Batteiie
7Zw Bu sintes i?/ Innovation
Environmental Technology
Verification Program
Advanced Monitoring
Systems Center
Test/QA Plan for
Verification of
Enzymatic Test Kits
EtVeT&Tv

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TEST/QA PLAN
for
Verification of
Enzymatic Test Kits
September 21, 2005
Prepared by
Battelle
505 King Avenue
Columbus, OH 43201-2693

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A2 TABLE OF CONTENTS
Section	Page
A	PROJECT MANAGEMENT
A1	Title Page	 1
A2	Table of Contents	 2
A3	Distribution List	 5
A4	Verification Test Organization 	 6
A5	Background 	 11
A6	Verification Test Description and Schedule 	12
A7	Quality Objectives	 14
A8	Special Training/Certification	14
A9	Documentation and Records 	15
B	MEASUREMENT AND DATA ACQUISITION
B1	Experimental Design	16
B2	Reference Sample Collection	25
B3	Sample Handling and Custody Requirements	26
B4	Laboratory Reference Methods	27
B5	Quality Control Audits and Requirements	29
B6	Instrument/Equipment Testing, Inspection, and Maintenance 	32
B7	Instrument/Equipment Calibration and Frequency 	32
B8	Inspection/Acceptance of Supplies and Consumables	34
B9	Non-Direct Measurements	34
BIO	Data Management 	34
C	ASSESSMENT AND OVERSIGHT
CI	Assessments and Response Actions 	37
C2	Reports to Management	39
D	DATA VALIDATION AND USABILITY
D1	Data Review, Validation, and Verification Requirements 	40
D2	Validation and Verification Methods 	40
D3	Reconciliation with User Requirements 	41
E	HEALTH AND SAFETY
El	Standard/Test Sample Preparation	42
E2	Sample Handling during Verification Testing	42
E3	Testing with VX, GB, and GD	42
F	REFERENCES 	43

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List of Figures	Page
Figure 1 Organization Chart	 7
List of Tables
Table 1	Verification Test Performance Parameters 	 16
Table 2 Lethal Dose of Target Contaminants	18
Table 3	Summary of Test Samples for Enzymatic Test Kits Verification	21
Table 4 Reference Methods for Target Analytes 	28
Table 5 Reference Methods for Interferents	28
Table 6 Physio-chemical Characterization of Drinking Water	28
Table 7 Quality Control Measures for Reference Methods 	30
Table 8 Calibration Procedures for Reference Methods	33
Table 9	Summary of Data Recording Process 	35

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ETV Advanced Monitoring Systems Center
Test/QA Plan for Verification of
Enzymatic Test Kits
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APPROVAL:
Name	
Company
Enzymatic Test Kits
Test/QA Plan
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Date

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A3 DISTRIBUTION LIST
Karen Riggs

Amy Dindal
James Blundi
Stephanie Buehler
Severn Trent Services
Zachary Willenberg
3000 Advance Lane
Chris Fricker
Colmar, PA 18915
Battelle

505 King Ave.
Fernando Rubio
Columbus, OH 43201
Abraxis LLC

54 Steamwhistle Drive

Warminster, PA 18974

Ken Hayes

Aqua Survey, Inc.

469 Point Breeze Rd.

Flemington, NJ 08822

Tristan Ruysschaert, Ph.D.

Protein-BioSensor

Incubateur Midi-Pyrenees

29, rue Jeanne Marvig

France- 31400 Toulouse

Elizabeth A. Betz
U.S. Environmental Protection Agency -
HEASD
National Exposure Research Laboratory
E205-01 EPA Mailroom
Research Triangle Park, NC 27711
Robert Fuerst
U.S. Environmental Protection Agency -
HEASD
National Exposure Research Laboratory
D205-05 EPA Mailroom
Research Triangle Park, NC 27711
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SECTION A
PROJECT MANAGEMENT
A4 VERIFICATION TEST ORGANIZATION
The verification test will be conducted under the auspices of the U.S. Environmental
Protection Agency (EPA) through the Environmental Technology Verification (ETV) Program. It
will be performed by Battelle, which is managing the ETV Advanced Monitoring Systems (AMS)
Center through a cooperative agreement with EPA. The scope of the AMS Center covers
verification of monitoring technologies for contaminants and natural species in air, water, and
soil.
The day to day operations of this verification test will be coordinated and supervised by
Battelle personnel, with the participation of the vendors who will be having the performance of
their enzymatic test kits verified. The testing will be conducted at Battelle in Columbus and West
Jefferson, Ohio. Battelle staff will operate the test kits during the verification test. Each vendor
will provide sufficient supplies to allow for completion of the test (as described in this document)
and training to Battelle staff on the use of the enzymatic test kit. Quality assurance (QA)
oversight will be provided by the Battelle Quality Manager and the EPA AMS Center Quality
Manager at his/her discretion. The organization chart in Figure 1 identifies the responsibilities of
the organizations and individuals associated with the verification test. Roles and responsibilities
are defined further below.

