Environ. Sci. Technol. 1998, 32, 163-168
Evaluation of Microtiter-Plate Enzyme-Linked Immunosorbent Assay for the Analysis of Triazine and Chloroacetanilide Herbicides in Rainfall M I C H A E L L . P O M E S , * ,† E. MICHAEL THURMAN,† D I A N A S . A G A , †,‡ A N D DONALD A. GOOLSBY§ U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, Kansas 66049, and U.S. Geological Survey, Denver Federal Center, Building 25, Lakewood, Colorado 80225
Triazine and chloroacetanilide concentrations in rainfall samples collected from a 23-state region of the United States were analyzed with microtiter-plate enzyme-linked immunosorbent assay (ELISA). Thirty-six percent of rainfall samples (2072 out of 5691) were confirmed using gas chromatography/mass spectrometry (GC/MS) to evaluate the operating performance of ELISA as a screening test. Comparison of ELISA to GC/MS results showed that the two ELISA methods accurately reported GC/MS results (m ) 1), but with more variability evident with the triazine than with the chloroacetanilide ELISA. Bayes’s rule, a standardized method to report the results of screening tests, indicated that the two ELISA methods yielded comparable predictive values (80%), but the triazine ELISA yielded a false-positive rate of 11.8% and the chloroacetanilide ELISA yielded a false-negative rate of 23.1%. The falsepositive rate for the triazine ELISA may arise from cross reactivity with an unknown triazine or metabolite. The false-negative rate of the chloroacetanilide ELISA probably resulted from a combination of low sensitivity at the reporting limit of 0.15 µg/L and a distribution characterized by 75% of the samples at or below the reporting limit of 0.15 µg/L.
The relatively widespread occurrence of herbicides in rainfall is beginning to be recognized. Several studies have documented the occurrence of herbicides in fog (10) and rainfall in the midwestern (11, 12) and eastern United States (13, 14) and in Europe (15-17). However, information on the spatial and temporal distribution of triazine and chloroacetanilide herbicides in rainfall on a regional or multistate scale was deficient. The purpose of the U.S. Geological Survey’s Regional Assessment of Herbicides in Atmospheric Wet Deposition Study was to study the occurrence and deposition patterns of herbicides in precipitation over a large geographic area (7). The study area included the midcontinent and northeastern United States from Kansas north to North Dakota and Kansas east to Virginia, with control sampling sites in Colorado, Wyoming, Montana, and Alaska. Analysis of herbicides in rainfall began in March 1990 and continued until September 1991. Such a large-scale study had the potential to generate hundreds, if not thousands, of samples. GC/MS analysis of all the samples would be prohibitive, so a low-cost, easily accomplished screening method was needed. Enzymelinked immunosorbent assay (ELISA) was chosen as the method to analyze these samples because of its low per sample cost, and the microtiter-plate format allowed up to 72 duplicate atrazine and alachlor analyses of rainfall samples per day. The ability to maintain a high sample throughput was of great concern in a large-scale reconnaissance study that lasted for 18 months. This paper will describe and evaluate the ELISA method used to analyze for triazine and chloroacetanilide herbicides in rainfall with commercially available microtiter-plate kits. Included in this paper will be refinements made to the microtiter-plate ELISA method published in Pomes et al. (18). Information will be included on cross reactivity of triazine and chloroacetanilide herbicides and selected degradation products with the antibodies bound to the microtiter plates. Additionally, this paper will address the comparison of microtiter-plate ELISA results to GC/MS results and the use of Bayes’s rule to evaluate the distribution of positives, negatives, false-positives, and false-negatives. Used in the clinical sciences (19), Bayes’s rule is a standardized procedure for the evaluation of screening methods. Goolsby et al. (20) detail the concentrations and deposition patterns of triazine, and chloroacetanilide herbicides in rainfall and relate the consequences of their transport.
