Environmental Technology Verification Program Advanced Monitoring Systems Center Generic Verification Protocol for Test Kits for Detection of Atrazine in Water


Baltelle

The Business of Innovation

Environmental Technology
Verification Program

Advanced Monitoring
Systems Center

Generic Verification Protocol
for Test Kits for Detection of
Atrazine in Water

EWETVEW

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GENERIC VERIFICATION PROTOCOL

FOR

TEST KITS FOR DETECTION OF
ATRAZINE IN WATER

August 2004

Prepared by

Battelle

505 King Avenue
Columbus, OH 43201-2693

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FOREWORD

This generic verification protocol is based upon a peer-reviewed specific test/quality assurance
(QA) plan entitled "Test/QA Plan for Verification of Test Kits for Detection of Atrazine in
Water," Version 1.0 (dated September 15, 2003). The test/QA plan was developed with vendor
and stakeholder input by the ETV Advanced Monitoring Systems (AMS) Center. Peer reviewers
for the test/QA plan were AMS Center stakeholders Kenneth Wood and Marty Link, and Elin
Ulrich of EPA's National Exposure Research Laboratory. In preparing this generic verification
protocol, specific names of individuals involved, technology vendors and technologies, test
dates, and similar details in the test/QA plan were revised to be generic. The experimental
design in the protocol is the same as that in the peer-reviewed test/QA plan.

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Table of Contents

Page

1.0	INTRODUCTION	1

1.1	Test Description	1

1.2	Test Objective	1

1.3	Scope of Work	2

1.4	Organization and Responsibility	3

1.4.1	Battelle	3

1.4.2	Vendors	7

1.4.3	EPA	7

1.4.4	Analy ti cal Lab oratory	8

2.0	VERIFICATION APPROACH	8

2.1	Experimental Design	8

2.2	Test Samples	9

2.2.1	QC Samples	9

2.2.2	PT Samples	9

2.2.3	Environmental Samples	11

2.3	Reference Method	12

3.0	MATERIALS AM) EQUIPMENT	 12

3.1	Field Supplies	13

3.2	Laboratory Supplies	13

4.0	PROCEDURES	14

4.1	Environmental Sample Collection and Storage	14

4.2	Equipment Calibration	15

4.3	Test Sample Preparation and Storage	16

4.3.1	Physicochemical Characterization	16

4.3.2	Sample Preparation	16

4.4	Sample Identification	17

4.5	Sample Analysis	17

4.5.1	Test Kits	17

4.5.2	Reference Method	18

4.6	Schedule	18

5.0	DATA HANDLING AM) REPORTING	 19

5.1	Data Acquisition and Review	19

5.2	Data Analysis	20

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5.2.1	Accuracy	20

5.2.2	Precision	21

5.2.3	Linearity	21

5.2.4	Method Detection Limit	22

5.2.5	Cross Reactivity	22

5.2.6	Matrix Interferences	22

5.2.7	False Positives/False Negatives	22

5.3 Reporting	23

6.0	QUALITY ASSURANCE /QUALITY CONTROL	24

6.1	Sample Chain-of-Custody Procedures	24

6.2	Audits	24

6.2.1	Techni cal Sy stem s Audit	24

6.2.2	Data Quality Audit	25

6.2.3	Performance Evaluation Audit	25

6.3	Reference Method QC	25

6.4	QA/QC Reporting	27

6.5	Corrective Action	27

7.0	REFERENCES	27

LIST OF TABLES

Table 1. Sample Summary for Verification of Test Kits for Atrazine in Water	10

Table 2. Calibration Procedures for Sample Preparation and Physicochemical

Characterization	15

Table 3. Approximate Schedule of Verification Test Days	19

Table 4. Summary of Data Recording Process	20

LIST OF FIGURES

Figure 1. Generic Organization Chart for Verification Test	4

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ACRONYMS

AMS

Advanced Monitoring Systems

DOC

dissolved organic carbon

ELISA

enzyme-linked immunosorbent assay

EPA

Environmental Protection Agency

ETV

Environmental Technology Verification

ID

identification

LFB

laboratory fortified blank

MCL

maximum contaminant level

MDL

method detection limit

NIST

National Institute of Standards and Technology

PE

performance evaluation

ppb

parts per billion

PT

performance test

QA

quality assurance

QC

quality control

QMP

Quality Management Plan

RB

reagent blank

RPD

relative percent difference

RSD

relative standard deviation

TFE

polytetrafluoroethylene

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1.0 Introduction

1.1 Test Description

This protocol provides generic procedures for a performance verification test of test kits
for the analysis of atrazine in water. Verification tests are conducted under the auspices of the
U.S. Environmental Protection Agency's (EPA) Environmental Technology Verification (ETV)
program. The purpose of ETV is to provide objective and quality-assured (QA) performance data
on environmental technologies so that users, developers, regulators, and consultants can make
informed decisions about purchasing and applying these technologies.

Verification tests of monitoring technologies are coordinated by Battelle, of Columbus,
Ohio, which is EPA's verification partner for the ETV Advanced Monitoring Systems (AMS)
Center. As such, Battelle will be referred to as the testing laboratory in this document. The
scope of the AMS Center covers verification of monitoring methods for contaminants and
natural species in air, water, and soil. In performing verification tests, Battelle follows the
procedures specified in this protocol and complies with quality requirements in the "Quality
Management Plan for the ETV Advanced Monitoring Systems Center" (QMP).(1)

1.2 Test Objective

The purpose of a verification test generated from this protocol is to provide quantitative
verification of the performance of test kits for the detection of atrazine in water. These
technologies are typically based on immunoassay methods, where specific antibodies may be
used to detect and measure the analytes of interest. Immunoassay-based test kits may utilize a
competitive enzyme-linked immunosorbent assay (ELISA), where atrazine in the sample
competes with enzyme-labeled atrazine for a limited number of antibody binding sites specific to
atrazine. After an incubation period, a magnetic field may be used to separate the bound and
unbound atrazine. A simple wash step is typically followed by a color development step. The
sample concentration is inversely proportional to color development, with a darker color
indicating a lower sample concentration and a lighter color indicating a higher concentration.

