Science Inventory



Shoemaker, J A. METHOD DEVELOPMENT FOR ALACHLOR ESA AND OTHER ACENTANILIDE HERBICIDE DEGRADATION PRODUCTS. Presented at 224th American Chemical Society National Meeting, Boston, MA, August 18-22, 2002.


Develop an analytical method for the analysis of alachlor ESA and other acetanilide pesticide degradation products. The draft method, suitable for use in assessing occurrence of the pesticide degradation products as part of the Unregulated Contaminant Monitoring Rule (UCMR), will be delivered to OGWDW by February 2004. The final peer-reviewed method was delivered in September 2004.


Introduction: Acetanilide herbicides are frequently applied in the U.S. on crops (corn, soybeans, popcorn, etc.) to control broadleaf and annual weeds. The acetanilide and acetamide herbicides currently registered for use in the U.S. are alachlor, acetochlor, metolachlor, propachlor, flufenacet and dimethenamid. Acetanilide degradation products are generally more water soluble and mobile than the parent herbicide, thus there is greater potential for these degradates to be found in ground waters and surface waters. The most common acetanilide degradation products are the ethanesulfonic acid (ESA), oxanilic acid (OA) and sulfinylacetic acid (SAA) derivatives of the parent herbicides. The ESA and OA degradates of alachlor, metolachlor, and acetochlor have been reported in U.S. Midwestern surface and ground waters at typical concentrations of 0.1- 20 g/L [1-5].

High priority is given to this research project in EPA's Office of Research and Development because acetanilide degradation products are identified on the 1998 Drinking Water Contaminant Candidate List (CCL) [6]. Specifically, analytical methodology is needed to gather occurrence data on the acetanilide degradation products. While several methods have been reported in the literature [3,7], these methods do not address issues specific to analyzing compounds in drinking water, such as preservatives and internal and surrogate standards. In addition, the reported methods do not contain all the target analytes listed in Table 1. The objective of this research is to develop an accurate and precise analytical method to detect and quantitate the ESA, OA and SAA acetanilide pesticide degradates in drinking water matrices. This will include two steps: 1) evaluating the capability of solid phase extraction (SPE) techniques to concentrate the acetanilide ESA, OA, and SAA degradates from drinking water, and 2) evaluating the capability of liquid chromatography/mass spectrometry (LC/MS) techniques to separate and detect ESA, OA, and SAA acetanilide herbicide degradates in the concentrated sample extracts. Future occurrence data gathered with this developed method can then be used in determining whether to study the health effects of the acetanilide herbicide degradation products, and ultimately whether to regulate these compounds or remove them from the CCL.

Materials and Methods

Reagents/Standards. The ESA, OA, and SAA degradation products of alachlor and propachlor were obtained from Monsanto Co. (St. Louis, MO). The ESA and OA degradation products of metolachlor were obtained from Novartis (Greensboro, NC). The ESA and OA degradation products of dimethenamid were obtained from BASF Corp. (Research Triangle Park, NC) and the acetochlor ESA and OA degradates from Zeneca (Berkshire, UK). (Note: Novartis and Zeneca Crop Protection Units merged in 2001 to form Syngenta.) Flufenacet ESA and OA were obtained from Bayer Corp. (Stilwell, KS). The internal standard, 4-phenoxybenzoic acid (PBA), and surrogate, 2-benzoylbenzoic acid (BBA), were purchased from Sigma-Aldrich (Milwaukee, WI). The preservatives, copper(II) sulfate pentahydrate, tris(hydroxymethyl)nitromethane (trisnitro), diazolidinylurea (DZU), and trizma (tris(hydroxymethyl)aminomethane+tris hydrochloride) were purchased from Sigma-Aldrich and sodium sulfite from Fisher (Fair Lawn, NJ). Optima grade methanol and ACS grade ammonium acetate were purchased from Fisher. Deionized water was used from a four-stage Milli-Q water system (Millipore; Bedford, MA). Ten millimolar ammonium acetate was prepared by adding 0.77 g of ammonium acetate to 1 L of deionized water. The acetanilide, internal standard, and surrogate spiking mixes were prepared in methanol and calibration standards were prepared in the 10 mM ammonium acetate. Three or four point linear calibration curves were generated daily using PBA as an internal standard and BBA as the surrogate standard each at 200 pg/ L. Calibration points were generated at 10, 20, 50, 100, and 200 pg/ L.

