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Degradation of ethylenethiourea (ETU) in oxic and anoxic sandy aquifers
FEMS Microbiology Reviews 20 (1997) 539^544
Degradation of ethylenethiourea (ETU) in oxic and anoxic sandy aquifers Ole Stig Jacobsen a *, Rossana Bossi ;
a b
b
Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark National Environmental Research Institute, Frederiksborgvej 399, DK-4000 Roskilde, Denmark
Abstract Ethylenethiourea is an important degradation product of ethylenebisdithiocarbamate fungicides, which are widely used in different kinds of crops. The ethylenebisdithiocarbamate group includes maneb, zineb and mancozeb. The ethylenebisdithiocarbamates are not highly toxic and degrade rapidly in the presence of moisture and oxygen, forming different compounds. One of these is the polar ethylenethiourea, which is relatively stable. Thus, this compound appears to be a potential contaminant for groundwater. Batch experiments were carried out under biotic as well as abiotic conditions to study the degradation dependence of concentration, temperature and organic matter. The decomposition of ethylenethiourea under abiotic conditions was found to be less than 5% of the degradation under biotic conditions. Further, ethylenethiourea showed to be stable over a period of 150 days at 20³C in tap water as well as in batch with soil sterilized with NaN3 . The degradation of ethylenethiourea depends on the concentration in the water implying first order reaction kinetics. The microbial degradation of ethylenethiourea is highly temperature dependent with aerobic Q10 between 2.9 and 4.2, and an anaerobic between 2.1 and 2.5. A minor increase in degradation rates was observed by application of nitrate and manure to the batches. The experiments show extremely complete degradation of ethylenethiourea in the presence of microbial nitrate reduction with pyrite which occurs in deeper parts of the aquifers. Keywords :
Ethylenethiourea; Groundwater; Degradation ; Microbiology ; Fungicide ; Kinetics
Contents 1. Introduction . . . . . . . . . . . . . . 2. Materials and methods . . . . . . 2.1. Analytical method . . . . . . 2.2. Experimental methods . . . 2.3. Batch experiments . . . . . . 2.4. Pure culture isolation . . . . 3. Results and discussion . . . . . . . 3.1. Biotic-abiotic batch . . . . . 3.2. Oxic-anoxic batch . . . . . . . 3.3. Temperature dependence . . 3.4. Concentration dependence
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* Corresponding author. Tel.: +45 (3814) 2000; Fax: +45 (3814) 2050; E-mail: [email protected] 0168-6445 / 97 / $32.00 ß 1997 Published by Elsevier Science B.V. All rights reserved PII S 0 1 6 8 - 6 4 4 5 ( 9 7 ) 0 0 0 3 2 - 6
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3.5. Reduction processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction
Ethylenethiourea (ETU) (2-imidazolidinethione) is an important degradation product of ethylenebisdithiocarbamate (EBDC) fungicides, which are widely used in Denmark for potato plant protection with a treatment frequency of up to 6 times during the growing season. ETU is also present as impurity (up to 10%) in EBDC formulations. The EBDCs are not highly toxic and degrade rapidly in the presence of moisture and oxygen, forming di¡erent compounds. One of these is ETU, which is relatively stable and polar. These two characteristics make this compound a potential contaminant for groundwater. The relative stability of ETU and its classi¢cation as a probable human carcinogen [1] make environmental studies on its persistence and mobility in soil urgently needed. ETU is only weakly adsorbed to soil particles and therefore its high soil mobility makes it a potential contaminant for groundwater. ETU can be degraded by microorganisms to ethyleneurea (EU), which is further degraded to CO2 (Fig. 1). Low soil temperatures, anaerobic conditions and/ or absence of microorganisms will result in a substantial reduction in the degradation rate of ETU. Although some ETU degradation studies have been reported [2^5], the kinetics of microbial degradation have not been studied in detail. This study describes degradation kinetics of di¡erent concentrations of ETU in a sandy soil under the in£uence of various temperatures and redox conditions. Further, the in£uence of the presence/absence
543 543 543 544
of nitrate and manure was tested. Soil samples were taken from a sandy locality in western Jylland (Fladerne B×k) where EBDC fungicides were in use. 2. Materials and methods
2.1. Analytical method
Analyses of ETU residues were performed with high performance liquid chromatography (HPLC) and ultraviolet detection (UV). The method proposed by Hogendoorn et al. [6] for the analysis of ETU in aqueous samples was employed. This method is based on the use of two C18 analytical columns with column switching and allows direct injection of water samples without clean-up. The column switching method was applied in this study for the detection of ETU in nearly all water samples from the batch experiments, when the concentration of ETU was high enough (10 Wg l31 ) to be detected without any pre-concentration step. When the concentration of ETU was expected to be lower than the detection limit of the column switching method, the samples were extracted with the liquidliquid extraction method used by Hogendoorn et al. [6] and the extracts were analyzed by HPLC with one analytical column. The HPLC system (Waters, Milford, USA) consisted of two Model 510 reciprocating pumps, a WISP 712 autosampler and a UV detector Model 440 equipped with extended UV module containing a cadmium lamp for detection at 229 nm wavelength.
