PROGRAMS AND PLANS--Immunoassay Field Screening Tests for Pesticides In Reply Refer To: April 21, 1992 Mail Stop 412 OFFICE OF WATER QUALITY TECHNICAL MEMORANDUM 92.09 Subject: PROGRAMS AND PLANS--Immunoassay Field Screening Tests for Pesticides INTRODUCTION Within WRD, awareness and interest are increasing in the use of immunoassay field screening tests for pesticides. Accordingly, the purposes of this memorandum are to: (a) provide a brief description of the development and procedures for immunoassay tests, (b) describe uses and limitations of the tests for WRD projects, and (c) provide details for storing test results in WATSTORE. Immunoassay tests for pesticides are currently available (1992) for atrazine/triazines, cyanazine/triazines, alachlor/acid amides, metolachlor/acid amides, carbofuran/carbamates, aldicarb/ carbamates, aldrin/cyclodienes, 2,4-D/phenoxy acids, and paraquat. At the current time within WRD, the two most popular are the atrazine/triazine and alachlor/acid amide tests. DEVELOPMENT AND PROCEDURES FOR IMMUNOASSAY TESTS Immunoassays work on the principles that: (a) target analytes bind to biologically-derived antibodies, and (b) the extent of this reaction can be quantified by a chemical colorimetric test. The first and most important step in the development of these tests is the selection of an antibody for each target chemical. Antibodies are protein molecules which are produced by an organism as a reaction to the exposure to a foreign chemical. For the development of an immunoassay test, an animal produces antibodies in response to an injection of the target compound. The antibodies are then isolated and tested. If one is found which can serve as a basis for an assay, the antibody is produced in mass culture. Most of the antibodies which are incorporated into the commercial pesticide screening test are "polyclonal." Polyclonal antibodies are composed of proteins and protein fragments containing non-selective binding sites which impart multiple chemical specificity. Polyclonal antibodies can lead to the isolation of monoclonal antibodies, which have specificity and greater sensitivity for a single chemical. However, most of the commercial pesticide immunoassay tests are based on polyclonal antibodies and, therefore, exhibit chemical specificity for families of compounds instead of individual compounds (i.e., triazine herbicides rather than atrazine only). Once the antibody is produced, a very small amount can be coated to a magnetic particle or a plastic surface, such as the inside of a tube or the inside of a well in a microtiter plate. The environmental water sample and an analyte-enzyme conjugate are added to the tube and allowed to competitively react with the antibodies. After an incubation time, this solution is rinsed from the tube or plate. The ratio of the analyte to analyte- enzyme conjugate which has bound to the antibodies is in direct proportion to the amount of analyte in the water sample. The quantification step occurs with the addition of two reactants (substrate and chromogen), which results in a color change in the solution. The intensity of the color change can be related to the analyte's concentration in the water sample. Principles of the Tube Enzyme Immunoassay are detailed in Office of Water Quality Technical Memorandum No. 89.02. Immunoassay tests are fairly simple to perform, and can be done in a field or laboratory setting. The field format for immunoassay tests are individual test tubes and a hand held spectrophotometer. The field test can be performed on one to five water samples at a time and takes about 15 minutes. Analyzing more than five samples at a time is not recommended because the reaction kinetics are rapid. For the laboratory, there are two general formats for immunoassay tests. One is the magnetic particle format in which up to 50 water samples, standards, and blanks can be analyzed in each set. This takes between 3 to 4 hours to complete. The second format is a 96-well microtiter plate with a microtiter plate reader. The 96-well plate has space to accomodate required standards and blanks, and 42 duplicate environmental samples. About 3 hours are required to analyze the 96 samples. The National Water Quality Laboratory (NWQL) plans to offer an immunoassay screening schedule for water samples, provided there is enough interest by District project chiefs. If you would like to see immunoassay screening tests offered as a laboratory schedule, please respond by letter to the NWQL Chief or by EDOC to "DENORG". USEFULNESS OF IMMUNOASSAY TESTS TO WRD Immunoassay tests can be useful additions to some WRD investigations. The primary function of the tests is to provide a qualitative to semiquantitative screening to detect the presence or absence of a targeted chemical or chemical family. For example, if the purpose of a study is to examine atrazine in a specific hydrologic setting where it is the dominant herbicide applied, then the immunoassay test can be used with great benefit to screen water samples over time and space. A water sample could be screened in the field or District laboratory to decide whether or not it should be sent to the NWQL for quantitative analysis. In this way, a large percentage of the samples below the target analyte's detection limit (by the standard laboratory analytical method) can be eliminated. The immunoassay method cost is about $10 to $15 for each water sample test compared to several hundred dollars for laboratory quantitative analysis of a pesticide group. The errors inherent in immunoassay tests may give false positives (i.e., detection by immunoassay, but not by laboratory analysis), but seldom give false negatives (i.e., non-detection by immunoassay, but detection by laboratory analysis). In practice, several investigators have already shown that the triazine concentrations measured by immunoassay tests are equal to or greater than the sum of concentrations for individual triazine compounds as analyzed by gas chromatography/mass spectrometry. This characteristic of immunoassay tests makes them a conservative screening tool for pesticides and does not diminish their usefulness. 