Analytical methods using high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) were developed for the analysis of the following chloroacetanilide herbicide metabolites in water: acetochlor ethanesulfonic acid (ESA), acetochlor oxanilic acid (OXA), alachlor ESA, alachlor OXA, metolachlor ESA, and metolachlor OXA. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. The mean HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.50, and 2.0 mg/L (micrograms per liter) ranged from 84 to 112 percent, with relative standard deviations of 18 percent or less. The mean HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.20, and 2.0 mg/L ranged from 81 to 125 percent, with relative standard deviations of 20 percent or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 mg/L, whereas the LOQ using the HPLC/MS method was 0.05 mg/L. These metabolite-determination methods are valuable for acquiring information about water quality and the fate and transport of the parent chloroacetanilide herbicides in water.

The chloroacetanilide herbicides-acetochlor, alachlor, and metolachlor-are an important class of herbicides in the United States. Together with the triazine compounds, chloroacetanilide herbicides compose the majority of pesticides applied in the Midwestern United States for control of weeds in corn, soybeans, and other row crops (Gianessi and Anderson, 1995). Alachlor and metolachlor have been used extensively for more than 20 years, whereas acetochlor application is relatively recent, having been applied extensively since March 1994 (Kolpin, Nations, and others, 1996). Chloroacetanilide herbicides have been shown to degrade more rapidly in soil than other herbicides, with half-lives from 15 to 30 days, whereas triazine half-lives are typically 30 to 60 days (Leonard, 1988).

Recent studies have reported the occurrence of chloroacetanilide metabolites in surface and ground water (Aga and others, 1996; Kolpin, Thurman, and Goolsby, 1996; Thurman and others, 1996; Kolpin and others, 1998). Kolpin and others (1998) found that metabolite concentrations in ground water may be at similar or even higher concentrations than the parent compounds, whereas in surface water the parent compounds are more abundant in the spring after application and are replaced gradually by metabolites during the remaining growing season. In understanding the fate and transport of parent compounds, reliable methods for the analysis of metabolites are vital. These methods also are important for analytical verification of the metabolites in toxicological studies.

High-performance liquid chromatography (HPLC) is needed for the analysis of chloroacetanilide herbicide metabolites because they are ionic compounds and are not sufficiently volatile for analysis by gas chromatography. HPLC-diode array detection (DAD) may be used in determining metabolite concentrations, especially when the water sample is relatively free of humic materials and ionic surfactants that can cause chromatographic interference. Coupling HPLC with mass spectrometry (MS) yields more qualitative data and lower detection limits than HPLC-DAD analysis alone.

This report describes the development of reliable HPLC-DAD and HPLC/MS methods for the analysis of ethanesulfonic acid (ESA) and oxanilic acid (OXA) metabolites of acetochlor, alachlor, and metolachlor in surface water and ground water. The HPLC-DAD method was derived from an analytical method for the analysis of alachlor ESA and alachlor OXA as reported by Macomber (1992). For application to the acetochlor and metolachlor metabolites, several modifications to that method were necessary to achieve chromatographic separation of metabolite peaks. The HPLC/MS method was derived from Ferrer and others (1997), with a minor modification to resolve co-eluting peaks on the chromatogram, and was reported in Hostetler and Thurman (1999). Similarly, the analysis of the ESA and OXA metabolites of the newly registered acetanilide herbicide dimethenamid could be added to these methods of analysis, but performance data for these compounds are not included in this report. These methods supplement other methods of the U.S. Geological Survey (USGS) and have been implemented by the USGS Organic Geochemistry Research Group in Lawrence, Kansas.

The HPLC-DAD method of analysis described in this report has been assigned the method number "O-2133-00." This unique code represents the HPLC-DAD automated method of analysis for organic compounds as described in this report and can be used to identify the method.

The HPLC/MS method of analysis described in this report has been assigned the method number "O-2134-00." This unique code represents the HPLC/MS automated method of analysis for organic compounds as described in this report and can be used to identify the method.

This report provides a detailed description of the methods, including the apparatus, reagents, instrument calibration, and the solid-phase extraction (SPE) procedure required for sample analysis. Estimated method detection limits, recoveries, and relative standard deviations for six chloroacetanilide herbicide metabolites for the HPLC-DAD and the HPLC/MS methods are presented.

