RESIDUES AND TRACE ELEMENTS

Determination of Atrazine in Water by Magnetic Particle

Immunoassay: Collaborative Study
HAYES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 79, NO. 2, 1996
MARY C. HAYES, SCOTT W. JOURDAN, and DAVID P. HERZOG
Ohmicron Environmental Diagnostics, 375 Pheasant Run, Newtown, PA 18940
Collaborators: P. Barnes; C. Charan; J. Fleeker; H. Francis; D.W. Litke; C. Hall; P. Luitweiler; A. Lucas; L. Marti; C. Mihaliak;
R. Mumma; T. Spittler; J. Strauss; E.M. Thurman

A collaborative study was performed to determine

mean recovery and precision for analysis of
atrazine in drinking and surface waters by immunoassay. The study design was based on the blind
duplicate test plan for collaborative studies. Three
blank waters (municipal drinking water, well water,
and surface water) were spiked at 3 atrazine levels.
Two water samples with naturally incurred atrazine
loads were also spiked with atrazine at 3 levels. In
the enzyme-linked immunoassay method, the
water sample is mixed with a pesticideenzyme
conjugate and added to paramagnetic particles
with triazine-specific antibodies attached. After
separation of antibody-bound atrazine and
atrazineenzyme conjugate from free components,
the bound enzyme conjugate catalyzes a reaction
producing a colored end product. The color developed is inversely proportional to the original concentration of atrazine in the water sample. Fourteen
laboratories participated in the collaborative study.
Data were analyzed for repeatability and reproducibility, and average recoveries at the spike levels
were calculated. Over the concentration range
tested, the mean recovery of atrazine spiked into
blank and pesticide-contaminated waters was
104%. Overall RSDR averaged about 40% for
atrazine concentrations near the method detection
limit (0.05 g/L) and about 15% at concentrations
above 5 times the detection limit (0.25 g/L). Corresponding single-analyst RSDr values were 24 and
10%. Recovery and precision for the 3 blank water
matrixes and the waters that had been naturally
contaminated with atrazine showed no significant
differences. The magnetic particle immunoassay
Submitted for publication October 20, 1994.
The recommendation was approved by the Methods Committee on
Environmental Quality and was adopted by the Official Methods Board of
the Association. See Official Methods Board Actions (1995) J. AOAC Int.
78, 88A, and Official Methods Board Actions (1995) The Referee 19,
June issue.

for determination of atrazine in water has been

adopted first action by AOAC INTERNATIONAL.

ressure for increased testing of pesticide residues in food

and the environment, as well as the expense and delays
associated with traditional chemical methods, has focused attention on immunochemical detection methods that are
sensitive, reliable, simple, and cost effective as well as rapid.
The benefits of immunoassays for residue analysis have been
described previously (1, 2). Atrazine and related triazines are
widely used selective herbicides for the control of annual
grasses and broad-leaf weeds and selective weed control in
crops. Residues of triazines have been found in surface water
and groundwater, especially in farming regions during the summer months (3). Atrazine has been the most heavily used herbicide in the United States in the past 30 years (4). An atrazine
immunoassay was chosen for evaluation in this collaborative
study because levels of this pesticide are regulated by the U.S.
Environmental Protection Agency (EPA) and other international regulatory organizations and are actively monitored in
the international water supply.
An enzyme-linked immunosorbent assay (ELISA) for
atrazine and related triazines was developed at Ohmicron Environmental Diagnostics and has been commercially available
as a test kit since 1990. Although the properties and performance of this immunoassay have been thoroughly characterized
elsewhere (5), an AOAC collaborative study has never been
reported for a pesticide immunoassay. In October 1993, Ohmicron invited 15 laboratories including industrial, regulatory,
university, and commercial representatives, to participate in
this interlaboratory study for the atrazine immunoassay. Fourteen laboratories agreed to participate. All laboratories were familiar with immunoassay techniques and had some prior experience with the Atrazine RaPID Assay test kit. Study samples
and test kits were mailed to the participants in November 1993,
and all results were returned in December 1993.
The objective of this study was to characterize multilaboratory performance of the magnetic-particle-based ELISA in
terms of recovery, overall and single-analyst precisions, and the
effect of water type on atrazine recovery, and precision of

analysis. The study was conducted entirely by Ohmicron according to AOAC requirements for collaborative studies (6).

