Environ. Sci. Technol. 1993, 27, 562-564
COMMUNICATIONS Immunoassay Screens for Alachlor in Rural Wells: False Positives and an Alachlor Soil Metabolite David B. Baker,**+Rodney J. Bushway,$ Stephen A. Adams,§ and Carol Macomber11
Water Quality Laboratory, Heidelberg College, Tiffin, Ohlo 44883, Department of Food Science, University of Maine, Orono, Maine 04469, The Agricultural Group of Monsanto, 700 Chesterfield Village Parkway, St. Louis, Missouri 63198, and ImmunoSystems, Inc., 4 Washington Avenue, Scarborough, Maine 04074 Introduction Enzyme-linked immunosorbent assay (ELISA) techniques are coming into widespread use as screening methods for pesticides in various media, including water, soils, and foods ( I , 2). Relative to traditional chromatographic techniques for pesticides, ELISA offers the advantages of simplicity, high sensitivity,low cmt, and speed. However, ELISA may be subject to false positives if the antibodies used in the test cross-react with pesticides of similar chemistry or various analogues of the targeted pesticide (3). Interpretation of negative ELISA results is unambiguous. These samples lack not only the target compound, at concentrations above its detection limit, but also any other compounds which cross-react with the ELISA system, at concentrations above their detection limits. However, if the results of the ELISA test are positive, the reactivity may be due to (1) the targeted compound for which the system is calibrated, (2) compounds known to cross-react with the system, (3) an unknown cross-reactivematerial, or (4)a combination of the above. If the reactivity is due to any of the last three possibilities, the concentration of the target compounds is uncertain. In any case, confirmation by alternative analytical techniques is needed to determine the cause of the positive response. The Water Quality Laboratory (WQL) at Heidelberg College operates a rural private well testing program in which ELISA testing for the herbicide alachlor has been conducted on 8137 private rural wells, primarily from Indiana, Kentucky, and Ohio. Alachlor is manufactured by The Agricultural Group of Monsanto Co. and sold under the trade name Lasso. For alachlor screening, the WQL uses the EnviroGard Alachlor Plate Kit developed by Immunosystems, Inc. and sold by Millipore, Inc. False positives for alachlor have frequently been encountered in this program. Studies were undertaken to investigate the frequency of false positives and to identify the Cross-reactive material. A known soil metabolite of alachlor, 2-[ (2,6-diethylphenyl)(methoxymethyl)amino]-2-oxoethanesulfonate,has been identified as a cross-reactive material that accounts for many of the false positive responses. To our knowledge, the ethanesulfonate metabolite (ES) has not previously been found or identified in water, although it has previously been noted to cross-react with alachlor antibodies (4). Analyses for ES were not included in the US. EPA's +HeidelbergCollege. of Maine. The Agricultural Group of Monsanto. 11 ImmunoSystems, Inc.
1 University
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National Pesticide Survey (5), in Monsanto's National Alachlor Well Water Study (6),or in monitoring programs conducted by midwestern states (7). Experimental Procedures Water samples used in the rural well testing program and for subsequent GC confiiation testing were collected by residents and shipped to the WQL. The WQL subsequently shipped sets of samples to ImmunoSystems and the University of Maine for ES testing. Samples analyzed for ES by the Agricultural Group of Monsanto were collected by Monsanto staff according to good laboratory practices. ELISA testing for alachlor and atrazine was conducted by the WQL according to the instructions contained in the EnviroGard plate kits for alachlor and a second herbicide, atrazine. Final absorbance readings were measured using a microplate reader. Confirmation testing for alachlor by the WQL used solid-phase extraction and analysis by GC, using dual capillary columns (DB5 and DB1701) and nitrogen-phosphorus detectors. Confirmation testing by Monsanto was conducted using GC-MS. An HPLC method for the analysis of ES was developed at ImmunoSystems and modified at the University of Maine (8). This method involves extraction of ES with a C18 solid-phase cartridge, alcohol elution, and quantitation using HPLC with UV detection at 205 nm. A slight modification of this method was used by Monsanto for independent ES testing in well water samples. Results and Discussion The results of the WQL's ELISA-based testing program differ greatly from results of previous rural well testing studies where GC and GC-MS techniques have been used. In the previous studies alachlor was detected much less frequently than atrazine and rarely exceeded drinking water standards, except in cases of obvious point source contamination (6, 7, 9). In the WQL program, positive responses for alachlor were observed more frequently than for atrazine, and the raw results suggested that alachlor exceeded its drinking water standard of 2 pg/L in 1.9% of the wells sampled. These discrepancies are illustrated in Table I, where the results from a Monsanto study of rural wells (6)are compared with the results of this ELISA testing. Not only are the ratios of alachlor to atrazine different but also the general levels of both herbicides are considerably higher in the ELISA testing. The nonrandom nature of samples submitted to the WQL for testing may account for the higher frequency of atrazine detections. ELISA positive responses for alachlor were particularly prevalent in some counties of northern Indiana (Table 11).
