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Journal of Environmental Quality 30:1271-1277 (2001)
© 2001 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORT
Organic Compounds in the Environment

Anaerobic Degradation of Atrazine and Metolachlor and Metabolite Formation in Wetland Soil and Water Microcosms

C.A. Seybolda, W. Mersie*,b and C. McNameeb

a USDA-NRCS, Oregon State Univ., ALS Bldg., Rm. 3017, Corvallis, OR 97331
b Agric. Res. Stn., P.O. Box 9061, Virginia State Univ., Petersburg, VA 23806

* Corresponding author (wmersie{at}vsu.edu)

Received for publication April 14, 2000.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The half-lives, degradation rates, and metabolite formation patterns of atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] were determined in an anaerobic wetland soil incubated at 24°C for 112 d. At 0, 7, 14, 28, 42, 56, and 112 d, the soil and water were analyzed for atrazine and metolachlor, and their major metabolites. The soil oxidation–reduction potential reached -200 mV after 14 d. Degradation reaction rates were first-order for atrazine in anaerobic soil and for metolachlor in the aqueous phase. Zero-order reaction rates were best fit for atrazine in the aqueous phase and metolachlor in anaerobic soil. In anaerobic soil, the half-life was 38 d for atrazine and 62 d for metolachlor. In the aqueous phase above the soil, the half-life was 86 d for atrazine and 40 d for metolachlor. Metabolites detected in the anaerobic soil were hydroxyatrazine and deethylatrazine for atrazine, and relatively small amounts of ethanesulfonic acid and oxanilic acid for metolachlor. Metabolites detected in the aqueous phase above the soil were hydroxyatrazine, deethylatrazine, and deisopropylatrazine for atrazine, and ethanesulfonic acid and oxanilic acid for metolachlor. Concentrations of metabolites in the aqueous phase generally peaked within the first 25 d and then declined. Results indicate that atrazine and metolachlor can degrade under strongly reducing conditions found in wetland soils. Metolachlor metabolites, ethanesulfonic acid, and oxanilic acid are not significantly formed under anaerobic conditions.

Abbreviations: DEA, deethylatrazine • DIA, deisopropylatrazine • ESA, metolachlor ethanesulfonic acid • HA, hydroxyatrazine • HPLC, high performance liquid chromatography • OA, metolachlor oxanilic acid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
ATRAZINE and metolachlor are widely used herbicides to control annual grasses and broad leaf weeds in various crops. Their widespread use has resulted in their detection in shallow ground waters of 20 major hydrologic basins of the United States (Kolpin et al., 1998). In addition, metabolites of atrazine (Lerch et al., 1995) and metolachlor (Kalkhoff et al., 1998) have been detected in surface and ground water. In wetland sediments, residues of metolachlor and atrazine have also been detected (Albanis et al., 1994; Gueune and Winnett, 1994; Fletcher et al., 1994). Once an herbicide enters a wetland soil ecosystem, its fate is uncertain because most of the research has been conducted on well-drained agricultural soils. The oxidation–reduction status of soils and sediments can strongly influence the persistence or degradation of organic chemicals (Gambrell and Patrick, 1988).

Differing results have been obtained for atrazine degradation under anaerobic conditions in wetland soils (Papiernik and Spalding, 1998). Chung et al. (1995) showed about 50% degradation of atrazine in 38 wk under anaerobic wetland soils and waters. Gu et al. (1992) indicated that when soils were maintained under methanogenic conditions atrazine was stable. DeLaune et al. (1997) suggested that atrazine degradation is rapid at soil redox levels representing aerobic conditions and much slower at redox levels depicting anaerobic or reducing conditions. They suggested that it is unlikely that any degradation would occur at low redox or methanogenic conditions. However, strongly reducing (-250 to -300 mV) conditions, where only strict anaerobic microbial processes occur, were not evaluated by DeLaune et al. (1997). Goswami and Green (1971) indicated that in anaerobic sediments, biotransformations of atrazine would generally be slower than under aerobic conditions. Conversely, Kearney et al. (1967) indicated that atrazine disappears more rapidly under anaerobic conditions than under aerobic conditions. In a comparison study, Kruger et al. (1993) showed that at a depth of 90 to 120 cm in an Iowa soil, atrazine half-life was 231 d under unsaturated conditions and 87 d under saturated conditions. It has been shown that atrazine primarily degrades to hydroxyatrazine (HA) [2-hydroxy-4-ethyl- amino-6-(1-methylethyl)-1,3,5-triazine-2,4-diamine] and to a lesser extent to deethylatrazine (DEA) [6-chloro-N-(1-isopropyl)-1,3,5-triazine-2,4-diamine] in anaerobic sediment–water systems (Mersie et al., 1998; Jones et al., 1982). Additional information establishing the fate of atrazine in anaerobic and strongly reducing wetland soil environments is critical to predicting its environmental effect.

