Environ. Sci. Technol. 2000, 34, 1117-1121
Role Played by Basic Hydrolysis in the Validity of Acid Herbicide Data: Recommendations for Future Preparation of Herbicide Performance Evaluation Standards MARGARITA V. BASSETT* AND BARRY V. PEPICH IT Corporation, 26 West Martin Luther King Drive, Cincinnati, Ohio 45219 DAVID J. MUNCH United States Environmental Protection Agency, Office of Groundwater and Drinking Water, 26 West Martin Luther King Drive, Cincinnati, Ohio 45219
Water Supply (WS) 41 herbicide performance evaluation (PE) ampules were designed by the U.S. Environmental Protection Agency (EPA) to test whether laboratories were performing necessary basic hydrolysis in their sample preparation for acid herbicide analysis. This was accomplished by the inclusion of the butyl ester as well as the acid form of 2,4-dichlorophenoxy acetic acid (2,4-D). The results reported for WS 41 did show a significantly lower percentage of labs passing the PE acceptance criteria, suggesting that many labs do not hydrolyze before analysis, but were clustered around a higher than expected value. An in-house study of the WS 41 ampules indicated that the acid and ester concentrations of WS 41 had changed since the time of manufacture and helped explain the bias in reported laboratory results. Extended storage stability studies of acid-catalyzed methylation and transesterification were conducted to investigate the stability of an acid herbicide standard made to mimic WS 41. These results are presented together with recommendations for PE sample preparation.
Introduction The phytotoxic effects of phenoxyacid herbicides toward broadleaf plants were first reported in 1944 (1). Since their discovery, they have been widely used in the agricultural industry, with 2,4-dichlorophenoxyacetic acid (2,4-D) experiencing the most pervasive use and being the most frequently detected in well waters (2). Phenoxyacid herbicides are generally applied as mixtures of salts of the parent acid and esters (2). Ester formulations of 2,4-D offer the advantage of having greater herbicidal activity as compared to the acid formulations, presumably because the ester form is more rapidly absorbed by the plants (1). In addition, the higher molecular weight esters such as the isooctyl ester of 2,4-D have lower volatility and thereby reduced vapor drift (3). It is well established that the hydrolysis of esters is catalyzed by acid or base. However, a fair amount of * Corresponding author phone: (513)569-7182; fax: (513)569-7837; e-mail:
[email protected]. 10.1021/es991043m CCC: $19.00 Published on Web 02/04/2000
2000 American Chemical Society
seemingly disparate data can be found in the literature regarding the rates of hydrolysis of the esters of 2,4-D (411). Zepp et al. published a comprehensive study on several esters of 2,4-D in waters (4). They measured half-life values at pH 6 and 28 °C of 44 days for the methyl ester of 2,4-D and of 26 days for the butoxyethyl ethyl. Calculated half-life values for other alkyl esters ranged up to 1500 days. Similarly, reported half-life values for esters of the acid herbicide 2,4,5trichlorophenoxyacetic acid also differed widely, ranging from 3.4 to 450 days (5). Studies of ester hydrolysis in water agree that hydrolysis rates depend on factors such as pH, ester structure, photolysis, and temperature (5, 6) as well as biotic factors, such as the presence of fish (7). U.S. Environmental Protection Agency (EPA) Methods 515.1, 515.2, and 555 are currently approved for analyzing drinking waters for acid herbicides in conjunction with State and Federal compliance monitoring requirements. Because esters of 2,4-D can be stable in waters for extended periods under appropriate conditions, each method requires a hydrolysis step at pH greater than 12 for 1 h to convert all ester forms of the phenoxyacid herbicide to its corresponding free acid. The free acid may then be analyzed by highperformance liquid chromatography or derivatized to a methyl ester and analyzed by gas chromatography with electron capture detection. All forms of the acid herbicide are reported as a total of the acid form. Confusion regarding the need for basic hydrolysis together with the understanding