Environmental Immunochemical Methods - American Chemical Society

Chapter 13

A First Application of Enzyme-Linked Immunosorbent Assay for Screening Cyclodiene Insecticides in Ground Water 1

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Downloaded by STANFORD UNIV GREEN LIBR on October 8, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch013

Tonya R. Dombrowski , Ε. M. Thurman , and Greg B. Mohrman 1

U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, KS 66049 Rocky Mountain Arsenal, 72nd and Quebec Street, Building 111, Commerce City, CO 80022 2

A commercially available enzyme-linked immunosorbent assay (ELISA) plate kit for screening of cyclodiene insecticides (aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor) was evaluated for sensitivity, cross reactivity, and overall performance using ground -water samples from a contaminated site. Ground-water contaminants included several pesticide compounds and their manufacturing by -products, as well as many other organic and inorganic compounds. Cross-reactivity studies were carried out for the cyclodiene compounds, and results were compared to those listed by the manufacturer. Data obtained were used to evaluate the sensitivity of the ELISA kit to the cyclodiene compounds in ground water samples with a contaminated matrix. The method quantitation limit for the ELISA kit was 15 μg/L (as chlordane). Of the 56 ground-water samples analyzed using the ELISA plate kits, more than 85% showed cyclodiene insecticide contamination. The E L I S A kit showed excellent potential as a screening tool for sites with suspected ground -water contamination by insecticides.

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The Rocky Mountain Arsenal (RMA), a 70 km tract of land 14.5 km northeast of Denver, Colorado, has been the site of several intensive chemical research and production projects. From the 1940s to the 1960s chemical agents, rocket fuels, and weapons were manufactured at the site by the U.S. Army. In the mid-1940s, production facilities at the Arsenal were leased to private industry, and agricultural pesticides were manufactured by what is now a division of Shell Chemical Company. Waste and other industrial by-products of the manufacturing processes from both the U.S. Army and Shell Chemical Company were disposed of according to the accepted protocols of that time period. Since the first contaminant problems were reported in the mid-1950s, innovative containment and clean-up measures have been undertaken on a massive scale by all organizations associated with R M A property (7). Target analytes and "fingerprint" compounds (materials known to be unique to the R M A ) have been identified. Aggressive monitoring policies have resulted in the establishment of a network of over 1200 ground

This chapter not subject to U.S. copyright Published 1996 American Chemical Society In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

13. DOMBROWSKI ET AL.

ELISA & Screening Cyclodiene Insecticides

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water wells, extending throughout the R M A property and into the surrounding areas. The number of analyses required to support these monitoring programs combined with sampling constraints due to well volume, hazard and accessibility, and the complex matrix present in many areas of the R M A present a major challenge in the areas of time, expense, and overall analysis complexity. Because a method for rapidly identifying areas of significant interest or for reliably screening a large number of samples (many potentially in the field) would be beneficial when dealing with such a large and complex site, the cyclodiene enzyme-linked immunosorbent assay (ELISA) plate kit (Millipore Corporation , Bedford M A ) seemed applicable to the R M A as it would provide specific, rapid results with minimal sample preparation and volume. ELISA utilizes the highly specific binding sites of antibodies, which recognize a single compound or class of compounds, to provide information on the presence and concentration of those compounds in a sample. Because of the specific nature of this technique, many other contaminants present in the same sample can be effectively "screened out," and the analyte(s) of interest reproducibly determined. The specificity of the binding site, however, is based on chemical structure, and needs to be adequately characterized to determine the type or configuration of compounds to which the kit will respond, particularly when dealing with a complex and highly uncharacterized sample matrix (2,3). Specific compounds of interest to this study conducted by the U.S. Geological Survey in cooperation with the U.S. Department of the Army from March 1994 to October 1995 were the cyclodiene insecticides: aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor (see Figure 1). The purpose of this paper is to discuss the results obtained from this study, in which the cyclodiene ELISA kit was evaluated as a screening tool for insecticide contamination in highly complex ground-water matrices. The cyclodiene ELISA kit used in this study was rated by the manufacturer as having a linear concentration range of 5 to 100 μg/L (as chlordane). Historical gas chromatography (GC) data from the R M A wells showed cyclodiene concentrations that ranged from non-detectable levels to about 100 μg/L (composite values from the four cyclodienes, aldrin, chlordane, dieldrin, and endrin, were evaluated separately) with the majority of the wells showing levels below 10 μg/L. Given this information, and the linear concentration range specified for the cyclodiene ELISA, this kit appeared well suited as a screening tool for ground-water samples at the R M A . In several areas of the Arsenal, however, ground water containing both insecticides and herbicides in combination with many other organic and inorganic compounds was present; therefore, it was possible that compounds with structural similarities to the cyclodiene insecticides were present. As the ELISA kit had not been used previously with samples in such a complex ground-water matrix, the sensitivity, and cross reactivity of the kit, as well as the possibility of matrix interferences, had to be fully evaluated.