995.08 Atrazine in Water, Magnetic Particle

Immunoassay Method

Collaborative Study

First Action 1995

The study was organized according to the blind duplicate

design with balanced replicates of spiked and untreated water
samples for evaluation of repeatability, reproducibility, and recovery. In this design, 2 samples, identical in atrazine concentration, were numbered randomly among 40 other samples and
analyzed by the laboratory. Analysts were instructed to test the
42 randomly numbered samples for atrazine in the sequence
provided by Ohmicron. Only one value was to be reported for
each unknown sample. Generation of a new calibration curve
was suggested after analysis of a series of 20 samples. Analyses
of the 2 spiked drinking waters were included to demonstrate
suitability of the method for quantitation of atrazine in regulated drinking waters.
Stock spiking solutions of atrazine (Riedel de Haen, Hanover, Germany) were prepared in 100% methanol. Final concentrations of atrazine (0.154.5 g/L) were made by adding microvolumetric amounts of the stock solution to 100 mL water
sample such that insignificant levels of solvent persisted. Water
samples used in the study were obtained from the following
sources: (1) reagent water was deionized by a NANOpure ultrapure water system (Barnstead/Thermolyne, Dubuque, IA);
(2) municipal water was collected from a residence in Henrietta, NY; (3) well water was obtained from a pump that provided drinking water in Chester County, PA; (4) surface water
was collected from a pond in a state park also in Chester
County, PA. Two environmental waters that were naturally contaminated with atrazine (naturally incurred) were obtained
from a water treatment facility in an agricultural area near
Cameron, KS (low level), and from deep groundwater in
Plains, GA (high level).
One milliliter of natural or spiked water solution was dispensed into glass vials with Teflon-lined screw caps. The vials
were stored under refrigeration until they were shipped to collaborators. Each collaborator received 42 sample vials, an excess antigen sample, one 100-test Atrazine RaPID Assay kit
containing 4 atrazine calibrators, a quality control (QC) sample
with acceptance limits specified by Ohmicron, and reporting
forms for recording results. Thirteen participants used the Ohmicron RPA-I photometric analyzer, a laboratory benchtop
spectrophotometer that measures absorbance through 12
75 mm plastic test tubes. One participant used another laboratory spectrophotometer that was set at 450 nm and that read the
1 mL optical solutions in cuvettes. Data were transformed in all
cases by linear regression of loglogit standard curves (7, 8).
Analysts were instructed to keep all reagents and samples refrigerated until use and to conduct all analyses with strict adherence to the directions given in the kit package insert. Only
one analyst per laboratory was to perform all analyses for this
study. Return results were requested within 30 days of receipt
of materials.

(Presumptive test for determination of 0.154.00 g

atrazine/L of drinking, well, and surface waters. Assay also responds to presence of other entities; all presumptive positive
results must be confirmed by definitive assay.)
Caution: See Safety Appendix: Laboratory Safety for Safe
Handling of Acids and Safe Handling of Alkalies. See material safety data sheets on reagents specified. Dispose of waste
solvents in an appropriate manner compatible with applicable
environmental rules and regulations.
Method Performance:
See Table 995.08A for method performance data and Table
995.08B for method selectivity data.

A. Principle
Water sample is mixed with horseradish peroxidase-labeled
atrazine conjugate, and then paramagnetic particles with attached triazine-specific antibodies are added to the sample mixture. Atrazine present in sample competes with enzyme-labeled
atrazine for binding sites on antibodies. After 30 min incubation, a magnetic field is applied to hold paramagnetic particles
in tube while excess reagents and other unbound material are
washed away. After separation of antibody-bound atrazine and
atrazineenzyme conjugate from free components, particles are
washed with wash solution. Atrazine bound to paramagnetic
particles is identified by adding substrate and a chromogen (hydrogen peroxide and 3,3,5,5-tetramethylbenzidine [TMB]
chromogen solution) that generates blue color. After another
(20 min) incubation period, reaction is stopped. Intensity of
color is proportional to enzyme-labeled atrazine and inversely
proportional to initial concentration of atrazine in test sample.
Absorbance is measured at 450 nm. Atrazine concentration is
determined from standard curve.

B. Apparatus
(a) Test tubes.12 75 mm, polystyrene.
(b) Micropipets.Capable of accurately delivering
200 L; used for test samples, atrazine standard solutions, and
diluent/zero standard solution.
(c) Repeater pipet.Capable of accurately delivering 250,
500, and 1000 L; used for enzyme conjugate, antibody particles, wash, and stop solutions.
(d) Spectrophotometer.Capable of measuring absorbance at 450 nm through 12 75 mm polystyrene test tubes
containing 1 mL sample.
(e) Magnetic separation rack.2-piece plastic unit consists of test tube holder that fits over magnetic separation rack
containing rare earth magnets (available from Ohmicron Environmental Diagnostics).
(f) Vortex mixer.
(g) Absorbent pad.About 5 layers of paper towels.
(h) Filter paper.0.2 m.
(i) Glass tubes.Borosilicate, holding 50 mL.

C. Reagents
Items C(a)(j) are available as Atrazine RaPID Assay Kit
(Ohmicron Environmental Diagnostics, Newtown, PA; Part
No. A00002, 30 tests/kit, or Part No. A00071, 100 tests/kit).
(a) Atrazineantibody-coupled magnetic particles.Rabbit antitriazine antibodies covalently bound to paramagnetic
particles and suspended in buffered saline containing antimicrobial preservatives and protein stabilizers.
(b) Atrazineenzyme conjugate.Horseradish peroxidase-labeled atrazine analog diluted in buffered saline containing antimicrobial preservatives and protein stabilizers.
(c) Atrazine standard solutions.0.1, 1.0, and 5.0 g/L;
prepared in buffered saline containing antimicrobial preservatives and protein stabilizers.
(d) Quality control (QC) solution.Approximately 3 g
atrazine/L in buffered saline containing antimicrobial preservatives and protein stabilizers.
(e) Diluent/zero standard solution.Buffered saline with
antimicrobial preservatives and protein stabilizers, containing
no detectable atrazine.
(f) Hydrogen peroxide.0.02% H2O2 in citric acid buffer.
(g) TMB chromogen solution.3,3,5,5-Tetramethylbenzidine, 0.4 g/L, in N,N,-dimethylformamide.
(h) Stop solution.2M H2SO4. Provided in ready-to-use
form in 30-test kit. When using 100-test kit, prepare stop solution before analysis by pouring contents of stop solution bottle
into stop solution diluent and mixing. Some warming of solution will occur.
(i) Wash solution.Preserved deionized H2O.
(j) Loglogit graph paper.