DETERMINATION OF CHLOROACETANILIDE HERBICIDE METABOLITES IN WATER USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-DIODE ARRAY DETECTION

Water samples are filtered at the collection site using baked, glass-fiber filters with 0.7-mm pore diameter to remove suspended particulate matter. In the laboratory, filtered water samples are passed through a preconditioned C-18 column. The C-18 column is rinsed with ethyl acetate to remove interferring compounds. The adsorbed chloroacetanilide metabolites are removed from the C-18 with methanol. The eluted solution is spiked with an internal standard, evaporated under nitrogen, and reconstituted. The sample components are separated, identified, and measured by injecting an aliquot of the concentrated extract into an HPLC equipped with a DAD. Compounds eluting from the LC columns are identified by comparing their retention times obtained by the measurement of control samples under the same conditions used for the collected samples. The concentration of each identified compound is measured by relating the DAD response produced by that compound to the DAD response produced by the internal standard.

Samples with high concentrations of humic materials and ionic surfactants may cause chromatographic interference. Compounds that elute from the LC at the same time and are detected at the same wavelengths as the metabolites of interest also may interfere.

• Analytical balances-Capable of accurately weighing 0.0100 g ±0.0001 g.
• Autopipettes-20- to 200-µL, variable-volume autopipettes with disposable tips (Rainin, Woburn, Massachusetts, or equivalent).
• Millilab 1A workstation-Automated SPE workstation with an online computer. The two syringe pumps on the fluidics module are equipped with a 5- and a 1-mL syringe (Waters, Milford, Massachusetts).
• Multiple intake accessories (MIAs): Two MIAs are attached to the 5-mL syringe to increase sample capacity from 3 to 14 samples.
• Software: Millilab 1A software, version 3.0 (Waters, Milford, Massachusetts).
or

• Tekmar six-position AutoTrace-Automated SPE workstation, (Tekmar-Dohrmann, Cincinnati, Ohio).
• Software: Tekmar AutoTrace Extraction software, version 1.33 (Tekmar-Dohrmann, Cincinnati, Ohio).
• Automated solvent evaporator-The heated bath temperature needs to be maintained at 50 °C, and the nitrogen gas pressure at 15 lb/in² (TurboVap LV Zymark Inc., Hopkinton, Massachusetts, or equivalent).
• Mechanical vortex
• Analytical columns-Phenomenex (Torrance, California) 5-mm, 250- x 3-mm C-18 column coupled to a Keystone (Bellefonte, Pennsylvania) 3-mm, 250- x 4.6-mm C-18 column.
• HPLC benchtop system-Hewlett Packard (Wilmington, Delaware), model 1090 with autoinjector and DAD detector.
• LC oven conditions: constant 60 °C.
• LC mobile phase: 60 percent, pH 7.0, 25-mm phosphate buffer, 35 percent methanol, and 5 percent acetonitrile solution with a flow rate of 0.6 mL/min.
• DAD conditions: Acquiring wavelengths 190 to 300 nm.
• Data system-Computer and printer compatible with the HPLC system.
• Software-HPLC 3D ChemStation rev.A.03.03 (Hewlett Packard, Wilmington, Delaware) is used to acquire and store data and for peak integration.

• Sample bottles-Baked 4-oz. amber glass bottles (Boston round) with Teflon-lined lids.
• Sample filters-0.7-mm glass-fiber filters (Gilson, Middleton, Wisconsin, or equivalent).
• Reagent water-Generated by purification of tap water through activated charcoal filtration and deionization with a high-purity, mixed-bed resin, followed by another activated charcoal filtration, and finally distillation in an autostill (Wheaton, Millville, New Jersey, or equivalent).
• Analytical standards-Standards of the chloroacetanilide herbicide metabolites and the internal standard.
• SPE columns-C-18 Sep-Pak Plus, containing 360 mg of 40-µm C-18 bonded-silica packing (Waters, Milford, Massachusetts).
  or
  SPE columns-C-18 Sep-Pak Vac 6 cm&sub3;, containing 500 mg of 50- to 105-µm C-18 bonded-silica packing (Waters, Milford, Massachusetts).
• Disposable snap-cap finish centrifuge tubes-10 mL (Kimble, Vineland, New Jersey, or equivalent).
• Solvents-
• Acetonitrile, American Chemical Society (ACS) and HPLC grade.
• Ethyl acetate, HPLC grade.
• Methanol, ACS and HPLC grade.
• Sodium phosphate, dibasic anhydrous, ACS grade.
• Gas for evaporation-Nitrogen, ultrapure grade.
• Pasteur pipettes-Kimble, (Vineland, New Jersey), or equivalent.
• 0.1-mL autosampler vials-Plastic vial with glass cone insert and cap (Wheaton, Millville, New Jersey).
• Gases-
• LC solvent degasser-Helium, zero grade.
• LC autosampler gas-Air, zero grade.