A variety of quality control (QC), performance test, and environmental water samples
shall be analyzed to assess the capabilities of the test kits relative to an accepted reference
laboratory method.

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1.3 Scope of Work

This generic protocol intends to provide information related to verification of
immunoassay test kits that provide qualitative, semi-quantitative, or quantitative measurements
of atrazine in water. Test kits that provide qualitative results may only indicate the presence or
absence of the target analyte relative to a regulatory or health-based concentration level. Test kit
methods that provide semi-quantitative results determine the presence or absence of the analyte
within a specified concentration interval. Quantitative results may be obtained using test kits
that generate a calibration curve using a range of analyte concentrations. The vendor shall
specify the type of the measurement provided by the test kit to be verified.

Qualitative test kits are typically designed to be operated by non-technical users. Semi-
quantitative and quantitative immunoassay test kits will yield more consistent and reliable results
when operated by an experienced user. In order to minimize the error introduced by operator
inexperience, an analyst with previous experience in using immunoassay test kits may analyze all
test samples.

Test kits that are specific for atrazine may be cross-reactive for a variety of triazine
analogues, some of which are degradation products of atrazine. Cross-reactivity information is
typically provided in the test kit documentation. It is assumed that most users of these kits will
perform laboratory analysis of samples that provide a positive immunoassay response to confirm
the presence and concentration of atrazine in the sample (i.e., the test kits will be used primarily
as a screen for the possible presence of atrazine). As such, this test will not verify all vendor-
provided information on cross-reactivity; however, the effect of two cross-reactive atrazine
degradation products (such as hydroxyatrazine and desethyl atrazine) on test kit performance
shall be verified.

Natural and atrazine-fortified water samples shall be analyzed using both the
immunoassay test kits and an appropriate reference method. A variety of sample matrices shall
be tested: This generic protocol assumes that ASTM Type I water, fresh pond water, brackish
pond water, shallow (i.e., alluvial) groundwater, and chlorinated drinking water will be tested.
These matrices are examples of water types that are typically monitored using immunoassay test
kits to give a sense of the performance using a variety of matrices. However, this is not intended
to be an exhaustive study nor to represent all possible water types that could be tested.

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The performance of each test kit shall be evaluated quantitatively by comparing the test
kit and reference method results. Each test kit also shall be qualitatively evaluated for ease of
use, cost, and sample throughput. The test kits shall be evaluated for the following parameters:

•	accuracy

•	precision

•	linearity

•	method detection limit

•	cross reactivity of cross-reactive atrazine degradation products (such as
hydroxyatrazine and desethyl atrazine)

•	matrix interference effects, and

•	occurrence of false positive and false negative results.

Qualitative technologies should not be evaluated for linearity or method detection limit
because they only provide a positive or negative result relative to a specified concentration level.
The experimental design is described further in Section 2.0.

1.4 Organization and Responsibility

The organizational chart provided in Figure 1 identifies the responsibilities of the
organizations and individuals associated with the verification test. Roles and responsibilities are
defined further below.

1.4.1 Battelle

Battelle staff will direct and support atrazine test kit verification in the following
capacities: Verification Test Coordinator, Verification Testing Leader, AMS Center Manager,
Quality Manager, and technical staff. The activities and responsibilities of each of these positions
are described below.

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Figure 1. Generic Organization Chart for Verification Test

The Verification Test Coordinator will have overall responsibility for ensuring that the
technical, schedule, and cost goals established for the verification test are met, and will:

• Assemble a team of qualified technical staff to conduct the verification test.

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• Direct the team in performing the verification test in accordance with the procedures in
this protocol.

• Ensure that all quality procedures specified in this protocol and in the AMS Center QMP
are followed. Prepare the draft and final verification reports and verification statements.

• Revise the draft verification reports and verification statements in response to reviewers'
comments.

• Respond to any issues raised in assessment reports and audits, including instituting
corrective action as necessary.

• Serve as the primary point of contact for vendor representatives.

• Coordinate distribution of the final verification reports and statements.

• Establish a budget for the verification test and monitor staff effort to ensure that budget is
not exceeded.

• Ensure that confidentiality of vendor information is maintained.

The Verification Testing Leader will provide technical guidance and oversee the various
stages of verification testing, and will:

• Support the Verification Test Coordinator in organizing the test.

• Review the draft and final verification reports and verification statements.

The Battelle AMS Center Manager will:

• Review the draft and final verification reports and verification statements.

• Ensure that necessary Battelle resources, including staff and facilities, are committed to
the verification test.

• Ensure that vendor confidentiality is maintained.

• Support the Verification Test Coordinator in responding to any issues raised in
assessment reports and audits.

• Maintain communication with EPA's technical and quality managers.

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Technical staff will conduct sample collection and the testing of the atrazine test kits
during the verification test. The responsibilities of the technical staff include:

• Assist in the collection of environmental water samples and ship them to the testing
laboratory.

• Assist in the receipt, and storage of environmental samples.

• Filter environmental water samples, as required.