that hydrolysis adds a time-consuming step to sample preparation led the EPA to question whether laboratories were performing the necessary hydrolysis step. The EPA began formulating a plan to address this question about one year prior to the end of their Water Supply (WS) Performance Evaluation (PE) Program, which was established in the mid-1970s to help the various states assess laboratory performance and administer their programs under the Safe Drinking Water Act. Historically, WS samples had been formulated to contain the target 2,4-D at some concentration solely as the free acid. To address the hydrolysis issue, the EPA designed WS 41 to contain both the acid and some additional n-butyl ester of 2,4-D at an approximate molar ratio of 1:3. This approach was preferred over a formulation that contained only the n-butyl standard because the EPA felt it would provide good separation between laboratories that were not performing the required hydrolysis step and those that were but would still yield a positive result when hydrolysis was skipped, thereby not alarming the laboratories causing them to look more deeply into the matter. In this paper, results obtained for 2,4-D in the EPA’s last WS PE study, WS 41, are presented and compared to the laboratory community’s performance to several earlier WS samples formulated with only the 2,4-D acid. The Agency’s treatment of these data is briefly discussed, and the effect the new standard had on the percentage of laboratories meeting acceptance criteria is presented. Methanol, a common solvent used by commercial standard manufacturers for acid herbicide standards, was inadvertently used as the solvent for WS 41. While this did not effect how the data were treated or the validity of WS 41, storage in methanol altered the initial concentrations of the free acid and n-butyl ester of 2,4-D, somewhat complicating the interpretation of the distribution of laboratory data. Stability studies were conducted on a simulated acid herbicide standard in methanol. The susceptibility of 2,4-D acid toward methylation and its n-butyl ester toward transesterification to the methyl ester are discussed. Finally, because of the Agency’s decision to VOL. 34, NO. 6, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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privatize its WS and WP (Water Pollution) study programs as announced in the Federal Register in 1997 (12) after the WS41 sample, recommendations for the design of future acid herbicide PE standards are presented.
Experimental Section Chemicals and Standard Materials. PE ampules from WS 41 were provided by the EPA. The acid herbicide standards 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenoxyacetic acid n-butyl ester (2,4-D n-butyl ester), dicamba, dinoseb, picloram, acifluorfen, pentachlorophenol, dalapon, and silvex were 97-99% pure (ChemService, West Chester, PA). 2,4-D was also purchased as certified solutions: 2,4-D acid, 0.2 mg/mL in methanol (Accustandard, New Haven, CT), 2,4-D acid; methyl ester, 100 µg/mL in methanol (Ultra Scientific, North Kingstown, RI). Chemicals used for derivatization were prepared as described in the EPA methods (13, 14). Solvents were HPLC grade, except for methanol, which was purge-and-trap grade (B&J, Muskegon, MI). Equipment. The storage stability studies were conducted in a thermostated water bath that regulated temperature within (0.1 °C. Analyses were performed using an HP 6890 gas chromatograph (GC) equipped with an electron capture (ECD) detector and a 30 m, 0.25 mm i.d. (0.25 µm film thickness) J&W DB-5 capillary GC column. Sample Preparation and Analysis. Analyses of water samples spiked with WS 41 PE ampules were conducted as described in EPA Method 515.2, with derivatization of sample extracts as outlined in EPA Method 515.1. (Although not suitable for dalapon analysis, 515.2 is suitable for 2,4-D analysis and was chosen because of its use of solid-phase extraction and GC/ECD analysis.) To help explain the unexpected results compiled from external laboratory analysis of WS 41, sample preparation was performed at three different pH values: neutral (pH 7), pH 10.3, and pH 12.5. Samples were acidified to pH e 