2. A second function of immunoassay tests is to provide almost real-time information in the field about the presence or absence of a chemical or chemical family. The field analysis takes about 15 minutes to complete, and thus has the potential to guide the selection of sampling locations and frequency for a project. Even for this purpose, however, these field tests are not meant to be quantitative and do not replace standard laboratory analyses for samples which show that the target analyte(s) is/are present. LIMITATIONS OF IMMUNOASSAYS IN WRD There are a few significant limitations of immunoassay screening tests. These are: 1. The tests are designed to react with dissolved chemicals; therefore, only water samples can be screened semi-quantitatively. Some immunoassay test manufacturers sell water extraction kits for screening pesticides in soils. The soil extraction immunoassay test should be considered purely qualitative. 2. The user can not be certain which particular compound of the chemical family has elicited the response, because the test is chemically nonspecific. Although each test is designed to be most sensitive to a particular chemical, it has some level of sensitivity to the whole family of chemicals and their metabolites. As an example, a positive result in the triazine test could come individually from atrazine, simazine, other triazine herbicides, a metabolite, or from some combination of compounds. If the immunoassay's positive response is from more than one compound, it is possible that a laboratory analysis would report all target chemicals below the reporting limit. 3. Many, but not all, of the current immunoassay tests for pesticides have detection limits which are greater than the reporting limits at the NWQL for the target analytes. If an immunoassay test has a greater detection limit than the NWQL, it is probably not suitable even as a screening test in WRD. As immunoassay tests are improved, this limitation will diminish. 4. To calculate a cost savings of immunoassays compared to NWQL, both the tests and the personnel time must be considered. There definitely is a substantial savings if one immunoassay test is performed. However, if more than two or three immunoassay tests are performed for different compounds in one water sample, the cost savings rapidly diminishes if all of the compounds are available from the NWQL in one schedule. As an example, three different immunoassay tests are presently required to screen for atrazine, alachlor and metolachlor; however, all three compounds are available in one quantitative analysis from the NWQL. 5. Immunoassay tests are probably not appropriate to be used in general reconnaissance or pesticide occurrence studies. In these studies, generally the project goal is to determine which pesticides are present, where they are present, and at what concentration levels. To use the immunoassay test as a screening tool in this type of study, the first question to ask is which pesticide (and immunoassay test) should be used as a surrogate for all pesticides. In most hydrologic settings throughout the Nation, it is impossible to use one pesticide as a surrogate for all others. In some portions of the country, such as the upper Midwest (Iowa, Minnesota), it may be possible to use atrazine as a surrogate for many (not all) other pesticides in ground water, but this has been concluded only after many pesticide occurrence studies have been conducted. The extrapolation of the usefulness of the triazine immunoassay test in the Midwest can not be made to other parts of the country without scientific justification. DATA STORAGE IN WATSTORE WATSTORE/EPA parameter codes exist for the atrazine/triazine herbicides immunoassay. The same codes are used regardless of the manufacturer of the test. These codes are 34756 for filtered water, 34757 for unfiltered water, 34758 for bed material, and 34759 for suspended sediments. Given the solid/water distribution characteristics of atrazine and other triazine herbicides and the constraints of the immunoassay test (only the dissolved fraction of the analyte is "seen" by the antibodies), the bed material and suspended sediments codes are meaningless and misleading. Accordingly, these two (34758 and 34759) are being removed from the Parameter Code Dictionary. Results for filtered and unfiltered water samples should be identical owing to the factors cited above. However, both of these codes will be kept because Division projects run immunoassay tests on both filtered and unfiltered samples. Results appropriate for storage under parameter codes 34756 or 34757 should be entered by Districts into WATSTORE. At present, there are no WATSTORE/EPA parameter codes for the other immunoassay tests. As the need arises, the Office of Water Quality will request new parameter codes for other immunoassay test results. CONTACT PERSON If there are questions about the issues covered in this memo, or about other issues concerning immunoassay tests, please contact Paul Capel by phone at (612) 471-0438 or by EDOC at CAPEL. David A. Rickert Chief, Office of Water Quality Key words: Immunoassay, pesticides, organic compounds, enzyme This memo refers to Office of Water Quality Technical Memorandum No. 89.02. Distribution: A, B, S, FO, PO