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Battelle
Management
Amy Dindal
Verification
Testing Leader
Vendor
Representatives
Karen Riggs
Battelle AMS
Center Manager
Robert Fuerst
EPA AMS Center
Manager
Zachary Willenberg
Battelle AMS Center
Quality Manager
Elizabeth Betz
EPA AMS Center
Quality Manager
Dr. Stephanie Buehler
Verification
Test Coordinator
Battelle
Technical Staff
Figure 1. Organization Chart

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A4.1 Battelle
Dr. Stephanie Buehler is the AMS Center's Verification Test Coordinator for this test. In
this role, Dr. Buehler will have overall responsibility for ensuring that the technical, schedule, and
cost goals established for the verification test are met. Specifically, Dr. Buehler will:
•	Assemble a team of qualified technical staff to conduct the verification test.
•	Direct the team in performing the verification test in accordance with the test/QA plan.
•	Ensure that all quality procedures specified in the test/QA plan and in the AMS Center
Quality Management Plan1 (QMP) are followed.
•	Prepare the draft and final test/QA plan, verification reports, and verification
statements.
•	Revise the draft test/QA plan, 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 test/QA plan, verification reports, and verification
statements.
•	Establish a budget for the verification test and manage staff to ensure the budget is not
exceeded.
•	Ensure that confidentiality of sensitive vendor information is maintained.
Ms. Amy Dindal is a Verification Testing Leader for the AMS Center. Ms. Dindal will
provide technical guidance and oversee the various stages of verification testing. She will
•	Support Dr. Buehler in preparing the test/QA plan and organizing the testing.
•	Review the draft and final test/QA plan.
•	Review the draft and final verification reports and verification statements.
•	Support Dr. Buehler in responding to any issues raised in assessment reports and
audits.
Ms. Karen Riggs is Battelle's manager for the AMS Center. Ms. Riggs will

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•	Review the draft and final test/QA plan.
•	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 confidentiality of sensitive vendor information is maintained.
•	Maintain communication with EPA's project and quality managers.
•	Facilitate a stop work order if Battelle or EPA QA staff discovers adverse findings that
will compromise test results.
Battelle Technical Staff will conduct the testing of the enzymatic test kits during the
verification test. The responsibilities of the technical staff will be to:
•	Assist in the collection, receipt, and storage of drinking water samples.
•	Maintain and operate the test kits after proper training is provided by the vendor.
•	Perform the verification testing as described in the test/QA plan, including the
preparation of all test samples.
•	Make qualitative observations about the operation of the enzymatic test kits.
•	Record the results for each enzymatic test kit and transmit them to the Verification
Test Coordinator on a daily basis.
•	Perform reference method measurements indicated in this test/QA plan.
•	Troubleshoot any problems with the enzymatic test kits and communicate them to the
Verification Test Coordinator immediately.
Mr. Zacharv Willenberg is Battelle's Quality Manager for the AMS Center. Mr.
Willenberg will:
•	Review the draft and final test/QA plan.
•	Conduct a technical systems audit at least once during the verification test, or
designate other QA staff to conduct the audit.
•	Audit at least 10% of the verification data.
•	Prepare and distribute an assessment report for each audit.

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•	Verify implementation of any necessary corrective action.
•	Notify Battelle's AMS Center Manager to issue 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 test/QA plan is followed.
A4.2 Vendors
The responsibilities of the vendor representatives are as follows:
•	Review the draft test/QA plan.
•	Approve the test/QA plan prior to test initiation.
•	Provide enough off-the-shelf enzymatic test kits for evaluation of all test samples
during the verification test.
•	Provide all other equipment/supplies/reagents/consumables needed to operate their test
kits for the duration of the verification test.
•	Provide training to Battelle staff in the operation of their enzymatic test kit.
•	Provide written instructions for operation of enzymatic test kits.
Review and provide comments on the draft verification report and statement.
A4.3 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:
Ms. Elizabeth Betz is EPA's AMS Center Quality Manager. For the verification test, Ms.
Betz will:
•	Review the draft test/QA plan.
•	Perform at 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.