2 prior to extraction using C18 columns. The amount of the acid, methyl, and n-butyl ester forms of 2,4-D in the WS 41 ampules was determined by analyzing three ampules of WS 41 in triplicate by GC/ECD after direct dilution of 250 µL of WS 41 into 5 mL of methyl tert-butyl ether (MTBE). Calibration curves were prepared for both methyl and n-butyl ester forms of 2,4-D. One set of triplicates was analyzed unesterifiedsin this way quantitating the 2,4-D content that had already methylated to methyl ester as well as the butyl ester present. Another set of triplicates was esterified before analysis as described in method 515.1 to determine the amount of 2,4-D acid present. The apparent methylation of 2,4-D acid and partial transesterification of the n-butyl 2,4-D to the methyl form in the WS 41 ampules was initially investigated by preparing a mixture of analytes to mimic the WS 41 ampule analytes and concentrations in methanol in a class A 100-mL volumetric flask. Another mixture of a similar composition was prepared in acetone. Samples were partitioned into amber 2-mL screw-cap vials with Teflon-lined lids and stored in the dark at room temperature. Triplicate vials for the standards both in methanol and in acetone were analyzed by direct dilution into MTBE as described above at times equal to 0, 1, 2, 4, 8, 14, and 45 days in the same manner as the WS 41 ampules. A second storage stability study using heat and acid to catalyze the reactions in methanol was prepared at concentrations similar to the WS 41 ampules. One set of vials was prepared and stored in the dark at 40 ( 1 °C, and the second set was stored at room temperature (about 20 °C) in the dark. Solutions were prepared to investigate four different levels of acidification: 3.56 × 10-3 mol/L HCl (HCl/2,4-D, 11:1), 1.78 × 10-3 mol/L HCl (HCl/2,4-D, 5.4:1), 3.56 × 10-4 mol/L HCl (HCl/2,4-D, 1.1:1), and unacidified. A sufficient 1118
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FIGURE 1. Laboratory results for EPA PE sample WS 41. number of vials were formulated to allow triplicate measurement at each temperature on each day of the storage stability study. Vials were analyzed on days 0, 1, 3, 7, 14, and 28 by direct dilution as described above. For all experiments, the QC recommendations of EPA Methods 515.1 and 515.2 were followed. Additional QC samples, such as additional calibration checks, were added as needed to ensure data quality.
Results and Discussion WS 41 PE Results. To test the hypothesis that some of the laboratories might be skipping the required basic hydrolysis step in EPA 500 series methods, WS 41 was prepared with 2,4-D in both acid and n-butyl ester forms. Target concentrations for acid and n-butyl ester were established at 18 and 54 µg/L (as free acid), respectively, because it was believed that the 1:3 molar ratio would provide ideal separation of laboratories not following the method-required hydrolysis step from the data set. The WS 41 acid herbicide ampules were prepared during November 1997. True values for the 2,4-D acid and n-butyl forms were determined to be 18.3 and 54.8 µg/L by HPLC shortly after preparation. This corresponds to total acid concentration of 2,4-D equal to 73.1 µg/L. If the hypothesis held true, WS 41 should have produced results clustered about two values: 73.1 µg/L for laboratories that performed the required hydrolysis step and 18.3 µg/L for laboratories that did not. A total of 298 laboratories participated in WS 41 during the period June-August 1998. The resulting distribution of laboratory results presented in Figure 1 seemed to indicate two populations of data that were separated by an intermediate region where no laboratories reported results. When treated as separate populations (separated by the region without results), the lower group of 128 labs yielded a median value of 30.6 µg/L and the higher group of 170 labs yielded a median value of 67.4 µg/L. While the higher group was in good agreement with the true value, the lower population of data was centered about a median nearly twice the expected concentration. Comparison of WS 41 Results to Previous PE Results. Results for formulations WS 32-36, prepared to contain only the 2,4-D acid, were readily available in electronic format. This facilitated their use in a statistical comparison to WS 41. Laboratory results for 2,4-D in WS 36 are plotted in Figure 2. The distribution of data for WS 36 appears much more normal (or bell-shaped) than WS 41. WS studies 32-35 were similar in shape to WS 36. Acceptance criteria used by the EPA to determine the range of acceptable results for WS PEs were established based on historical performance of each analyte in their respective methods. For 2,4-D, the acceptance range was established as the true value ( 50%. In practice,