Fig. 1. Simpli¢ed decay path the EBDC fungicides.
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Column switching analyses were performed with a six-port valve from Rheodyne. The analytical column was a C18 Hypersil ODS (Phenomenex), 0.4U250 mm. The operating conditions were: mobile phase, ammonium acetate bu¡er 0.025 M (pH 7.5) +0.05% tetrahydrofurane; £ow rate, 1 ml min31 ; injection volume, 100 Wl. 2.2. Experimental methods
Soil samples were collected using piston coring (Nordrill, Denmark) while drilling wells in the Fladerne B×k watershed. The samples were used for chemical and microbial investigations in the laboratory and for ETU degradation experiments. All samples were stored at maximum 5³C until use. Samples collected below the water table were kept in air-tight containers in order to prevent oxidation of reduced compounds. In order to sterilize the soil an addition of sodium azide (NaN3 ) has proved to be adequate for inhibition of the microbial activity. The ¢nal concentration was 50 Wg ml31 and had no e¡ect on ETU analyses. Inorganic analyses were performed by ion chromatography (Dionex, CA, USA). As Na-azide interfered with the quanti¢cation of nitrate, the determination of nitrate was made by £ow injection colorimetry using a FIA colorimeter (Tecator, Sweden).
4.2) or air to maintain anoxic or oxic conditions. After an initial £ushing for 10 min a periodic £ush (30 s h31 ) was established and maintained during the batch run. The air £ush performed a circulation in the batch without changing the CO2 content signi¢cantly. Collection of anoxic water samples was done during constant argon £ush excluding oxygen to enter the system. The batches were incubated in the dark at constant temperature (5, 10 and 23³C). All experiments were done in duplicate or triplicate and were supplemented in di¡erent experimental runs. Water samples for ETU analyses were preserved with Na-azide and kept at 4³C until analysis, whereas samples for inorganic constituents were analyzed immediately. 2.4. Pure culture isolation
The isolation was performed by Dr. Vinther, at the Danish Institute of Plant and Soil Science. The soil was suspended in 10 ml sterile demineralised water, and shaken for 30 s for preparation of dilution series. Dilution plating was performed on four replicate plates and inoculated from each soil dilution. The plates were incubated for 2 weeks at 25³C before counting. The assay method has been described by Smithgrenier and Adkins [9]. The inoculation method in liquid medium on microtiter plates has been described by Koëlbel-Boelke et al. [10].
2.3. Batch experiments
Batch experiments were carried out in glass containers (jars) of 1, 3 or 5 l with a gas-tight lid. Three ports for sampling and gas £ushes were built into the lid. Depending on the volume needed for analyses the experiments were set up in di¡erent sizes using a rate of 100^200 g soil l groundwater31 . Soil and groundwater were transferred to the jars, which were instantly closed and £ushed with either argon (grade Table 1 Microbial decay rates for ETU in batch experiments Temperature Oxic decay (³C) (Wg/kg soil day) 5 1.93^2.30 10 2.22^3.03 23 11.4^13.3
3. Results and discussion
3.1. Biotic-abiotic batch
In order to determine whether ETU decomposition is generated by microorganisms or by an abiotic process, the ¢rst batch was set up using fresh soil and sterilized soil. The stability of ETU in a groundwater solution was also tested.
Anoxic decay (Wg/kg soil day) 0.78^0.94 1.01^1.39 2.41^3.80
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Anoxic+manure (Wg/kg soil day)
Anoxic+nitrate (Wg/kg soil day)
2.80^3.16
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Fig. 2. Degradation of ETU in experimental batch (n =2). Initial concentration is 125 Wg l31 .
