of aldrin, which also had the lowest Z value, or greatest fugacity. From the vapor pressure and solubility data obtained from the literature, endosulfan I has a Henry’s constant of 0.72, approximately 18 times that of endosulfan II (H=0.04), which correlates well with the greater rate of disappearance from the nonPTFE-sealed vessels (consistent with volatilization and absorption mechanisms of loss). Aldrin had the highest Henry constant of 4.95, while dieldrin was lower at 0.53. 3.8. Detection and analysis of potential hydrolysis products of endosulfan Trace levels of the hydrolysis product, endosulfan diol, were detected after 30 days incubation in flasks containing either parent isomer of endosulfan at the beginning of the experiment. The recovery treatments showed that this degradation product was extracted when spiked into zero time vessels. The identity of endosulfan diol was confirmed using two different gas chromatographic columns (Guerin and Kennedy, 1992). The highest concentrations of endosulfan diol were detected in the PTFE-sealed incubations. With endosulfan I, these concentrations were 0.08–0.1 ppm of endosulfan diol after 30 days. This rate of endosulfan diol formation correlates well for the calculated half-life of endosulfan I of approximately 22 days in the sterile media. This rate of formation, however, was not stoichiometric, as approximately 0.5 ppm of endosulfan diol would have been expected to form over this period
  10. 10. 228 T.F. Guerin / Environmental Pollution 115 (2001) 219–230 if there was complete transformation. Some of the difference between the amount of endosulfan diol expected and that which was observed, may have been due to the reduced extraction efficiency of the endosulfan diol. Higher concentrations of 0.1–0.15 ppm endosulfan diol were detected in the endosulfan II incubations under the same conditions, consistent with its lower chemical stability. Much lower amounts of endosulfan diol (0.01 ppm) were detected in the endosulfan sulfate incubations after 30 days, and then only in PTFE-sealed incubations. These findings correlate well with the observed stability of this compound under these conditions. The potential hydrolysis products of dieldrin, cis- and trans-aldrin diol, were not detected in any of the treatment incubations containing dieldrin, although the underivatized standard compounds were chromatographed successfully under the conditions described for analyzing the parent compounds (Guerin et al., 1992). Given the highly recalcitrant nature of dieldrin, and the mild incubation conditions of water and growth medium, no hydrolysis products were expected to form. Polytetrafluoroethylene (PTFE) exhibits chemical and physical properties which, when coated onto butyl or silicone rubber, make it suitable for sealing aqueous media that is in contact with semi-volatile or hydrophobic compounds such a the cyclodiene pesticides. These characteristics of PTFE are its high resistance to heat, inertness to chemical attack over a wide range of temperatures, low moisture absorption and permeability (0.01% in 24 h), high physical strength, very high thermal stability, and flexibility. Because of its high resistance to temperature, it is also autoclavable (Schlanger and Baumgartner, 1980; Guerin, 1993). However, dry heat and radiation may also sterilize it. These properties are listed in Table 6. Since this material is impermeable to gases, including N2, O2, H2, and CO2, it can also be used to maintain anaerobic conditions in flasks containing media for the growth of microorganisms. Of these properties, its high chemical resistance is of greatest importance in biodegradation studies as it prevents absorption of the compounds under study, into the sealing material. 4. Conclusions This study reports that the endosulfan isomers can be dissipated from simple aqueous systems at neutral pH in the absence of biological material or chemical catalysts. When the incubation vessels are sealed with PTFE, then endosulfan II is more readily degraded than endosulfan I, a phenomenon already observed in various aqueous systems. The study also showed that under PTFE-sealed conditions, but in oxygen-limited conditions, the halflives are more than doubled indicating that the parent isomers of endosulfan are more stable under these conditions. This result is in contrast to that obtained in unsealed systems, where the loss of endosulfan I is greater than that of endosulfan II. Half-lives determined from the data indicate that the parent isomers are much Table 6 Physico-chemical properties of polytetrafluoroethylene (PTFE) Inertness to chemical attack Low moisture absorption and permeability Impermeable to N2, O2, H2 and CO2 High physical strength and high thermal stability Low coefficient of friction Very low dielectric constant and excellent electrical insulator Non-stick (anti-adhesion) surfaces Flexible, making suitable for sealing vessels in biodegradation or dissipation studies Table 7 Implications and recommendations from the current study 1. The endosulfan isomers, like aldrin and dieldrin, volatilize readily from incubation vessels containing microbial growth media, which are unsealed, or sealed with butyl rubber. Published values for half-lives of volatile and semi-volatile values should be critically reviewed prior to use in modelling their degradation to see whether the study has taken bi-phasic effects into account and use the data accordingly. Biodegradation assays should include sterilized cells as a control to minimize glass surface binding effectsa. Half-life values generated in the current study can be used in modelling the dissipation of the endosulfan and related compounds from aqueous systems. When endosulfan (or aldrin and dieldrin) are added to aqueous media at levels higher than their solubility in water, adsorption effects are likely to retain the pesticide at the glass-media interfaces until microbial growth becomes significant, and causes it to desorb. Therefore in sterile treatments, entire vessels should be extracted without prior subsampling, particularly with compounds that have low water solubility. Anaerobic biodegradation assays should be sealed with Teflon-lined butyl rubber to avoid pesticide absorption, while maintaining an oxygen impermeable seal. 2. 3. 4. 5. 6. a Particularly in aerobic studies where the medium surface has direct contact with the atmosphere through a cotton wool plug.
