Journal of Environmental Quality 30:18-23 (2001)
© 2001 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORT
BIOREMEDIATION AND BIODEGRADATION
In Vitro Pesticide Degradation in Turfgrass Soil Incubated under Open and Sealed Conditions
Toshinari Suzuki,
Kumiko Yaguchi,
Sukeji Suzuki and
Tetsuya Suga
Dep. of Clinical Biochemistry, Tokyo Univ. of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0355, Japan
Corresponding author (tsuzuki{at}tokyo-eiken.go.jp)
Received for publication November 12, 1999.
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ABSTRACT
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Degradation of selected pesticides was conducted in a turfgrass soil from a golf course under open (i.e., allowing gas exchange with atmosphere) and sealed systems. The time required for 50% of the initial dose of fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate), diazinon (O,O-dimethyl O-2-isopropyl-6-methylpyrimidin4-yl phosphorothioate), iprodione [3-(3,5-dichlorophenyl)-N-isopropyl-2,4-dioxo-imidazolidine-1-carboxamide], mecoprop [(RS)-2-(4-chloro-o-tolyloxy)propionic acid], and asulam (4-aminophenylsulfonyl-carbamate) to dissipate (half-life, t1/2) was less than 2 wk under both conditions. The t1/2 values of dithiopyr (S,S'-dimethyl 2-difluoromethyl-4-isobutyl-6-trifluoro-methylpyridine-3,5-dicarbothioate) were 324 and 185 d under the open and sealed conditions, respectively. The t1/2 values of isoprothiolane (di-isopropyl 1,3-dithiolan-2-ylidene-malonate), flutolanil (
,,-trifluoro-3'-isopropoxy-o-toluanilide), and benefin (N-butyl-N-ethyl-,,-trifluoro-2,6-dinitro-p-toluidine) under the open conditions were 154, 336, and 47 d, respectively. The t1/2 values of these pesticides increased slightly under the sealed conditions. The t1/2 values of terbutol (2,6-di-tert-butyl-4-methylphenyl N-methycarbamate) and one of the major degradation products, N-demethyl-terbutol (2,6-di-tert-butyl-4-methylphenyl carbamate), were 182 and 291 d under the open conditions and increased by six- and threefold under the sealed conditions, respectively. The degradation system under the sealed conditions could characterize the persistence of terbutol and N-demethyl-terbutol, which were the most persistent in the field.
Abbreviations: DMSO, dimethyl sulfoxide • GC, gas chromatograph • IC, ion chromatograph • MS, mass spectrometer • TCD, thermal conductivity detector
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INTRODUCTION
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THE extent of water pollution derived from pesticides applied to golf courses has been of interest in Japan since the 1980s. Many kinds of fungicides, insecticides, and herbicides are used on golf courses for turf maintenance. Pesticides in drainage waters from golf courses have been reported by many researchers (Tsuji et al., 1991; Ebise, 1994; Fushiwaki et al., 1993; Suzuki et al., 1998; Toba et al., 1997). The fungicides (isoprothiolane and flutolanil), insecticides (diazinon and fenitrothion), and herbicides (asulam, simazine, triclopyr, dithiopyr, MCPP, and terbutol) appeared in the drainage water that originates mainly from leaching and runoff water on golf courses. The concentrations of almost all these pesticides detected in the drainage waters decreased by 100- to 1000-fold within 2 or 3 mo after the application. In contrast, the phenylcarbamate herbicide, terbutol (2,6-di-tert-butyl-4-methylphenyl N-methyl-carbamate) and its degradation products were detected in golf course drainage and ground water at the parts per billion concentration level more than 6 yr after application (Suzuki et al., 1998). Therefore, terbutol is regarded as a suitable compound for the development of laboratory screening methods as this represents a worst-case scenario and the persistence of terbutol is useful baseline against which to select pesticides for use on golf courses.