• Measure physicochemical parameters on environmental water samples prior to
preparation of atrazine-fortified samples.

• Prepare the atrazine spiking solution and fortify the test samples as required.

• Analyze test samples using the atrazine test kits.

• Split and ship water samples to the analytical laboratory for measurement of atrazine and
dissolved organic carbon (DOC) as part of the physicochemical characterization.

The Battelle Quality Manager will:

• Conduct a technical systems audit once during the verification test, or designate another
QA Manager to conduct the audit.

• Audit at least 10% of the verification data.

• Prepare and distribute an assessment report for each audit.

• Verify implementation of any necessary corrective action.

• Notify Battelle's AMS Center Manager about the need for a stop work order if self audits
indicate that data quality is being compromised.

• Provide a summary of the QA/QC activities and results for the verification reports.

• Review the draft and final verification reports and verification statements.

• Assume overall responsibility for ensuring that the protocol is followed.

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1.4.2	Vendors

The responsibilities of the vendor representatives are as follows:

•	Provide off-the-shelf test kits for analysis of all verification test samples.

•	Provide all other equipment needed to complete the immunoassay analyses, including a
sp ectrophotometer.

•	If desired, provide training to Battelle on operating the test kits and associated equipment
prior to testing.

•	Review the draft verification statement and report.

1.4.3	EPA

EPA's responsibilities in the AMS Center are based on the requirements stated in the
"Environmental Technology Verification Program Quality Management Plan" (EPA QMP).2
The roles of the specific EPA staff are as follows:

The EPA AMS Center Quality Manager will:

•	Perform at his or her option one external technical system audit during the verification
test.

•	Notify the EPA AMS Center Manager of the need for a stop work order if external audit
indicates that data quality is being compromised.

•	Prepare and distribute an assessment report summarizing results of external audit.

•	Review draft verification reports and statements.

The EPA AMS Center Manager will:

•	Review the draft verification reports and statements.

•	Oversee the EPA review process of the verification reports and statements.

•	Coordinate the submission of verification reports and statements for final EPA approval.

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1.4.4 Analytical Laboratories

The analytical laboratories at Battelle, a subcontractor, and/or a partnering organization
will analyze DOC in environmental samples as part of the physicochemical characterization.
The analytical laboratory will also conduct reference analysis of atrazine in water. All DOC and
atrazine results will be QA-reviewed by the generating analytical laboratory before used in
verification test data analysis.

2.0 Verification Approach

2.1 Experimental Design

The verification shall involve challenging each test kit with a variety of water samples
that are intended to represent those typically monitored using the test kits. Sample matrices may
include fresh pond water, brackish pond water, alluvial groundwater, and chlorinated drinking
water. Natural and atrazine-fortified (i.e., unspiked and spiked) samples of each matrix shall be
analyzed. ASTM Type I (or equivalent) water samples fortified with atrazine or an atrazine
degradation product also shall be analyzed. Physicochemical parameters (pH, temperature,
salinity, conductivity, alkalinity, and DOC) shall be measured in environmental samples to
provide supporting characterization data.

All samples shall be analyzed by each test kit and by the reference method. Each sample
shall be analyzed in triplicate with each test kit. Samples shall be given to the analyst blind and
in random order.

Test kit and reference method results shall be used to assess test kit accuracy and
linearity. Replicate sample results shall be used to assess test kit precision. Results for replicates
of a low-level spiked sample shall be used to evaluate the method detection limit. Cross-
reactivity of hydroxyatrazine and desethyl atrazine shall be assessed by evaluating the test kit
results for samples that contain only the degradation compound, but not atrazine. Potential
matrix effects shall be assessed by comparing accuracy and precision results for environmental
samples (i.e., chlorinated drinking water, fresh surface water, brackish surface water and
groundwater) to those for ASTM Type I water samples. Performance parameters, such as ease

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of use and reliability, shall be based on documented observations of the analyst. Sample
throughput shall be estimated based on the time required to analyze a sample set.

2.2 Test Samples

Test samples to be used in this verification test shall include QC samples, performance
test (PT) samples, and environmental water samples. Table 1 lists the number and type of each
sample to be analyzed in the verification test. Each type of test sample is described further
below.

2.2.1 QC Samples

QC samples will include reagent blank (RB) and calibration check samples. The RB
samples will be prepared from ASTM Type I water and will be exposed to identical sample
preparation and analysis procedures as the test samples, including the addition of all reagents.
These samples will be used to help ensure that no sources of contamination are introduced in the
sample handling and analysis procedures. At least 10% of the test samples will be RB samples.

Calibration check samples may be used to verify that the test kits are properly calibrated
and reading within defined control limits. Calibration check samples will be analyzed in
accordance with the vendor-provided test kit protocol using standards or calibration check
samples supplied by the vendor.

2.2.2 PT Samples

PT sample types are listed in Table 1. The first type of PT sample consists of ASTM
Type I water spiked at five different atrazine concentration levels. The PT sample
concentrations will span the calibration range of the immunoassay test kits. This range includes
the EPA maximum contaminant level (MCL) for atrazine in drinking water, which is 3 parts per
billion [ppb]3. Three replicates of each PT sample will be analyzed using the test kits.