D. General Instructions
Reagents are stable until the expiration date when stored at
4C. Do not use reagents after expiration date.
Bring all reagents to room temperature (2025C) before
analysis. Mix thoroughly antibody-coupled paramagnetic particles before use. Do not transfer antibodies to glass containers.
Treat each test tube in identical manner to obtain optimal
performance and precision.
Add reagents directly to the bottom of test tube; avoid contact between reagents and pipet tip to ensure consistent quantities of reagent in test mixture.
To avoid cross-contaminations and carryover of reagents,
use new pipet tips for each addition of test sample. Avoid contact of pipet tips with droplets of reagent on walls of test tubes.
Magnetic separation rack consists of (1) upper rack that securely holds test tubes and (2) lower separator containing magnets used to attract antibody-coupled paramagnetic particles.
Before incubation, upper rack is removed from lower separator
to allow paramagnetic particles to remain suspended during incubation. For separation steps, rack and separator are combined
to pull paramagnetic particles to sides of test tubes.
Mix antibody-coupled paramagnetic particles just before
pipetting and add mixture to all test tubes within 2 min to ensure consistent results.

Calibration of micropipet and operator technique are critical

and will affect accuracy of results.

E. Preparation of Sample
Filter through filter paper, B(h), water samples containing
gross particulate matter. If acid was added to environmental
water sample to preserve analyte, neutralize sample with strong
base (e.g., 6N NaOH) before analysis.

F. Preparation of Color Reagent

Prepare just before analysis. Mix equal volumes
(0.33 mL/test tube) of hydrogen peroxide, C(f), and TMB
chromogen solution, C(g), in clean glass tube, B(i). Color reagent should remain clear.

G. Determination
(1) Before beginning assay, label polystyrene test tubes for
standards, QC solution, and test samples. See Table 995.08C
for labeling pattern of test tubes.
(2) Separately place 200 L atrazine standard solutions,
C(c), QC solution, or test sample into labeled tubes.
(3) Add 250 L atrazineenzyme conjugate, C(b), to each
tube.
(4) Mix thoroughly atrazine antibody-coupled paramagnetic particles, C(a). Add 500 L to each tube.
(5) Mix contents of test tubes for 12 s on Vortex mixer.
Minimize foaming by gently pressing test tube to Vortex mixer
preset at low speed.
(6) Incubate test tubes 15 min at room temperature (20
25C).
(7) Allow antibody-coupled paramagnetic particles to
separate from test mixture for 2 min in magnetic separation
rack, B(e). Note: To obtain optimum precision of assay, perform separation steps carefully and consistently.
(8) Decant excess reagent from tubes. Gently blot test tubes
in a consistent manner, as follows: Invert rack away from analyst by using smooth turning action, so liquid flows consistently along only one side of test tubes. While rack is still inverted, blot all tubes briefly on absorbent pad. Lift rack and
place it gently onto absorbent pad several times to remove liquid completely from rim of test tubes.
(9) Add 1 mL wash solution, C(i), to each tube and allow
tubes to remain in magnetic separation unit for 2 min.
(10) Decant wash solution from test tubes, and then gently
and briefly blot tubes on absorbent pad in consistent manner.
(11) Repeat steps (9) and (10).
(12) Remove upper rack from separator and add 500 L
color reagent from F to each tube.
(13) Mix contents of test tubes for 12 s on Vortex mixer.
Minimize foaming as in (5).
(14) Incubate tubes 20 min at room temperature.
(15) Add 500 L stop solution, C(h), to each test tube.
Note: No mixing on Vortex mixer is required.
(16) Read absorbance, A, for each test tube at 450 nm
wavelength 15 min after adding stop solution.

H. Manual Calculations
(1) Calculate mean A for each atrazine standard solution.
(2) Calculate A/ADZ ratio (%) for each atrazine standard solution, where ADZ = mean absorbance for diluent/zero standard
solution.
(3) Construct standard curve on loglogit graph paper, by
plotting data as y-axis = A/ADZ ratio for each atrazine standard
solution, %; and x-axis = corresponding atrazine concentration, g/L.
(4) Calculate A/ADZ ratios for QC solution and test samples
using corresponding A values. Read atrazine concentration in
QC solution and test samples using standard curve from (3).
If atrazine concentration in test sample is >5 g/L, dilute
sample at least 10-fold (e.g., 50 + 450 L) with diluent/zero
standard solution, C(e), and repeat analysis, beginning at G. It
is not recommended to use <50 L sample for dilution. Mix
solution thoroughly before analysis. Calculate atrazine concentration by multiplying obtained value by dilution factor (e.g., 10).

