Environ. Sci. Technol. 1996, 30, 592-597
Identification of a New Sulfonic Acid Metabolite of Metolachlor in Soil D. M. L. T.
S. E. R. D.
A G A , * ,† E . M . T H U R M A N , † YOCKEL,† ZIMMERMAN,† AND WILLIAMS‡
U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, Kansas 66049, and Mass Spectrometry Laboratory, University of Kansas, Lawrence, Kansas 66045
An ethanesulfonic acid metabolite of metolachlor (metolachlor ESA) was identified in soil-sample extracts by negative-ion, fast-atom bombardment mass spectrometry (FAB-MS) and FAB tandem mass spectrometry (FAB-MS/MS). Product-ion fragments from MS/MS analysis of the deprotonated molecular ion of metolachlor ESA in the soil extract can be reconciled with the structure of the synthesized standard. The elemental compositions of the (M - H)- ions of the metolachlor ESA standard and the soil-sample extracts were confirmed by high-resolution mass spectrometry. A dissipation study revealed that metolachlor ESA is formed in soil under field conditions corresponding to a decrease in the concentration of the parent herbicide, metolachlor. The identification of the sulfonated metabolite of metolachlor suggests that the glutathione conjugation pathway is a common detoxification pathway shared by chloroacetanilide herbicides.
Introduction Alachlor [2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide] and metolachlor [2-chloro-N-(2-ethyl6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] are structurally related chloroacetanilide herbicides that are used extensively in the United States for the control of some annual grasses and broadleaf weeds in crop and noncrop areas. Although alachlor use is twice that of metolachlor for corn and soybeans, metolachlor use has increased substantially since 1980 (1). Recent pesticidemonitoring studies revealed the occurrence of a sulfonic acid metabolite of alachlor in ground water. The 2-[(2,6diethylphenyl)(methoxymethyl)amino]-2-oxoethanesulfonic acid (alachlor ESA) was detected in samples collected from rural private wells in the Midwest at concentrations ranging from 1.2 to 74 µg/L (2). This finding is important * Author to whom correspondence should be addressed: e-mail address:
[email protected]; telephone: (913) 832-3561; fax: (913) 832-3500. † U.S. Geological Survey. ‡ University of Kansas.
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because alachlor ESA is relatively persistent in surface waters (3) and has high water solubility; thus, it is highly leachable to the ground water. The occurrence of the sulfonic acid metabolite of alachlor in surface and ground waters suggests that the formation of the metolachlor sulfonic acid metabolite also might be important. Both alachlor and metolachlor are detoxified rapidly by nonsensitive plants via conjugation with glutathione and/ or homoglutathione (4). The glutathione conjugate of alachlor further degrades to the sulfonic acid derivative as a major metabolite. Although the sulfonic acid derivative is reported to be a major soil metabolite of other chloroacetanilide herbicides, such as acetochlor (5), alachlor (6), and propachlor (7), the presence of the metolachlor sulfonic acid (metolachlor ESA) metabolite in soil, plants, or animals has not been reported. The objectives of this study were to identify and confirm the formation of metolachlor ESA in soil under field conditions.