Limited data exists for metolachlor on degradation rates and pathways in anaerobic and strongly reducing conditions (Stamper et al., 1997; Konopka, 1994). Metolachlor was shown to transform to metolachlor ethanesulfonic acid (ESA) [2-((2-ethyl-6-methylphenyl)(2-methoxy-1-methylethyl)amino)-2-oxoethanesulfonic acid] and metolachlor oxanilic acid (OA) [2-(2-ethyl-6-methylphenyl)(2-methoxy-1-methylethyl) amino oxoacetic acid] in aerobic aquatic field mesocosms (Graham et al., 1999). Whether ESA and OA can form under anaerobic conditions and their persistence has not be evaluated. Knowledge concerning the effect of strongly reducing and anaerobic conditions on the rate of transformation and intermediate product formation is needed to predict the fate of metolachlor and other agricultural chemicals that move into wetland areas through runoff; wetlands are important environmental buffers in the hydrologic cycle.

Degradation rates and half-lives for specific herbicides in actual anaerobic wetland soil environments are fundamental parameters in assessing their fate. However, in situ monitoring of herbicides in tidal wetland soils is difficult because of the dense vegetation, high water table, constant tides, and lack of control of environmental parameters. To overcome these problems, degradation of atrazine and metolachlor was determined in microcosms under anaerobic soil conditions. The objectives of this study were to determine the half-lives (t1/2), degradation rates (k), and metabolite formation patterns of atrazine and metolachlor in anaerobic wetland soils under strongly reducing conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil
Soil samples for the study were collected from a tidal freshwater wetland site along the James River, in Prince George County, Virginia. The soil was a Levy silt loam (fine, mixed, superactive, acid, thermic Typic Hydraquents), which formed in clayey fluvial sediments on tidal marshes along creeks and rivers. The site is inundated twice daily by freshwater. Soil samples were collected from the 0- to 30-cm depth and sieved through a 2-mm sieve in a cold room (5°C) to remove roots. Organic matter content of the soil was 14.1% (Walkley–Black procedure; Nelson and Sommers, 1982), soil pH was 6.3 (1:1 soil to water ratio; pH in 0.01 M CaCl2; McLean, 1982), and soil texture was a silty clay loam (57% silt, 37% clay, and 6% sand; pipet method; Sheldrick and Wang, 1993).

Experimental Setup
Fresh sieved soil (6.7 g dry weight basis) was placed in 125-mL serum bottles (microcosms) and the volume of the slurry was brought to 100 mL with water collected from the same site. The serum bottles were sealed with a septum and crimp caps. Into each serum bottle containing soil, a 16-gauge needle equipped with a luer-lock valve was inserted through the septum into the head space to release accumulated gases. Serum bottles with soil slurries were equilibrated in a water bath at 24°C for 7 d. The soil settled, leaving a water column above it (referred to as the aqueous phase). A 60-mL syringe was used to remove any accumulated gas and then nitrogen gas was bubbled into each bottle through the 16-gauge valve. To each serum bottle, 1 mL solution containing 374 µg of technical grade atrazine (98% purity) or metolachlor (97% purity) was added using a syringe. Bottles were then shaken for 24 h at 220 rpm and placed in the dark in water baths set at 24°C. Triplicate bottles with soil treated with atrazine or metolachlor were removed at 0, 7, 14, 28, 42, 56, and 112 d and placed in a freezer at -22°C until extraction and analysis. Three serum bottles containing soil were fitted with either an Ag–AgCl combination pH electrode or a combination redox electrode or a water-resistant thermometer that was maintained throughout the study. The area around the electrodes was sealed with a silicone sealant. At each sampling time the redox potential, pH, and soil temperature were measured with these permanently installed electrodes in the serum bottles. Accumulated gas in the head space of each bottle was released through the valves and replaced with nitrogen gas every week for the duration of the study.