I. Calculation by Spectrophotometric Method

If using spectrophotometer with ability to calculate calibration curves and to store them in memory, refer to operating
manual for detailed instructions. Final concentrations of
atrazine are determined within 0.055.0 g/L by comparing
absorbances of test sample to linear regression loglogit standard curve prepared from atrazine standard solutions.

J. Quality Control
Results of analysis are valid when the following QC requirements are met:
(1) Absorbance of diluent/zero standard solution measured
at 450 nm wavelength must be >0.8.
(2) Correlation coefficient (r) of standard curve must be
>0.990.
(3) Concentration of atrazine in QC solution must fall
within 3.0 0.6 g/L.
When routinely testing environmental water samples, QC
measurements might involve inclusion of replicate or multilevel kit control samples in the middle and at the end of test tube
sequence; use of blanks, spikes, or spike duplicates in each
batch; and tracking ratio or distribution of positive and negative
results in stable test sample population.
Before performing analyses, analyst should practice procedure. Video tapes with detailed instructions are available from
Ohmicron Environmental Diagnostics.
Ref.: J. AOAC Int. 79, 529(1996)
Results and Discussion

Treatment of Data
The data from 14 laboratories (Table 1) were reviewed for
completeness and acceptability according to the minimum QC
criteria described earlier. No data points were missing and all
reported runs met the acceptability criteria. All nondetectable
concentrations of atrazine (<0.05 g/L) were converted to zero
before data entry. Results were grouped by water type in sub-

sets of 3 or 4, depending on spiking levels applied. Data were

evaluated for precision with a Lotus 1-2-3 software for evaluating balanced and unbalanced blind replicates available from
AOAC INTERNATIONAL (9). Outliers in all spiked and incurred residue samples were evaluated by the software according to critical values for the Cochran test and the single- and
double-value Grubbs test. Blank water data were not subjected
to outlier analysis.
Only 8 of 588 data points (1.4%) were rejected. Six were
identified by the Cochran test as having abnormally high
within-laboratory variation on duplicates. Four of these data
points were from laboratory 4 and 2 were from laboratory 7. A
pair of data points from laboratory 6 was rejected because one
of the 2 points exceeded the range of the calibrators
(>5.0 g/L).
Mean recoveries for blank waters were calculated with
overall statistics to compare spiked concentrations with observed concentrations of atrazine. Recovery for waters with incurred atrazine was calculated by subtracting the original
atrazine level of the sample from the observed concentration
before comparing with the spiked concentration. Regression
analysis was done on 15 values for comparison of spiked
amount and average concentration measured by immunoassay.

Method Recovery
Summary statistics, after removal of outliers, are shown in
Table 995.08A. Mean recovery at each concentration level in
each water matrix was calculated as the percentage of added
atrazine. Waters with naturally incurred atrazine were tested in
the authors laboratory to estimate the initial atrazine content so
spiking concentrations would allow total measured atrazine to
remain within the assay calibration curve. Blank waters used to
prepare study samples were collected in sufficient quantity and
prescreened in the authors laboratory with the atrazine immunoassay to ensure that atrazine-free starting material was used.
In all cases, results were below the detection limit of 0.05 g/L
atrazine. This detection limit is recommended by Ohmicron to
its kit users on the basis of validation studies performed with
multiple lots of kit reagents with different operators over more
than 20 standard curve runs. The 0.05 g/L detection limit represents the analyte level predicted by a regression analysis of
an average standard curve at 10% displacement from the zero
standard response, i.e., 90% B/Bo. When the 4 blank samples
were analyzed by the collaborators, 9 of 112 determinations
(8%) were reported as detectable, ranging from 0.07 to
0.54 g/L. Seven of the 9 detectable results were from analysis
of municipal or surface water blanks. We have frequently observed 0.05 to 0.20 g/L levels of atrazine in randomly sampled environmental waters. It is possible that these blanks contained very low background concentrations of atrazine such
that they did not behave as a true zero in the system and were
therefore detectable in a small percentage of the results. Another explanation for the positive results on blank waters is
found by further analysis of data from laboratory 5. Four of the
9 detectable blank results occurred in a single run performed in
this laboratory. Comments from the operator suggest that some
delays in timing of reagent addition and reading of optical den-