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•	Prepare and distribute an assessment report summarizing results of external audit.
•	Review draft verification reports and statements.
Mr. Robert Fuerst is EPA's manager for the AMS Center. Mr. Fuerst will:
•	Review the draft test/QA plan.
•	Approve the final test/QA plan.
•	Review the draft verification reports and statements.
•	Oversee the EPA review process for the verification reports and statements.
•	Coordinate the submission of verification reports and statements for final EPA
approval.
•	Contact the AMS Center Manager to issue a stop work order if an EPA assessment
indicates that data quality is being compromised.
A4.4 Subcontract Laboratory
Any laboratory providing reference measurements will follow the requirements of the
reference methods as well as the QC requirements as stated in this test/QA plan. A subcontract
laboratory will provide reference measurements for the drinking water characterization as well as
confirmation of the interferent solution concentrations. The responsibilities of this laboratory will
include:
•	Proper receipt and handling of sample material.
•	Accurate measurement of the target analyte(s) or target parameter(s).
•	Submission of data and any supporting documents to Battelle.
•	Participation in audit by Battelle AMS Quality Manager or EPA AMS Quality
Manager, if requested.
A5 BACKGROUND
The ETV Program's AMS Center does third-party verification testing of commercially
available technologies that detect natural species and contaminants in air, water, and soil. A
stakeholder committee of buyers and users of such technologies recommend the technology

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categories and technologies within those categories as priorities for testing. Enzymatic test kits
for detection of chemical agents were identified as a priority technology category through the
AMS Center stakeholder process.
A6 VERIFICATION TEST DESCRIPTION AND SCHEDULE
A6.1 Summary of Technology Category
Enzymatic test kits are qualitative technologies that can detect the presence of chemical
agents, carbamate pesticides, and/or organophosphate (OP) pesticides. These kits are generally
designed to be handheld and portable. This verification test will assess the ability of the
enzymatic test kits to detect these compounds in water.
Enzymatic test kits generally rely on the presence or absence of color or a color change to
indicate that a chemical agent, carbamate pesticide, or OP pesticide has been detected. These test
kits cannot determine the specific contaminant that is present, nor can they determine the amount
of contaminant(s) present. However, some technologies can indicate the concentrations range of
chemical agent, carbamate pesticide, or OP pesticide that is present (e.g., high, medium, or low)
through a sliding color scale.
To detect chemical agents, carbamate pesticides, and OP pesticides, enzymatic test kits
rely on the reaction of the cholinesterase enzyme. Under regular conditions, the enzyme reacts
normally with other chemicals that might be present in the test kit. When a chemical agent,
carbamate pesticide, or OP pesticide is present, the activity of the enzyme is inhibited by these
compounds. The effects of this inhibition will then generally lead to a color change in the test kit,
indicating the presence of a chemical agent, carbamate pesticide, or OP pesticide. For some kits,
a color change may indicate the absence of the contaminant.
This verification test will assess the performance of each enzymatic test kit relative to key
verification parameters including accuracy, precision, and false positive and negative rates in
detecting selected chemical agents, carbamate pesticide and OP pesticide in American Society of
Testing and Materials (ASTM) Type II deionized (DI) water, in the presence of possible
interferents added to ASTM Type II DI water, and in drinking water obtained from a variety of
geographically dispersed U.S. water utilities that use various water treatment processes.