  11. 11. T.F. Guerin / Environmental Pollution 115 (2001) 219–230 less persistent than the related cyclodienes, aldrin and dieldrin. However, the major oxidation product of endosulfans I and II, endosulfan sulfate, is less volatile and can persist longer than either of the parent isomers. Endosulfan sulfate was not formed in any of the treatments in the current study. This suggests that endosulfan sulfate would not be formed in aerated waters in the absence of microbial activity or strong chemical oxidants. Since endosulfan sulfate is formed in many natural environments through biological oxidation, and is only slowly degraded (both chemically in sterile media and biologically), it represents a predominant residue of endosulfan in aerobic aquatic environments. Both the endosulfan isomers dissipated from the incubation vessels at faster rates when the vessel was sealed with butyl rubber, than when they were sealed with PTFE. Conversely, the relatively inert PTFE seals greatly reduced losses from volatilization and absorption, thus providing the necessary conditions for studying the chemical degradation of the cyclodienes. Analysis of the data on the dissipation of the cyclodienes has indicated that the loss of the more volatile cyclodienes, aldrin, dieldrin and endosulfan I, from unsealed and butyl rubber sealed treatments, is bi-phasic. The fact that there was very little difference between the rates of dissipation of aldrin and dieldrin from media sealed with butyl rubber and that which were unsealed, showed that butyl rubber sealing was ineffective. As known from previous biodegradation studies, such a rubber seal is therefore unsuitable for microbial degradation studies when endosulfan or other volatile/ semi-volatile compounds are studied. Although butyl rubber has a very low permeability towards oxygen, it has a high affinity for organic compounds (e.g. hexane and volatile organochlorine pesticides). Conversely, PTFE, due to its very low coefficient of friction and resistance to chemical reaction, has an extremely low porosity to volatile/semi-volatile organic compounds. PTFE-lined rubber therefore provides an ideal seal for anaerobic degradation studies with compounds of high volatility, for example see Guerin (1999a). A significant finding was the complete absence of the formation of endosulfan sulfate. This is a toxic degradation product (Table 1), and is the major oxidative product of endosulfan in the environment. This was true for both sterile incubations and incubations containing soil extracts in the well defined liquid media used for cultivating anaerobic and aerobic bacteria. From the current study, it is unlikely that endosulfan sulfate forms in naturally occurring waters under anaerobic conditions, either with or without microorganisms present. However, endosulfan diol was formed in the sterile incubations, indicating that this degradation product may be formed in the absence of any microbial activity. Furthermore, under the conditions described, there was 229 no interconversion between the parent isomers of endosulfan during the study period. Further implications for studying the behaviour of chlorinated organic compounds in aqueous systems are also given (Table 7). It is imperative that in any aqueous incubation containing volatile/semi-volatile organic compounds, such as endosulfan, aldrin or dieldrin, special precautions must be taken to reduce volatilization. An important demonstration in this study is that of the necessity to seal aqueous incubation vessels with Teflonlined butyl rubber seals to prevent volatilization which would have otherwise reduced the apparent half-lives of the compounds under study. For these volatile organochlorines, unlined butyl rubber was shown to be ineffective as a vessel stopper, and may even enhance the loss of these compounds from sterile aqueous media. These findings are fundamental to the design of future biodegradation experiments as losses of these compounds due to volatilization, as well as from chemical hydrolysis, are also likely to occur. These losses can confound the results of biodegradation experiments, making it difficult to determine which losses are actually a result of biological activity. 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The relative significance of biodegradation and physico-chemical dissipation of endosulfan from water and soil. Department of Agricultural Chemistry and Soil Science University of Sydney, Sydney, New South Wales, Australia, pp. 240. Guerin, T.F., 1995. Anaerobic biodegradation of the chlorinated hydrocarbon endosulfan. In: Hinchee, R.E., Hoeppel, R.E., Anderson, D.B. (Eds.), Bioremediation of Recalcitrant Organics, Vol. 7. Battelle Press, Ohio, pp. 157–164. Guerin, T.F., 1999a. The anaerobic degradation of endosulfan by indigenous microorganisms from low-oxygen soils and sediments. Environmental Pollution 106, 13–21. Guerin, T.F., 1999b. Natural attenuation of a low mobility chlorinated insecticide in low-level and high-level contaminated soil: a feasibility study. Remediation 9, 51–63. Guerin, T.F., Kennedy, I.R., 1991. The biodegradation of endosulfan in cotton growing soils. The Australian Cotton Grower 12, 13–15.
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