sity may have occurred in this run. Delays of more than about
2 min in addition of antibody-coupled particles to sample tubes
compared with calibrator tubes will cause sample results to
drift higher. This occurs because the antibodyantigen reaction
proceeds to a greater extent in the calibrator tubes when the
reaction is stopped, and the sample tubes show relatively lower
optical density translated as higher analyte concentrations.
When other nonblank samples included in the same batch by
this laboratory were checked for this trend, results on most
samples were among the highest reported by all collaborators.
This observation supports a kinetic drift explanation for blank
samples. Data from this run were not excluded from the database, because the QC criteria were met and no data points qualified for removal as outliers. However, the result of 0.54 g/L
on the reagent water blank reported by laboratory 9 appears to
be a true outlier. When the rule of the huge error (10) is applied
to the B/Bo ratios of reagent water results from all laboratories,
the 0.54 g/L result is obviously eliminated.
Average recovery for all waters was 104%. Average recoveries for individual spiked matrixes ranged from 80 to 120%,
with one exception: 134% for natural water with background
atrazine at 0.47 g/L. The precision of measurement on this
sample was good, suggesting that the spiking procedure may
have produced the high bias in the results. Recoveries from
samples containing atrazine at 5 g/L are expected to fall below
100% as the loglogit fit of the calibration curve becomes
poorer when binding of unlabeled atrazine is in great excess of
binding of atrazineenzyme conjugate. The average calculated
concentration by regression for the 5.0 g/L calibrator was
4.54 g/L in 41 runs by 14 collaborators. Absorbance data in
this range can be corrected by use of an excess antigen tube
whose optical density is subtracted from each unknown absorbance, producing a more linear response in the calibration
curve at elevated concentrations. Such a reagent was provided
to the collaborators, and it was included in each run for this
purpose. When the naturally incurred waters spiked with
4.0 g/L were corrected for nonspecific antigen binding, recovery of the incurred low sample was not affected (81% recovery), but results on the high sample improved from 89 to 96%.
In the authors experience, if a matrix interference is present in
an environmental sample, high recoveries typically would be
observed, with the lowest spiking concentrations returning to
normal recoveries as more analyte is added. This was not the
case. The water type did not appear to influence the ability of
the immunoassay to recover added atrazine. Regression analysis of spiked atrazine versus measured atrazine in 15 samples
ranging from 0.15 to 4.47 g/L predicts X = 0.91C + 0.17 (r =
0.974).
Results for the QC sample are shown in Table 2. Mean concentration recovered was 3.13 g/L, or 104% of the true (spiked)
value for 14 collaborators in 41 immunoassay batch runs.

Precision
Precision parameters are reported in Table 3. The method
detection limit (MDL) is estimated at 0.05 g/L or 90% binding of the optical density of the zero concentration reaction tube
(maximum-binding tube). Precision of immunoassay results

typically is optimal between about 20 and 80% maximum binding. Because collaborative studies of chromatographic methods for pesticides have been evaluated for precision at concentrations with reference to the MDL, precision parameters for
this study were assessed in this way.
Samples with atrazine concentrations less then 5 times the
MDL (up to 0.25 g/L) exhibited an average single-analyst
relative standard deviation (RSDr) of 23.3 (range, 16.629.9)
and an average overall relative standard deviation (RSDR) of
39.4 (range, 35.843.4). In samples with atrazine concentrations above 5 times MDL (0.25 to 5.0 g/L), the RSDr averaged
10.1 (range, 7.7420.1) while RSDR averaged 15.7 (range,
9.0830.8). Water type did not appear to affect measurement
precision.
Conclusions
The magnetic-particle-based immunoassay is accurate and
precise in the detection range defined by the kit calibrators.
Overall and single-analyst relative standard deviations are
comparable with similar parameters for chromatographic
methods for pesticides (11, 12) approved by EPA for analysis
of drinking water and by AOAC at concentrations above
5 times MDL. The quality of the returned data was excellent,
with less than 2% of data points qualifying as outliers. The simplicity of the immunoassay method, which requires no laborious sample pretreatment steps such as solvent extraction or
analyte concentration, contributes to its robustness in a variety
of laboratory types. The majority of the collaborators were
regular users of immunoassay tests and were familiar with the
pipetting and decanting techniques and reaction timing issues
which can most substantially influence the precision of results.
Proper training in micropipetting techniques, monitoring of
quality control results, and understanding of assay kinetic properties must be established for routine achievement of good results.
This collaborative study format using relatively clean
atrazine-spiked waters has shown the atrazine immunoassay
performance comparable with traditional methods in accuracy
and precision. The method had previously been shown by the
developers to exhibit no interference with 250 mg/L of the ions
of calcium, copper, nickel, magnesium, sulfate, nitrate, and
thiosulfate. Test results were not affected by 100 mg/L of added
humic acid, iron, sulfide, and sulfite or by 0.65M sodium chloride. Reactivity of compounds that are either metabolites of
atrazine or similar in chemical structure and that might appear
in the sample need to be understood if the immunoassay is to
be properly applied. Similar interpretation would be necessary
when coelutants are potentially present in a chromatographic
procedure. This atrazine immunoassay was developed with an
antibody possessing broad specificity for triazine residues. The
analyst must incorporate this facet of immunochemical technology into the interpretation of results from unknown samples
and the design of a QC system. The most practical use for the
method may be as a screening test for evaluation of large numbers of water samples or for monitoring drinking water supply
systems with greater frequency for presence of atrazine at regu-

lated levels. Application of reproducibility characteristics, like

those generated in this study, allows projection of confidence
intervals around a desired action level that can conveniently
screen out the high percentage of samples negative in pesticide
content while identifying true positives with a high degree of
certainty. The Atrazine RaPID Assay was developed to maximize performance around 3.0 g/L, the regulatory limit for
drinking water established by the EPA.