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Qualitative characteristics of each enzymatic test kit, such as ease of use, sample throughput, and
cost, will also be assessed and reported. While most of the testing will occur in a laboratory, the
enzymatic test kits that are designed for field use will be tested outside of the laboratory. In
performing the verification test, Battelle will follow the technical and QA procedures specified in
this test/QA plan and will comply with the quality requirements in the AMS Center QMP1.
A6.2 Verification Schedule
The verification test of enzymatic test kits will begin in September 2005 and last through
October 2005. At the close of testing, individual reports will be drafted for each technology,
reviewed, and submitted to EPA for final signature. All reports will be submitted to EPA in
electronic (Word and Adobe portable document format [pdf]) and hard copy formats.
A6.3 Test Facility
The verification test will take place at Battelle facilities in Columbus and West Jefferson,
Ohio. A portion of the test (field portability) will also be conducted in a non-laboratory
environment. Testing with chemical agents will take place at the Hazardous Materials Research
Facility (HMRC). The HMRC, located in the North area of Battelle's West Jefferson Campus, is
an ISO 9001 certified facility. The HMRC and its personnel have demonstrated the capability for
storing and safely handling chemical agents, Class A poisons, toxins, agent simulants, and other
hazardous materials.
The HMRC research laboratories meet or exceed all requirements for the safe use, storage,
decontamination, and accountability of chemical agents as defined by Army Regulation 50-6
(Chemical Surety).3 Operations withing the laboratories always are conducted in accordance with
Battelle's Chemical Safety Information Program.4 Any non-disposable equipment that is part of a
particular technology and that comes into contact with chemical agent(s) will not be able to be
returned to the vendor because of restrictions on the release of such equipment.5
The pesticides to be evaluated in this test are not required to be used in a specially
designed facility as are the chemical agents. Thus, testing of these chemicals will take place in
qualified laboratories in the Battelle Columbus Operations facilities.

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A7 QUALITY OBJECTIVES
This verification test will evaluate the performance of enzymatic test kits for the
identification of chemical agents, carbamate pesticides, and OP pesticides in water. Only
qualitative results (e.g., presence/absence of contaminant at specified levels) will be considered
for each technology. In the instance where semi-quantitative (e.g., percent inhibition) measures
are used in determining the results for a particular technology, a qualitative result will be reported
(i.e., presence or absence of the contaminant of interest) as with the other technologies, and the
semi-quantitative measure used to determine that result will also be reported for that sample but
will not be used in any other data analyses as described in Section B1.2.
Method blank and positive and negative control samples will be included as QC samples
for each technology. The quality of the reference measurements will be monitored using QC
samples and procedures, as described in the testing laboratory's procedures or the method. These
requirements are further discussed in Section B. Performance evaluation audit samples will also
be used to confirm the accuracy of the reference solution concentrations (see Section Cl.l).
The Battelle Quality Manager or his designee will perform a technical systems audit
(TSA) at least once during this verification test and will audit at least 10% of the verification data
acquired. The EPA AMS Center Quality Manager also may conduct an independent TSA, at her
discretion.
A8 SPECIAL TRAINING/CERTIFICATION
Battelle staff will have appropriate training and experience. Documentation of training is
maintained for all Battelle technical staff in training files at their respective locations. Chemical
warfare agent testing will be conducted by staff with the appropriate training that will be
documented in their training record. Any use of respirators will be conducted by staff with the
appropriate training. The Battelle Quality Manager will verify the presence of appropriate
training records prior to the start of testing. The technology vendor will be required to train
Battelle technical staff in the operation of his/her technology prior to the start of testing. Battelle
will document this training with a consent form, signed by the vendor, that states which specific
Battelle staff have been trained to use the enzymatic test kit.

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A9 DOCUMENTATION AND RECORDS
The records for this verification test include the test/QA plan, the protocols, chain of
custody forms, laboratory record books (LRB), data collection forms, electronic files (both raw
data and spreadsheets), and the final verification report and statement. All of these records will
be maintained in the Verification Test Coordinator's office or at the testing locations during the
test and will be transferred to permanent storage at Battelle's Records Management Office at the
conclusion of the verification test. All Battelle LRBs are stored indefinitely, either by the
Verification Test Coordinator or Battelle's Records Management Office. EPA will be notified
before disposal of any files. The results from the reference measurements made by the Battelle or
subcontractor laboratory will be submitted to Battelle after making the measurement and
obtaining the results of the analyses. Section BIO further details the data recording practices and
responsibilities. QA documents generated over the course of this verification test include audit
and assessment reports and will be maintained by the Battelle Quality Manager.
All written records must be in ink. Any corrections to notebook entries, or changes in
recorded data, must be made with a single line through the original entry. The correction is then
to be entered, initialed, and dated by the person making the correction. In all cases, strict
confidentiality of the raw data from each vendor's technology, and separation of data from
different technologies, will be maintained throughout the verification test. Separate files
(including manual records, printouts, and/or electronic data files) will be kept for each instrument.