Following USGS protocol, sampling methods capable of collecting water samples that accurately represent the water-quality characteristics of the surface water or ground water at a given time or location are used. Detailed descriptions of sampling methods used by the USGS for obtaining depth- and width-integrated surface-water samples are given in Edwards and Glysson (1988) and Ward and Harr (1990). Similar descriptions of sampling methods for obtaining ground-water samples are given in Hardy and others (1989).

Briefly, sample-collection equipment are free of tubing, gaskets, and other components made of nonfluorinated plastic material that might leach interferences into water samples or sorb the pesticides and metabolites from the water. The water samples from each site are composited in a single container and filtered through a 0.7-mm glass-fiber filter using a peristaltic pump. Filters are leached with about 200 mL of sample prior to filtration of the sample. The filtrate for analysis is collected in baked 125-mL amber glass bottles with Teflon-lined lids. Samples are chilled immediately and shipped to the laboratory within 3 days of collection. At the laboratory, samples are logged in, assigned identification numbers, and refrigerated at 4±2 °C until extracted and analyzed.

• Stock standard solutions-Obtain chloroacetanilide herbicide metabolites and internal standard as pure materials from commercial vendors or chemical manufacturers. Prepare solutions of 1.00 mg/mL (corrected for purity) by accurately weighing, to the nearest 0.001 g, 50 mg of the pure material in a 50-mL volumetric flask and dilute with methanol. Store at less than 0 °C. This solution is stable for about 24 months.
• Primary fortification standard-Prepare a 1.23ng/µ/L concentration, primary fortification standard by combining appropriate volumes of the individual chloroacetanilide herbicide metabolites stock solutions in a 1-L, volumetric flask. Dilute with methanol. Store at less than 0 °C. This solution is stable for 24 months.
• Internal standard solution-Prepare a solution of 2,4-dichlorophenoxyacetic acid in methanol at a concentration of 2.0 ng/µL. Store at less than 0 °C. This solution is stable for about 12 months.
• Calibration solutions-Prepare a series of calibration solutions using the primary fortification standard in reagent water (at concentrations ranging from 0.2 to 2.0 µg/L (0.2, 0.5, 1.0, and 2.0 µg/L). These will be processed through the extraction procedure (described in the "Procedure" section).

LC performance is evaluated by background absorbance readings, peak shape, and pressure. Background absorbance signals should remain balanced and low and indicate that the columns have equilibrated with the mobile-phase flow and the absence of interferents. If peak shape deteriorates, the columns may need to be replaced. If the pressure reading is high, there may be a clog in the mobile-phase flow path, or the column compartment thermostat may not have reached the required temperature yet. A variable DAD background signal indicates that the lamp may need replacement.

Two automated extraction systems are used in the laboratory. One method uses an automated Millilab lA workstation (Waters, Milford, Massachusetts), and the SPE cartridges (Sep-Pak) are obtained from Waters (Milford, Massachusetts). The SPE cartridges contain 360 mg of 40-µm C-18- (C₁₈H₃₇) bonded silica.

• The expected retention time (RT) of the peak of the selected metabolite of interest needs to be within ±2 percent of the expected retention time on the basis of the RRT_c obtained from the internal-standard analysis. The expected retention time is calculated as follows:
  

  (2),
  RT = (RRT_c) (RT_i) ,
  where RT= expected retention time of the selected metabolite,
  where RRT_c = relative retention time of the selected metabolite, and
  where RT_i = uncorrected retention time of the internal standard.
  

