Immunochemical Technology for Environmental Applications

Chapter 18

Evaluation of Immunoassay for the Determination of Pesticides at a Large-Scale Groundwater Contamination Site Downloaded by NORTH CAROLINA STATE UNIV on October 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch018

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1

T. R. Dombrowski , E. M . Thurman , and G. B. Mohrman

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U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, KS 66049 Rocky Mountain Arsenal, Headquarters Building, Commerce City, CO 80022 2

Pesticide concentrations in ground water at Rocky Mountain Arsenal (RMA) near Denver, Colorado, were determined using solid-phase extraction (SPE) gas chromatography/mass spectrometry (GC/MS) procedures and enzyme-linked immunosorbent assay (ELISA) for cyclodiene insecticides and triazine herbicides. Matrix interferences resulted in inconclusive results for some GC/MS analyses due to baseline disturbances and co-elution, but ELISA analyses consistently gave definitive results in a minimum amount of time. ELISA was used initially as a screening method, and pesticide concentrations and plume extents identified by ELISA were confirmed by SPE-GC/MS. A high degree of correlation was seen between results from GC/MS and ELISA methods for the triazine herbicides (correlation coefficient (R ) = 0.99). All areas with high pesticide concentrations were found to be within the boundaries of R M A . 2

Enzyme-linked immunosorbent assay (ELISA) has proven to be an increasingly important technique for the analysis of an expanding list of environmentally significant compounds. One major example of this growing field of research is the application of immunoassay techniques to the evaluation of pesticide concentration and transport in ground and surface water in the central United States (1,2,3). Within these evaluations, ELISA has been demonstrated to perform in a consistent, reliable manner in the characterization of agricultural pesticide residues in water and soil. The major part of the contamination described in these evaluations, however, usually is confined to the natural water systems present in agricultural areas, and the potential contaminants likely to be encountered (pesticides and fertilizers) are fairly well known. Matrix interferences usually are due to naturally occurring dissolved organic matter (such as humic and fulvic acids) and pesticide degradation products and metabolites, which have been characterized to a broad extent (4). The use of ELISA to characterize extensively contaminated ground water has not been as well documented, and the performance of the technique in these situations is not as well understood. Rocky Mountain Arsenal (RMA), a 70-km tract of land 14.5 km northeast of Denver, Colorado (Figure 1), has been the site of several intensive chemical research and production projects. From the 1940's to the 1960's the R M A was the principal center for the production of chemical agents and their constituents and for emptying 2

This chapter is not subject to U.S. copyright. Published 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

Downloaded by NORTH CAROLINA STATE UNIV on October 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch018

A *

Rocky Mountain / Arsenal i

( ] Denver

Colorado

0

50

100 kilometers

Freshwater reservoirs 0

1

2 kilometers

Figure 1. Location of the study area and significant features at the Rocky Mountain Arsenal near Denver, Colorado.