0013-936X/93/0927-0562$04.00/0
0 1993 American Chemical Society
Table I. Comparison of Alachlor and Atrazine Concentrations in Private Rural Wells in Programs Using GC-MS and ELISA Analytical Procedures % of wells at or
study NAWWS"
above anal. 0.1 0.5 1.0 herbicide method pgfL pgfL pgfL alachlor GC-MS atrazine GC-MS
0.36 0.06 2.32 0.23
0.03 0.16
cooperative well alachlor ELISA testing programb atrazine ELISA
11.02 5.09 6.31 2.62
3.46 1.53
"National Alachlor Well Water Study (6); 1430 randomly selected wells representing 6 million wells in the alachlor use area. More than 8100 wells tested as part of a voluntary program.
+*+.-
Table 11. Summary of Alachlor ELISA Results"
N 8137 all samplesb Indiana samples 5513 5 northern Indiana countiesC 1166 673 drilled wells 418 driven wells
+
+
. .1
1
10
+I 100
ELISA Alachlor, p g L
est % of wells at or above 0.2 1.0 2.0 rglL rglL PgIL 3.5 1.9 8.5 7.9 3.3 1.7 19.8 9.3 4.5 12.4 4.4 2.5 32.3 17.0 7.4
B
"Most of these alachlor detections are false positives. bEntire cooperative private rural well testing program. Counties are LaPorte, St. Joseph, Marshall, Jasper, and Pulaski.
Aquifers within these counties are highly vulnerable due to very permeable soils and shallow water tables. Many of the private drinking water supplies in these counties utilize driven wells rather than drilled wells, and alachlor positive results are particularly evident in these driven wells. The average depth of the driven wells is 13.1 m while that of the drilled wells is 26.5. Confirmation testing for alachlor was initiated by Monsanto for several cases where the ELISA testing suggested alachlor exceeded its drinking water standard. Using GC-MS, Monsanto determined that alachlor was not present in the samples. Subsequent GC testing of these samples at the WQL detected neither alachlor nor metolachlor or metalaxyl, two cross-reacting compounds identified in the EnviroGard Alachlor Plate Kits. To determine the prevalence of false positives for alachlor, the WQL offered a confirmation program to residents whose initial ELISA results suggested alachlor at 6.5 pg/L or higher. In response, 157 samples were submitted for retesting by both ELISA and GC. Samples were obtained primarily from Indiana, but also from 11linois, Kentucky, New Jersey, Ohio,and Virginia. Samples for the confirmation testing were collected between late January and early May 1992,prior to 1992 herbicide applications. In 136 of these samples, the ELISA tests indicated alachlor concentrations greater than 0.2 pg/L. Alachlor was detected by GC in only 33 of the 136 samples that were positive in the ELISA test, and it was not detected in any of the 21 samples that were negative on the ELISA test. Even where alachlor was detected by GC, ita concentxation was generally much lower than that suggested by ELISA (Figure lA), indicating that cross-reactingsubstances were present in most of the samples. False positive samples were identified from all six states. Metolachlor was detected by GC in 22 of the 136 samples, but in only 5 cases was the metolachlor concentration sufficiently high to account for the ELISA response.
0
20
40
60
80
ELISA Alachlor, &$
C
0
20
40
60
80
ELISA ES, pLg/L Flgure 1. Summary of alachbr confirmation testing and ES analyses. (A) Comparlson of alachlor GC analyses with alachbr ELISA analyses for 136 well samples with alachlor ELISA greater then 0.2 pg/L. The 1:1 line shows the expected relationship If all of the ELISA response were due to alachlor. (E) Comparison between ES concentrations as measured by HPLC and alachlor ELISA results for 17 samples that were false positives for alachlor. (C)Comparison between ES determined by HPLC and ES determined by ELISA alachlor kits calibrated with ES for the above 17 samples. The solid lines in (8) and (C) represent the regression relationship between the results given by the two methods.