For a period of 120 days ETU was shown to be stable in groundwater at 20³C, whereas in the fresh soil batch the initial concentration of 105 Wg l31 declined to 16þ 4 Wg l31 . The abiotic batches decreased to about 88 Wg l31 , whereas the groundwater batch with no soil showed no change in concentration, which con¢rms an earlier work by Herrchen [7]. The decomposition of ETU seems to be mostly biotic in an oxic environment as earlier suggested by Miles and Doerge [2]. A number of bacterial isolates from the soil samples representing di¡erent depths were tested for their ability to use ETU as sole source of carbon and energy [8] (Table 1). The results showed that a relatively high number of isolates were able to use ETU as electron donor, indicating that a large proportion of the microbial biomass may be involved in the degradation.
The decomposition of ETU approximated a ¢rst order decay, which has been suggested by Satterthwaite [11] (Fig. 2). Over 50 days the oxic decomposition was higher than the anoxic decomposition (4.5 Wg ETU kg soil31 day31 vs. 3.2 Wg ETU kg soil31 day31 ). Anaerobic decomposition was formerly believed to be non-existent [12]. Further, abiotic decay under oxic conditions was detected to be less than 5%, whereas no decay was observed under anoxic conditions.
3.2. Oxic-anoxic batch
The oxygen status in the groundwater may be of major importance to the microbial decomposition of ETU. Parallel sets of biotic and abiotic batches were therefore incubated at 20³C for 50 days under oxic and anoxic conditions. The initial concentration was 125 Wg l31 ETU corresponding to a residue concentration in soil water after one spray event with mancozeb, e.g. 2 kg active ingredient ha31 .
Fig. 3. Decay rate of ETU in experimental batch (n = 3) under oxic and anoxic condition as a function of temperature.
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matter or additional oxidation equivalents on the decay rate. However, the results showed only a slight increase of the decay rate (Table 2). 3.5. Reduction processes
Fig. 4. Decay rate of ETU as a function of initial concentration.
Soil samples from the deeper part of the aquifer contain minor amounts of pyrite and lignite. By reaction with nitrate the pyrite will be oxidized by microbial processes [13,14]. Incubation under anoxic conditions showed that ETU was degraded to 70% in 20 days in the absence of nitrate, whereas ETU was totally depleted if nitrate reduction by pyrite was present.
3.3. Temperature dependence
The temperature dependence was tested at 5, 10 and 23³C in both oxic and anoxic batches. A rather high Q1 was estimated from the results under oxic conditions. Q10 was calculated to be in the range of 2.9^4.5. Under anoxic conditions the Q10 was found to be 2.1^2.5, which is normally found in pesticide decay experiments (Fig. 3). A more detailed study to describe the mechanisms behind the high Q10 has to be carried out. 3.4. Concentration dependence
The dependence of the initial ETU concentration on the decay was determined at 23³C under oxic conditions. Three levels of ETU concentrations were tested, 20, 125 and 500 Wg l31 . The results showed a nearly linear correlation between decay rate and concentration, which supports a ¢rst order reaction for estimating the decay (Fig. 4). Addition of manure or nitrate to anaerobic batches was carried out to study the e¡ect of organic
4. Conclusions
In summary, it may be concluded that ETU decomposition is mainly microbial and may under certain conditions be coupled to other microbial processes in the aquifer. Further, this study has shown that the degradation might not depend on very specialized microorganisms, and that it takes place in oxic as well as in anoxic reducing environments. The results are in agreement with analyses of ETU in soil water and groundwater under potato ¢elds at Fladerne B×k, as only very small amounts have been detected in spite of a previously applied amount of up to 16 kg EBDC ha31 to the ¢elds. Acknowledgments
We wish to thank the Pesticide Foundation of the Danish EPA for ¢nancial support, Grant 7041-0002. Also we appreciate microbial counting done by Dr. Vinther.
Table 2 Bacterial isolates able to use ETU or MCPP as sole source of carbon and energy (from [7]) Depth (m below surface) Total number of isolates ETU positive isolates % positive 0.20 24 15 62 0.50 24 13 54 0.90 24 7 29 4.00 22 5 23 13.0 24 8 33
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MCPP positive isolates 11 12 9 10 7
% positive 46 50 38 42 29
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References
lected Pesticides in a Sandy Agricultural Watershed, Fladerne B×k, Jylland. Completion report (Jacobsen, O.S., Ed.). DCP,
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the rat. J. Agric. Food Chem. 21, 324^329. [2] Miles, C.J. and Doerge, D.R. (1991) Fate of ethylenethiourea in Hawaiian soil and water. J. Agric. Food Chem. 39, 214^ 217.