In vitro laboratory studies are often used to estimate pesticide persistence in the field (Kuwatsuka, 1981; Anderson et al., 1996). Under laboratory conditions it is simple to keep experimental conditions constant. It is, however, difficult to approximate field conditions. Microbial populations and species vary with soil depth (Alexander, 1977; Lavy et al., 1973) and microbial activity related to pesticide degradation in soil is affected by the soil atmosphere composition, moisture content, and temperature (Hewleg, 1993; Fournier et al., 1997). The concentrations of CO2 and O2 in the soil atmosphere vary with soil depth and time of year. At a soil depth of 1.8 m, the O2 content decreased to less than 10%, and CO2 increased to more than 10% in the soil atmosphere (Baver et al., 1972). The O2 and CO2 concentrations of unsaturated turfgrass thatch and soil at depths of 2 to 6 cm ranges from 10 to 15% and 0.1% to 1%, respectively (Thompson et al., 1983). In a previous study, degradation of terbutol in the soil was measured in open- and closed-incubation jars in the laboratory (Suzuki et al., 1996). Dissipation rates of terbutol decreased markedly in the closed incubation jar but comparative studies with other pesticides were not performed under the same conditions.
The objectives in this study are to (i) evaluate dissipation rates of several pesticides in soil incubated under open and sealed conditions and (ii) examine the conditions of the two systems by monitoring anion levels in soil and gas compositions of headspace in the incubation jars.
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MATERIALS AND METHODS
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Degradation of Pesticides in Soil
Soil samples under turfgrass (Zoisia japonica Steudel) were taken with a core sampler from golf course fairways in Tokyo (Tokyo Kokusai Country Club). The characteristics of the surface soil and subsoil are listed in Table 1. Turfgrass in surface soil samples was sieved using a 2-mm sieve. The surface soil and subsoil were combined in equivalent dry weights, and 10 g (dry weight) of the mixed soil sample was placed into 60-mL amber narrow-mouth jars (35 mm o.d. x 90 mm height). The water content of each sample was adjusted to 30% of soil dry weight by adding water sterilized through a membrane filter (pore size 0.22 µm). The jars containing the soil samples were loosely covered with aluminum foil, which allowed gaseous exchange with the atmosphere (open conditions), or tightly sealed with silicone rubber (sealed conditions) as described previously (Suzuki et al., 1996). The jars were pre-incubated in the dark at 25°C for 2 wk, then 50 µL (55.9 mg) of 0.6 mg mL-1 pesticide listed in Table 2 in dimethyl sulfoxide (DMSO) as carrier was added to each soil and then mixed by rolling the jars. In the case of analysis of gas composition and anions during incubation, 50 µL of only DMSO was added to each soil. The soil samples were incubated in the dark at 25°C for up to 180 d. Sterilized water was added to the foil-covered jar at 1-wk intervals to maintain the 30% water content of soil dry weight. This degradation study was conducted with two or three replications for each pesticide.
Extraction of Pesticides and Anions in Soils and Sampling of Gases in the Jars
Pesticides in soils were extracted with solvent and concentrations were determined by gas chromatography–mass spectrometry (GC–MS) (Suzuki et al., 1996, 1998). Soil samples (10 g dry weight) were extracted three times with 20 mL of acetone by vigorously shaking for 5 min, sonicating for 5 min, and centrifuging at 3000 rpm for 10 min. The acetone solution was filtered and the filtrate concentrated to about 10 mL with a rotary evaporator at 40°C. The acetone solution was added to 100 mL of 5% NaCl and extracted with 10 mL of CH2Cl2. The CH2Cl2 solution extract was dehydrated with Na2SO4 and evaporated to dryness under a stream of N2. The residue was dissolved in acetone-n-hexane (25:75) and then analyzed by GC–MS. For asulam, MCPP, MCPA, triclopyr, and terbutol metabolites containing the carboxyl group, the acetone solution was added to 100 mL of 0.1 M HCl and extracted twice with 10 mL of CH2Cl2. The CH2Cl2 solution was dehydrated with Na2SO4 and evaporated to dryness under a stream of N2. The residue was subjected to methylation with diazomethane, which was generated by adding N-methyl-N'-nitro-N-nitrosoguanidine (Sigma–Aldrich, Tokyo, Japan) into 10% sodium hydroxide, for asulam, MCPP, MCPA, and triclopyr and with HCl–methanol for terbutol metabolites containing the carboxyl group (Suzuki et al., 1996) and then analyzed with GC–MS. The recovery of pesticides from 10 g of dry weight of soil samples spiked with 30 g of pesticides ranged from 92 to 102%.