FIGURE 2. Laboratory results for EPA PE sample WS 36. this usually results in about 75% of the laboratories submitting acceptable results in a given WS study. Summary statistics for WS 32-36 and 41 are presented in Table 1. For the PEs prepared solely from the acid, the percent of laboratories generating acceptable results ranged from 71.8 to 82.0% with an average of 76.2%. For WS 41, this dropped significantly to 62.8%. In-House Evaluation of WS 41 Ampules. An in-house study was initiated in November 1998 in an attempt to explain the disparity between the observed populations of data and the true values of n-butyl and acid forms of 2,4-D in WS 41. Triplicate analyses of reagent water spiked with WS 41 were performed at three different hydrolysis pH values (7, 10, and 12) to compare the effect that no hydrolysis and hydrolysis at pH lower than recommended had on 2,4-D values. Concentrations paralleled those reported by the WS 41 participants, with the unhydrolyzed samples resulting in an average concentration of 35.4 µg/L and the hydrolyzed samples, both at pH 10 and at pH 12, yielding average concentrations of 75.2 and 79.1 µg/L, respectively. Because in-house results were in reasonable agreement with the observed populations of data in WS 41, the concentrations of the n-butyl ester and acid forms of 2,4-D in WS 41 were reevaluated to determine whether the original true values had changed since their time of manufacture. In this experiment, three ampules of WS 41 were analyzed in triplicate by direct dilution of the underivatized standards into MTBE and injection onto the GC/ECD system. These results are summarized in Table 2, where the ampule concentrations are reported in mg/L and do not take into account the 1000-fold dilution factor that comes from spiking reagent water as directed with the WS standard. Because the overall average of the total 2,4-D concentration in Table 2 is 102% of the true value, it was concluded that essentially all the 2,4-D acid had been converted to the methyl ester. In addition, the concentration of 2,4-D methyl ester was approximately 20 mg/L higher than the original 2,4-D acid spiked (18.3 mg/L), leading to the conclusion that some of the n-butyl ester had transesterified to the methyl ester form. This is supported by the 20 mg/L decrease in the n-butyl ester concentration, from an initial spiked concentration of 54.8 mg/L to about 35.6 mg/L. In other words, about 35% of the 2,4-D n-butyl ester had transesterified to the methyl ester form. It is possible to conclude that laboratories that reported an amount for WS 41 near the value of 39 mg/L determined in-house for the methyl ester belong to one of three groups: laboratories that followed the method, but demonstrated poor accuracy; laboratories that did not hydrolyze before extraction; or laboratories that did not perform an extraction of the PE sample at all, instead choosing to dilute and
derivatize the standard directly. Because WS 41 was prepared in methanol, the molar ratio of the n-butyl to acid forms of 2,4-D had changed from 3:1 at the time of preparation to one closer to unity at the time of their analysis by the laboratory community. While this change decreased the separation between data populations in the Figure 1, it still resulted in a significant decrease in the number of laboratories passing WS 41 for 2,4-D. Given the variability of acid herbicide formulations and hydrolysis rates found in the literature, an analyst does not have enough information to conclude that all possible acid herbicides found in a sample have already undergone hydrolysis. To do so risks compromising