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[7] Herrchen, M. (1987) Aerobic bio-degradation of the EBDCfungicide maneb in soil. Report PW-102, Frauenhofer-Institut
[12] Herrchen,
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[10] Ko ë lbel-Boelke, J., Anders, E.-M. and Nehrkorn, A. (1988)
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Matter
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Degradation of ethylenethiourea (ETU) in oxic and anoxic sandy aquifers Ole Stig Jacobsen a *, Rossana Bossi ;
a b
b
Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark National Environmental Research Institute, Frederiksborgvej 399, DK-4000 Roskilde, Denmark
Abstract Ethylenethiourea is an important degradation product of ethylenebisdithiocarbamate fungicides, which are widely used in different kinds of crops. The ethylenebisdithiocarbamate group includes maneb, zineb and mancozeb. The ethylenebisdithiocarbamates are not highly toxic and degrade rapidly in the presence of moisture and oxygen, forming different compounds. One of these is the polar ethylenethiourea, which is relatively stable. Thus, this compound appears to be a potential contaminant for groundwater. Batch experiments were carried out under biotic as well as abiotic conditions to study the degradation dependence of concentration, temperature and organic matter. The decomposition of ethylenethiourea under abiotic conditions was found to be less than 5% of the degradation under biotic conditions. Further, ethylenethiourea showed to be stable over a period of 150 days at 20³C in tap water as well as in batch with soil sterilized with NaN3 . The degradation of ethylenethiourea depends on the concentration in the water implying first order reaction kinetics. The microbial degradation of ethylenethiourea is highly temperature dependent with aerobic Q10 between 2.9 and 4.2, and an anaerobic between 2.1 and 2.5. A minor increase in degradation rates was observed by application of nitrate and manure to the batches. The experiments show extremely complete degradation of ethylenethiourea in the presence of microbial nitrate reduction with pyrite which occurs in deeper parts of the aquifers. Keywords :
Ethylenethiourea; Groundwater; Degradation ; Microbiology ; Fungicide ; Kinetics
Contents 1. Introduction . . . . . . . . . . . . . . 2. Materials and methods . . . . . . 2.1. Analytical method . . . . . . 2.2. Experimental methods . . . 2.3. Batch experiments . . . . . . 2.4. Pure culture isolation . . . . 3. Results and discussion . . . . . . . 3.1. Biotic-abiotic batch . . . . . 3.2. Oxic-anoxic batch . . . . . . . 3.3. Temperature dependence . . 3.4. Concentration dependence
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* Corresponding author. Tel.: +45 (3814) 2000; Fax: +45 (3814) 2050; E-mail: [email protected] 0168-6445 / 97 / $32.00 ß 1997 Published by Elsevier Science B.V. All rights reserved PII S 0 1 6 8 - 6 4 4 5 ( 9 7 ) 0 0 0 3 2 - 6
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3.5. Reduction processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction
Ethylenethiourea (ETU) (2-imidazolidinethione) is an important degradation product of ethylenebisdithiocarbamate (EBDC) fungicides, which are widely used in Denmark for potato plant protection with a treatment frequency of up to 6 times during the growing season. ETU is also present as impurity (up to 10%) in EBDC formulations. The EBDCs are not highly toxic and degrade rapidly in the presence of moisture and oxygen, forming di¡erent compounds. One of these is ETU, which is relatively stable and polar. These two characteristics make this compound a potential contaminant for groundwater. The relative stability of ETU and its classi¢cation as a probable human carcinogen [1] make environmental studies on its persistence and mobility in soil urgently needed. ETU is only weakly adsorbed to soil particles and therefore its high soil mobility makes it a potential contaminant for groundwater. ETU can be degraded by microorganisms to ethyleneurea (EU), which is further degraded to CO2 (Fig. 1). Low soil temperatures, anaerobic conditions and/ or absence of microorganisms will result in a substantial reduction in the degradation rate of ETU. Although some ETU degradation studies have been reported [2^5], the kinetics of microbial degradation have not been studied in detail. This study describes degradation kinetics of di¡erent concentrations of ETU in a sandy soil under the in£uence of various temperatures and redox conditions. Further, the in£uence of the presence/absence
543 543 543 544
of nitrate and manure was tested. Soil samples were taken from a sandy locality in western Jylland (Fladerne B×k) where EBDC fungicides were in use. 2. Materials and methods
2.1. Analytical method
Analyses of ETU residues were performed with high performance liquid chromatography (HPLC) and ultraviolet detection (UV). The method proposed by Hogendoorn et al. [6] for the analysis of ETU in aqueous samples was employed. This method is based on the use of two C18 analytical columns with column switching and allows direct injection of water samples without clean-up. The column switching method was applied in this study for the detection of ETU in nearly all water samples from the batch experiments, when the concentration of ETU was high enough (10 Wg l31 ) to be detected without any pre-concentration step. When the concentration of ETU was expected to be lower than the detection limit of the column switching method, the samples were extracted with the liquidliquid extraction method used by Hogendoorn et al. [6] and the extracts were analyzed by HPLC with one analytical column. The HPLC system (Waters, Milford, USA) consisted of two Model 510 reciprocating pumps, a WISP 712 autosampler and a UV detector Model 440 equipped with extended UV module containing a cadmium lamp for detection at 229 nm wavelength.