In order to evaluate the state in the incubation jar spiked only with DMSO (control jar), gas compositions and dimethyl sulfide in the headspace of the incubation jar and anions in the soil samples were determined. Nitrogen, O2, CH4, and CO2 in the incubation jars were determined by a gas chromatograph equipped with a thermal conductivity detector (GC–TCD). Dimethyl sulfide in the incubation jars was measured by directly injecting the headspace gas with a gas-tight syringe into the GC–MS. Water extractable anions in the soils (10 g dry weight) were extracted with 50 mL of distilled water by vigorously shaking for 120 min and centrifuging at 3000 rpm for 10 min, and then filtered through a membrane filter (pore size 0.45 µm). The filtrate was analyzed by an ion chromatograph (IC) system equipped with an electrical conductivity detector.
Apparatus and Conditions
The GC–MS conditions for pesticides were as follows: HP-5890 Series II gas chromatograph (Hewlett–Packard, Palo Alto, CA); 200°C injector temperature; 80-kPa column head pressure; helium carrier gas; HP-7673 auto sampler; 2-µL sample size (splitless injection, glass wool was not stuffed in the splitless insert of the GC injector); DB-5 analytical column (0.25 mm i.d. x 30 m, 0.25-µm film thickness, J&W Scientific, Folsom, CA); GC oven temperature program: held at 50°C for 1 min, then increased from 50 to 180°C at 20°C min-1 and from 180 to 270°C at 4°C min-1; Automass II mass spectrometer (JEOL, Akishima, Japan); 70-eV ionization potential; 305-µA ionization current; 200°C ion source temperature; 250°C temperature of transfer line between GC and MS. Pesticides were determined by single ion monitoring using the base peak and the molecular-ion peak of each pesticide. In the case of dimethyl sulfide, GC–MS conditions were the same as described above except for the following conditions: 50-kPa column head pressure; 300-µL sample volume (split injection, ratio 1:10). The GC oven temperature was programmed as follows: held at 35°C for 1 min; increased from 35 to 100°C at 10°C min-1. Dimethyl sulfide was analyzed by scanning mode at the mass range from 15 to 200 (m/z) and then determined using the base peak of dimethyl sulfide.
The GC–TCD conditions were as follows: GC-9AM model (Shimadzu, Kyoto, Japan); 120°C injection temperature; 100°C detector temperature; He carrier gas (50 mL min-1); molecular sieve 13X column (60 mesh, 3 mm i.d x 2 m); 40°C column temperature for N2, O2, CH4, and active carbon (60 mesh, 3 mm i.d x 1 m); 100°C column temperature for CO2; 300-µL sample volume.
The IC conditions were as follows: Model 2010I (Dionex, Sunnyvale, CA); AS4A column (Dionex); 2.2 mM sodium carbonate and 0.75 mM sodium bicarbonate buffer as elution solvent (flow rate, 1 mL min-1); 50-µL sample volume.
Methods of Statistical Analysis
Pesticides degradation data obtained from the GC–MS analysis were conventionally analyzed assuming the following first-order kinetics:
where t is the sampling time in days, C0 (100%) is the initial amount of pesticide, C is a percent of the initial amount remaining at time t, and k is a rate constant in this equation.
Parameter estimates were obtained for each of the degradation conditions using linear regression. Regression analyses were performed with the software package STATISTICA Version 5j (StatSoft Japan, 1996). Formal F-tests for goodness of fit was performed by the analysis of variance of the regression analysis. The analysis of covariance procedure used incubation condition as the qualitative indicator variable and sampling time as the quantitative covariate to test parallelism of the two regression lines. This analysis was conducted to detect differences in the effect of the incubation conditions on pesticide dissipation rates. Results for which the reported P was less than 0.05 indicate statistical significance at the 5% level.
The time for the dissipation of 50% of the initial amount (half-life, t1/2) of the pesticides was calculated by the following equation:
If the t1/2 calculated was beyond the range of sampling time in the experiment, the t1/2 was estimated by extrapolation of the regression model.
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RESULTS
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Changes in Control Incubation Jar
The changes in the gas composition of the headspace gas in the incubation jars without pesticide addition are shown in Table 3. The O2 level of the sealed jars decreased to <10% within 15 d of initial incubation and then remained at <5% up to 180 d. Carbon dioxide increased to about 5% during the initial 15-d incubation; thereafter, it ranged from 2.0 to 5.4% up to 180 d in the sealed jars. Carbon dioxide under the open jars was less than 0.05%, the detection limit under these GC conditions, during the 180-d incubation. Methane was not detected (limit > 0.1%) during the incubation under both conditions.
