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