Experimental Procedures Soil-Extraction Procedure. Soil samples were collected from an experimental corn field near Topeka, KS, where alachlor and metolachlor were applied as preemergent herbicides at a rate of 1.5 kg/ha (active ingredient). Soil samples were extracted according to the method described by Mills and Thurman (8), with slight modification. Briefly, 20 g of soil was extracted twice with a methanol (15 mL)/ water (5 mL) mixture at 75 °C for 30 min. The soil extracts were combined and subjected to a solid-phase extraction (SPE) procedure described by Aga and co-workers (9) using a C-18 Sep-Pak cartridge (Waters, Milford, MA) to separate the parent herbicides from their more polar metabolites. In this procedure, the soil extracts were evaporated to less than 10 mL to remove the methanol. Then, the remaining aqueous extracts were passed through a preconditioned C-18 resin by using an automated Millilab Workstation (Waters-Millipore, Milford, MA). The C-18 resins were eluted sequentially with 3 mL of ethyl acetate followed by 3 mL of methanol. The ethyl acetate fractions contained the parent herbicides, which were analyzed by enzymelinked immunosorbent assay (ELISA). Random samples were selected for confirmation by gas chromatography/ mass spectrometry (GC/MS). The methanol fractions contained the ESA metabolites and were analyzed by highperformance liquid chromatography (HPLC). Samples for fast-atom bombardment mass spectrometry (FAB-MS) and tandem mass spectrometry (FAB-MS/MS) were prepared by using the preceding SPE procedure, except that the methanol fractions were combined and concentrated by evaporation to 100 µL to facilitate detection and confirmation by FAB-MS/MS. Identification of Metolachlor ESA by FAB-MS and FABMS/MS. Negative-ion FAB analyses were conducted on a Fisions/VG AUTOSPEC-Q tandem mass spectrometer (Fisions/VG Analytical Ltd., Manchester, UK) of EBEqQ configuration. FAB experiments were performed using a cesium-ion gun operated at 20 keV of energy and 1 µA of emission. Samples as methanol solutions were added to a glycerol matrix on the FAB probe. Exact mass FAB determinations were conducted at a resolving power of 10 000 using linear voltage scans. Spectra were acquired
0013-936X/96/0930-0592$12.00/0
1996 American Chemical Society
A
B
FIGURE 1. FAB-MS/MS spectrum of the negatively charged molecular ion m/z 314 from the alachlor sulfonic acid standard (A) and the soil-sample extract (B).
in continuum mode, and 10 scans were integrated for the exact mass determination. Homologous fatty acid standards served as bracketing calibrant ions. FAB-MS/MS was conducted by using a linked scan at constant B/E (10). The collision gas was argon-adjusted to a pressure that attenuated the precursor ion 80% in order to maximize production yield (11). Synthesis of Alachlor ESA and Metolachlor ESA. Alachlor ESA and metolachlor ESA were synthesized on the basis of the procedure described by Feng (5). Either alachlor or metolachlor was refluxed with excess sodium sulfite in 10% ethanol in water for 3-4 h or until the mixture became homogeneous. Following acidification with sulfuric acid, the product was extracted into methylene chloride. The methylene chloride was evaporated, and the reaction products were dissolved in hot ethanol. The hot ethanol
mixture was filtered and allowed to stand undisturbed for recrystallization of the ESA. The white crystals that formed were collected and washed several times with cold ethanol. The chemical structures of the white crystals were confirmed as alachlor ESA or metolachlor ESA by mass spectrometry. Analysis of Metolachlor and Metolachlor ESA in Soil. Concentrations of metolachlor in the ethyl acetate fractions of the SPE eluate were measured by using ELISA kits obtained from Idetek/Quantix Systems (Sunnyvale, CA). The ethyl acetate was first evaporated to dryness and then reconstituted with 5 mL of water. Samples with concentrations greater than the highest calibration standard were diluted first with distilled water until they were within the linear range of the ELISA. The recommended procedure indicated in the ELISA kits was followed.
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A
B
FIGURE 2. FAB-MS/MS spectrum of the negatively charged molecular ion m/z 328 from the metolachlor sulfonic acid standard (A) and the soil-sample extract (B).
Concentrations of metolachlor ESA in the methanol fractions were measured by HPLC using an HP Model 1090 series II liquid chromatograph with a photodiode array detector (Hewlett-Packard, Palo Alto, CA). A reversed-phase ODS-Hypersil column (Keystone Scientific, Bellefonte, PA) with dimensions of 100 × 4.6 mm (3 µm particle size and 120 Å pore size) was used. The mobile phase was composed of 40:60 methanol/phosphate buffer (10 mM, pH 7.0) and was run under isocratic mode at 1.2 mL/min. The column temperature was controlled and maintained at 60 °C.