Chemicals
All reagents were American Chemical Society (ACS) or high performance liquid chromatography (HPLC) grade from Fisher Scientific (Pittsburgh, PA). Reference standards used in this study were atrazine (98%), DEA (94%), deisopropylatrazine (DIA) [6-chloro-N-(1-ethyl)amino-1,3,5-triazine-2,4-diamine] (98%), ESA (95.7%), OA (95.1%), HA (97%), metolachlor (97%), metribuzin [4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one] (99%), terbuthylazine (2-tert-butylamino-4-chloro-6-ethylamino-1,3,5-triazine) (99%), and 2,4-D [(2,4-dichlorophenoxy)acetic acid] (98%). Atrazine and terbuthylazine were obtained from Accustandard (New Haven, CT). Metolachlor, metribuzin, and 2,4-D were received from Sigma–Aldrich (Milwaukee, WI). Hydroxypropazine [2-hydroxy-4,6-di(isopropylamino)-1,3,5-triazine] (98%) was purchased from Riedel-delhaen (Seelze, Germany). The remaining standards were obtained from Novartis Crop Protection (Greensboro, NC). Terbuthylazine was used as an internal standard for atrazine, DEA, and DIA. Hydroxypropazine was used as an internal standard for HA, while 2,4-D was added as an internal standard for ESA and OA. Metribuzin was included as an internal standard for metolachlor.

Extraction of Herbicides from Soil and Aqueous Phases
Frozen samples were thawed overnight and placed in 250-mL Teflon centrifuge bottles and centrifuged at 5200 x g for 15 min. The aqueous portion was decanted into 100-mL glass jars and filtered using 0.7-µm glass-fiber filters, and the volume was measured. The soil fraction was extracted with 20 mL of methanol and water (4:1, v/v) for a total of three times. At each of the three steps, the mixture was shaken on a wrist-action shaker for 30 min and centrifuged at 5200 x g for 15 min. Supernatant from the three extractions was combined and evaporated in a vacuum oven at 50°C to remove methanol. Dried samples were redissolved in 100 mL distilled water. Samples containing atrazine were spiked with 2 mL terbuthylazine (1 µg mL-1). In addition, extracts from atrazine-treated samples received 1 mL of 2-hydroxypropazine (10 µg mL-1). Both the aqueous and soil extracts were extracted using 0.5-g, C18 Sep-Pak cartridges (Whatman, Clifton, NJ). The C18 cartridges were then preconditioned sequentially with 3 mL of methanol, ethyl acetate, methanol, and distilled water under negative pressure (-20 kPa). The samples were then passed through the cartridges under vacuum and dried for 30 min by pulling air. Analytes were then eluted from cartridges with 2 mL of ethyl acetate. In a preliminary study (data not shown), we found no significant difference between the elution of HP and HA from the C18 cartridges using ethyl acetate. Cartridges used for metolachlor were dried and eluted again with methanol for analysis using HPLC.

Analysis of Atrazine and Metabolites
Ethyl acetate eluates from cartridges containing atrazine residues were dried under nitrogen and resuspended in 1 mL of 10% methanol in 5 mM ammonium acetate (v/v) at pH 5.2. A reverse-phase Waters (Milford, MA) HPLC system was used to analyze atrazine and its metabolites. The system consisted of two Model 510 pumps, a Model 996 Photo Diode Array detector and a Model 717+ autosampler. All instrumentation and data collection were controlled using Waters Millinium 2.1 software. Samples of 30 µL were injected into a Waters Nova-Pak C18 (3.9 x 150 mm, 60 Å, 4 µm) column and the chromatography was monitored at 230 nm. The organic portion of the mobile phase (Solvent A) was methanol. The aqueous portion of the mobile phase (Solvent B) was 10% methanol in 5 mM ammonium acetate (v/v) at pH 5.2. Atrazine and hydroxyatrazine were baseline-separated with a gradient elution. The first gradient was with Solvent B for 2 min, then it was linearly increased to 100% Solvent A in 20 min, then decreased to 100% Solvent B for 6 min and held at this point for another 6 min. The retention time for DIA was 9.9 min, for DEA 12.1 min, for HA 13 min, for hydroxypropazine 15.3 min, for atrazine 17 min, and for terbuthylazine 19.8 min. The instrument detection limits for atrazine, HA, DIA, and DEA, were 22, 90, 40, and 30 µg L-1, respectively.