Experimental Procedures Introduction Between 1987 and 1989, approximately 65 million kg/yr of alachlor, atrazine, cyanazine, and metolachlor were applied in 12 agricultural midwestern States (1). Use of herbicides has caused considerable concern because their moderate water solubility and mobility can cause them to leach into groundwater and surface water, be transported in the air, and fall with precipitation (2). Several large-scale reconnaissance studies have been carried out to address the distribution of herbicides in groundwater (3), surface water (4-6), and rainfall (7, 8) as part of the Toxic Substances Hydrology Program of the U.S. Geological Survey (9). * Corresponding author telephone: 785-832-3564; fax: 785-8323500; e-mail
[email protected] or
[email protected]. † U.S. Geological Survey, Lawrence, KS. ‡ Present address: Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-8600 Dubendorf, Switzerland. § U.S. Geological Survey, Lakewood, Colorado 80225. S0013-936X(97)00462-8 Not subject to U.S. Copyright. Publ. 1997 Am. Chem. Soc. Published on Web 01/01/1998
Sample Collection. The National Atmospheric Deposition Program (NADP)/National Trends Network (NTN) Program maintains a national network of wet- and dry-fall collectors to monitor the extent of acid rain in the United States. As detailed in Goolsby et al. (20), site representatives collected weekly rainfall samples in wet-fall buckets from the NADP/ NTN sites and sent the samples to the Central Analytical Laboratory (CAL) at the Illinois State Water Survey in Champaign. Subsamples consisting of as much as 125-mL aliquots from the wet-fall samples were obtained from CAL. A total of 88 subsamples from sites in the study area was then sent each week to the U.S. Geological Survey laboratory in Lawrence, KS, for herbicide analysis. Standard Preparation. Atrazine (obtained from Supelco, Bellefonte, PA) and alachlor standards (U.S. Environmental Protection Agency Pesticide Chemical Repository, Research Triangle Park, NC) were prepared by spiking appropriate volumes of 1 mg/mL stock solutions into 100-mL volumes of distilled water to yield 0.1, 0.5, and 5.0 µg/L concentrations. VOL. 32, NO. 1, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Distilled water was used as the blank. The atrazine, prometon, propazine, and simazine stocks used in the triazine cross reactivity study were obtained from Supelco (Bellefonte, PA), and deethylatrazine was obtained from Ciba Geigy (Greensboro, NC). Finally, the chloroacetanilide herbicide and metabolite stocks were obtained from Monsanto Agricultural Company (St. Louis, MO). Microtiter-Plate Method. The immunoassay kits used for the ELISA analyses consisted of 96-well polystyrene microtiter plates that were coated with polyclonal antibodies (Res-I-Quant kits, Immunosystems, Scarborough, ME). Separate kits with different antibody coatings were used to analyze for atrazine and alachlor. A 160-µL aliquot of sample or standard was transferred to each well of a blank (uncoated) plate. Sample and standard identifications were entered into a template file created with the Softmax operating software of the VMAX microtiter-plate reader (Molecular Devices, Palo Alto, CA). Standards were placed on blank plates in triplicate or quadruplicate; samples were in duplicate. Eighty microliters (80 µL) of sample or standard was then transferred to the test plate (coated with antibodies) with a 12-channel pipet followed by the addition of 80-µL aliquots of enzyme conjugate to each well. Use of the blank plate and the placement of quadruplicate standards in alternating rows reduced within plate variation, because less time was taken to pipet eight rows individually than the time required to pipet 48 sets of duplicate wells. The microtiter plate was covered with a paraffin film and allowed to incubate for 1 h in a microplate incubator at approximately 32 °C while being shaken at 200 rpm on an orbital shaker. During the incubation period, the target analyte in the sample and the enzyme conjugate competed for antibody binding sites. After 1 h, the plate was emptied, flushed five times with deionized water, and tapped dry. Next, 160 µL of a substrate and chromogen mixture was transferred to each well using the 12-channel pipet. Then, the microtiter plate was covered with paraffin film and incubated for 30 min while being shaken at 200 rpm. Finally, 40 µL of 2.5 N sulfuric acid was added to each well to stop the reaction. The finished plate was placed on the plate reader, which determined the optical densities of each well at the preset wavelength of 450 nm. Results were quantified with three solutions of known atrazine or alachlor concentrations that ranged from 0.10 to 5.0 µg/L and blanks (no herbicides). Using the calibration curves, optical densities associated with calibration standards were measured. Wells with optical densities producing calculated values greater than 5% different than actual standard values were deleted from the plate template. The optical density of a well was not determined if it was deleted from the template. Following this operation, the calibration curve was recalculated by rereading the plate. Samples were analyzed in duplicate, and the results were averaged. The reporting limits for ELISA were 0.10 