Results and Discussion Analysis of the soil extracts by negative-ion FAB-MS showed the presence of the (M - H)- ions of alachlor ESA (m/z 314) and metolachlor ESA (m/z 328). The mass spectra of the soil extracts were compared with the spectra of the alachlor
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ESA standard obtained from Monsanto Agricultural Co. (St. Louis, MO) and of the metolachlor ESA synthesized in the laboratory. FAB-MS/MS spectra of precursor ions (m/z 314 and 328) derived from standard samples (Figures 1A and 2A) and soil extracts (Figures 1B and 2B) revealed characteristic fragment ions consistent with the structures of the alachlor ESA and metolachlor ESA. Figure 3 is a structural interpretation of the fragmentation observed for the two suspected sulfonic acid metabolites. The product ions that are common to both metabolites are m/z 80 [SO3], 94 [CH2SO3], and 121 [COCH2SO3 - H]. Their source is indicated in Figure 3A,B. A distinguishing ion unique for alachlor ESA is m/z 270, which represents the removal of the methoxymethyl group. Similarly, m/z 256 results from the analogous removal of the 2-methoxy-1-methylethyl group in metolachlor ESA. The m/z 256 ion in metolachlor
A
TABLE 1
Negative-Ion FAB Exact Mass Determinations calculated standard massa soil-sample massa mass 314 C14H20NO5S 314.1062 314.1060 ( 0.6 314.1057 ( 1.6 328 C15H22NO5S 328.1219 328.1222 ( 0.9 328.1234 ( 4.6 ion
formula
a Mass measurement error with respect to the calculated mass, expressed as parts per million.
B
FIGURE 3. Structural interpretation of the fragmentation of alachlor ESA and metolachlor ESA by FAB-MS/MS. (A) Alachlor sulfonic acid, molecular weight 314. (B) Metolachlor sulfonic acid, molecular weight 328.
ESA is noticeably less abundant than the equivalent m/z 270 ion in alachlor ESA. This may be attributed to the more favorable cleavage of the C-N bond in alachlor ESA, where C is adjacent to an O atom (12). On the other hand, the C-N bond in the methoxymethylethyl group of metolachlor ESA is one carbon away from the O atom, resulting in less favorable cleavage of the C-N bond, as is evident in the lower abundance of the m/z 256 ion. The favored cleavage of the C-C bond adjacent to the O atom in the metolachlor ESA molecule results in the formation of m/z 282. The other prominent ions observed in both ESA metabolites is the loss of one (M - 16) or two (M - 32) oxygen atoms in the sulfonate group. These ions are the m/z 298 and 282 in alachlor ESA and the equivalent m/z 312 and 296 in metolachlor ESA. There was significant ion current in ions adjacent to the (M - H)- ion in the crude soil extracts. This gave rise to a suite of precursor ions transmitted from the samples and, thus, a busy B/E-linked scan spectrum (Figures 1B and 2B). Precursor-ion resolution is generally modest using B/E linked scans (10). This can be observed in the clear natural abundance isotope clusters associated with the major product ions in the MS/ MS spectra of the standards. The agreement between the fragmentation pattern of the authentic ESA standards and the suspected ESA metabolites in the soil extracts is strong evidence of the identification of metolachlor ESA and alachlor ESA in the samples. Soil extracts and standard-derived anions were subjected to exact mass determinations. The (M - H)- ions derived from the soil extracts and standard samples have the same mass units within experimental error and matched the calculated formula mass of the sulfonic acid metabolites within less than 5 ppm error (Table 1). The exact mass and MS/MS results indicate that both alachlor ESA and metolachlor ESA were present in the soil samples. Additional evidence of the formation of the metolachlor ESA metabolite in the metolachlor-treated soil is provided by the HPLC chromatograms in Figure 4. The peak at 5.947 min in Figure 4A (soil extract) matched the retention time of the metolachlor ESA standard peak (5.945 min) in Figure 4B. The UV spectrum of the unknown peak at 5.947 min in the soil extract also gives a 100% match with the UV spectrum of the metolachlor ESA standard.
FIGURE 4. HPLC chromatograms of the soil extract (A) and ESA standards (B) and comparison of the UV spectrum of the unknown peak in the soil extract with that of metolachlor ESA.