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SECTION B
MEASUREMENT AND DATA ACQUISITION
B1 EXPERIMENTAL DESIGN
This verification test will specifically address verification of enzymatic test kits for the
detection of GB, GD, and VX (chemical agents); aldicarb (carbamate pesticide); and dicrotophos
(OP pesticide) in drinking water. The contaminants were selected based on the capabilities of the
technologies being tested and the importance of these contaminants. The enzymatic test kits
participating in this test will be evaluated on qualitative results (as noted in Section A7),
indicating only the presence or absence of the contaminants within a specified concentration
interval (see Section B1.1). The performance of the enzymatic test kits will be verified based on
the parameters identified in Table 1. These parameters are discussed in detail in Section B 1.2.
Table 1. Verification Test Performance Parameters
Performance
Parameter
Method of Evaluation
Accuracy
Frequency of positive responses in the presence of nominal
concentrations of each contaminant in ASTM Type IIDI water
Precision
Determined by repeatability of response from triplicate
measurements of each test sample
False Positive/Negative
Rates
Determined by response in the presence or absence of a contaminant
in various matrices
Potential Matrix and
Interference Effects
Evaluation of performance in various matrices and in the presence of
potential interferents
Portability
Evaluation of operational factors obtained in a non-laboratory setting
Operational Factors
Observational factors such as operator observations, ease of use, and
sample throughput

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Bl.l Test Procedures
The following sections describe the test procedures that will be used to evaluate each of
the enzymatic test kit performance parameters listed in Table 1. Performance testing will be
conducted on each kit separately, and will follow the operational procedures specified by the
vendor, including analysis of quality control (QC) samples.
This verification test will determine the performance capabilities of enzymatic test kits to
detect GB, GD, VX, aldicarb, and dicrotophos in three types of samples—performance test (PT),
drinking water (DW), and QC. Each sample type is described in detail in the following sections.
Contaminants will be tested individually on each technology. Stock solutions of each contaminant
will be prepared in the ASTM Type IIDI water or appropriate reagents from certified standards.
Samples will then be prepared in the appropriate matrix using these stock solutions. Samples will
be prepared and analyzed on the same day, with samples being prepared as close in time to their
use with a test kit as possible. To minimize the loss of analytes to hydrolysis, contaminant stock
solutions prepared in DI water will be made on a daily basis unless the stability of the analyte in
water has been previously demonstrated. Aliquots of each stock solution will be diluted to the
appropriate concentration using volumetric glassware and volumetric or calibrated pipettes. In
some cases, reference solutions will be prepared in ASTM Type II DI water using the stock
solutions to determine their concentration; otherwise, the concentration of the stock solutions will
be confirmed. Reference methods for confirming solution concentrations are discussed in Section
B4 (see Tables 4 and 5). All DW samples will be dechlorinated prior to their use to minimize the
hydrolysis of the contaminants. The DW samples will also be characterized for typical water
quality parameters (see Sections B1.1.2 and B4) prior to use.
Bl. 1.1 PT Samples
PT sample types will be prepared in ASTM Type II DI water. The first type of PT sample
will consist of ASTM Type II DI water spiked with a contaminant at five concentrations.
Concentrations will include the lethal dose concentration given in Table 2 for each contaminant
along with dilutions at approximately 10, 100, 1,000, and 10,000 times less than the lethal dose.
The lethal dose of each contaminant was determined by calculating the concentration at which

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250 mL of water is likely to cause the death of a 70-kg person based on human oral LD50 data.6'7
Human oral LD50 data was not available for aldicarb, so rat oral LD50 data was used instead.8
Each concentration level for the PT samples will be analyzed in triplicate.
Table 2. Lethal Dose of Target Contaminants
Contaminant (common
name)
Oral Lethal Dose
Concentration
Contaminant Class
VX
2.1 milligrams (mg)/Liter
Chemical agent

(L)

GB (sarin)
20 mg/L
Chemical agent
GD (soman)
1.4 mg/L
Chemical agent
Aldicarb
260 mg/L
Carbamate pesticide
Dicrotophos
1400 mg/L
Organophosphate
pesticide
The second type of PT sample will be potential interferent samples. Three replicates of
each interferent PT sample will be analyzed to determine each enzymatic test kit's susceptibility
to these commonly found interferents in DW. There will be two types of interferent PT samples.
One will contain calcium and magnesium from carbonates spiked into ASTM Type IIDI water,
and the other will contain humic and fulvic acids obtained from the International Humic
Substances Society spiked into ASTM Type II DI water. Each of these interferent mixtures will
be prepared at two different concentration levels. One concentration will be near the upper limit
of what would be expected in drinking water (250 mg/L total concentration for calcium and
magnesium, 5 mg/L total concentration for humic and fulvic acids) and one concentration at a
mid-low range of what would be expected (50 mg/L total concentration for calcium and
magnesium, 1 mg/L total concentration for humic and fulvic acids). These spiked interferent
levels will be confirmed through analysis of aliquots by the subcontract laboratory. Also, each
contaminant will be added to these samples along with the potential interferent, at a concentration
consistent with a lOx dilution of the lethal dose, and analyzed in triplicate.