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Experimental Methods A l l sample analyses were performed using the ELISA plate kit for cyclodienes according to manufacturer's instructions (Millipore Corporation, Bedford M A ) using reagents included with the plate kits. Cross-reactivity and sensitivity studies were carried out using standards obtained through the Army Standard Analytical Reference Materials (SARM) repository. Separate standard solutions for aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor were prepared 1

The use of brand names in this paper is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey.

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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ENVIRONMENTAL IMMUNOCHEMICAL METHODS


CI

ALDRIN

CI

CI

CI CI

ENDRIN

CI CI

ENDOSULFAN CI

DIELDRIN CI CI

CI

HEPTACHLOR CI

Figure 1. Chemical structures of the cyclodiene insecticides. Heavy lines indicate bridge position above the plane of the paper. Dashed lines indicate bridge position below the plane of the paper.




ELISA & Screening Cyclodiene Insecticides

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from neat or solid standard material. Appropriate amounts of each of these materials were weighed out and dissolved in hexane (Fisher Scientific, Pittsburgh PA). These standard solutions were then diluted to 1,000.0, 100.0, 10.0, 1.0, and 0.1 μg/L concentrations using distilled water as the final diluent. Stock standard concentrations were 2,000,000 μg/L to ensure that dilute standards would contain less than 0.5% organic solvent. Two blank solutions were evaluated in the cyclodiene kit study, distilled water produced in-house, and the negative control included with the kit reagents. The analyses of ground water samples were performed using a chlordane standard at concentrations of 0.0, 5.0, 25.0, and 100.0 μg/L. The linear concentration range of the ELISA kit was determined from a plot of B / B Q versus the log of the corresponding standard concentration. B / B values represent the optical density of the sample solutions divided by the optical density of the negative control solution. Sensitivity information on each compound was obtained by using the IC (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. These were calculated from the standard curve of B / B versus the log of the corresponding standard concentration. A l l 56 ground-water wells involved in this study were sampled from May 1994 thru June 1994. A l l wells sampled were in the unconfined aquifer system and represented a geographical distribution that included a range of contaminant concentrations from relatively uncontaminated to very contaminated water. Sampling was carried out by personnel from the U.S. Geological Survey. Duplicate samples were taken at a frequency of 1 in 10, (duplicates equaled 10% of the total samples). Sample and trip blanks were collected with the same frequency. A l l pertinent geological information (well depth, screened interval, pumping method, and water level) was noted at the time of sample collection. Samples were collected in clean, baked, amber glass jars with Teflon-lined lids, stored at or below 4 C., shipped within 48 hours of sampling, and analyzed on receipt. Q