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Table 3. Changes in gas composition, dimethylsulfide, and anions in the incubation bottle without pesticide addition.
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Suzuki et al. (1996) reported that the principal constituent of a smell like that of a sulfur-containing compound was identified as dimethyl sulfide when DMSO was used as a solvent to spike pesticide into the soil sample. Dimethyl sulfide was detected only from 70 to 80 µg per bottle after 15 d under the open jars (Table 3). Under the sealed jars, dimethyl sulfide increased markedly to about 400 µg per bottle during the initial 15 d and then decreased gradually to less than 1 µg per bottle at 180 d.
The initial concentrations of nitrate in the soils were high levels from 540 to 580 mg kg-1 due to application of N fertilizer before soil sampling. The concentration of nitrate in the soils under the open jars decreased to about 6% of the initial concentration after 30 d and then increased gradually to about 1.5-fold of the initial concentration at 180 d (Table 3). The concentration of nitrate in the soils under the sealed jars decreased steeply after the 30-d incubation and was lower than 1 mg kg-1 dry soil at 180 d. Nitrite was observed at a concentration of about 5 mg kg-1 at 60 d under the open jars. The initial concentration of sulfate in the soils was from 100 to 110 mg kg-1. The concentration of sulfate in the soils under the open jars increased steeply by more than 40-fold the initial concentration within the initial 60 d of incubation and then gradually decreased to about 2600 mg kg-1 dry soil at 180 d. The concentration of sulfate in the soils under the sealed jars gradually increased to about 2000 mg kg-1 dry soil up to 180 d. The concentration of chloride did not change throughout the incubation period under both degradation systems. Other anion peaks were not detected on the ion chromatograms under these chromatographic conditions.
From these results on the control incubation jars, the conditions under the open and sealed jars differed in O2 and CO2 composition and in the behavior of nitrate, nitrite, sulfate, and dimethyl sulfide during the incubation periods.
Dissipation of Pesticides in Soil in the Jars
The degradation rates of pesticides from the open and sealed jars are shown in Table 4. Exponential first-order regression gave a good fit to the dissipation of all the pesticides examined under the two systems (formal F-tests, < 0.01). For fenitrothion, diazinon, iprodione, mecoprop, and asulam, there were no significant differences between the degradation rates in soils under the sealed jars and those under the open jars. These pesticides were degraded within 2 wk of incubation (Table 4). The degradation rate of dithiopyr in the soils under the sealed system increased significantly (P < 0.01) in comparison with that in the soils under the open system. The estimated t1/2 of dithiopyr in the soils under the open and sealed systems was 324 and 185 d, respectively. The degradation rates of isoprothiolane and benefin in the soils under the sealed system decreased significantly (P < 0.05 and P < 0.01, respectively) in comparison with those in the soils under the open system. The t1/2 of isoprothiolane and benefin in the soils was 154 and 47 d, respectively, under the open system and 197 and 75 d, respectively, under the sealed system. The degradation rate of terbutol in the soils under the sealed system decreased significantly (P < 0.001) in comparison with that in soils under the open system. The estimated t1/2 of terbutol under the sealed system was 182 d and increased by more than sixfold compared with the open system.
For degradation of terbutol metabolites, the degradation rate of NH2–terbutol in the soils under the sealed system decreased significantly (P < 0.001) in comparison with that in soils under the open system. The estimated t1/2 of NH2–terbutol under the open and sealed systems was 291 and 852 d, respectively (Table 4). The BHT and 4–COOH–BHT disappeared within 30 d under both the open and sealed conditions. In contrast, 4–COOH– and 4–COOH–NH2–terbutol decreased rapidly within the initial 60 d and then scarcely decreased during further incubation under both the open and sealed systems. The t1/2 values of 4–COOH– and 4–COOH–NH2–terbutol under the open and sealed systems were estimated to be between 173 to 190 d.