the accuracy of the analysis by reporting concentrations that are lower than actual values and increases the risk of false negative results during compliance monitoring. Yet comparison of the inhouse WS 41 results to those reported by the labs participating in WS 41 leads to the conclusion that a significant percentage of these labs are skipping hydrolysis as part of their acid herbicide sample preparation. This conclusion emphasizes the continued need for performance monitoring and improved bench-level training of chemists and technicians. Methylation and Transesterification Studies of 2,4-D in WS 41. The conversion of acids to esters is an equilibrium process that can be catalyzed by acid or base (15, 16). In the presence of small amounts of strong mineral acids, like HCl or H2SO4, carboxylic acids react with alcohols to form esters and water. The acid-catalyzed formation of an ester proceeds through a nucleophilic acyl substitution known as the Fischer esterification reaction (15). The same catalyst that catalyzes the forward reaction also catalyzes the reverse reactions ester hydrolysis. Equilibrium concentrations may be shifted to favor the production of either ester or acid with esters being favored in the presence of large excesses of the alcohol and ester hydrolysis being favored in aqueous solutions. The presence of bulkier groups near the reaction site for either the acid or the alcohol slows both ester formation and hydrolysis through steric hindrance (16). Transesterification, which is also catalyzed by acid or base, follows a directly analogous mechanism. Speculation that the new equilibrium concentration in WS 41 occurred as a result of using methanol as the solvent prompted an experiment in which two mixtures of acid herbicide standards were prepared at concentrations close to that of the WS 41 ampule standard, one in methanol and one in acetone. Care was taken to mimic WS 41 composition because of the possibility that one of the other compounds in the mix was acting as a catalyst for the 2,4-D reactions. The vials were monitored at room temperature over time. The methanol standard showed 42% methylation (8.0 mg/L out of 19.0 mg/L) by the 45th day of storage at room temperature. The acetone mixture showed no sign of methylation. Neither set of standards exhibited any sign of transesterification of the n-butyl ester over the same period. Apparently, the mix of analytes was insufficient to catalyze the kind of composition changes seen in the WS 41ampules. Acid and heat were added to the experimental conditions after discovering that the standard vials could have been stored in a warm warehouse before shipment to participating labs and after consulting with standard manufacturers that had similar observations that they attributed to trace levels of acid in their methanol. A series of storage stability experiments were next conducted at ambient (∼20 °C) and elevated (40 °C) temperature over a 28-day period, with three different catalytic amounts of HCl (molar ratios of HCl/2,4-D were 1.1:1, 5.4:1, and 11:1) added to an acid herbicide standard formulated at concentrations similar to WS 41 in methanol. These samples were prepared and analyzed in triplicate as described in the Experimental Section. A fourth set of samples VOL. 34, NO. 6, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Comparison of Water Study Performance Evaluation Sample Results for 2,4-D true value 2,4-D as acid (µg/L) total no. of results reported acceptance rangea (µg/L) % of labs passing 2,4-D criteria a
WS 32
WS 33
WS 34
WS 35
WS 36
WS 41
18.6 537 9.3-27.9 75.4
7.57 438 3.78-11.4 76.7
32.8 522 16.4-49.2 71.8
52.4 447 26.2-78.6 75.2
38.6 528 19.3-57.9 82.0
73.1 298 36.6-110 62.8
Acceptance ranges are (50% of true value.
TABLE 2. WS 41 Analysis by Direct Dilution into MTBE without Derivatization sample
av concn methyl estera (mg/L)
% RSD
av concn butyl estera (mg/L)
% RSD
av concn total 2,4-Da concn (mg/L)
av % of true valueb
% RSD
WS 41 A WS 41 B WS 41 C all samples
39.9 39.4 37.7 39.0
2.09 2.80 0.61 2.99
33.5 34.9 38.3 35.6
2.32 2.63 1.74 6.85
73.4 74.3 75.9 74.6
100 102 104 102
2.12 2.69 1.18 1.72
a Concentration is reported as acid. 54.8 mg/L n-butyl 2,4-D).