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Table 1. Sample Summary for Verification of Test Kits for Atrazine in Water

Type of sample

Description

Replicates
for test kit
analysis

Total No.
laboratory
reference
analyses

Performance Factor3

Quality Control

Reagent blanks (10%)

minimum 10% frequency

20

1

False positive/negative

Calibration check samples

As required by the test kit protocol

TBD



-

Performance Test

Performance test # 1

0.1 ppb atrazine

3

1

Accuracy, precision,
linearity, false
positive/negative

Performance test #2

0.5 ppb atrazine

3

1

Performance test #3

1 ppb atrazine

3

1

Performance test #4

3 ppb atrazine

3

1

Performance test #5

5 ppb atrazine

3

1

Method detection limit

Atrazine concentration 2X vendor-stated detection limit

7



Method detection limit

Cross-reactivity test #1

3 ppb hydroxyatrazine

3

1

Cross-reactivity, false
positive/negative

Cross-reactivity test #2

3 ppb desethyl atrazine

3

1

Environmental

Fresh water

Fresh surface water, unspiked

3

1

Accuracy, precision, matrix
effects, false
positive/negative

Fresh water spike # 1

Fresh surface water with 1 ppb atrazine spike

3

1

Fresh water spike #2

Fresh surface water with 3 ppb atrazine spike

3

1









Brackish water

Brackish water, unspiked

3

1

Brackish water spike #1

Brackish water with 1 ppb atrazine spike

3

1

Brackish water spike #2

Brackish water with 3 ppb atrazine spike

3

1









Groundwater

Groundwater, unspiked

3

1

Groundwater spike # 1

Groundwater with 1 ppb atrazine spike

3

1

Groundwater spike #2

Groundwater with 3 ppb atrazine spike

3

1









Chlorinated drinking water

Chlorinated drinking water

3

1

Chlorinated drinking water spike #1

Chlorinated drinking water with 1 ppb spike

3

1

Chlorinated drinking water spike #2

Chlorinated drinking water with 3 ppb atrazine spike

3

1

Performance Evaluation Sample



-

1

-

Total



84

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a Other performance factors to be evaluated qualitatively include ease of use and reliability.

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The second type of PT sample is a low-level atrazine fortified sample. Seven replicates
of this sample will be analyzed for the method detection limit determination. This sample will
be spiked at a level of two times the vendor-stated test kit detection limit.

The third type of PT sample is a cross-reactivity check sample. Two samples will consist
of ASTM Type I water spiked with two different cross-reactive atrazine degradation products
(hydroxyatrazine and desethyl atrazine) at a level of 3 ppb. Three replicates of each cross-
reactivity check sample will be analyzed using the test kits. One replicate will be analyzed by
the reference method to confirm the absence of atrazine in the samples.

All PT samples will be prepared by Battelle using certified, commercially-available
standards. PT sample results will be used to assess test kit accuracy, precision, linearity, method
detection limit, cross-reactivity, and occurrence of false positive and false negative results using
the data analysis methods described in Section 5.2.

2.2.3 Environmental Samples

Environmental samples will be collected from a variety of sources to evaluate the
performance of the test kits with various sample matrices. For this generic protocol, it is
assumed that samples will be collected from the following sources:

• Fresh surface water

• Brackish surface water

• Groundwater

• Chlorinated drinking water.

It is recognized that the composition of surface water, groundwater, and chlorinated drinking
water will vary considerably depending upon the sampling location, source, and/or chlorination
method. Use of surface water from two sources (fresh and brackish), groundwater from one
source, and chlorinated drinking water from one source will provide a single example (not a
comprehensive overview) of possible matrix effects associated with these environmental
samples.

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Some test kit protocols recommend filtration for removal of gross particulate matter prior
to sample analysis. Therefore, the fresh and brackish pond water samples will be filtered with a
0.45 |im filter prior to sample spiking/preparation and analysis. After filtration, the following
physicochemical parameters will be measured in each environmental water sample to
characterize the sample matrix: pH, temperature, salinity, conductivity, and alkalinity. An
aliquot of each environmental sample will be collected and sent to the analytical laboratory for
DOC analysis. The physicochemical parameters will be measured in the testing laboratory
instead of in the field to provide information about the sample matrix immediately prior to test
kit analysis.

As shown in Table 1, each environmental water sample also will be fortified with
atrazine at two spike levels. The fortified samples will be spiked to increase the analyte
concentration by the amount shown in Table 1. The spike solution will be prepared from a
certified, commercially-available atrazine standard. Three replicates of each sample will be
analyzed. The data for the environmental samples will be used to assess accuracy, precision,
potential matrix effects, and occurrence of false positives and false negatives following the data
analysis procedures provided in Section 5.2.

2.3 Reference Method

The laboratory reference method to be used for this verification test is an EPA standard
method for the analysis of drinking water. Each sample will be analyzed using gas
chromatography / mass spectrometry (GC/MS) according to EPA Method 525.24.

3.0 Materials and Equipment

In general, this verification test will rely on test kit materials and equipment provided by
the vendors. Battelle will provide the following equipment and materials for the collection,
preparation, storage, and shipment of test samples.

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3.1 Field Supplies

The following supplies will be needed for the collection of field samples:

•	1-L or 2-L certified clean amber glass bottles fitted with polytetrafluoroethylene
(TFE)-lined screw caps

•	Solvent-rinsed foil to keep bottle caps contaminant-free during sampling

•	Disposable latex gloves

•	Sample labels

•	Clear packing tape (to affix the sample label to bottle and seal the cooler for sample
shipment)

•	Chain-of-custody forms

•	Bubble wrap to prevent sample breakage

•	Coolers and blue ice packs for sample storage and shipping

•	Address labels for the coolers.

3.2 Laboratory Supplies

The following laboratory supplies will be needed for the preparation of the PT and
laboratory-fortified environmental samples and sample shipment. Vendors will provide all
equipment needed to perform the immunoassay test kit analyses.