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DISCUSSION
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Degradation of terbutol in soils was previously investigated in the open and closed jars system, but the parameters that affected pesticide degradation were not quantified (Suzuki et al., 1996). In this study, the O2 percentages in jars sealed with silicon rubber were maintained at below 5% after 15 d. The conditions under the sealed system would be microaerophilic in comparison with aerobic conditions of the open system. Inorganic compounds containing N and S are a source of energy for growth of bacteria and were oxidized or reduced by many living microorganisms, such as Nitormonas, Nitrobacter, and Thiobacillus thiooxidans (Suzuki, 1974), and sulfate/sulfur- and nitrate/nitrite-reducing bacteria (Thauer, 1977). The nutrients in the soil samples in the jars might be changed during incubation, but data on these nutrients in soil samples during pesticide degradation are rarely published. Under the open system, the steep increase in sulfate during the first 60 d may be attributed to the oxidation of DMSO, a solvent used to spike pesticides into soils, by some microorganisms. The weight of 50 µL of DMSO spiked initially into the jar is 55.9 mg (0.715 mmol). The quantity of sulfate produced during the first 60 d under the open system was approximately 50 mg (0.526 mmol). Therefore, it is estimated that approximately 73% of DMSO was converted to sulfate under the open jars. A wide range of microorganisms has the capability to reduce DMSO to dimethyl sulfide under atmosphere and nitrogen gas (Alef and Kleiner, 1989; Sparling and Searle, 1993). The formation of dimethyl sulfide and the depletion of nitrate under the sealed system indicated that reduction of sulfur and denitrification occurred in the soil sample in the jars. The results in this study suggested that microorganisms arising in the soil sample under the sealed system would be different from those occurring under the open system.
For the degradation of the pesticides under the two conditions, fenitrothion, diazinon, iprodione, mecoprop, and asulam did not change under both the open and sealed conditions, probably due to O2 concentrations greater than 5% in the incubation jar during the first 15 d under the sealed system. The degradation rate of terbutol under the open system was lower than that of fenitrothion, diazinon, iprodione, isoprothiolane, benefin, mecoprop, and asulam and was greater than that of flutoranil and dithiopyr. In the previous field study, the pesticides used in this study except for terbutol were detected in the drainage water at high frequency but were not more persistent than terbutol (Suzuki et al., 1998). Although the open upland conditions are suitable for turfgrass (Branham and Wehner, 1985), the results in this study suggest that the persistence of terbutol in the field could not be estimated by the open system alone. The previous studies on effects of O2 on in vitro pesticide degradation in soils reported that the degradation rates of pesticides in soils decreased under parts per million levels of O2 in comparison with normal aeration (Jones et al., 1982; Shaler and Klecka, 1986). In the sealed conditions in this study, the degradation rate of terbutol in soil decreased markedly in comparison with the other pesticides. The sealed system might offer useful data for predicting the persistence of pesticides, especially those whose degradation rates decrease markedly under microaerophilic conditions. In situ experiments reported that degradation of pesticides in soils decreased with increasing soil depth (Johnson and Lavy, 1994; Sharon and Roy, 1998). Additional studies for vertical distribution and changes of O2 and CO2 gases in soils at golf course turf and for dissipation of pesticides in different soil depths will be desirable.
The terbutol metabolites NH2–, 4–COOH–, and 4–COOH–NH2–terbutol were detected at parts per billion levels in the drainage from golf courses for a long period together with the parent compound terbutol (Suzuki et al., 1998). The degradation of NH2–terbutol in soil was similar to that of terbutol under both the open and sealed conditions. BHT and 4–COOH–BHT, (hydrolysis products of the corresponding carbamates) rapidly disappeared from soil under both the open and sealed conditions. 4–COOH– and 4–COOH–NH2–terbutol were degraded more slowly than BHT and 4–COOH–BHT. Terbutol, NH2–, and 4–COOH–terbutol were scarcely hydrolyzed in 50 mM potassium phosphate buffer (pH 7.0) even after standing for approximately 3 mo (data not shown). Several alkyl phenyl N-methylcarbamates such as 2-sec-butylphenyl N-methylcarbamate (BPMC) (Ogawa et al., 1976) and 2-isopropylphenyl N-methyl carbamate (MIPC) (Ogawa et al., 1977) did not persist in soils. These results for terbutol metabolites suggest that insertion of the carbamate ester substituent of the phenol group between the two bulky tert-butyl groups would lead to persistence of the pesticide in the field.
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ABSTRACT
INTRODUCTION