The evidence that metolachlor ESA is formed under the same field conditions in which alachlor ESA is formed is important in several aspects. First, the formation of metolachlor ESA indicates that both chloroacetanilide herbicides follow a common metabolic pathway in soil. Feng (5) and Lamoureux and Rusness (7) have proposed glutathione conjugation as an initial pathway of acetochlor and propachlor metabolism in soil, resulting in the formation of sulfonic acid metabolites. The evidence obtained from this research suggests that alachlor and metolachlor undergo the same glutathione conjugation as a metabolic pathway in soil, which ultimately results in the substitution of chlorine with a sulfonic acid group as shown in Figure 5. It is thought that conjugation with glutathione is a common mechanism utilized by living organisms to detoxify electrophilic xenobiotics such as pesticides, drugs, and other chemical pollutants (13). An understanding of the formation and final metabolism of the sulfonic acid metabolites of chloroacetanilide herbicides is necessary if glutathione conjugation is to be evaluated as a detoxification process or if the fate of pesticides is to be considered. Secondly, because the sulfonic acid metabolites have high water solubility, they are expected to leach through the soil. A study on the mobility and transport of these metabolites, therefore, would become an important contribution to the understanding of the fate and transport of chloroacetanilide herbicides in the environment.
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FIGURE 5. Formation of ESA metabolites of alachlor and metolachlor via glutathione (GSH) conjugation.
FIGURE 6. Disappearance of metolachlor and formation of metolachlor sulfonic acid metabolite in soil from an experimental field plot.
To determine the rate of formation of metolachlor ESA relative to the disappearance of the parent herbicide, soil samples were collected from a metolachlor-treated corn field. Sampling was performed weekly for the first month and then at 2 week intervals thereafter until the 19th week. Then, samples were collected again 300 days after herbicide application to determine the persistence of the ESA metabolite in the topsoil. The soil consisted of a Eudora siltloam, with a pH ranging from 6.8 to 7.8 and an organic carbon content of 0.45% (top 15 cm). Figure 6 shows the average concentrations of metolachlor and metolachlor ESA in soil (0-15 cm depth) with respect to the number of days after application. Each point in the graph represents the average of three soil samples analyzed in duplicate. The concentrations observed varied greatly at each sample location with time, resulting in coefficients of variation ranging from 23% to 110%. This is attributed to the difficulty of applying herbicides uniformly due to variations in wind velocity and oscillations of the sprayer booms during herbicide application. Similar problems have been encountered by other researchers when measuring pesticide contents under field conditions (14). However, for the purpose of this study, the data obtained are useful in showing the trend in the formation of the metolachlor ESA in soil with respect to the dissipation of metolachlor under natural field conditions. The concentration of metolachlor in soil declined exponentially with time, with a disappearance half-life of
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approximately 25 days based on the equation described by Hurle and Walker (15) for first-order kinetics. A corresponding rapid increase in metolachlor ESA was observed for the first 5 weeks after application. The highest concentration of metolachlor ESA, which was observed between 12 and 13 weeks after application, occurred after harvest and reached an average of 120 µg/kg. It is possible that corn plants took up the ESA metabolite and then released it back into the soil after harvest, resulting in a considerable increase in metolachlor ESA concentration in the soil. A similar observation has been reported for propachlor ESA (7), which was taken up by soybean plants grown in soil treated with propachlor. The initial rapid formation of metolachlor ESA indicates that sulfonation is an important detoxification process for metolachlor in soil. It has been previously reported that microbial dechlorination of metolachlor by mixed and isolated microbial cultures results in the formation of sulfurcontaining metolachlor derivatives as intermediate and end products (16, 17). However, in those laboratory studies the sulfonic acid metabolite was not identified as one of the metabolic products. Under field conditions, where the natural microbial community exists, formation of the sulfonated metabolite of metolachlor appears to be an important product of microbial activity. The gradual disappearance of the metolachlor ESA could either be due to its leaching through the unsaturated zone because of its high water solubility or due to further degradation in the topsoil. This could be an important area of research that needs to be addressed further. Because of increasing public concern about the quality of drinking water, renewed pesticide-monitoring programs are being implemented, and the toxicological significance of extensively used herbicides is receiving closer examination. Increased knowledge about the degradation of herbicides and the dissipation of their metabolites in the environment could include consideration of herbicide metabolites as part of the basis for the establishment of health advisories and water-quality regulations.