•	ASTM Type I water

•	Class A volumetric flask

•	10-|iL to 500-|iL adjustable pipette and disposable tips

•	Repeating pipette and disposable tips (volume appropriate for each test kit)

•	Certified, commercially-available atrazine, hydroxyatrazine and desethyl atrazine
standards

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•	Performance evaluation (PE) atrazine standard

•	Dichloromethane, acetone, and/or methanol, as applicable (pesticide-grade or equivalent)

•	Glass vials fitted with TFE-lined screw caps for test kit aliquots

•	Pre-cleaned glass container of sufficient size to prepare atrazine-fortified samples

•	1-L certified clean amber glass bottles fitted with TFE-lined screw caps for reference
sample aliquots

•	Pre-cleaned glass container fitted with TFE-lined screw caps for DOC samples (provided
by the laboratory subcontractor)

•	Sample filtration equipment; 0.45 |im cellulose filters

•	Conductivity meter

•	Salinity refractometer

•	Alkalinity meter

•	pH meter capable of reading pH levels of 1 to 14

•	Thermometers to measure air and water temperature

•	Sample labels

•	Clear packing tape (to affix sample label to bottles and seal cooler for sample shipment)

•	Chain-of-custody forms

•	Bubble wrap to prevent sample breakage

•	Coolers and blue ice packs for sample shipping and address labels for coolers.

4.0 Procedures

4.1 Environmental Sample Collection and Storage

Before environmental sample collection, the total volume of sample material needed to
complete all required analyses for both the test kit and reference methods at all required spike

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levels will be determined. Environmental samples will be collected within 21 days of the
preparation of atrazine-fortified samples for the verification test.

The chlorinated drinking water sample will be collected directly from a tap into certified
clean amber glass bottles. The drinking water source for the tap (city, state, chlorination method)
will be documented. These samples will be stored in the dark at 4°C until test sample
preparation (see Section 4.3). Fresh and brackish pond water samples will be collected directly
into certified clean amber glass bottles. The samples will be collected at the surface of the water
near the shoreline by submerging the containers no more than one inch below the surface of the
water. The groundwater sample will be collected directly from a tap prior to any pretreatment.

Each sample will be assigned a unique sample identification (ID) number (see
Section 4.4). The sample ID number, date, name of person collecting the sample, sample
location, and time of collection will be recorded on a chain-of-custody form for all field samples.
All environmental samples collected in the field will be stored at 4°C and shipped to the testing
laboratory on the day of collection following chain-of-custody procedures (see Section 6.1).

4.2 Equipment Calibration

Table 2 identifies the laboratory equipment that requires calibration. All equipment will
be calibrated according to the criteria provided in Table 2. The spectrophotometer for
immunoassay analyses will be provided by the vendor and will be calibrated in accordance with
the manufacturer's recommended procedures.

Table 2. Calibration Procedures for Sample Preparation and Physicochemical
Characterization

Equipment

Calibration Frequency

Acceptance Criteria

Thermometer

Glass - annually

Electronic - quarterly

at two temperatures that bracket target

temperature(s) against an NIST

traceable thermometer

Appropriate correction factors applied

Variable
volume pipettes
(i.e.. Eppendorf)

Monthly

3% of known of true value.

Salinity
(refractometer)

In accordance with manufacturer's
instructions

In accordance with manufacturer's instructions

Conductivity
meter

Daily

In accordance with manufacturer's instructions

pH meter

Prior to the analysis

In accordance with manufacturer's instructions

Alkalinity meter

Prior to the analysis

In accordance with manufacturer's instructions

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4.3 Test Sample Preparation and Storage

Prior to test kit analysis, environmental samples will be filtered and characterized, and
fortified PT samples will be prepared. These procedures are described below.

4.3.1 Physicochemical Characterization

Prior to the physicochemical characterization, the fresh and brackish pond water will be
filtered with a 0.45 |im cellulose filter. All environmental samples will be measured for
temperature using a thermometer, for salinity using a refractometer, for conductivity using a
conductivity meter, for alkalinity using an alkalinity meter, and for pH using a pH meter. All
measurements will be recorded manually by the analyst on data sheets designed specifically for
this verification test. All instruments used to measure physicochemical parameters will be
calibrated prior to use (Table 2). Instrument model, serial number, and calibration information
will be recorded on data sheets, and calibration records will be maintained in the test files. An
aliquot of each environmental sample will be collected and sent to the analytical laboratory for
DOC analysis according to Method 90605.

4.3.2 Sample Preparation

The PT and fortified environmental samples will be prepared from certified,
commercially-available standard solutions. The purchased standards will be diluted to the
appropriate concentration using pesticide-grade or equivalent solvent in Class A volumetric
glassware. All samples will be stored in the dark at 4°C until use. No other preservatives will be
added to the samples because atrazine has been shown to be stable in water for up to two years
when samples are stored under refrigerated conditions6. The PT and fortified environmental
samples will be analyzed within 14 days of sample preparation.

Each sample will be split into 1-L and 100-mL aliquots. The 100-mL aliquots will be
retained for test kit analysis and stored in the dark at 4°C until use. Two 1-L aliquots will be sent
to the reference laboratory for reference analysis. The chlorinated water sample will be treated
with sodium sulfite according to Method 525.2 at the analytical laboratory prior to analysis. The
samples for reference analysis will be stored in the dark in amber glass bottles at 4°C until

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extraction. The reference method sample extraction will be performed within 14 days of sample
preparation (i.e., spiking), and analysis will be performed within 30 days of sample extraction.