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


18. DOMBROWSKI ET AL.

Pesticide Concentrations in Groundwater 223

canisters of unused mustard gas by the U.S. Department of the Army (5). In the late 1940's, production facilities at R M A were leased to private industry and were used to manufacture agricultural pesticides. Waste and other industrial byproducts of the manufacturing processes from both the U.S. Department of the Army and private industry were disposed of in accordance with the accepted protocols of that time period. Since the first contaminant problems were recognized in the mid-1950's, innovative containment and cleanup of these wastes have been undertaken on a massive scale by all organizations associated with R M A property (6). Extensive barrier systems have been installed on the downgradient boundaries of R M A (Figure 1). These systems consist of an impermeable, physical barrier (bentonite) to the ground water flowing off post to the north and northwest of R M A , combined with an activated-carbon filtration system that intercepts ground water. The ground water is pumped through a series of activatedcarbon filters and then recharged back into the subsurface on the opposite (downgradient) side of the barrier system. The emplacement of these and other related measures has resulted in the reduction of contaminants moving offsite to concentrations less than the maximum acceptable levels established by the U.S. Environmental Protection Agency (EPA) and the Colorado Department of Health (7). Target analytes and "fingerprint" compounds (materials known to be unique to RMA) were identified early in characterization and remediation activities to allow the specific contaminant-plume boundaries to be positively identified and mapped. Aggressive monitoring policies have resulted in the establishment of a network of more than 1,200 wells that extends throughout R M A property and into the surrounding areas. The volume of analytical work required to support the monitoring network activities combined with sampling constraints due to well volume, hazard and accessibility, and the complex chemical matrix present in many areas of R M A , present a major challenge intime,expense, and overall complexity of analysis. The application of ELISA as a rapid screening technique for the identification of pesticide contaminants at R M A was examined. ELISA was selected to be evaluated as a screening technique at R M A because several characteristics of the immunoassay may serve to minimize the constraints listed above. ELISA techniques have several advantages over conventional instrumentation methods currently in use at R M A in that ELISA has very small sample-volume requirements, is fast, field-portable, and is highly specific. The small sample volumes required by ELISA (usually 300 \iL or less) are a benefit where sample volumes are limited and where several analyses may need to be done on a single sample. The small sample-volume requirement is also beneficial when the samples are highly contaminated with toxic materials. Small volumes ntinimize the risk of exposure to these compounds during handling, transport, and storage of the samples. The specificity of the technique comes from the inherent physiologically selective requirements for antibodies produced in a living system. Immunoassay utilizes the specific binding sites of antibodies to recognize and bind to a single compound or members of a class of compounds. This "targeted" binding can be used in conjunction with a labeled enzyme conjugate of the target analyte (competitive binding assay) or a separate, labeled antibody specific to the structural conformation of the antibody-analyte complex (noncompetitive sandwich assay) to provide information on the presence and concentration of target analytes in a sample. Because of the highly specific nature of the ELISA technique, many other contaminants present in the same sample can be effectively "screened out," and the analyte(s) of interest reproducibly determined (8). The specificity of the binding site is based on chemical structure and conformation, however, and allows ELISA to exhibit some cross reactivity to compounds with a chemical conformation similar to the target analyte. The extent of this cross reactivity, therefore, needs to be adequately characterized to identify the configuration of the compounds to which the ELISA kit will respond, particularly when dealing with a highly complex, uncharacterized sample


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CI

CI

CI

Atrazine

CI

CI

CI

CI

CI

Cyanazine

Figure 2. Chemical structures of target cyclodiene and triazine pesticides. Note: For dieldrin and endrin, heavy lines indicate bond planes above the plane of the illustration. Narrow lines indicate bond planes below the plane of the illustration.




Pesticide Concentrations in Groundwater 225

matrix. A separate analytical method for validation also needs to be used, both in the initial evaluation of the kit performance and in routine analyses after the kit response has been fully characterized. Positive identification methods such as gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS) often are used for this purpose. Initially, all samples are usually analyzed by both the ELISA and the validation methods. After routine analysis procedures and responses have been established, a designated fraction or percentage of the samples are analyzed by the validation method for quality-assurance purposes. In several areas of R M A , a highly complex chemical matrix, including pesticides and a variety of complex organic and inorganic salts, was present in ground water. Some of the organic compounds were present at parts-per-thousand concentration levels (5). As it was possible that compounds with structural similarities to the target analytes were present, it was unknown how the ELISA would perform in this complex matrix and what possible interferences might be encountered. The ELISA kits selected for the research described herein have been used extensively in evaluations of herbicides in the water of the midwestern United States (1,2,3). Concentration ranges for the triazine and cyanazine ELISA kits used in these evaluations were found to be adequate for the concentration levels resulting from the agricultural application of atrazine and cyanazine. These and other evaluations have shown a high degree of correlation between the data obtained from both the triazine and cyanazine ELISA analyses and that obtained from concurrent GC/MS analyses (9). This previous research, combined with the ease of operation and small sample-volume requirements of the ELISA kits, was a major factor in selecting ELISA as a screening technique for current research. This study was conducted by the U.S. Geological Survey in cooperation with the U.S. Department of the Army from August 1994 through October 1995. Specific compounds of interest to this research were the cyclodiene insecticides—aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor, and the triazine herbicides, atrazine and cyanazine (Figure 2). The specific objectives of this study were: (1) The identification of pesticide contamination (both current location and potential path) at R M A using ELISA and (2) the evaluation of ELISA as a rapid screening technique for a large-scale, ground-water contamination site with a highly complex chemical matrix. This paper focuses mainly on the second objective. Experimental Methods A l l wells involved in this study were sampled from May 1994 through October 1995. The sampled wells were selected from either the confined and unconfined aquifer systems (with the majority in the unconfined system) and represented a geographical distribution that included a range of contaminant concentrations from generally uncontaminated to very contaminated water. Duplicate samples were collected at a frequency of 1 in 10. Sample and trip blanks were collected with the same frequency. Well depths, screened intervals, pumping methods, and water levels were noted at the time of sample collection. Samples were collected in clean, baked, 125-mL amber i glass jars with Teflon -lined lids, stored at or below 4 C, shipped within 48 hours of sampling, and analyzed (ELISA) or extracted (solid-phase extraction (SPE) GC/MS) upon receipt at the U.S. Geological Survey laboratory in Lawrence, Kansas. E L I S A Analyses. A l l ELISA analyses were performed according to the manufacturer's instructions (Millipore Corporation, Bedford, M A , for the cyclodiene and triazine kits, and Ohmicron Corporation, Newton, PA, for the cyanazine kit) using reagents included with the kits. Cross-reactivity and sensitivity studies were carried out using standards obtained through National Institute of Standards and Technology (NIST) traceable sources. The separate standard solutions required for each kit were prepared from neat standard materials. Appropriate amounts of each of these materials 0