• If there are multiple peaks that agree in retention time to a metabolite, then additional qualitative indentification can be made using spectral information obtained by the 3D ChemStation software on the HPLC-DAD instrument.

• The dilution factor is calculated as follows:
  

  (3),
  DF = (123/123 - V_mp) (123/123 + V_a) ,
  where DF= dilution factor,
  where V_np = volume not pumped = volume, in mL, not pumped through the SPE column, and
  where V_a volume added = volume, in mL, of distilled water added to a sample that contains less than 123 mL.
  

The dilution factor is incorporated into the calculation for determining the final concentration in the sample.

• If a selected metabolite has passed the aforementioned qualitative identification criteria, the concentration in the sample is calculated as follows:
  

  (4),
  C = ((H_c/H_i) (m) + y) (DF) ,
  where C = concentration of the selected metabolite in the sample, in micrograms per liter;
  where H_c= peak height, in milliabsorbance units, for the selected metabolite identified;
  where H_i = peak height, in milliabsorbance units, for the internal standard;
  where m = slope of correlation curve between the selected metabolite and the internal standard from the original calibration data;
  where y = y intercept of correlation curve between the selected metabolite and the internal standard from the original calibration data; and
  where DF = dilution factor as calculated in equation 3.
  

Chloroacetanalide herbicide metabolites are reported in concentrations ranging from 0.2 to 3.0 µg/L. If the concentration is greater than 3.0 µg/L, the sample is re-extracted with a 1:10 (sample:distilled water) dilution and re-analyzed for those compounds that were greater than 3.0 µg/L. Linearity experiments have been conducted showing linear response curves from 0.2 to 10 µg/L.

Water samples are filtered at the collection site using baked, glass-fiber filters with 0.7-µm pore diameter to remove suspended particulate matter. In the laboratory, filtered water samples are passed through a preconditioned C-18 column. The C-18 column is rinsed with ethyl acetate to remove interferring compounds. The adsorbed chloroacetanilide metabolites are removed from the C-18 with methanol. The eluted solution is spiked with an internal standard, evaporated under nitrogen, and reconstituted. The sample components are separated, identified, and measured by injecting an aliquot of the concentrated extract into an HPLC equipped with a DAD and a mass spectrometer detector. Compounds eluting from the LC columns are identified by comparing their retention times obtained by the measurement of control samples under the same conditions used for the collected samples. Compounds are identified further by selected fragment ions for compounds that can produce fragment ions. The concentration of each identified compound is measured by relating the MS response produced by that compound to the MS response produced by the internal standard.

Compounds that elute from the LC at the same time and have identical ions as the metabolites of interest may interfere. Samples with considerable humic materials can cause interference with the ionization of the internal standard if they are eluting from the LC column at the same time.

• Analytical balances-Capable of accurately weighing 0.0100 g ± 0.0001 g.
• Autopipettes-5- to 200-µL, variable-volume autopipettes with disposable tips (Rainin, Woburn, Massachusetts, or equivalent).
• Millilab 1A workstation-Automated SPE workstation with an online computer. The two syringe pumps on the fluidics module are equipped with a 5- and a 1-mL syringe (Waters, Milford, Massachusetts).
• Multiple intake accessories (MIAs): Two MIAs are attached to the 5-mL syringe to increase sample capacity from 3 to 14.
• Software: Millilab 1A software, version 3.0 (Waters, Milford, Massachusetts).
or

• Tekmar six-position AutoTrace-Automated SPE workstation (Tekmar-Dohrmann, Cincinnati, Ohio).
• Software: Tekmar AutoTrace Extraction software, version 1.33 (Tekmar-Dohrmann, Cincinnati, Ohio).
• Automated solvent evaporator-The heated bath temperature needs to be maintained at 50 °C, and the nitrogen gas pressure at 15 lb/in² (TurboVap LV, Zymark Inc., Hopkinton, Massachusetts, or equivalent).
• Mechanical vortex
• Analytical columns-two Phenomenex 5-µm, 250- x 3-mm C-18 columns coupled to one (or two, if within backpressure limitations) Phenomenex 3-m, 150- x 2.0-mm C-18 column.
• HPLC/MS benchtop system-Hewlett Packard (Wilmington, Delaware), model 1100 HPLC with autoinjector and MS detector.
• LC oven conditions: constant 70 °C.
• LC mobile phase: 0.3 percent acetic acid, 24 percent methanol, 35.7 percent distilled water, and 40 percent acetonitrile solution with a flow rate of 0.3 to 0.4 mL/min.
• MS detector mode: electrospray in negative ion mode.
• Drying gas: flow was set at 6 L/min.
• Nebulizer pressure was 25 lbs/in², the drying gas temperature was 300 °C, the capillary voltage was 3,100 V, and the fragmenter voltage was 70 V.
• Data system-Computer and printer compatible with the HPLC system.
• Software-HP LC/MSD ChemStation rev.A.06.03 (Hewlett Packard, Wilmington, Delaware) is used to acquire and store data and for peak integration.