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Bl.1.2 Drinking Water (DW) Samples
Drinking water samples will be collected from four geographically distributed municipal
sources (Ohio, New York, California, and Florida) to evaluate the performance of the enzymatic
test kits with various DW matrices. These samples will vary in their source and treatment and
disinfection process. All samples will have undergone either chlorination or chloramination
disinfection prior to receipt. Samples will be collected from water utility systems with the
following treatment and source characteristics:
•	Chlorinated filtered surface water source
•	Chlorinated unfiltered surface water source
•	Chlorinated filtered groundwater source
•	Chloraminated filtered surface water.
All samples will be collected in new, pre-cleaned high density polyethylene (HDPE)
containers. After sample collection, to characterize the DW matrix, an aliquot of each DW
sample will be sent to a subcontract laboratory to determine the following water quality
parameters: concentration of trihalomethanes, haloacetic acids, total organic halides, calcium,
magnesium, pH, conductivity, alkalinity, turbidity, organic carbon, and hardness. The DW
samples will be dechlorinated prior to their use with sodium thiosulfate pentahydrate to prevent
the degradation of some of the target contaminants by chlorine. Also, some test kits require
dechlorination of DW prior to analysis. The dechlorination of the DW will be qualitatively
confirmed by adding a diethyl-p-phenylene diamine (DPD) tablet to an aliquot of DW. If the
water does not turn pink, the dechlorination process will be determined to be successful. If the
water does turn pink, additional dechlorinating reagent will be added and the dechlorination
confirmation procedure repeated. Each DW sample will be analyzed without adding any
contaminant, as well as after fortification with each individual contaminant at a single
concentration level (lOx dilution of the lethal dose). Aliquots of each contaminant stock solution
will be diluted with DW samples to the appropriate concentration. Each sample will be tested in
triplicate.

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Bl.1.3 Quality Control (QC) Samples
QC samples will include method blank (MB) samples consisting of ASTM Type IIDI
water, and positive and negative controls, if applicable and as provided by the vendor. All of the
MB QC samples will be exposed to identical sample preparation and analysis procedures as the
test samples. Positive and negative controls will be provided by the vendor or prepared and used
according to the protocol provided by the vendor. The MB samples will be used to ensure that no
sources of contamination are introduced in the sample handling and analysis procedures. At least
10% of the test samples (7 samples for each contaminant) will be MB samples. The vendor
provided control samples will indicate to the operator whether the enzymatic test kit is
functioning properly and will be added to each set of samples analyzed by the technology, as
applicable, or added per batch of samples as indicated by the vendor. The test samples and MB
samples will be analyzed blindly by the operator in that the samples used for analysis will be
marked with a non-identifying number.
Bl. 1.4 Portability Samples and Operational Factors
Those enzymatic test kits that are designed to be field portable will be tested outside of the
laboratory by a non-technical operator. A non-technical operator is considered to be someone
with little to no laboratory experience who would be representative of a first responder. The non-
technical operator will be trained in the use of the enzymatic test kit by the vendor or by another
Battelle staff person who was trained by the vendor. Because many of the contaminants being
tested are highly toxic and unsafe to be handled outside of a special facility, method blank
samples and non-toxic positive and negative control samples will be analyzed as portability
samples. The non-toxic positive and negative control samples will be provided by the vendor or
prepared and used according to the vendor's protocol.. Because no samples spiked with the
contaminants of interest will be used, only the operational aspects of each enzymatic test kit will
be evaluated as part of the portability testing. As these technologies are anticipated to be used by
first-responders, their performance in a non-laboratory setting will be evaluated under simulated
first response conditions by having the operator dressed in a Level B protective suit, butyl gloves,
boots, and either a self contained breathing apparatus (SCBA) or an air purifying respirator,