'NOTE:

Please see Acknowledgments page 232.



226


were weighed and dissolved in methanol (HPLC grade, Fisher Scientific, Pittsburgh, PA). The standard solutions then were diluted to working concentrations using distilled water generated through activated charcoal filtration and deionization with a high purity, mixed-bed resin, followed by a second activated charcoal filtration step, and finally distillation in an automatic still. Stock standard concentrations were calculated to ensure that dilute standards would contain less than 0.5% organic solvent to minimize interference with the antibodies in the immunoassay. Cyclodiene ELISA analyses were performed using chlordane as the calibration standard. Cyanazine ELISA analyses used cyanazine as the calibration standard. Triazine ELISA analyses used atrazine as the calibration standard. Standard solutions and negative controls were analyzed with all sample sets. All standards and negative controls were analyzed in duplicate; samples were analyzed in duplicate or triplicate. Calibration curves were generated for each sample set. Plots of the B / B values (B/B values represent the optical density of the sample concentration (B) divided by the optical density of the blank solution (B )) versus log of the corresponding standard concentration were used to identify the linear concentration range of each standard and to identify I C (concentration of the compound required to give a B / B value of 50%) and L D D (least-detectable dose, defined as B / B of 0.90) values. S P E - G C / M S Analyses. Validation of the data obtained from the triazine and cyanazine ELISA analyses was obtained through a SPE-GC/MS method established for the determination of pesticide residues in water samples (10-13). A brief overview of this method follows. Pesticide-grade methanol and ethyl acetate (Fisher Scientific, Pittsburgh, PA) were used. Triazine herbicide (atrazine, cyanazine, propazine, simazine) standard materials were obtained from Supelco (Bellefonte, PA); terbuthylazine (the surrogate recovery standard) was obtained from EPA Pesticide Chemical Repository (Research Triangle Park, NC); phenanthrene-d (the internal standard) was obtained from UltraScientific (North Kingstown, RI); deethylatrazine and deisopropylatrazine (triazine metabolites) and cyanazine amide (a cyanazine metabolite) were obtained from CibaGeigy Agricultural Division (Greensboro, NC). A l l standard materials were obtained at >97% purity for standard preparation. Concentrated stock and spiking solutions were prepared in methanol, except for phenanthrene-d which was prepared in ethyl acetate. Distilled water was generated as discussed previously. As the isolation and analysis procedures used have been published previously (10-13), only a brief summary of the method is outlined below. Herbicides (triazine class and cyanazine) and metabolites were isolated from water samples using C-18 SPE cartridges (Sep-Pak plus, Waters, Milford, MA). Prior to extraction, each water sample was spiked with terbuthylazine as a surrogate standard. A 100-mL volume of each sample then was pumped at 20 mL/min through a SPE cartridge that had been conditioned by a Millilab 1A Workstation (Waters-Millipore, Milford, M A ) . The SPE cartridge then was eluted with 2.5 mL ethyl acetate, and the eluate spiked with internal standard, phenanthrene-d . The ethyl acetate eluate then was evaporated to a volume of approximately 100 uL under a nitrogen stream and transferred to a 200-|xL glasslined polystyrene crimp-top vial. A l l sample extracts were stored at -10 °C until analysis by GC/MS. GC/MS analyses were performed on a Hewlett-Packard 5890A G C with a 5970A mass selective detector (MSD) (Palo Alto, CA). Chromatographic separation was accomplished by a Hewlett Packard (Palo Alto, CA), 12-m x 0.2-mm-i.d., HP-1 or Ultra-1 capillary column with 0.33-|xm methylsilicone film. Quantification for each analyte was based on the internal standard, phenanthrene-di . Identification of target analytes was based on the presence and relative response ratios of the base peak and one or two confirming ions (molecular ion wherever possible) and with a retention time 0