• Sample bottles-Baked 4-oz amber glass bottles (Boston round) with Teflon-lined lids.
• Sample filters-0.7-µm glass-fiber filters (Gilson, Middleton, Wisconsin, or equivalent).
• Reagent water-generated by purification of tap water through activated charcoal filtration and deionization with a high-purity, mixed-bed resin, followed by another activated charcoal filtration, and finally distillation in an autostill (Wheaton, Millville, New Jersey, or equivalent).
• Analytical standards-Standards of the chloroacetanilide herbicide metabolites and the internal standard.
• SPE columns-C-18 Sep-Pak Plus, containing 360 mg of 40-µm C-18 bonded-silica packing (Waters, Milford, Massachusetts).
  or
  SPE columns-C-18 Sep-Pak Vac 6 cm³, containing 500 mg of 50- to 105-µm C-18 bonded-silica packing (Waters, Milford, Massachusetts).
• Disposable snap-cap finish centrifuge tubes-10 mL (Kimble, Vineland, New Jersey, or equivalent).
• Solvents-
• Acetonitrile, ACS and HPLC grade.
• Ethyl acetate, HPLC grade.
• Methanol, ACS and HPLC grade.
• Acetic acid, glacial-ACS grade.
• Gas for evaporation-nitrogen, ultrapure grade.
• Pasteur pipettes-(Kimble, Vineland, New Jersey, or equivalent).
• 0.1-mL autosampler vials-Plastic vial with glass cone insert and cap (Wheaton, Millville, New Jersey).
• Nebulizer gas-nitrogen, ultrapure grade.

Following USGS protocol, sampling methods capable of collecting water samples that accurately represent the water-quality characteristics of the surface water or ground water at a given time or location are used. Detailed descriptions of sampling methods used by the USGS for obtaining depth- and width-integrated surface-water samples are given in Edwards and Glysson (1988) and Ward and Harr (1990). Similar descriptions of sampling methods for obtaining ground-water samples are given in Hardy and others (1989).

Briefly, sample-collection equipment are free of tubing, gaskets, and other components made of nonfluorinated plastic material that might leach interferences into water samples or sorb the pesticides and metabolites from the water. The water samples from each site are composited in a single container and filtered through a 0.7-µm glass-fiber filter using a peristaltic pump. Filters are leached with about 200 mL of sample prior to filtration of the sample. The filtrate for analysis is collected in baked 125-mL amber glass bottles with Teflon-lined lids. Samples are chilled immediately and shipped to the laboratory within 3 days of collection. At the laboratory, samples are logged in, assigned identification numbers, and refrigerated at 4 ±2 °C until extracted and analyzed.

• Stock standard solutions-Obtain chloroacetanilide herbicide metabolites and internal standard as pure materials from commercial vendors or chemical manufacturers. Prepare solutions of 1.00 mg/mL (corrected for purity) by accurately weighing, to the nearest 0.001 g, 50 mg of the pure material in a 50-mL volumetric flask and dilute with methanol. Store at less than 0 °C. This solution is stable for about 24 months.
• Primary fortification standard-Prepare a 1.23-ng/µL concentration, primary fortification standard by combining appropriate volumes of the individual chloroacetanilide herbicide metabolites stock solutions in a 1-L volumetric flask. Dilute with methanol. Store at less than 0 °C. This solution is stable for about 24 months.
• Internal standard solution-Prepare a solution of 2,4-dichlorophenoxyacetic acid in methanol at a concentration of 2.0 ng/µL. Store at less than 0 °C. This solution is stable for about 12 months.
• Calibration solutions-Prepare a series of calibration solutions using the primary fortification standard in reagent water at concentrations ranging from 0.05 to 2.0 µg/L (0.05, 0.10, 0.20, 0.50, 1.0, and 2.0 µg/L).