µg/L for atrazine and 0.15 µg/L for alachlor based on comparisons with GC/MS analyses. Poor correspondence between ELISA and GC/MS analyses occurred at concentrations less than 0.10 µg/L for atrazine and less than 0.15 µg/L for alachlor. Evaluation of Cross Reactivity. Distilled water blanks and herbicide and metabolite standards in increasing concentrations were analyzed in triplicate on a single row of a microtiter plate. Optical density values were measured and processed as individual standard curves. From the optical density data, values of Bo (blank) and B (absorbance due to standard) were determined. The IC50 (inhibition concentration obtained at 50% absorbance) obtained at B/Bo ) 0.5 measured the sensitivity of the immunoassay test. Specifically, IC50 concentrations were obtained from line equations of plots of B/Bo versus the logarithms of analyte concentrations. For each analyte, solution of the line equation for the condition of B/Bo ) 0.5 gave the IC50 at 50% 164
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absorbance. Lower IC50 concentrations resulted because the ELISA was sensitive to a particular analyte. The leastdetectable dose (LDD) measured the lowest quantified concentration and was obtained at B/Bo ) 0.9. Gas Chromatography/Mass Spectrometry Procedure. Randomly selected samples, including positive and negative detections of the triazine and chloroacetanilide herbicides as analyzed by ELISA, were analyzed using GC/MS according to the method described by Thurman et al. (21) and Meyer et al. (22). Briefly, the method featured extraction of herbicides from 125-mL water samples with C18 solid-phase extraction cartridges (Sep-Pak Millipore, Cambridge, MA), elution of the cartridges, and spiking of extracts with phenanthrene-d10 using an automated workstation. The volume of the extract containing the herbicides was reduced to 100 µL with a stream of nitrogen. Concentrated extracts were analyzed using a Hewlett Packard (Palo Alto, CA) Model 5890 gas chromatograph and a Model 5970 mass selective detector. Quantitation of herbicides was based on the analyte response ratioed to the response generated by the ion fragment of the internal standard, phenanthrene-d10, with a mass-to-charge ratio of 188 mass units. Confirmation of analytes relied on the detection of the molecular ion, two confirming ions, and on the matching retention of times relative to the internal standard. The quantitation limit for GC/MS analysis was 0.05 µg/L for all analytes of interest. Method for Application of Bayes’s Rule to ELISA Results. Bayes’s rule provides a means to evaluate the operating performance of screening or diagnostic procedures (19) by evaluating the performance of the screening method against that of the confirmatory method for the same set of samples. Tabulations were performed to find numbers of confirmed positives (detections by ELISA and GC/MS), confirmed negatives (nondetections by ELISA and GC/MS), falsepositives (detection by ELISA, but nondetection by GC/MS), and false-negatives (nondetection by ELISA, but detection by GC/MS). Results of a tabulation of confirmed positives, confirmed negatives, false-positives, and false-negatives were placed in a matrix from which values of prevalence rate, specificity, false-positive rate, sensitivity, false-negative rate, and yield were calculated (23).
Results and Discussion Cross Reactivity. The antibody used in the triazine ELISA procedure has a cross reactivity with deethylatrazine, prometon, propazine, and simazine because of the affinity that the antibodies have for similar molecular structures common to that class of herbicides. Thus, the procedure is nonspecific for atrazine but is for triazines in general (24). Other workers reporting on triazine cross reactivity include Dunbar et al. (25), Thurman et al. (26), and Schultze et al. (27). Although widely used in Europe, cross reactivity for terbutylazine was not evaluated because it was not applied within the study area and, at present, has not been registered for use in the United States (28). Of the triazine herbicides under consideration in this study, the antibody used by Immunosystems had the most affinity for atrazine as signified by the lowest IC50 concentration of 0.41 µg/L and one of the lowest LDD of 0.02 µg/L (Figure 1). Probable points of attachment for the antibody include the 5-isopropyl secondary amine and the 3-ethyl secondary amine groups as well as the 1-chloro group. Experience with surface-water samples has shown that the 5-isopropyl secondary amine is primarily responsible for reactivity in the triazine ELISA (23). Modifications to the atrazine molecular structure lead to the decreased cross reactivity observed for deethylatrazine, prometon, propazine, and simazine. Only seven out of 2072 samples detected as positive by triazine ELISA (0.34%) yielded GC/MS detections of deethylatrazine, prometon, propazine, and simazine
FIGURE 2. Selected chloroacetanilide herbicides and metabolites in order of decreasing cross reactivity. Atrazine is included for comparison purposes. IC50 is the 50% inhibition concentration, and LDD is the least- detectable dose, both in µg/L. m ) 1 linear trend. Such scatter may indicate that the triazine ELISA yields enhanced response in some samples.