Analysis of Metolachlor and Metabolites
Concentrations of metolachlor in the ethyl acetate fraction of cartridge eluate were analyzed using a Hewlett–Packard (Wilmington, DE) Model 6890 gas liquid chromatograph equipped with a Hewlett–Packard 7673A autosampler and a Hewlett–Packard 5972 mass selective detector. A J & W Scientific (Folsom, CA) DB-5 column (30-m long, 250-µm diam., 0.25-µm film thickness) was used. The ethyl acetate fraction was evaporated under nitrogen and the internal standard, metribuzin (1 µg mL-1), was added. The volume was then brought to 2 mL with ethyl acetate. From each sample, a 2-µL extract was injected into the gas chromatograph with mass detector. A temperature ramp was used such that the initial temperature of 80°C was held for 1 min after injection, increased to 175°C at a rate of 30°C min-1 and held for 5 min, increased to 212°C at 6°C min-1 and held for 2 min, and finally increased to 280°C at 3°C min-1 and held for 2 min. Helium was used as a carrier gas at a flow rate of 1.1 mL min-1. The mass selective detector source temperature was 186°C and was run in a scan mode, 300 to 500 amu. The electron multiplier offset was set 400 volts over autotune. Detection limit for metolachlor and terbuthylazine was 41 and 24 µg L-1, respectively. The methanol fraction of cartridge eluate was used to determine the concentrations of the two metabolites, ESA and OA. Methanol was evaporated under a stream of nitrogen and 2,4-D (1 µg L-1) was added to each vial. The volume was then brought to 1 mL with methanol before a 30-µL sample was injected into the HPLC. Analysis was performed on the Waters HPLC system used for atrazine with the following modifications. A reversed-phase Keystone ODS-Hypersil column (Analytical Sale and Service, Mahwah, NJ) with a length of 250 mm, a 4.6-mm diameter, 3-µm particle size, and 12-nm pore size was connected to another column, Waters Nova Pak C18 (3.9 x 150 mm, 4 µm, 6 nm). A mobile phase (40:60 methanol to 10 mM sodium phosphate buffer, pH 7) was run under isocratic mode at 1.2 mL min-1. The temperature around the columns was maintained at 60°C. Retention times for 2,4-D, OA, and ESA were 6.5, 10.9, and 11.9 min, respectively. The detection limits for ESA and OA were 67 µg L-1 and 63 µg L-1, respectively.

Data Analysis
Linear regressions were applied to the data to determine degradation rate constants (k) and half-lives (t1/2) in either log of concentration or concentration vs. time plots. Best-fit linear regressions as indicated by the coefficient of determination (r2) were used to determine zero- and first-order reaction rates. Confidence intervals at the 95% probability level were calculated for herbicide half-lives.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
The redox potential (Eh) in the soil at the start of the study was 124 mV, which decreased to about -200 mV within 14 d (Fig. 1). This was suggested as the necessary time for the sequential biological reduction of all electron acceptors (O-2, NO3, Mn4+, Fe3+, SO2-4) before reduction of CO2 to CH4, methanogenesis (Patrick and DeLaune, 1977). A continuing decrease in Eh was observed as the incubation continued to Day 112, indicating an increasing activity of anaerobic microorganisms in the soil (Wang et al., 1993). The low redox values are consistent with values that would be obtained in a system with organic matter contents greater than 3% (Ponnamperuma, 1972). The effects of such highly reducing conditions will include increased solubility of iron, aluminum, and release of hydrogen sulfide, methane, and hydrogen.



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  		Fig. 1. The oxidation–reduction potential (Eh) and soil pH of a tidal wetland soil incubated at 24°C in the laboratory for 112 d.