Solid phase extraction. Samples were extracted using a Zymark AutoTrace SPE WorkStation (Hopkinton, MA) and Supelco (Bellefonte, PA) ENVI-CARB carbon cartridges (6 mL, 250 mg). The deionized water samples were fortified with BBA at 200 pg/ L and the preservatives at the concentrations noted in Table 1. The carbon cartridges were conditioned with 20 mL of 10 mM ammonium acetate in methanol (prepared by adding 0.77g of ammonium acetate to 1 L of methanol) followed by 30 mL of deionized water. Water samples were passed through the cartridges at a flowrate of 10 mL/min. The target analytes were eluted from the carbon cartridges with 10 mL of 10 mM ammonium acetate prepared in methanol at a flowrate of 5 mL/min. All extracts were evaporated to dryness with a nitrogen stream in a 70 C water bath, spiked with 20 L of 10 ng/ L PBA and reconstituted to 1 mL with 10 mM ammonium acetate prepared in deionized water.

Liquid chromatography/mass spectrometry. Extracts were analyzed on a ThermoFinnigan (San Jose, CA) LCQ Deca ion trap mass spectrometer equipped with an atmospheric pressure ionization source and an Agilent (Palo Alto, CA) HP1090 LC. The target analytes were quantitated by negative ion electrospray using the peak area of the [M-H]- for each target analyte. The sheath gas (80, unitless), auxillary gas (30, unitless), and heated capillary temperature (300?C) were optimized on m/z 314 of alachlor ESA (0.25 mg/L) infused at 0.4 mL/min. An Agilent Hypersil (2.1 x 100 mm, 5 m) C18 analytical column was used to separate the target analytes at a flowrate of 0.4 mL/min and column temperature of 70?C. The injection volume was 100 L. The binary mobile phase gradient composition was (A)10 mM ammonium acetate (ammonium acetate in deionized water, pH = 7.0 unadjusted) and (B) methanol. The initial mobile phase composition was 90:10 A:B, with a linear ramp to 80:20 A:B in 7 minutes followed by a linear ramp to 75:25 A:B in 3 minutes. The column was held at 75:25 A:B for 7 minutes and allowed to re-equilibrate to initial conditions for 15 minutes prior to the next injection.

Results and Discussion

Previous research has demonstrated chromatographic separation of 12 target acetanilide degradates, including chromatographic separation of the alachlor/acetochlor ESA and OA structural isomers [8,9]. Flufenacet ESA, flufenacet OA, a potential internal standard, and a surrogate standard have been added to the analysis. Figure 1 shows the chromatographic separation achieved using a 10 mM ammonium acetate/methanol gradient and heating the analytical column to 70 C. Previous internal standards used were characterized by poor day-to-day precision resulting in frequent calibration. PBA is currently being evaluated as a potential internal standard and BBA as a potential surrogate. The internal calibration using PBA is linear with r2>0.995 for all target analytes. Long-term stability of the calibration using PBA as the internal standard is currently being studied.

EPA drinking water regulatory methods typically use preservatives to prevent microbial degradation (e.g., acid, copper(II) sulfate, DZU, trinitro) and to dechlorinate (e.g., sodium sulfite, trizma) the sample. Microbial degradation of the target analytes cannot be predicted in all types of matrices containing various types of microbiological contaminants, thus an anti-microbial agent is desirable. While chlorine may not adversely affect the stability of acetanilide degradates, it can interfere in the solid phase extraction, thus the residual chlorine should be removed. A number of preservative combinations were investigated based on research conducted by Winslow and colleagues [10]: copper(II) sulfate/trizma, DZU/trizma, trisnitro/trizma, hydrochloric acid (pH=2)/sodium sulfite. Table 1 lists the recoveries and relative standard deviations (RSDs) obtained with the various preservatives. The recovery and precision goals for EPA methods are typically 70-130% recovery with <30% RSDs. Only the hydrochloric acid (pH=2)/sodium sulfite combination met these recovery and precision goals with the exception of alachlor SAA and the surrogate. Extractions performed using only trizma (no anti-microbial) resulted in acceptable recoveries indicating that in 200 mL samples, the trizma is not the problem. In the case of copper(II) sulfate, the sulfate anion may be interfering with the adsorption of the target acids onto the carbon SPE sorbent. In the case of DZU, retention of the highly concentrated DZU on the carbon cartridges is probably exceeding the capacity of the cartridge, thereby preventing retention of the target analytes. The extracts produced using the trisnitro/trizma combination were yellow, indicating retention of the trisnitro on the carbon sorbent similar to DZU. Analysis of the trisnitro/trizma extracts overwhelmed the LC/MS and yielded poor target analyte recoveries (not shown), thus studies with trisnitro were discontinued. The acid/sodium sulfite combination demonstrates the most promise as potential preservatives for this method. Further studies are in progress to study the effect of water quality parameters, such as humic material and hardness, on the recovery of these analytes using this procedure. In addition, holding time studies will be performed to ensure the target analytes will survive typical shipping conditions and times.

Record Details:

Product Published Date: 08/18/2002
Record Last Revised: 06/21/2006
Record ID: 62104