Fig. 1. Simpli¢ed decay path the EBDC fungicides.
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Column switching analyses were performed with a six-port valve from Rheodyne. The analytical column was a C18 Hypersil ODS (Phenomenex), 0.4U250 mm. The operating conditions were: mobile phase, ammonium acetate bu¡er 0.025 M (pH 7.5) +0.05% tetrahydrofurane; £ow rate, 1 ml min31 ; injection volume, 100 Wl. 2.2. Experimental methods
Soil samples were collected using piston coring (Nordrill, Denmark) while drilling wells in the Fladerne B×k watershed. The samples were used for chemical and microbial investigations in the laboratory and for ETU degradation experiments. All samples were stored at maximum 5³C until use. Samples collected below the water table were kept in air-tight containers in order to prevent oxidation of reduced compounds. In order to sterilize the soil an addition of sodium azide (NaN3 ) has proved to be adequate for inhibition of the microbial activity. The ¢nal concentration was 50 Wg ml31 and had no e¡ect on ETU analyses. Inorganic analyses were performed by ion chromatography (Dionex, CA, USA). As Na-azide interfered with the quanti¢cation of nitrate, the determination of nitrate was made by £ow injection colorimetry using a FIA colorimeter (Tecator, Sweden).
4.2) or air to maintain anoxic or oxic conditions. After an initial £ushing for 10 min a periodic £ush (30 s h31 ) was established and maintained during the batch run. The air £ush performed a circulation in the batch without changing the CO2 content signi¢cantly. Collection of anoxic water samples was done during constant argon £ush excluding oxygen to enter the system. The batches were incubated in the dark at constant temperature (5, 10 and 23³C). All experiments were done in duplicate or triplicate and were supplemented in di¡erent experimental runs. Water samples for ETU analyses were preserved with Na-azide and kept at 4³C until analysis, whereas samples for inorganic constituents were analyzed immediately. 2.4. Pure culture isolation
The isolation was performed by Dr. Vinther, at the Danish Institute of Plant and Soil Science. The soil was suspended in 10 ml sterile demineralised water, and shaken for 30 s for preparation of dilution series. Dilution plating was performed on four replicate plates and inoculated from each soil dilution. The plates were incubated for 2 weeks at 25³C before counting. The assay method has been described by Smithgrenier and Adkins [9]. The inoculation method in liquid medium on microtiter plates has been described by Koëlbel-Boelke et al. [10].
2.3. Batch experiments
Batch experiments were carried out in glass containers (jars) of 1, 3 or 5 l with a gas-tight lid. Three ports for sampling and gas £ushes were built into the lid. Depending on the volume needed for analyses the experiments were set up in di¡erent sizes using a rate of 100^200 g soil l groundwater31 . Soil and groundwater were transferred to the jars, which were instantly closed and £ushed with either argon (grade Table 1 Microbial decay rates for ETU in batch experiments Temperature Oxic decay (³C) (Wg/kg soil day) 5 1.93^2.30 10 2.22^3.03 23 11.4^13.3
3. Results and discussion
3.1. Biotic-abiotic batch
In order to determine whether ETU decomposition is generated by microorganisms or by an abiotic process, the ¢rst batch was set up using fresh soil and sterilized soil. The stability of ETU in a groundwater solution was also tested.