4.4 Sample Identification

All samples will be assigned a unique sample ID number at the time of preparation. The
sample ID will include a survey code and sequential number, and will not contain information
about the nature of the sample (i.e., the samples will be blind to the test kit analyst). A master
log of the sample description, sample ID number, and preparation date will be maintained by
Battelle. Each sample container will be labeled with the sample ID, container number (e.g. 1 of
3), preparation date, and initials of the person preparing the sample.

4.5 Sample Analysis

4.5.1 Test Kits

Each vendor will provide test kits and other necessary equipment (e.g. vortexer,
spectrophotometer) for the analysis of all samples. The full set of samples listed in Table 1 will
be analyzed by each test kit. The vendors are encouraged to train the analyst prior to test kit
analysis. A technical staff member with previous experience in performing immunoassay
analyses will analyze the complete set of samples for each technology. The analyses will be
performed according to the vendor's recommended procedures as described in the test kit
instructions or user manual. Calibration and maintenance of the test kits will be performed as
specified by the vendor.

Test kit results will be recorded manually by the analyst on data sheets designed
specifically for this verification test. In addition to the test kit results, the data sheets will include
records of the time required for sample analysis and operator observations concerning the use of
the test kit (e.g. ease of use, maintenance, etc.).

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4.5.2 Reference Method

The reference method for analysis of atrazine will be performed on a Hewlett-Packard
5971 GC/MS or equivalent. The reference instrument will be operated according to the
recommended procedures in the instrument operating manual, and samples will be analyzed
according to EPA Method 525.2, "Determination of Organic Compounds in Drinking Water by
Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry"4.

Results from the reference analyses will be recorded electronically and compiled by the
analytical laboratory into a report format, including the sample ID and the analyte concentration
for each sample.

4.6 Schedule

The sample collection, preparation, and analysis are expected to take place over a four-
week period. The time period for each activity should be considered approximate. Table 3 lists
the activities to be conducted and a nominal schedule.

All samples will be analyzed by one test kit before proceeding to the next test kit if more
than one technology is involved in the verification test. Test conditions will be kept as similar as
possible throughout the duration of the test (e.g., air temperature and lighting conditions) to
minimize error introduced by variable test conditions.

Participating vendors must provide the test kits and other sample analysis equipment to
Battelle one week before the start of sample analysis, so that technical staff may become familiar
with the kits and equipment prior to test initiation. This period will also be used to clarify any
questions about the test kit's operation. Vendor staff may be present for this familiarization
stage and may provide training in the operation of the test kits. Vendors are encouraged to stay
and observe during the duration of the tests. Unused test kits and associated equipment will be
returned to the vendors at the completion of testing.

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Table 3. Approximate Schedule of Verification Test Days

Testing Period

Test Location

Activity

Days 1-21

Field locations

Collection of environmental samples and shipment to
testing laboratory

Days 14-21

Testing laboratory

Receive test kits and supplies from vendor; test kit
familiarization

Day 18

Testing laboratory

Environmental sample filtration

Day 21

Testing laboratory

Training, environmental sample physicochemical
characterization, test sample preparation, shipment of
reference samples and DOC samples to appropriate
analytical laboratory

Days 22-28

Testing laboratory

Analysis of all samples using test kits

Days 22-52

Analytical laboratory

Analysis of reference samples

Days 22-52

Analytical laboratory

Analysis of environmental water samples for DOC

5.0 Data Handling and Reporting

5.1 Data Acquisition and Review

Various types of data will be acquired and recorded electronically or manually by
Battelle technical staff during this verification test. Table 4 summarizes the types of data to be
recorded. All data and observations will be documented on data sheets or in laboratory record
books. Results from the laboratory reference instruments will be compiled in electronic format.

Records received by or generated by Battelle in the verification test will be reviewed by a
more senior Battelle staff member within two weeks after receipt or generation, respectively,
before the records are used to calculate, evaluate, or report verification results. This review will
be performed by a Battelle technical staff member involved in the verification test, but not the
staff member that originally received or generated the record. The review will be documented by
the person performing the review by adding his/her initials and date to a hard copy of the record
being reviewed. This hard copy will then be returned to the Battelle staff member who will be
storing the record.

In addition, data calculations performed by Battelle will be spot-checked by Battelle
technical staff to ensure that calculations are performed correctly. Calculations to be checked
include and statistical calculations described in this protocol.

The data obtained from this verification test will be compiled and reported independently
for each test kit. Results for test kits from different vendors will not be compared with each
other.

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Table 4. Summary of Data Recording Process

Data to be
Recorded

Responsible
Party

Where
Recorded

How often
Recorded

Disposition of data(a)

Dates and times of
test events

Battelle

ETV data sheets

Start/end of test, and
at each change of a
test parameter

Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary

Calibration
information and
results for
physicochemical
parameters
(temperature,
salinity, pH,
conductivity, etc.)

Battelle

ETV data sheets

Prior to sample
preparation

Manually incorporated
in data spreadsheets as
necessary

Sample collection
and preparation
information,
including chain-of-
custody

Battelle

ETV data sheets
and chain-of-
custody forms

At time of sample
collection and
preparation

Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary

Test kit procedures
and sample results

Battelle

ETV data sheets

Throughout test
duration

Manually incorporated
in data spreadsheets

Reference method
procedures and
sample results

Analytical
laboratory

ETV data sheets,
or data
acquisition
system, as
appropriate

Throughout sample
analysis process

Transferred to
spreadsheets

DOC analysis
procedures and
sample results

Analytical
laboratory

ETV data sheets,
or data
acquisition
system, as
appropriate

Throughout sample
analysis process

Transferred to
spreadsheets

(a) All activities subsequent to data recording are carried out by Battelle.