b
Based on true value at time of manufacture for total 2,4-D as acid (73.1 mg/L: 18.3 mg/L acid 2,4-D and
FIGURE 3. Decrease in 2,4-D acid over time at room temperature with varying molar ratios of HCl. that contained no acid were also analyzed each day in triplicate. Data from these studies are presented in Figures 3-5. Figure 3 shows the decrease in the concentration of the acid form of 2,4-D in standards held at room temperature. The unacidified methanol standard was relatively stable. Acidified standards all showed a quick drop in the acid form of 2,4-D, with the highest level of HCl showing the fastest drop. Even samples with the lowest catalytic amount of HCl showed only about half of the 2,4-D acid remaining on the third day (19.8-10.4 mg/L), and in each case no 2,4-D acid remained at the end of 28 days. Heated standards showed similar results, but at an accelerated rate as was expected. At 3 days, even in the samples containing the lowest levels of acid (1.1/1), the 2,4-D acid had decreased in concentration by 86% (from 19.8 to 2.8 mg/L). The heated but unacidified standard also displayed a significant reduction of 52% in the 2,4-D acid concentration by day 28. This compares to a 42% loss (reported as methylation) seen at day 45 in the experiment discussed above. Figure 4 shows the decrease in the n-butyl ester form of 2,4-D in standards held at room temperature. The n-butyl ester concentrations were stable in the nonacidified methanol standards over the 28-day time window. Even the heated standards showed no loss of the n-butyl ester without acid catalysis. Similar to the behavior of the acid form of 2,4-D, the rate of the decline for the n-butyl ester increased with increasing acid concentration; however, the disappearance of the n-butyl ester was more gradual. By day 3, in the sample containing the lowest level of acid, only 9.4% of the 2,4-D 1120
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FIGURE 4. Decrease in n-butyl ester 2,4-D over time at room temperature with varying molar ratios of HCl.
FIGURE 5. Increase in 2,4-D methyl ester over time at room temperature with varying molar ratios of HCl. n-butyl ester had disappeared (from 50.0 to 45.3 mg/L). At the end of the 28-day study, about 60% of the n-butyl ester remained at room temperature with the 1.1:1 HCl. Heated standards showed similar but accelerated results with about 20% of the n-butyl ester remaining after 28 days with the lowest level of acid catalyst. The appearance of the 2,4-D methyl ester, which is formed through methylation of the acid and transesterification of the n-butyl ester, in the room temperature studies is presented in Figure 5. These results come from the analysis of the standards by direct dilution without derivatization. The disappearance of the acid and n-butyl ester forms of 2,4-D in Figures 3 and 4 correlate well with the formation of
the methyl ester. For example, by day 28 the methyl ester concentration of the 1.1:1 molar ratio of HCl is 40.5 mg/L, which is 20.7 mg/L more than the original 19.8 mg/L 2,4-D acid concentration. This extra 2,4-D methyl ester comes from the transesterification of the n-butyl ester, which at day 28 is 33.5 mg/L, or 19.0 mg/L less than the original 52.5 mg/L concentration. Even without the addition of acid, the heated samples contained about 10 mg/L of the methyl ester after 28 days. It is clear that acid herbicide standards formulated to contain the acid form of the molecule in methanol are not stable. The equilibrium reaction of 2,4-D with methanol is driven toward completion over long periods of time in methanol due to its great molar excess. This reaction is accelerated by the addition of acid catalyst and increases with increasing temperature. As expected, transesterification of the n-butyl 2,4-D proceeds more slowly than the methylation of 2,4-D acid due to steric hindrance; however, over long periods of time this equilibrium is also driven toward formation of the methyl ester in methanol. These experiments help explain the changes encountered in WS 41 from the time it was formulated. Recommendations for Future Standard Preparation. The purpose of any PE program is the validation of laboratory analytical methods and techniques. PE studies are important tools for screening out labs with poor proficiency. As the PE program evolves into the externalized proficiency testing studies program, good PE sample design and administration will remain an important way to ensure data reliability. Results reported from the laboratory community participating in WS 41 demonstrated a significant decrease in the performance of the laboratory community when an acid herbicide PE standard was prepared that, for the first time, tested their bench-level sample preparation steps in addition to the determinative analytical step. These results stress the important role that PE sample design plays in the outcome of the study. When designing PE standards for target lists such as acid herbicides, several factors should be considered, including how the target analytes are applied in the field and their relative stability. Potential stumbling blocks in the method like difficult or cumbersome steps in the analytical procedure that could be omitted or poorly performed should be identified and tested whenever possible. And finally, performance evaluation materials must be well characterized and demonstrated to be stable. Quality control testing of the ampules should be performed just prior to distribution and periodically repeated during the recommended holding time of the vials. Applying this approach to the design of acid herbicide PEs, we conclude that they should be formulated to contain at least one stable ester like the n-butyl ester of 2,4-D in a suitable solvent like MTBE to prevent transesterification. Methanol (or other alcohols) should not be used as a formulation solvent. While established criteria currently
specify that acid herbicide standards must contain 2,4-D formulated with at least half butyl ester (17), higher molar ratios of the butyl ester to the acid (3:1, for example) will increase the separation between the laboratories that are not performing this step from the rest of the participants and is therefore desirable. The use of ester forms of other target compounds, such as silvex, would increase this separation even further.