Metolachlor was not detected in any of 21 samples that were ELISA negative. In 13 samples, both alachlor and metolachlor were present. Thus,in 94 of the samples that Environ. Scl. Technol., Vol. 27, No. 3, 1993 563
were positive in the ELISA test, neither alachlor nor metolachlor was detected. ES analyses by HPLC at the University of Maine were completed on 30 well water samples, of which 5 contained alachlor confirmed by GC, 24 were false positives, and 1 was negative for alachlor by ELISA. ES was detected in all 24 of the false positive samples but in none of the other 6 samples. ES concentrations ranged from 1.2 to 74 pg/L with a median value of 14 kg/L. In the initial six samples, all of which were false positives for alachlor, the presence of ES was confirmed by LC-MS-MS (8). Of the 24 false positive samples, 17 were analyzed by the ELISA system calibrated with ES instead of alachlor. These 17 samples were collected during the middle portion of the study. The correlation between ES, as measured by HPLC, and the ELISA alachlor results for these 17 samples is shown in Figure l B , while the relationship between the HPLC ES and ELISA calibrated with ES is shown in Figure 1C. It is clear that the alachlor ELISA test kits do cross-react with ES and that the ES concentrations, as measured by HPLC, are similar to the concentrations predicted by the ELISA kits with ES calibration. Monsanto independently Collected samples for ES analyses by HPLC at 12 sites which comprised a subset of the 30 sites where samples were analyzed for ES by the University of Maine. There was excellent agreement in the ES concentrations, as measured by HPLC, between the two laboratories. The relatively high concentrations of ES in private well samples, coupled with its detection in samples collected during winter and spring months prior to spring herbicide applications, suggest that ES is relatively persistent and mobile in groundwater. ES has also been identified by HPLC in surface water samples from Ohio tributaries to Lake Erie (8). ELISA false positive results for alachlor have been noted in surface water samples from the Mississippi River (Don Goolsby, USGS, personal communication, April 1992). Toxicity data for ES available from Monsanto and submitted to the US. EPA show ES is nonmutagenic, does not bioconcentrate, and does not undergo significant metabolic transformations when fed to animals. A structural isomer of ES has an LD,, of X O O O mg/kg in short-term animal tests. Additional toxicity testing of ES is now underway. The US.EPA has been notified of the detections of ES in groundwater samples, as required under Section 6(a)(2) of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). The EnviroGard Alachlor Plate Kit now contains a statement regarding cross-reactivity with ES. Other com-
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mercially available ELISA kits for alachlor have varying sensitivities to ES. Recent studies by the WQL indicate that the Alachlor 2.0 kits distributed by Agri-Diagnostics have much less cross-reactivityto ES than do EnviroGard alachlor plates. Alachlor test kits distributed by OHMICRON have cross-reactivity to ES that is similar to that of EnviroGard plates (Robert Sandberg, OHMICRON, personal communication, November 2, 1992).
Summary Unexpectedly high frequencies of positive responses have been observed in ELISA screening for alachlor in private rural wells, when samples were analyzed using the EnviroGard Alachlor Plate Kit. The majority of positive responses are false positives with respect to alachlor. In the false positive samples which have been tested, most of the positive response appears to be due to the ethanesulfonate metabolite of alachlor. The concentrations of ES are relatively high, even in samples collected months after alachlor application, suggesting that ES is relatively persistent and mobile in soil. Additional studies of ES are warranted, and ELISA alachlor screens may be useful in its study. Literature Cited (1) Bushway, R. J.; Perkins, B.; Savage, S. A.; Lekousi, S. J.; Ferguson, B. S. Bull. Environ. Contam. Toxicol. 1988,40, 647-654. (2) Van Emon, J. M.; Lopex-Avila, V. Anal. Chem. 1992,64, 79-88. (3) Lee, S. M.; Richman, S. J.Assoc. U f f . Anal. Chem. 1991, 74, 893. (4) Feng, P. C.; Wratten, S. J.; Horton, S. R.; Sharp, C. R.; Logusch, E. W. J . Agric. Food Chem. 1990,38, 159-163. (5) National Pesticide Survey: Survey Analytes; U.S. Environmental Protection Agency, Fall, 1990. (6) Holden, L. R.; Graham, J. A.; Whitmore, R. W.; Alexander, W. J.; Pratt, R. W.; Liddle, S. K.; Piper, L. L. Enuiron. Sci. Technol. 1992,26, 935-943. ( 7 ) Hallberg, G. R. Agric. Ecosyst. Environ. 1989,26,299-367. ( 8 ) Macomber, C.; Bushway, R. J.; Perkins, L. B.; Baker, D. B.; Fan, T. S.; Ferguson, B. S. J . Agric. Food Chem. 1992, 40, 1450-1452. (9) National Pesticide Survey: Project Summary; U.S. Environmental Protection Agency, Fall, 1990.
Received for review September 18, 1992. Revised manuscript received November 8,1992. Accepted November 23,1992. T h e GC confirmation testing was supported by grants to the Heidelberg College Water Quality Laboratory from ImmunoSystems, Inc., Millipore Corp., and T h e Agricultural Group of Monsanto CO .