5.2 Data Analysis

Procedures for analyzing the data produced in the verification test are described in this

section.

5.2.1 Accuracy

For test kits that provide quantitative results, PT sample accuracy will be assessed

relative to the spike level, and environmental sample accuracy will be assessed relative to the

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reference method results. The results for each set of analyses will be averaged, and the accuracy
will be expressed in terms of a percent recovery (R) as calculated from Equation 1:

R=C/Crx100	(1)

where C is the average concentration measured by the test kit and CR is the spike level for the
PT samples and the reference measurement for the environmental samples.

For qualitative results, accuracy will be assessed by determining whether the test kit
result agrees with the reference method result. An overall percent agreement will be determined
by dividing the number of correct responses to the overall number of analyses.

5.2.2 Precision

For test kits that provide quantitative results, the standard deviation (S) of the results for
the three replicate samples will be calculated for each sample using Equation 2:

S =

i n

1/2

(2)

where n is the number of replicate samples, Ck is the concentration measured for the kth sample,
and C is the average concentration of the replicate samples. The precision for each sample will
be reported in terms of the relative standard deviation (RSD) as calculated using Equation 3:

RSI)

S_

c

x 100

(3)

For test kits that provide qualitative results, precision will be assessed by calculating the
percentage of consistent responses.

5.2.3 Linearity

For test kits that provide quantitative results, linearity will be assessed by performing a
linear regression with the spiked analyte concentration as the independent variable, and the
individual test kit result as the dependent variable. Individual replicate results for the five PT

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samples will be used in the linear regression. Linearity will be expressed in terms of the slope,
intercept, and correlation coefficient (r).

5.2.4 Method Detection Limit

The method detection limit (MDL) for each test kit will be assessed using results from
seven replicate analyses of a sample spiked at a level of two times the vendor-stated test kit
detection limit. The standard deviation of the seven replicate samples will be calculated using
Equation 2. The MDL will be calculated using Equation 4:

MDL = txS (4)

where t is the Student's t value for a 99% confidence level and S is the standard deviation of the
seven replicate samples.

5.2.5 Cross Reactivity

The cross reactivity of the test kits to the atrazine degradation products hydroxyatrazine
and desethyl atrazine will be assessed qualitatively by evaluating the test kit results for samples
that contain only the degradation compound, but not atrazine. The reference analysis results will
be used to confirm the absence of atrazine in the samples.

5.2.6 Matrix Interferences

The potential effect of the sample matrix on the test kit performance will be evaluated
qualitatively by comparing the accuracy and precision results for the natural and atrazine-
fortified environmental samples to those for the PT samples.

5.2.7 False Positives/False Negatives

For quantitative and semi-quantitative technologies, the occurrence of false positive and
false negative results will be assessed relative to the test kit's lowest calibration standard. A
false positive is defined as a positive test kit result when reference method analysis indicates that
the atrazine concentration in the sample is below the test kit's lowest calibration standard. A

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false negative will be defined as a negative test kit result when the reference method analysis
indicates that the atrazine concentration in the sample is above the test kit's lowest calibration
standard. For qualitative technologies, a false positive is defined as a positive test kit result when
the reference method analysis indicates that the atrazine concentration in the sample is below the
threshold value (±10%) of the kit. A false negative is defined as a negative test kit result when
the reference method analysis indicates that the atrazine concentration in the sample is above the
threshold value (±10%) of the kit. The rate of false positives/false negatives will be expressed as
a percentage of total number of samples. Reagent blanks, PT samples, and environmental
samples will be included in the analysis.

5.3 Reporting

The data obtained in the verification test will be compiled separately for each test kit, and
the data evaluation methods described in Section 5.2 will be applied to each data set without
comparison to any other technology. At no time will data for test kits from different vendors be
intercompared or ranked. Following completion of the data evaluation, a draft verification report
will be prepared for each test kit. The verification report will describe the verification test
procedures and document the results. Each draft verification report will be submitted to the
corresponding vendor for review and comment. Each draft report will be revised in response to
the comments provided by the vendor. The revised reports will be submitted for external peer
review. The reports will be revised again to address the peer review comments, and then
submitted to EPA for final approval.

A verification statement will also be prepared for each test kit. The verification statement
is a 2- to 3-page summary of the technology, test procedures, and results. The verification
statement will follow the same review and revision process as the verification reports. Upon
final approval by EPA, each verification statement will be signed by a senior Battelle manager
and an EPA laboratory director. Final verification reports and statements will be posted on the
ETV website (http://www.epa.gov/etv). and original signed verification statements will be
provided to the vendor.

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6.0 Quality Assurance /Quality Control

The QA/QC activities associated with this verification test will focus primarily on sample
preparation and handling, data recording and analysis, and reference laboratory analysis. An
independent audit covering each of these areas will be performed by the Battelle Quality
Manager to ensure the quality of the verification test.

6.1 Sample Chain-of-Custody Procedures

Sample custody will be documented throughout collection, shipping, and analysis of the
samples following standard chain-of-custody procedures. The chain-of-custody form
summarizes the samples collected and analyses requested. The custody form will track sample
release from the field to the testing laboratory, and from the testing laboratory to the analytical
laboratory. Each sample custody form will be signed by the person relinquishing samples once
that person has verified that the custody form is accurate. The original sample custody forms
accompany the samples; the shipper will keep a copy. Upon receipt at the sample destination,
sample custody forms will be signed by the person receiving the samples once that person has
verified that all samples identified on the custody forms are present in the shipping container.
Any discrepancies will be noted on the form and the sample receiver will immediately contact
the Verification Test Coordinator to report missing, broken, or compromised samples.