5Q

Q

Q

Q

Results and Discussion The data obtained from the cross-reactivity studies are listed in Table I. The study concentrations show the same overall trends as noted in product information supplied by the manufacturer. The highest sensitivity is exhibited toward endosulfan, while the lowest is toward aldrin. A tentative hypothesis that has been proposed to explain this difference in sensitivity centers on the chlorinated bridge structure spanning the primary chlorinated ring as seen in the chemical structures of the compounds of interest (See Figure 1). It is believed that this bridge (common to all compounds investigated) may be the antibody binding site. The additional bridge on the secondary ring in aldrin, dieldrin, and endrin also may affect binding as may the presence of electronegative atoms or structural components. The presence of an oxygen atom near the chlorinated bridge (as in the ring oxygens in endosulfan and the oxygenated "bridge" structure of endrin) then would serve to stabilize the binding site and enhance binding capability. The oxygenated bridge in dieldrin is structurally removed from the binding site a considerable distance as this molecule is in a "trans" conformation, and therefore the added binding enhancement is not as readily apparent as in the "cis" conformation compound (endrin) and the sterically unhindered heptachlor. The presence of a chlorine atom or a double bond near the chlorinated bridge seems to destabilize the binding site, resulting in decreased binding efficiency and decreased sensitivity for aldrin, chlordane, and heptachlor. The double bond present in aldrin may affect the structural configuration of the binding site, which


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Table I. Comparison of manufacturer's cyclodiene ELIS A performance data with performance data from ground water monitoring study I C is 50% inhibition concentration, L D D is least-detectable dose.


50

Compound Name

30 \iglL

250 μg/L 34μ /ί

2μg/L 27 μg/L 6 μ g / L 0.6 μg/L 3 μ g / L 0.15 μg/L 4μg/L 33μg/L

12μg/L 17.5 μg/L 140 μg/L

84 μg/L

Aldrin Chlordane Dieldrin Endosulfan Endrin Heptachlor

τ-

Study Concentrations LDD

Manufacturer's Concentrations LDD

CM

β

48 2.0 1.8 1.7

μg/L μg/L μg/L μ ^ β

1.7 \igfh

2.1 μ / ί β

CO

Cyclodiene concentration ^ g / L ) Figure 2. Frequency of cyclodiene concentrations in ground-water samples from the Rocky Mountain Arsenal.




ELISA & Screening Cyclodiene Insecticides 153

results in the decreased sensitivity of the ELISA kit to this compound. The overall least detectable dose (LDD) of the ELISA kit was rated by the manufacturer at a cyclodiene concentration of 5 μg/L (as chlordane). In actual practice, however, the method quantitation limit (calculated as lOx the standard deviation of the blank) was set at 15 μg/L (as chlordane) by the authors, to assure that an appropriate level of confidence in the values reported was maintained. Given this linear range of approximately 15 to 100 μg/L the ELISA kit was suited for application at the R M A , although the less contaminated samples would fall in the lower end of the quantitation range and may require some preconcentration if remediation limits are established below 15 μg/L. As stated previously, historical GC data available for wells in this study showed cyclodiene concentrations which ranged from non-detectable levels to about 100 μg/L (composite values from the four cyclodienes, aldrin, chlordane, dieldrin., and endrin, were evaluated separately) with the majority of the wells showing levels below 10 \iglh. The linear range of the ELISA kit therefore approximated the concentration range expected in the R M A samples. Given the manufacturing and disposal (manufacturing wastes and by-products) history of the R M A , it is expected that the application of this ELISA kit to other sites of cyclodiene contamination would be successful. The toxicity of the individual cyclodiene insecticides range from about 5 to 500 mg/kg of body weight (oral dosage, rat) so the sensitivity of the ELISA kit is more or less adequate based on health considerations. (Because of the toxicity of these compounds, all personal precautions were taken when handling either sample or reagent solutions.) As low concentration samples may benefit from preconcentration methods, a solid-phase extraction (SPE) procedure is being developed for the R M A samples. Due to the highly contaminated matrix, standard SPE procedures (4,5) are being modified to increase selectivity in retained compounds. The linear range of each of the six cyclodiene compounds listed was investigated. Dieldrin, endosulfan, and heptachlor showed a linear range of roughly 3 to 100 μg/L while the ranges for aldrin, endrin and chlordane (the calibrator) were shifted to slightly higher values. The samples analyzed show a wide concentration range (see Figure 2), with more than 85% of the wells sampled exhibiting positive contamination values. The majority of contaminant concentrations identified were less than 120 μg/L, and less than 20% of the wells sampled having concentrations greater than 500 \igiL. Identification of potential interferents is currently underway for these high concentration samples. The relative percent difference (RPD) for all duplicate field samples was within 20%, and similarly, the RPDs for the analytical duplicates were all within 12% for the 56 samples analyzed. Quality control samples analyzed included both trip and sample blanks as well as the duplicate samples previously mentioned. A l l analysis blanks analyzed agreed to within 10% (coefficient of variance) of the negative control. GC/MS methods are currently under development to provide verification of the ELISA kit results. A l l analysis results were available within a short time, and at substantially reduced cost in terms of laboratory hours and equipment cost as compared to traditional GC and GC/MS analyses. When the sample data obtained were plotted with the appropriate geographical orientation, contaminant plume boundaries could be identified through concentration gradients defined by the data points and showed strong relative agreement with those wells for which historical G C cyclodiene data were available. These unpublished GC data are consistent with, though lower than the ELISA results.