Evaluation of High-Performance Liquid Chromatography/Mass Spectrometry Performance

LC performance is evaluated by background absorbance readings, peak shape, and pressure. Background absorbance signals should remain balanced and low and indicate that the columns have equilibrated with the mobile-phase flow. If peak shape deteriorates, the columns may need to be replaced. If the pressure reading is high, there may be a clog in the mobile-phase flow path, or the column compartment thermostat may not have reached the required temperature yet. A variable DAD background signal indicates that the lamp may need replacement.

• Tune the MS in electrospray in negative-ion mode before each HPLC/MS sample set (approximately 45 injections) using the procedure and software supplied by the manufacturer.
• For the first sample of a sample set, inject a solution of 0.3 percent acetic acid, 24 percent methanol, 35.7 percent distilled water, and 40 percent acetonitrile solution to check for contamination.

Two automated extraction systems are used in the laboratory. One method uses an automated Millilab 1A workstation (Waters, Milford, Massachusetts), and SPE cartridges (Sep-Pak) obtained from Waters (Milford, Massachusetts). The SPE cartridges contain 360 mg of 40-µm C-18- (C₁₈H₃₇) bonded silica.

• The expected retention time (RT) of the peak of the quantitation ion for the selected metabolite of interest needs to be within ±2 percent of the expected retention time on the basis of the RRT_c obtained from the internal-standard analysis. The expected retention time is calculated using equation 2.
• A metabolite is not identified unless it has the correct quantitation ion. If more than one ion is acquired for a metabolite, then additional verification is done by comparing the relative integrated abundance values of the significant ions monitored with the relative integrated abundance values obtained from the standard control samples. The relative ratios of the ions need to be within ±20 percent of the relative ratios of those obtained in the absence of any obvious interferences.

• The dilution factor is calculated using equation 3.
• If a selected metabolite has passed the aforementioned qualitative identification criteria, the concentration in the sample is calculated as follows:
  

  (6),
  C = ((A_c/A_i) (m) + y) (DF) ,
  where C = concentration of the selected metabolite in the sample, in micrograms per liter;
  where A_c= peak area of the quantitation ion for the selected metabolite;
  where A_i = peak area of the quantitation ion for the internal standard;
  where m = slope of correlation curve between the selected metabolite and the internal standard from the original calibration data;
  where y = y intercept of correlation curve between the selected metabolite and the internal standard from the original calibration data; and
  where DF = dilution factor as calculated in equation 3.
  

Chloroacetanalide herbicide metabolites are reported in concentrations ranging from 0.05 to 5.0 µg/L. If the concentration is greater than 5.0 µg/L, the sample extract is diluted (volume increased to approximately 150 µL with the reconstitution solution (0.3 percent acetic acid, 24 percent methanol, 35.7 percent distilled water, and 40 percent acetonitrile) and re-analyzed. If the concentration is greater than 10 µg/L, the sample is re-extracted with a 1:10 (sample:distilled water) dilution and re-analyzed for those compounds that were greater than 10 µg/L. Linearity experiments have been conducted showing linear response curves from 0.5 to 10 µg/L.

This report presents two methods for routine analysis of six chloracetanalide herbicide metabolites in natural water samples. From the data presented in this report, solid-phase extraction and determination by high-performance liquid chromatography-diode array detection (HPLC-DAD) or high-performance liquid chromatography/mass spectrometry (HPLC/MS) are shown to be sensitive and reliable methods for the determination of low concentrations. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. Method detection limits for the HPLC-DAD method ranged from 0.09 to 0.17 µg/L. Method detection limits for the HPLC/MS method ranged from 0.04 to 0.05 µg/L. The mean HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.50, and 2.0 µg/L ranged from 84 to 112 percent, with relative standard deviations of 18 percent or less. The mean HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.20, and 2.0 µg/L ranged from 81 to 125 percent, with relative standard deviations of 20 percent or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 µg/L, whereas the LOQ using the HPLC/MS method was 0.05 µg/L. Information about the fate and transport of the chloroacetanilide herbicides-acetochlor, alachlor, and metolachlor-in water can be acquired from the analysis of surface-water runoff and ground water from wells. These methods also can be useful for water-quality determinations and analytical verification in toxicological studies.