Acknowledgments The authors thank Mr. Robert Drake and Ms. Homigol Biesiada of the University of Kansas Mass Spectrometry
Laboratory (Lawrence, KS) for their efforts in acquiring the FAB spectra. The tandem mass spectrometer was purchased with the aid of a National Institutes of Health Grant S10 RRO 6294-01 (T.D.W.) and the University of Kansas. The metolachlor ELISA kits were gifts from Idetek/Quantix Systems (Sunnyvale, CA). The use of brand or trade names in this article is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey.
Literature Cited (1) Hogue, J. E. Preliminary Quantitative Usage of Metolachlor; Benefits and Use Division, Office of Pesticide Programs, U.S. Environmental Protection Agency: Washington, DC, 1986. (2) Baker, D. B.; Bushway, R. J.; Adams, S. A.; Macomber, C. S. Environ. Sci. Technol. 1993, 27, 562-564. (3) Goolsby, D. A.; Battaglin, W. A.; Fallon, J. D.; Aga, D. S.; Kolpin, D. W.; Thurman, E. M. Abstracts of the Technical Meeting; U.S. Geological Survey Toxic Substances Hydrology Program, September 1993, p 83. (4) Breaux, E. J.; Patanella, E. J.; Sanders, E. F. J. Agric. Food Chem. 1987, 35, 474-478. (5) Feng, P. C. C. Pestic. Biochem. Physiol. 1991, 40, 136-142. (6) Sharp, D. B. In Herbicides: Chemistry, Degradation and Mode of Action; Kearney, P. C., Kaufman, D. D., Eds.; Marcel Dekker, Inc.: New York, 1988; pp 301-333. (7) Lamoureux, G. L.; Rusness, D. G. Pestic. Biochem. Physiol. 1989, 34, 187-204. (8) Mills, M. S.; Thurman, E. M. Anal. Chem. 1992, 64, 1985-1990. (9) Aga, D. S.; Thurman, E. M.; Pomes, M. L. Anal. Chem. 1994, 66, 1495-1499.
(10) Jennings, K. R.; Mason, R. S. In Tandem Mass Spectrometry ; Mclafferty, F. W., Ed.; John Wiley & Sons: New York, 1983; Chapter 9, p 197. (11) Todd, P. J.; Mclafferty, F. W. In Tandem Mass Spectrometry; Mclafferty, F. W., Ed.; John Wiley & Sons: New York, 1983; Chapter 7, p 149. (12) Silverstein, R. M.; Bassler, G. C.; Morril, T. C. Spectrometric Identification of Organic Compounds, 5th ed.; John Wiley and Sons, Inc.: New York, 1991; pp 3-89. (13) Lamoureuax, G. L.; Rusness, D. G. In Sulfur Nutrition and Assimilation in Higher Plants; De Kok, L. J., et al., Eds.; SPB Academic Publishing: The Hague, The Netherlands, 1993; pp 221-237. (14) Taylor, A. W.; Freeman, H. P.; Edwards, W. M. J. Agric. Food Chem. 1971, 19, 832-836. (15) Hurle, K.; Walker, A. In Interactions between Herbicides and the Soil; Hance, R. J., Ed.; Academic Press Inc.: London, 1980; pp 83-122. (16) Krause, A.; Hancock, W. G.; Minard, R. D.; Freyer, A. J.; Honeycutt, R. C.; LeBaron, H. M.; Paulson, D. L.; Liu, S. Y.; Bollag, J. M. J. Agric. Food Chem. 1985, 33, 584-589. (17) McGahen, L. L.; Tiedje, J. M. J. Agric. Food Chem. 1978, 26, 414419.
Received for review May 26, 1995. Revised manuscript received September 6, 1995. Accepted September 6, 1995.X ES9503600 X
Abstract published in Advance ACS Abstracts, December 1, 1995.
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