R. Mumma, Pennsylvania State University, State College, PA

T. Spittler and L. Lavin, Cornell University, New York State
Agricultural Experimental Station, Geneva, NY
J. Strauss, Wisconsin State Laboratory of Hygiene, Madison, WI
E.M. Thurman, Organic Geochemistry Research Group,
U.S. Geological Survey, Lawrence, KS
References

Recommendation
On the basis of the results of this study it is recommended
that the magnetic particle immunoassay for determination of
atrazine in water be adopted first action.
Acknowledgments
We express appreciation to the following collaborators who
participated in this study:
P. Barnes, Kansas State University, Manhattan, KS
C. Charan and Viorica Lopez-Avila, Midwest Research Institute, Mountain View, CA
J. Fleeker, North Dakota State University, Fargo, ND
H. Francis and T. Haezlit, Kentucky Geological Survey,
Lexington, KY
D.W. Litke and D. Goolsby, U.S. Geological Survey, Denver, CO
C. Hall, K. Sagan, and K. Fuchs, University of Guelph,
Guelph, Ontario, Canada
P. Luitweiler and P. Staudte, Philadelphia Suburban Water
Co., Bryn Mawr, PA
A. Lucas, DuPont Corp., Wilmington, DE
L. Marti, U.S. Department of Agriculture, Tifton, GA
C. Mihaliak, Dow Elanco, Indianapolis, IN

(1) Hammock, B.D., & Mumma, R.O. (1980) in Pesticide Identification at the Residue Level, ACS Symposium Series 136,
American Chemical Society, Washington, DC, pp. 321352
(2) Van Emon, J.M., & Lopez-Avila, V. (1992) Anal. Chem. 64,
79A88A
(3) Drinking Water Health Advisory: Pesticides (1989) U.S.
EPA, Office of Drinking Water Health Advisories, Lewis
Publishers, Chelsea, MI
(4) Meister, R.G. (Ed.) (1987) Farm Chemicals Handbook, 3rd
Ed., Meister Publishing Co., Willoughby, OH
(5) Rubio, F.M., Itak, J.I., Scutellaro, A.M., Selisker, M.Y., &
Herzog, D.P. (1991) Food Agric. Immunol. 3, 113125
(6) Youden W.Y., & Steiner, E.M. (1975) Statistical Manual of
the AOAC, AOAC, Arlington, VA
(7) Rodbard, D. (1974) Clin. Chem. 2/1, 12551270
(8) Baud, M., Mercier, M., & Chatelain, F. (1990) Scand. J.
Clin. Lab. Invest., Suppl. 205, 120
(9) Statistical software, AOAC INTERNATIONAL, Arlington,
VA, AOACBUBR.WK1, 7/20/93 version
(10) Taylor, J.K. (1987) Quality Assurance of Chemical Measurements, Lewis Publishers, Inc., Chelsea, MI, pp. 3435
(11) Edgell, K.W., Erb, E.J., Longbottom, J.E., & Lopez-Avila, V.
(1992) J. AOAC Int. 75, 858871
(12) Edgell, K.W., Jenkins, E.L., Lopez-Avila, V., & Longbottom,
J.E. (1991) J. Assoc. Off. Anal. Chem. 74, 295309

Table 995.08A. Method performance for determination of atrazine in water by magnetic particle immunoassay
Water
Reagent
Municipal

Well

Surface

Contaminated
municipal water

Contaminated well
water

a
b
c
d
e

Spike, g/L

Na

_
xb, g/L

sr, g/L

sR, g/L

rc, g/L

Rd, g/L

RSDr, %

RSDR, %

Recovery,
%

0.00
0.00
0.15
1.00
3.00
0.00
0.15
1.00
3.00
0.00
0.15
1.00
3.00

14
14
14
13
14
14
13
14
14
14
14
14
13

0.02
0.02
0.16
1.13
2.85
0.00
0.15
1.05
2.88
0.02
0.17
1.06
3.44

0.10
0.04
0.03
0.10
0.26
0.02
0.03
0.10
0.29
0.06
0.05
0.09
0.40

0.10
0.06
0.06
0.18
0.26
0.02
0.07
0.12
0.29
0.07
0.07
0.17
0.66

0.28
0.11
0.08
0.28
0.73
0.06
0.08
0.28
0.81
0.11
0.14
0.25
1.12

0.28
0.17
0.17
0.50
0.73
0.06
0.20
0.34
0.81
0.20
0.20
0.48
1.85

19.1
9.09
9.08

16.6
9.53
9.95

27.7
8.22
11.7

39.6
15.6
9.08

43.3
11.5
9.95

39.0
16.2
19.1

107
113
95

100
105
96

113
106
114

0.00
0.60
2.00
4.00

14
14
14
14

0.24
0.93
2.19
3.48

0.07
0.09
0.20
0.29

0.09
0.17
0.30
0.32

0.20
0.25
0.56
0.81

0.25
0.48
0.84
0.90

29.9
9.27
9.13
8.29

35.8
17.9
13.8
9.28

115
98
81

0.00
0.80
2.00
4.00

14
13
14
14

0.47
1.28
3.15
4.03

0.09
0.13
0.24
0.38

0.14
0.25
0.58
0.50

0.25
0.36
0.67
1.06

0.39
0.70
1.62
1.40

20.1
10.0
7.74
9.52

30.8
19.5
18.4
12.5

101
134
89

N = Number of laboratories.
Mean recovery.
r = 2.8 sr.
R = 2.8 sR.
= not calculated.