 
Soil pH at the start of the study was 6.5 and slowly decreased to 5.6 over the 112-d study period (Fig. 1). Under anaerobic conditions, the redox and carbonate systems control the pH of waterlogged soils (Ponnamperuma, 1972). Most reduction reactions consume H+, causing a lower pH to increase toward 7. However, production of organic acids and CO2 may contribute to soil acidity and cause the pH to decrease (Patrick and DeLaune, 1977; Wang et al., 1993). The pH of most reduced soils is sensitive to the loss or gain of CO2 (Reddy and Patrick, 1975). As the partial pressure of CO2 increases in flooded soils, the pH decreases (Ponnamperuma et al., 1969).

Herbicide Dissipation Rates
The dissipation of atrazine and metolachlor in anaerobic soil and the aqueous phase is shown in Fig. 2. Each sampling point in the graphs is the mean of three replications. Initial concentrations of atrazine are much greater in the soil than in the aqueous phase. Atrazine concentrations in the anaerobic soil decreased over the 112-d study period (Fig. 2a). The rate of dissipation followed first-order kinetics (Table 1). The amount of time it took for 50% of atrazine (extractable amounts) to disappear in the anaerobic wetland soil was 38 d. Others have also reported the occurrence of anaerobic degradation of atrazine in sediments (Fletcher et al., 1994; Chung et al., 1996; Seybold et al., 1999). The addition of organic amendments in anaerobic wetland sediments was shown to enhance the biotransformation process of atrazine (Chung et al., 1996). In their study, atrazine half-lives ranged from 164 to 289 d depending on the organic amendment. Jones et al. (1982) showed rapid atrazine degradation rates for two estuarine sediments (aerobic or low oxygen) with half-lives of 15 to 20 d. In contrast, reported half-lives of atrazine in aerobic soils have been shown to range from 14 d to greater than 1 yr depending on the environmental conditions (Jones et al., 1982; Miller et al., 1997; Topp et al., 1994; Winkelmann and Klaine, 1991; Nicholls et al., 1982). The lower half-life of atrazine in our anaerobic soil compared with the average reported in the literature for aerobic conditions suggests that atrazine can degrade faster in anaerobic soils. Kearney et al. (1967) also indicated that atrazine disappears more rapidly under anaerobic conditions than under aerobic conditions, and Kruger et al. (1993) showed that atrazine half-lives under unsaturated conditions were about three times greater than under saturated soil conditions.



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  		Fig. 2. Dissipation of atrazine and metolachlor in (a) anaerobic soil and (b) water above the soil over 112 d of incubation at 24°C.

 

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  		Table 1. Degradation rate constants (k) and half-lives (t1/2) for atrazine and metolachlor in anaerobic wetland soil and water columns above soil after 112 d of incubation at 24°C.

 
The amount of atrazine in the aqueous phase above the soil decreased at a much slower rate than in the soil, and the reaction rate was zero-order or linear (Fig. 2b). In contrast, Widmer et al. (1993) reported first-order reaction rates for atrazine in ground water. The half-life of atrazine to disappear in the aqueous phase was 86 d (Table 1). Atrazine dissipation in the anaerobic soil was about twice the rate of that in the water above the soil. It is well established that degradation of atrazine is accelerated by adsorption to soil particles (Armstrong et al., 1967). In the absence of such adsorption in the aqueous phase, atrazine can persist for a relatively longer time. Solomon et al. (1996) reported, in a review, the half-lives of atrazine in aqueous systems to vary from 0.2 to 742 d depending on the temperature, light source, sediment type, water constituents, and concentration. In the presence of increasing dissolved organic matter and salt concentrations, atrazine half-lives were shown to decrease in aqueous systems (Khan, 1978; Li and Feldback, 1972). The rate of atrazine dissipation in water is highly dependent on environmental conditions and type and amount of constituents contained in the water.