0

Q

50

0

0

10

10

10

0



Pesticide Concentrations in Groundwater

match of ± 0.2% relative to the phenanthrene-di . compounds analyzed by this method was 0.05 |Xg/L. 0

227

The quantitation limit for all


Results and Discussion Quality-control samples analyzed included trip and sample blanks, duplicate samples (samples taken at the same site and time) and replicate analyses (repeated analyses of the remaining sample volume from a single sample). For the ELISA analyses, all method blanks agreed to within 10% (coefficient of variance) of the negative control, and duplicate samples agreed within 20% of the mean value. Replicate analyses agreed within 12% of the mean. All initial analyses that gave contaminant concentrations that exceeded the linear range of the ELISA kits were diluted with distilled water and reanalyzed. The data obtained from the cross-reactivity studies showed the same overall trends as noted in product information supplied by the manufacturer. Cyclodiene Results. The cyclodiene ELISA microtiter plate kit initially used in this study was rated by the manufacturer as having a quantitation range of 5.0 to 100 ug/L (as chlordane). The kit showed the highest sensitivity toward endrin and endosulfan (100% and 50%, respectively) The 56 ground-water samples analyzed showed a wide concentration range. Cyclodiene concentrations obtained by ELISA analyses ranged from i.O|ig/L

H
0.1

\ig/L

< 0.1 jig/L

Figure 3. Concentrations of cyanazine within the study area as identified by ELISA analyses. Note: Concentration intervals were extrapolated by hand from existing well data for visualization purposes only and do not represent a statistical calculation of probable cyanazine concentration for nonsampled wells falling within the concentration interval.




Pesticide Concentrations in Groundwater

229

wells prompted evaluation by GC/MS techniques and led to the identification of cyanazine as a contaminant at R M A . Samples exhibiting high triazine concentrations by ELISA were extracted and analyzed by the standard GC/MS herbicide method outlined previously to confirm the specific atrazine concentrations present. The results showed atrazine concentrations much higher than those commonly encountered in samples in agricultural areas but substantially less than the concentrations indicated by the triazine ELISA. Cyanazine concentrations in these samples, however, were found to exceed 5.00 |ig/L in all cases (average = 34.8 |ig/L, maximum = 97.3 \ig/L). Atrazine concentrations averaged 3.70 ug/L (maximum = 5.32 u.g/L) for samples from the same four wells. Because of the similarity in structural conformation of atrazine and cyanazine (Figure 2), the triazine ELISA displayed some sensitivity to both compounds; the response to cyanazine was 2.6% of the response to atrazine. It is theorized that the sensitivity of the triazine ELISA toward atrazine, when combined with the cross reactivity of this ELISA toward cyanazine when both atrazine and cyanazine were present at high concentrations, resulted in the increased triazine concentrations reported by the ELISA. These findings point out the potential benefits of a broad or classsensitive ELISA in which cross reactivity may lead to the identification of a previously unsuspected contaminant. Whereas the total response of this ELISA kit was due primarily to the presence of atrazine and cyanazine, GC/MS analysis of these samples showed that there were other cross-reacting triazine compounds present that also contributed to the overall response such as propazine, simazine, prometryn, and prometon. Cyanazine Results. With the confirmation of cyanazine by GC/MS methods in samples from 4 of the initial 56 wells sampled, a much larger scale ELISA screening program involving 353 wells was established to characterize the extent of cyanazine contamination at R M A , and cyanazine subsequently was evaluated separately for all RMA samples. The cyanazine ELISA kit used in this study had a magnetic particle format, rated by the manufacturer as having a quantitation range of 0.1 to 3.0 \ig/L. Cross reactivity of this ELISA toward compounds other than cyanazine was extremely low (with no significant response to atrazine), making it nearly compound specific. Cyanazine concentrations ranged from

Immunochemical Technology for Environmental Applications

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