Anoxic decay (Wg/kg soil day) 0.78^0.94 1.01^1.39 2.41^3.80
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Anoxic+manure (Wg/kg soil day)
Anoxic+nitrate (Wg/kg soil day)
2.80^3.16
2.80^3.02
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Fig. 2. Degradation of ETU in experimental batch (n =2). Initial concentration is 125 Wg l31 .
For a period of 120 days ETU was shown to be stable in groundwater at 20³C, whereas in the fresh soil batch the initial concentration of 105 Wg l31 declined to 16þ 4 Wg l31 . The abiotic batches decreased to about 88 Wg l31 , whereas the groundwater batch with no soil showed no change in concentration, which con¢rms an earlier work by Herrchen [7]. The decomposition of ETU seems to be mostly biotic in an oxic environment as earlier suggested by Miles and Doerge [2]. A number of bacterial isolates from the soil samples representing di¡erent depths were tested for their ability to use ETU as sole source of carbon and energy [8] (Table 1). The results showed that a relatively high number of isolates were able to use ETU as electron donor, indicating that a large proportion of the microbial biomass may be involved in the degradation.
The decomposition of ETU approximated a ¢rst order decay, which has been suggested by Satterthwaite [11] (Fig. 2). Over 50 days the oxic decomposition was higher than the anoxic decomposition (4.5 Wg ETU kg soil31 day31 vs. 3.2 Wg ETU kg soil31 day31 ). Anaerobic decomposition was formerly believed to be non-existent [12]. Further, abiotic decay under oxic conditions was detected to be less than 5%, whereas no decay was observed under anoxic conditions.
3.2. Oxic-anoxic batch
The oxygen status in the groundwater may be of major importance to the microbial decomposition of ETU. Parallel sets of biotic and abiotic batches were therefore incubated at 20³C for 50 days under oxic and anoxic conditions. The initial concentration was 125 Wg l31 ETU corresponding to a residue concentration in soil water after one spray event with mancozeb, e.g. 2 kg active ingredient ha31 .
Fig. 3. Decay rate of ETU in experimental batch (n = 3) under oxic and anoxic condition as a function of temperature.
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matter or additional oxidation equivalents on the decay rate. However, the results showed only a slight increase of the decay rate (Table 2). 3.5. Reduction processes
Fig. 4. Decay rate of ETU as a function of initial concentration.
Soil samples from the deeper part of the aquifer contain minor amounts of pyrite and lignite. By reaction with nitrate the pyrite will be oxidized by microbial processes [13,14]. Incubation under anoxic conditions showed that ETU was degraded to 70% in 20 days in the absence of nitrate, whereas ETU was totally depleted if nitrate reduction by pyrite was present.
3.3. Temperature dependence
The temperature dependence was tested at 5, 10 and 23³C in both oxic and anoxic batches. A rather high Q1 was estimated from the results under oxic conditions. Q10 was calculated to be in the range of 2.9^4.5. Under anoxic conditions the Q10 was found to be 2.1^2.5, which is normally found in pesticide decay experiments (Fig. 3). A more detailed study to describe the mechanisms behind the high Q10 has to be carried out. 3.4. Concentration dependence
The dependence of the initial ETU concentration on the decay was determined at 23³C under oxic conditions. Three levels of ETU concentrations were tested, 20, 125 and 500 Wg l31 . The results showed a nearly linear correlation between decay rate and concentration, which supports a ¢rst order reaction for estimating the decay (Fig. 4). Addition of manure or nitrate to anaerobic batches was carried out to study the e¡ect of organic
4. Conclusions
In summary, it may be concluded that ETU decomposition is mainly microbial and may under certain conditions be coupled to other microbial processes in the aquifer. Further, this study has shown that the degradation might not depend on very specialized microorganisms, and that it takes place in oxic as well as in anoxic reducing environments. The results are in agreement with analyses of ETU in soil water and groundwater under potato ¢elds at Fladerne B×k, as only very small amounts have been detected in spite of a previously applied amount of up to 16 kg EBDC ha31 to the ¢elds. Acknowledgments
We wish to thank the Pesticide Foundation of the Danish EPA for ¢nancial support, Grant 7041-0002. Also we appreciate microbial counting done by Dr. Vinther.
Table 2 Bacterial isolates able to use ETU or MCPP as sole source of carbon and energy (from [7]) Depth (m below surface) Total number of isolates ETU positive isolates % positive 0.20 24 15 62 0.50 24 13 54 0.90 24 7 29 4.00 22 5 23 13.0 24 8 33
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MCPP positive isolates 11 12 9 10 7
% positive 46 50 38 42 29
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