FIGURE 1. Selected triazine herbicides in order of decreasing cross reactivity. Alachlor is included for comparison purposes. IC50 is the 50% inhibition concentration, and LDD is the least-detectable dose, both in µg/L. without atrazine because of cross reactivity. Alachlor evoked no response from the triazine ELISA. Thus, the triazine ELISA showed the greatest reactivity for atrazine followed by decreased reactivity for propazine, prometon, simazine, and deethylatrazine. The lack of reactivity for alachlor shows that this ELISA reacts specifically with triazine herbicides and not chloroacetanilide herbicides. As described by Feng et al. (29), the antibodies used for the chloroacetanilide ELISA also demonstrated cross reactivity with metolachlor and other degradation products. The antibodies used by Immunosystems had the greatest affinity for alachlor as signified by the lowest IC50 concentration and LDD (Figure 2). Cross reactivity for chloroacetanilide herbicides appears to be based on the presence of (methoxymethyl)acetamide groups. Five (5) out of 2071 samples detected positive by chloroacetanilide ELISA (0.24%) yielded GC/MS detections of metolachlor without alachlor because of cross reactivity. Lacking (methoxymethyl)acetamide groups, atrazine evoked no response from the chloroacetanilide ELISA. Least-Squares Analysis: ELISA Compared to GC/MS. Results of ELISA and GC/MS analyses are listed in Goolsby et al. (8). Figure 3 illustrates plots of triazine and chloroacetanilide ELISA concentrations versus GC/MS concentrations for 1031 samples collected between April and August 1990-1991. Slopes (m) and correlation coefficients (r 2) obtained by comparison of ELISA to GC/MS results were m ) 0.99 and r 2) 0.77 for triazine herbicides and m ) 1.0 and r 2 ) 0.88 for chloroacetanilide herbicides. With slopes equal to 1.0, both ELISA methods accurately reported concentrations of triazine and chloroacetanilide herbicides. However, the triazine ELISA yielded a smaller r 2 value than the chloroacetanilide, reflecting greater scatter to the left of the
Cross reactivity can explain some instances of enhanced response. The ELISA antibodies may react with compounds possessing molecular structures similar to their target analytes but not detected by GC/MS. Such was the case for alachlor ethanesulfonic acid (ESA) in groundwater and surface water, which is responsible for positive detections of chloroacetanilides that were detected by GC/MS (30-32). Although ESA was not analyzed during the time of this study, little evidence exists for an enhanced response with the chloroacetanilide ELISA with a slope of 1.0 and r 2 ) 0.88. However, cross reactivity of the triazine ELISA with another triazine herbicide or metabolite not listed in Goolsby et al. (8) could account for the observed enhanced response. More commonly, cross reactivity can account for slopes less than 1.0 on plots of ELISA response versus GC/MS response. As demonstrated in Figures 1 and 2, ELISA antibodies had the greatest reactivity for their target analytes, but cross reactivity decreased when parts of the molecular structure were changed. Thus, a mixture of atrazine, deethylatrazine, prometon, propazine, and simazine in a given sample could produce a decreased ELISA response relative to the concentration of total triazine herbicides as determined by GC/MS. Least-squares analysis showed that decreased slopes resulted from the comparison of triazine ELISA results to total triazines determined by GC/MS in surface-water samples (18, 23). The decreased slope originated from the diminished response the triazine ELISA has for triazine herbicides other than atrazine. Application of Bayes’s Rule to Comparisons of ELISA with GC/MS Results. A tabulation of all confirmed positives, confirmed negatives, false-positives, and false-negatives was completed to evaluate operating performance of ELISA as a screening technique. Confirmed positives were samples that yielded concentrations greater than the reporting limits of the ELISA (greater than 0.1 µg/L for triazine herbicides and greater than 0.15 µg/L for chloroacetanilide herbicides) and the GC/MS (greater than 0.05 µg/L for both triazine and chloroacetanilide herbicides) analysis techniques. Included in the confirmed positive category were samples that had concentrations above the reporting limit of the ELISA technique but yielded only trace detections using GC/MS VOL. 32, NO. 1, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Bayes’s Rule Matrix for Evaluation of the Microtiter-Plate Triazine ELISA Listing the Distribution of Confirmed Positives and Negatives and False-Positives and False-Negatives herbicide detected by GC/MS