Table 995.08B. Selectivity of antitriazine antibody for

atrazine determination in water by magnetic particle
immunoassay
Compound
Atrazine
Triazines
Ametryn
Prometryn
Propazine
Prometon
Simazine
Terbutryn
Terbuthylazine
Cyanazine
Metabolites
Desethylatrazine
Desisopropylatrazine
Hydroxyatrazine
Didesalkylatrazine
a

IC50a, g/L
0.72

Reactivityb, %
100

0.39
0.64
0.74
2.22
4.90
5.50
15.5
>10000

185
113
97.0
32.4
14.7
13.1
4.6
<0.1

3.21
217
148
>10000

22.4
0.3
0.5
<0.1

Inhibitory concentration of compound that causes immunoassay

response to be displaced 50% from the absorbance of zero
standard.
Ratio of compound IC50 to atrazine IC50 multiplied by 100.

Table 995.08C. Setup of test tubes for atrazine

determination in water by magnetic particle
immunoassay
Tube No.
1, 2
3, 4
5, 6
7, 8
9
10
11
12

Tube content
Diluent/zero standard solution
Atrazine standard solution, 0.1 g/L
Atrazine standard solution, 1.0 g/L
Atrazine standard solution, 5.0 g/L
QC solution
Test sample 1
Test sample 2
Test sample 3

Table 1. Results of AOAC collaborative study of atrazine in water (

g/L)

ID No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37

Sample

Spike,
g/L

Reagent water blank

none
Duplicate
none
Municipal water blank
none
Duplicate
none
Municipal water level 1
0.15
Duplicate
0.15
Municipal water level 2
1.00
Duplicate
1.00
Municipal water level 3
3.00
Duplicate
3.00
Well water blank
none
Duplicate
none
Well water level 1
0.15
Duplicate
0.15
Well water level 2
1.00
Duplicate
1.00
Well water level 3
3.00
Duplicate
3.00
Surface water blank
none
Duplicate
none
Surface water level 1
0.15
Duplicate
0.15
Surface water level 2
1.00
Duplicate
1.00
Surface water level 3
3.00
Duplicate
3.00
Incurred low (IL)
IL plus zero
Duplicate
IL plus zero
Incurred low plus 3x
IL plus 0.60
Duplicate
IL plus 0.60
Incurred low plus 10x
IL plus 2.00
Duplicate
IL plus 2.00
Incurred low plus 20x
IL plus 4.00
Duplicate
IL plus 4.00
Incurred high (IH)
IH plus zero
Duplicate
IH plus zero
Incurred high plus 2x
IH plus 0.80

Laboratory No.
Sample
code

10

11

12

13

14

5
10
29
33
19
25
34
37
6
11
21
18
30
35
4
3
20
26
36
38
12
14
22
17
31
39
2
13
24
27
23
15
1
8
32
41
7

0.17
0.15
1.01
0.90
2.91
2.72

0.09
0.09
0.97
0.89
2.76
2.80

0.13
0.13
1.11
0.99
3.73
3.70
0.20
0.17
0.87
0.93
2.03
2.09
3.61
3.78
0.34
0.38
1.08

0.11
0.11
1.31
0.99
2.76
2.01

0.16
0.16
0.96
0.90
2.57
2.36

0.07
0.08
0.96
0.80
2.80
2.70
0.18
0.12
0.78
0.74
1.60
1.64
3.27
2.66
0.50
0.40
0.95

0.18
0.17
1.25
1.29
3.01
3.12

0.20
0.21
1.04
1.04
2.83
3.22

0.22
0.26
1.01
1.06
2.82
2.74
0.26
0.25
0.98
0.91
2.43
2.34
3.30
3.99
0.53
0.57
1.44

0.18
0.10
0.07
b
1.32b
b
2.67b
3.06
2.57

b
0.47b
b
0.28b
1.44
1.14
2.41
2.88

0.32
0.26
0.77
0.79
3.22
3.42
0.50
0.25
0.87
0.92
1.86
2.16
3.51
3.59
0.65
0.53
1.47

0.22
0.09
0.13
0.17
1.28
1.53
2.85
2.70
0.11

0.25
0.34
1.04
1.15
2.39
3.12
0.36
0.07
0.26
0.09
1.04
1.08
3.82
2.77
0.27
0.45
0.96
1.29
2.45
2.53
3.68
3.77
0.54
0.89
1.96

0.05
0.94
0.81
3.03
2.52

0.07
0.06
1.11
1.03
2.74
2.90

0.12
0.11
0.80
0.71
3.16b
5.11b
0.29
0.11
0.63
0.49
1.93
1.76
3.19
3.97
0.43
0.24
1.14

0.08

0.21
0.27
1.29
1.30
2.67
2.70

0.25
0.22
0.97
0.96
2.68
2.92

0.07
0.19
0.17
1.05
1.28
3.38
4.89
0.26
0.30
0.99
1.12
2.46
2.32
3.64
3.61
0.54
0.72
2.75b