6.2 Audits

6.2.1 Technical Systems Audit

The Battelle Quality Manager or designee will conduct a technical systems audit at least
once during the course of the verification test. The purpose of this audit is to ensure that the
verification test is being performed in accordance with this protocol and the AMS Center QMP1,
and that all procedures described in this protocol are being followed. This audit will review the
standards and methods used, compare actual test procedures to those specified in this protocol,
and review data acquisition and handling procedures. An independent technical systems audit

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may also be performed by EPA Quality Management staff during the verification test at EPA's
discretion.

6.2.2 Data Quality Audit

At least 10% percent of the data acquired during the verification test will be audited
during the verification test. Battelle's Quality Manager or designee will trace the data from its
initial acquisition, through reduction and statistical analysis, to final reporting, to ensure the
integrity of the reported results. All calculations performed on the data undergoing the audit will
be checked.

6.2.3 Performance Evaluation Audit

A performance evaluation (PE) audit will be conducted to assess the quality of the
reference measurements made in this verification test. A PE audit involves challenging the
reference instrument with a standard that is independent of the one used to calibrate the
instrument for the test. For the PE audit, a certified standard will be obtained from a commercial
supplier other than the one that supplied the standard for the preparation of PT and fortified
environmental samples, or the one used to calibrate the reference instrument. The PE sample
result must be within the certified range to be considered acceptable. Failure to achieve this
agreement will trigger recalibration of the reference instrument with the original QC standard,
and a repeat of the PE comparison. Failure in the second comparison requires obtaining another
set of standards, and repeating the performance audit.

6.3 Reference Method QC

The performance of the reference method will be demonstrated through the analysis of
QC samples. Laboratory RB samples will be analyzed to ensure that no sources of
contamination are present. If the analysis of a laboratory RB sample indicates a concentration
above the MDL for the reference instrument, then contamination will be suspected. Any
contamination source(s) will be corrected, and proper blank readings will be achieved, before
proceeding with the reference analyses.

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The accuracy of the reference method will be verified before the beginning and at the
conclusion of each testing day. The instrument to be used for reference analyses will be initially
calibrated according to the procedures specified in the reference method. The instrument
calibration will be verified using an appropriate calibration check sample. If the result for the
calibration check sample differs by more than ±20% from the value of the standard, then the
instrument will be recalibrated before continuation of the test.

Laboratory matrix spike samples will be analyzed at a frequency of at least 5% to assess
whether matrix effects potentially influence the results of the reference analyses. The percent
recovery (R) of the laboratory matrix spikes will be calculated from the following Equation 5:

C - C

R=—s—x 100 (5)

where Cs is the analyzed concentration of the spiked sample, C is the analyzed concentration of
the unspiked sample, and s is the concentration equivalent of the analyte spike. If the percent
recovery of a matrix spike sample falls outside the range from 70-130%, then a matrix effect will
be suspected.

Analytical duplicates will be analyzed at a frequency of at least 5% to assess analytical
precision. The relative percent difference (RPD) between the two duplicates should be within
30%.

A Laboratory Fortified Blank (LFB) sample will be analyzed with each set of samples to
determine whether the methodology is in control. If the recovery of the LFB falls out of the
range of 70-130%) of the actual concentration, then work should be stopped until the source of
the problem is identified.

Upon completion of the data package and draft final report, the analytical laboratory QA
staff will review all data and results for accuracy, precision, completeness, and
representativeness. The QA staff may at any time selectively review any data during tabulation,
and will be available for consultation with the analytical laboratory technical staff regarding any
deviations and corrective action.

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6.4 QA/QC Reporting

Each assessment and audit will be documented in accordance with Section 3.3.4 of the
AMS Center QMP1. The results of the technical systems audit will be submitted to EPA.
Assessment reports will include the following:

• Identification of any adverse findings or potential problems.

• Response to adverse findings or potential problems.

• Recommendations for resolving problems.

• Confirmation that solutions have been implemented and are effective.

• Citation of any noteworthy practices that may be of use to others.

6.5 Corrective Action

During the course of any assessment or audit, the Battelle Quality Manager will inform
the technical staff of any immediate corrective action that should be taken. If serious quality
problems exist, the Battelle Quality Manager will notify the Battelle AMS Center Manager of the
need to consider a stop work order. Once the assessment report has been prepared, the
Verification Test Coordinator will ensure that a response is provided for each adverse finding or
potential problem, and will implement any necessary follow-up corrective action. The Battelle
Quality Manager will ensure that follow-up corrective action has been taken.

7.0 References

1. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
Version 5.0. EPA Environmental Technology Verification Program, prepared by Battelle,
Columbus, Ohio, March, 2004.

2. Environmental Technology Verification Program Quality Management Plan, December
2002 (EPA/600/R-03/021).

3. National Primary Drinking Water Standards, 40 CFR Part 141.

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4.	U.S. EPA Method 525.2, Determination of Organic Compounds in Drinking Water by
Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry,
Revision 2.0, 1995.

5.	U.S. EPA Method 9060 Total Organic Carbon, Revision 0, September 1986.

6.	Ciba Crop Protection Report ABR-94094, Storage Stability of Atrazine, G-30033, G28279,
and G28273 in Water Under Refrigerator Storage Conditions, Greensboro, NC as cited in
"Interlaboratory Validation of an Atrazine Immunoassay." Journal of the American Water
Works Association, September 2001.

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