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Conclusions A wide range of contaminant concentrations at the R M A was identified using the ELISA cyclodiene kit. The data obtained exhibited a high degree of correlation among wells in close geographical proximity and with the available historical G C data for the wells sampled. It resulted in the identification of the current contaminant plume boundaries in a rapid and efficient manner. The only samplepreparation step required was dilution, which represents a considerable simplification of the many extraction, concentration, and analytical steps commonly required for the instrumental analysis of these cyclodiene insecticides. From the data generated through the sensitivity studies performed, the method quantitation limit was calculated as 15 μg/L (as chlordane). The cross reactivity of the ELISA kit as evaluated using the procedure previously described parallels that given by the manufacturer. Compounds to which this kit showed the greatest sensitivity were endosulfan and endrin. While direct GC/MS conformation was not available for the samples analyzed using the ELISA kit, the data obtained compares favorably with historical G C data available for some of these wells. The unpublished G C data available, are consistent with, though lower than the ELISA results. However, the relative concentration trends are similar for both sample sets, and contaminant plume boundaries outlined are essentially the same for both methods of analysis. The ELISA results did display a positive bias when correlated with the available G C data for the same wells. This may indicate an interfering compound, or may be inherent in the correlation due to the fact that GC data is available for aldrin, chlordane, dieldrin, and endrin only, while the ELISA kit is sensitive to several other compounds in this same family. However, in comparison, the ELISA data clearly revealed the same relative concentration trends and plume boundaries as the GC data. Acknowledgment: The authors acknowledge the U.S. Department of the Army for funding of this work.

Literature Cited: 1. Garlock, E.T. Ed.; Eagle Watch ; August 1992, Vol. 4, No. 8, p. 1-19. 2. Vanderlaan, M.; Watkins, B.E.; Stanker, L.; Environmental Monitoring by Immunoassay; Environmental Science & Technology; 1988, Vol. 22; No. 8; p. 247-254. 3. Van Emon, J.M.; Lopez-Avila, V.; Immunochemical Methods for Environmental Analysis; Analytical Chemistry, 1992, Vol. 64, No. 2; p. 79A-88A. 4. Aga, D.S.; Thurman, E.M.; Coupling Solid-Phase Extraction and EnzymeLinked Immunosorbent Assay for Ultratrace Determination of Herbicides in Pristine Water; AnalyticalChemistry;1993, Vol. 64; No. 20; p. 2894-2898. 5. Thurman, E.M.; Goolsby D.A.; Meyer, M.T.; Mills, M.S.; Pomes, M.L.; Kolpin, D.W.; A Reconnaissance Study of Herbicides and Their Metabolites in Surface Water of the Midwestern United States Using Immunoassay and Gas Chromatography/Mass Spectrometry; Environmental Science & Technology, 1992, Vol. 26; No. 12; p. 2440-2447.


Environmental Immunochemical Methods - American Chemical Society

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