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depending on the availability of personnel trained in the operation of such devices. The operator
will have prior experience in working in personal protective equipment (PPE). One set of MB
samples will be tested without the use of PPE. Ease of use from the perspective of the operator
will be documented both with and without the PPE.
Operational factors such as ease of use and sample throughput will be evaluated based on
observations recorded by Battelle staff. Operational factors will be noted for both the laboratory
and non-laboratory portions of the verification test. These observations will be summarized to
describe the operational performance of each enzymatic test kit in the verification reports.
Table 3 provides a summary of all of the samples to be tested for each enzymatic test kit
and each contaminate of interest.
Table 3. Summary of Test Samples for Enzymatic Test Kits Verification
Performance Test
(PT) Samples
Performance Factor
Evaluated
Sample Description
Reps
Contaminant Only
Accuracy, Precision,
False Negative
Contaminant PT sample @ lethal dose
3
Contaminant PT sample @ 10 times less than the
lethal dose
3
Contaminant PT sample @100 times less than the
lethal dose
3
Contaminant PT sample @ 1,000 times less than
the lethal dose
3
Contaminant PT sample @ 10,000 times less than
the lethal dose
3
Interferent
Interference Effects,
Precision, False
Positive/Negative
Responses
Fulvic and humic acids @ a total concentration of
1 mg/L
3
Fulvic and humic acids @ a total concentration of 1
mg/L + contaminant @ 10 times less than the lethal
dose
3
Fulvic and humic acids @ a total concentration of
5 mg/L
3
Fulvic and humic acids @ a total concentration of
5 mg/L + contaminant @ 10 times less than the
lethal dose
3
Calcium and magnesium @ a total concentration of
50 mg/L
3

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Calcium and magnesium @ a total concentration of
50 mg/L + contaminant @ 10 times less than the
lethal dose
3
Calcium and magnesium @ a total concentration of
250 mg/L
3
Calcium and magnesium @ a total concentration of
250 mg/L + contaminant @ 10 times less than the
lethal dose
3
Portability Samples
Performance Factor
Evaluated
Sample Description
Reps
Method Blank
Samples
Operational factors
Analysis in PPE by non-technical operator
3
Analysis not in PPE by non-technical operator
3
Drinking Water
(DW) Samples
Performance Factor
Evaluated
Sample Description
Reps
Filtered chlorinated
surface water
Potential Matrix and
Interference Effects,
Precision, False
Positive/Negative
Responses
Unspiked
3
Spiked with contaminant @ 10 times less than the
lethal dose
3
Unfiltered
chlorinated surface
water
Unspiked
3
Spiked with contaminant @ 10 times less than the
lethal dose
3
Filtered chlorinated
groundwater
Unspiked
3
Spiked with contaminant @ 10 times less than the
lethal dose
3
Filtered
chloraminated
surface water
Unspiked
3
Spiked with contaminant @ 10 times less than the
lethal dose
3
Quality Control
(QC) Samples
Performance Factor
Evaluated
Sample Description
Reps
Method Blank
Quality Check
DI water - 10% of all non-QC samples
7
Positive Control
Provided by vendor
Var(a)
Negative Control
Provided by vendor
Var(a)
An proximate total number of samples per contaminant
76
(a) Number of positive and negative controls will vary based on the protocol provided by the vendor.

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B1.2 Statistical Analysis
The technologies participating in this verification test will only be evaluated for
qualitative results (i.e., presence or absence of the contaminant of interest). All data analyses
will be based on these qualitative results. The statistical methods and calculations used for
evaluation of the qualitative performance parameters are described in the following sections. The
performance parameters will be determined for each target analyte (see Table 2) individually.
When semi-quantitative measures (e.g., percent inhibition) are used in determining the
presence or absence of a contaminant for a particular technology, the semi-quantitative measure
will be used to determine a qualitative result for the enzymatic test kit (i.e., presence or absence
of contaminant). A qualitative result will then be reported as with the other technologies and used
for the data analyses described in the following sections. The semi-quantitative measure will also
be reported for that sample but will not be used in any other data analyses.
B 1.2.1 Accuracy
The accuracy will be assessed by evaluating how often the enzymatic test kit result is positive
in the presence of a concentration above the LOD. Contaminant-only PT samples will be used for
this analysis. An overall percent agreement will be determined by dividing the number of positive
responses to the overall number of analyses of contaminant-only PT samples above the kit's LOD
(see Equation 1). If the LOD for a technology is not known or available, then all analyzed
contaminant-only PT samples above the concentration level where consistent negative results are
obtained will be used in estimating the accuracy for that enzymatic test kit.
# of positive contaminant only PT samples
Accuracy (% Agreement) =	——						x 100	(1)
total # oj contaminant only PT samples
B 1.2.2 False Positive/False Negative Rates
A false positive response will be defined as an enzymatic test kit response indicating the
presence of a contaminant when the ASTM Type II DI water (including interferent samples) or