Smaller amounts of metolachlor compared with atrazine partitioned to the soil after the 24-h equilibration period (time zero) (Fig. 2a). Atrazine has a greater adsorption capacity (Kd) than metolachlor on soil and sediments, which is most likely contributing to the greater partitioning of atrazine to the sediment (Seybold and Mersie, 1996; Weed Science Society of America, 1994). Metolachlor dissipation rate in anaerobic soil followed zero-order kinetics (Fig. 2a). The half-life of metolachlor in the anaerobic soil was 62 d (Table 1). Others have also reported the degradation of metolachlor in anaerobic sediments. Stamper et al. (1997) reported the transformation of metolachlor under sulfate-reducing conditions, and Konopka (1994) reported the occurrence of anaerobic metabolism of metolachlor in soils. In aerobic soils, Chesters et al. (1989) reported metolachlor half-lives ranging from 36 to 182 d. The moderate persistence of metolachlor in anaerobic soil is similar to that in aerobic soils. Konopka (1994) indicated metolachlor degradation in anaerobic soils to be equal to or slower than in aerobic conditions. In our study, the rate of dissipation in anaerobic soil was significantly slower for metolachlor than atrazine (Table 1). Conversely, in aerobic soil conditions metolachlor has been reported to have a shorter half-life than atrazine (Weed Science Society of America, 1994).

The half-life of metolachlor in the aqueous phase above the anaerobic soil was 40 d (Table 1). Similar results were reported by Graham et al. (1999). They found metolachlor half-lives in outdoor water mesocosms to range from 33 to 46 d. In our study, the reaction rate followed first-order kinetics. The rate of metolachlor dissipation was significantly faster in the water phase than in the anaerobic soil (Table 1).

Herbicide Metabolite Formation
In anaerobic soil, atrazine metabolites detected were HA and DEA, of which HA was detected in greater amounts (Fig. 3b). Concentrations of DIA were not detected in the soil. The level of HA increased with time and reached 7.6% of amount applied (in atrazine equivalent) on Day 112 (Fig. 3b). Hydroxyatrazine is more strongly adsorbed to soil and sediments compared with the other two metabolites and atrazine (Mersie and Seybold, 1996), and was shown to degrade (through microbial degradation) in anaerobic soil systems (Hance and Chesters, 1969). The persistence of HA is longer in soil than either atrazine or its chlorinated degradation products (Winkelmann and Klaine, 1991). Concentrations of DEA were first detected on Day 7 and amounts increased fivefold on Day 14 and then decreased (Fig. 3b). This decrease could be due to the degradation of DEA. The N-dealkylated metabolites may undergo dechlorination and form other hydroxylated atrazine metabolites. In anaerobic soil, HA appears to be the dominant and most persistent atrazine metabolite formed over time. Similar results were reported by Chung et al. (1996) in an anaerobic wetland sediment, in which most of the atrazine was transformed to HA. Under anaerobic soil conditions, HA did not mineralize (convert to CO2 and CH4) after 90 d of incubation (Goswami and Green, 1971). The buildup of HA in soil is due to its strong adsorption to soil and its relatively slow degradation and mineralization (Assaf and Turco, 1994).



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  		Fig. 3. Metabolite concentrations of (a) atrazine in water above the soil, (b) atrazine in anaerobic soil, (c) metolachlor in water above the soil, and (d) metolachlor in anaerobic soil. Concentrations were monitored over 112 d, while microcosms were incubated at 24°C.

 
In the aqueous phase, DEA was the major metabolite detected (Fig. 3a). Concentrations of DIA and DEA steadily increased to Day 28, then declined throughout the remainder of the study. Concentrations of DEA and DIA were shown to be relatively stable under low-oxygen aquifer conditions (Graham et al., 1999). In our study, concentrations of HA slowly increased throughout the study, but remained the lowest of the three metabolites detected (Fig. 3a). In a review by Solomon et al. (1996), two atrazine degradation studies in aqueous environments detected DEA, HA, and DIA, with slightly greater proportions observed under anaerobic than aerobic metabolism. In both studies, DEA and HA had the greatest concentrations and were similar in amounts detected. Hydroxyatrazine has been suggested as a primary degradation product of atrazine in water (Solomon et al., 1996; Ma and Spalding, 1997). However, the chemical hydrolysis of atrazine to HA is generally faster in the presence of soil than without (Jordan et al., 1970). Enhanced degradation of HA has been shown in the presence of a C source in soils (Assaf and Turco, 1994). Half-lives of atrazine degradates in aqueous environments are not available. Because HA has a relatively large capacity to adsorb to soil (Mersie and Seybold, 1996) and can persist in aqueous environments, it is suggested that adsorption to sediment was playing a bigger role in removing HA from the water column than degradation.