0.20
0.11
0.82
0.94
2.76
2.85

0.09
0.12
1.00
0.92
3.24
2.66

0.13
0.13
1.09
1.14
4.03
4.36
0.17
0.22
0.77
0.77
1.72
2.03
3.26
3.84
0.30
0.36
1.21

0.54

0.18
0.24
1.23
1.19
2.85
2.93

0.19
0.19
1.09
0.99
3.02
3.12

0.18
0.20
1.08
1.23
3.71
3.58
0.21
0.22
1.11
1.06
2.43
2.31
3.41
3.50
0.53
0.52
1.11

0.19
0.24
1.03
1.18
2.95
2.81

0.13
0.18
1.12
1.06
3.39
2.90

0.22
0.18
1.22
1.15
2.63
2.76
0.29
0.35
0.93
1.03
2.35
2.35
3.51
4.05
0.42
0.35
1.31

0.22
0.23
1.12
1.02
3.07
2.87

0.18
0.15
1.08
1.02
2.64
3.19

0.19
0.21
1.19
1.15
4.27
4.90
0.25
0.27
1.00
0.92
2.33
2.33
3.45
3.52
0.51
0.53
1.34

0.18
0.15
1.19
1.32
2.89
2.84

0.14
0.13
0.91
1.02
3.23
2.94

0.12
0.10
1.17
1.38
3.01
3.29
0.14
0.19
0.97
1.07
2.43
1.96
3.34
3.41
0.44
0.41
1.38

0.10
0.14
1.09
1.15
2.90
3.08

0.12
0.06
1.01
1.10
2.82
3.36

0.26
0.11
0.98
1.00
3.05
3.29
0.24
0.22
0.85
0.89
2.85
2.06
2.93
3.10
0.25
0.31
1.07

0.19
0.20
1.15
1.05
3.52
2.73

0.12
0.13
0.99
1.33
2.78
2.79

0.12
0.15
1.21
1.29
3.38
3.08
0.17
0.23
1.16
1.01
2.37
2.28
3.05
3.44
0.36
0.47
1.09

Table 1. (continued)

ID No.
38
39
40
41
42
a
b

Sample
Duplicate
Incurred high plus 5x
Duplicate
Incurred high plus 10x
Duplicate

, not detected
Excluded as outlier pair.

Laboratory No.

Spike,
g/L

Sample
code

10

11

12

13

14

IH plus 0.80
IH plus 2.00
IH plus 2.00
IH plus 4.00
IH plus 4.00

9
40
42
16
28

1.23
2.54
3.26
3.55
3.57

0.88
3.98
3.62
3.15
3.61

1.51
2.73
2.70
3.83
3.63

1.31
4.56
4.06
4.20
4.07

1.77
2.54
2.64
3.55
3.96

1.29
3.41
3.84
4.37
3.69

1.64b
4.09
3.64
3.62
3.60

1.39
3.03
2.77
4.98
3.87

0.85
3.13
3.09
4.70
4.55

1.34
2.39
2.44
4.26
3.90

1.30
3.18
3.23
4.60
4.45

1.28
2.89
3.17
4.15
4.80

1.29
3.23
2.91
4.66
4.02

1.42
2.43
2.77
3.91
3.40

Table 2. Results on quality control samplea by run

Lab. No.
1
2
3
4
5
6
7
b
8
9
10
11
12
13
14
a

Run 1, g/L

Run 2, g/L

Run 3, g/L

2.94
2.66
3.26
3.19
2.76
3.09
3.19
2.90
2.86
3.30
3.03
3.41
3.18
3.10

2.98
2.89
3.46
3.06
3.04
2.99
3.35
3.01
3.41
3.08
3.30
3.30
2.85
3.40

3.04
2.53
3.22
3.52
3.12
2.64
3.56

3.37
3.54
3.08
3.55
3.01
3.00

n = 41, mean = 3.13, sR = 0.26, RSDR = 8.30, true value = 3.00,

recovery = 104%. Sample acceptance range is 2.4 to 3.6 g/L.
Laboratory 8 completed all analyses in 2 immunoassay batch runs.

Table 3. Precision parameters for immunoassay of atrazine in water

Detection range
Low in detection range
(less than 5 MDL)

Atrazine spike or
incurred load, g/L

Sample matrix

RSDr

RSDR

0.15
0.15
0.15
0.24

Spiked municipal
Spiked well
Spiked surface
Naturally incurred load

19.1
16.6
27.7
29.9
23.3

39.6
43.4
39.0
35.8
39.4

0.47
0.60
0.80
1.00
1.00
1.00
2.00
2.00
3.00
3.00
3.00
4.00
4.00

Naturally incurred load

Spiked plus natural load
Spiked plus natural load
Spiked municipal
Spiked well
Spiked surface
Spiked plus natural load
Spiked plus natural load
Spiked municipal
Spiked well
Spiked surface
Spiked plus natural load
Spiked plus natural load

20.1
9.27
10.0
9.09
9.53
8.22
9.13
7.74
9.08
9.95
11.7
8.29
9.52
10.1

30.8
17.9
19.5
15.6
11.5
16.2
13.8
18.4
9.08
9.95
19.1
9.28
12.5
15.7

Average
High in detection range
(more than 5 MDL)

Average