Aga, D.S., Thurman, E.M., and Pomes, M.L., 1994, Determination of alachlor and its sulfonic acid metabolite in water by solid-phase extraction and enzyme-linked immunosorbent assay: Journal of Analytical Chemistry, v. 66, p. 1495-1499.

Aga, D.S., Thurman, E.M., Yockel, M.E., Zimmerman, L.R., and Williams, T.D., 1996, Identification of a new sulfonic acid metabolite of metolachlor in soil: Environmental Science and Technology, v. 30, p. 592-597.

Edwards, T.K., and Glysson, G.D., 1988, Field methods for measurement of fluvial sediment: U.S. Geological Survey Open-File Report 86-531, 118 p.

Ferrer, Imma, Thurman, E.M., and Barceló, Damià, 1997, Identification of ionic chloroacetanilide herbicide metabolites in surface water and groundwater by HPLC/MS using negative ion spray: Journal of Analytical Chemistry, v. 69, p. 4547-4553.

Gianessi, L.P., and Anderson, J.E., 1995, Pesticide use in U.S. crop production-national data report: Washington, D.C., National Center for Food and Agricultural Policy, unpaginated.

Hardy, M.A., Leahy, P.P., and Alley, W.M., 1989, Well installation documentation and ground-water sampling protocols for the pilot National Water-Quality Assessment Program: U.S. Geological Survey Open-File Report 89-396, 36 p.

Hostetler, K.A., and Thurman, E.M., 1999, Determination of chloroacetanilide herbicide metabolites in water using high-performance liquid chromatography-diode array detection and high-performance liquid chromatography/mass spectrometry, in Morganwalp, D.W., and Buxton, H.T., eds., U.S. Geological Survey Toxic Substances Hydrology Program-Proceedings of the Technical Meeting, Charleston, South Carolina, March 8-12, 1999, volume 2 of 3-Contamination of hydrologic systems and related ecosystems: U.S. Geological Survey Water-Resources Investigations Report 99-4018B, p. 345-353.

Kolpin, D.W., Nations, B.K., Goolsby, D.A., and Thurman, E.M., 1996, Acetochlor in the hydrogeologic system in the Midwestern United States, 1994: Environmental Science and Technology, v. 30, p. 1459-1464.

Kolpin, D.W., Thurman, E.M., and Goolsby, D.A., 1996, Occurrence of selected pesticides and their metabolites in near-surface aquifers of the Midwestern U.S.: Environmental Science and Technology, v. 30, no. 5, p. 335-340.

Kolpin, D.W., Thurman, E.M., and Linhart, S.M., 1998, The environmental occurrence of herbicides-the importance of degradates in ground water: Archives of Environmental Contamination and Toxicology, v. 35, p. 1-6.

Leonard, R.A., 1988, Herbicides in surface water, in Grover, R., ed., Environmental chemistry of herbicides-volume I: Boca Raton, Fla., CRC Press, p. 45-88.

Macomber, C., 1992, Determination of the ethane sulfonate metabolite of alachlor in water by high performance liquid chromatography: Journal of Agricultural and Food Chemistry, v. 40, p. 1450-1452.

Thurman, E.M., Goolsby, D.A., Aga, D.S., Pomes, M.L., and Meyer, M.T., 1996, Occurrence of alachlor and its sulfonated metabolite in rivers and reservoirs of the Midwestern U.S.: Environmental Science and Technology, v. 30, p. 569-574.

U.S. Environmental Protection Agency, 1992, Guidelines establishing test procedures for the analysis of pollutants (App. B, Part 136, Definition and procedures for the determination of the method detection limit): U.S. Code of Federal Regulations, Title 40, Revised as of July 1, 1992, p. 565-567.

Ward, J.R., and Harr, C.A., 1990, Methods for collection and processing of surface-water and bed-material samples for physical and chemical analyses: U.S. Geological Survey Open-File Report 90-140, 71 p.