Concentrations of metolachlor metabolites detected in soil ranged up to 0.4% of the amount applied (in metolachlor equivalent) (Fig. 3d). Concentrations of ESA and OA were detected in equal amounts on Day 7, and generally increased slightly throughout the study (Fig. 3d). Metolachlor can be detoxified in soil by conjugation with glutathione (Aga et al., 1996; Field and Thurman, 1996). The chlorine is removed through an enzyme-mediated reaction with glutathione. The glutathione conjugate can be further transformed to OA and ESA with the final step going through an oxidation process (Feng, 1991; Graham et al., 1999). Because the formation of OA and ESA requires an oxidation step, these two metabolites should not form under anaerobic conditions, unless a different pathway is used. However, the glutathione conjugation process is ubiquitous in aquatic and terrestrial environments, and can occur under reducing conditions (Field and Thurman, 1996). In our study, strict anaerobic conditions were not obtained until about Day 7 (Fig. 1). The largest significant increase in ESA and OA concentrations in the sediment was in the first 7 d (Fig. 3d). After Day 7, concentrations of ESA and OA were highly variable and did not increase much. Any increase could be due to adsorption on sediment or diffusion into the sediment from the water column, which shows a concomitant decrease in ESA and OA concentrations (Fig. 3c). In addition, the sediment–water interface may not have become anaerobic until some time later. This may have provided a temporary site for OA and ESA to form aerobically. In general, ESA and OA do not appear to be significant metabolites of metolachlor in highly reducing anaerobic sediments. In aerobic soil, Aga et al. (1996) indicated that sulfonation and formation of ESA was an important detoxification process for metolachlor. Also, metolachlor can be detoxified through reductive dechlorination (Liu et al., 1991). In our study, metabolites of reductive dechlorination were not quantified because we did not have the appropriate standards. The persistence of ESA and OA in the anaerobic soil after 112 d indicates that they do not degrade readily. The persistence of metolachlor metabolites has been reported to be four or more years after application in agricultural soils (Phillips et al., 1999).

In the aqueous phase, ESA and OA were also detected (Fig. 3c). Initially, there were higher concentrations of ESA than OA that formed. With time, ESA and OA concentrations become very similar to each other. The ESA pathway is indicated to be more important than the OA pathway in aquatic systems (Graham et al., 1999). In our study, the concentrations of ESA and OA generally increased to about Day 14 and then declined and remained constant through the remainder of the study (Fig. 3c). In contrast, ESA and OA concentrations at the end of the 112 d were at their lowest in the aqueous phase, and at their highest in the soil. The metabolite ESA has been shown to be relatively persistent in surface waters (Aga et al., 1996; Morton et al., 1997). As discussed previously, movement of OA and ESA between the aqueous phase and sediment could be occurring.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Both atrazine and metolachlor degraded under the highly reducing conditions (< -200 mV) obtained in this study. Half-lives of atrazine and metolachlor in anaerobic wetland soil were 38 and 62 d, respectively. In the aqueous phase above the soil, half-lives for atrazine and metolachlor were 86 and 41 d, respectively. Hydroxyatrazine was the major metabolite of atrazine formed in the anaerobic soil, and tended to accumulate over time. Two metabolites of metolachlor, ESA and OA, were not significantly produced in the anaerobic soil and are not considered major metabolites formed in these environments. Major metabolites of atrazine that are formed under aerobic conditions can be formed in strongly reducing soils. Once the metabolites HA, ESA, and OA are formed and get into these anaerobic and highly reduced environments, they can be very persistent.


   ACKNOWLEDGMENTS
 
This study was supported by a Capacity Building grant from the U.S. Department of Agriculture (no. 95-38814-1723) and the Agricultural Center of Excellence for Plants and Water Quality, located at Virginia State University. The Center is a joint venture between the Natural Resources Conservation Service of the U.S. Department of Agriculture and Virginia State University.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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G. K. Stearman, D. B. George, K. Carlson, and S. Lansford
Pesticide Removal from Container Nursery Runoff in Constructed Wetland Cells
J. Environ. Qual., July 1, 2003; 32(4): 1548 - 1556.
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