herbicide not detected by GC/MS
total
578 107 685
164 1222 1386
742 1329 2071
ELISA positive ELISA negative total
TABLE 2. Bayes’s Rule Matrix for Evaluation of the Microtiter-Plate Chloroacetanilide ELISA Listing the Distribution of Confirmed Positives and Negatives and False-Positives and False-Negatives herbicide detected by GC/MS
herbicide not detected by GC/MS
total
245 74 319
63 1690 1753
308 1764 2072
ELISA positive ELISA negative total
TABLE 3. Tabulation of Prevalence Rate, Sensitivity, Specificity, False-Positive Rate, False-Negative Rate, and Yield for Triazine and Chloroacetanilide Microtiter-Plate ELISA Methods parameter
triazine (%)
chloroacetanilide (%)
prevalence rate sensitivity specificity false-positive rate false-negative rate yield
33.1 84.4 88.2 11.8 15.6 77.9
15.4 76.8 96.4 3.6 23.2 79.5
all samples screened as negative by the ELISA are confirmed as negative by the GC/MS. No false-positives are found, so the false-positive rate of 0% results. A sensitivity of 100% requires that all samples screened as positive by the ELISA are confirmed as positive by the GC/MS. No false-negatives are identified, so a false-negative rate of 0% results. Yield is calculated by dividing the number of confirmed positives by the sum of confirmed positives and false-positives. In the absence of false-positives, yield equals 100%. FIGURE 3. Plots of concentrations of (A) triazines by ELISA versus concentrations of atrazine by GC/MS, and (B) chloroacetanilides by ELISA versus alachlor by GC/MS for 1031 rainfall samples collected between April and August 1990-1991. analysis. Trace detections were analyte concentrations less than the reporting limit of the GC/MS but present in sufficient quantities to prompt integration of the molecular and basepeak ions of respective analytes during data analysis [see Meyer et al. (22) for details]. Confirmed negatives were samples that yielded concentrations less than the reporting limits for the ELISA and GC/MS techniques. False-positives were samples yielding concentrations above the reporting limit for ELISA but for which nothing was detected by the GC/MS. Finally, false-negatives were samples that yielded concentrations greater than the reporting limit for GC/MS, but less than the reporting limit for ELISA. Tables 1 and 2 summarize the results of tabulations for the triazine and chloroacetanilide ELISA methods, respectively. Table 3 lists the prevalence rates, specificities, false-positive rates, sensitivities, false-negative rates, and yields of the respective microtiter-plate ELISA methods. As determined by Bayes’s rule, the ideal ELISA screening method produces values for specificity, sensitivity, and yield that approach 100% (23). A specificity of 100% requires that 166
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Values for specificity, sensitivity, and yield calculated for the triazine and chloroacetanilide ELISA methods vary from 100%. The triazine and chloroacetanilide ELISA gave different specificity values, 88.2 and 96.4%, respectively (Table 3), on the basis of relative occurrences of false-positives (Tables 2 and 3). With greater detections of false-positives, the triazine ELISA appears to have less ability to accurately predict positive responses than the chloroacetanilide ELISA. However, the chloroacetanilide ELISA has less ability to predict negative responses as indicated by respective sensitivity values (84.4 and 76.8%) and false-negative rates (15.6 and 23.2%). Despite differences in values specificity and sensitivity, the two ELISA methods gave comparable yields, 77.9% for triazine and 79.5% for chloroacetanilide herbicides, meaning that both methods have nearly the same ability to predict the presence of triazine and chloroacetanilide herbicides in rainfall. The larger false-positive rate may be attributed to the triazine ELISA antibody cross reacting with an unknown triazine or metabolite. Goolsby et al. (20) postulated that photochemical processes could transform atrazine to deethylatrazine in the atmosphere. Studies of the photocatalytic degradation and ozone oxidation of atrazine have established a pathway along which atrazine degrades to atrazine amide
TABLE 4. Summary of Herbicide Concentrations Measured by ELISA in 5297 Rainfall Samples (8, 20) and 1725 ELISA Storm Runoff Samples (36) concentration in µg/L, for indicated percentiles triazines percentile 1 10 25 50 (median) 75 90 95 99 100 (maximum) % detections
chloroacetanilides
rainfall (20) storm runoff rainfall (20) storm runoff