Acknowledgments Recommendations in this paper presented are solely those of the authors. We would like to acknowledge Steve Winslow, Chris Freebis, and Steve Wendelken for their technical assistance and Steve Winslow for his translation of ref 5. This work was funded by EPA under U.S. EPA Contract 68-C60040.
Literature Cited (1) Que Hee, S. S.; Sutherland, R. G. In The Phenoxyalkanoic Herbicides: Chemistry, Analysis, and Environmental Pollution; Zweig, G., Ed.; CRC Series in Pesticide Chemistry; CRC Press: Boca Raton, FL, 1981; Vol. I, pp 1-3, 233 (see also references therein). (2) Kamrin, M. A., Ed. Pesticide Profiles: Toxicity, Environmental Impact and Fate; CRC Press: Boca Raton, FL, 1997; p 304 (see also references therein). (3) Sirons, G. J.; Chau, A. S. Y.; Smith, A. E. Analysis of Pesticides in Water; Chau, A. S. Y., Afghan, B. K., Eds.; CRC Press: Boca Raton, FL, 1982; Vol. II, p 165. (4) Zepp, R. Z.; Wolfe, N. L.; Gordon, J. A.; Baughman, G. L. Environ. Sci. Technol. 1975, 9 (13), 1144-1149. (5) Struif, B.; Weil, L.; Quentin, K. E. Vom Wasser 1975, 45, 53-73. (6) Koshechkina, L. P.; Babicheva, A. F. Khim. Tekhnol. Vody 1981, 3 (4), 326-327. (7) Finlayson, B. J.; Verrue, K. M. Arch. Environ. Contam. Toxicol. 1985, 14, 153-160. (8) Smith, A. E. Weed Res. 1972, 12 (4), 364-72. (9) Stark, J. Weeds Weed Control 1983, 24 (1), 275-86. (10) Wilson, R. D.; Geronimo, J.; Armbruster, J. A. Environ. Toxicol. Chem. 1997, 16 (6), 1239-1246. (11) Wilson, R. G., Jr.; Cheng, H. H. J. Environ. Qual. 1978, 7 (2), 281-86. (12) Fed. Regist. June 12, 1997, 62 (113), 32112-32113. (13) Dressman, R. C.; Lichtenberg, J. J. EPA Method 515.1, Revision 1.0; U.S. Government Printing Office: Washington, DC, 1981. (14) Hodgeson, J. W. EPA Method 515.2, Revision 1.1; U.S. Government Printing Office: Washington, DC, 1995. (15) McMurry, J. Organic Chemistry; Brooks/Cole Publishing Co.: Belmont, CA, 1988; pp 748-750. (16) Morrison, R. T.; Boyd, R. N. Organic Chemistry; Allyn and Bacon, Inc.: Boston, MA, 1973; pp 602-603, 680-683. (17) U.S. EPA. National Standards for Water Proficiency Testing Studies Criteria Document; NERL-Ci-0045; U.S. Government Printing Office: Washington, DC, December 30, 1998.
Received for review July 26, 1999. Revised manuscript received December 13, 1999. Accepted December 15, 1999. ES991043M
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