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drinking water sample is not spiked at all. A false positive rate will be reported as the number of
false positive results out of the total number of unspiked samples (see Equation 2).
A false negative response will be defined as an enzymatic test kit response indicating the
absence of a contaminant when the sample is spiked with a contaminant at a concentration greater
than the enzymatic test kit's LOD. Spiked PT (contaminant and interferent) samples and spiked DW
samples will be included in the analysis. Contaminant-only PT samples above the technology' s LOD
or the level at which consistent negative responses are obtained (when the LOD is not known) will
be included in the analysis. A false negative rate will be evaluated as the number of false negative
results out of the total number of spiked samples for a particular contaminant (see Equation 3).
#	of positive results
False Positive Rate =	——			—-		—	(2)
total # oj unspiked samples
#	of negative results (3)
False Negative Rate =	——-—-—-		—	v '
total # oj spiked samples
B 1.2.3 Precision
Precision measures the repeatability and reproducibility of enzymatic test kit responses. The
precision of the three replicates of each sample set will be assessed. Responses will be considered
inconsistent if one or more of the three replicates differs from the response of the other samples in
the replicate set. The overall precision for each enzymatic test kit will be assessed by calculating the
overall number of consistent responses for all the sample sets. The results will be reported as the
percentage of consistent responses out of all replicate sets (see Equation 4).
# of consistent responses of replicate sets
Precision (% Consistent results) =	——				x 100
total # of replicate sets

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B1.2.4 Potential Matrix and Interferent Effects
The potential effect of the DW matrix on the enzymatic test kit performance will be evaluated
qualitatively by comparing the results for the spiked and unspiked DW samples to those for the PT
samples spiked with the contaminant at 100 times less than the lethal dose. Similarly, the potential
effect of interferent PT samples will be evaluated. The results indicating the correct or incorrect
reporting of the presence of a contaminant will be evaluated. The findings will be reported and
discussed.
B1.3 Reporting
The statistical comparisons described above will be conducted separately for each enzymatic
test kit being tested, and information on the additional performance parameters such as ease of use
and sample throughput will be compiled and reported. Separate verification reports will be prepared
for each kit that was tested. No intercomparison of the enzymatic test kit data will be performed at
any time. For each enzymatic test kit, the verification report will present the test procedures and test
data, as well as the results of the statistical evaluation of those data.
All use of the test kits by Battelle staff, including all operational aspects observed during
laboratory and field testing, will be documented at the time of the test and reported. The verification
report will briefly describe the ETV program, the AMS Center, and the procedures used in
verification testing. These sections will be common to each verification report. The results of the
verification test will then be stated, without comparison to any other technology tested or comment
on the acceptability of the technology's performance.
B2 REFERENCE SAMPLE PREPARATION AND ANALYSIS
An aliquot of the stock or reference solution (that will be prepared from the stock solution)
will be submitted for reference measurement (see Section B4 for reference methods). Reference or
stock solutions for each contaminant (see Table 2) will be prepared separately. Interferent stock

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solutions will contain multiple analytes in the same solution (calcium and magnesium or fulvic and
humic acids together). The concentration of the contaminant and interfering compound stock
solutions will be verified with standard analytical methods as described in Section B4. Up to four
aliquots of each stock or reference solution will be analyzed over the course of the verification test.
Aliquots to be analyzed by confirmatory methods will be preserved or extracted and stored as
prescribed by the standard method to be used. Reference solutions for the pesticides and chemical
agents will be prepared as close in time as possible to the test samples to minimize any differences
in the rate of sample degradation. A reference sample for these compounds may also be submitted
for reference measurement at the end of each day of testing to assess the stability of the analyte
throughout the testing day. Reference solutions known or suspected to undergo hydrolysis will be
preserved or extracted, according to the reference method, on the day of preparation. The time of
preparation, extraction or preservation, and analysis will be noted. Reference or stock solutions
(after extraction or preservation if prescribed in the reference method) will be stored according to the
reference method or laboratory protocols.
B3 SAMPLE HANDLING AND CUSTODY REQUIREMENTS
Sample custody will be documented throughout collection, shipping, and analysis of the
samples from the water utilities to Battelle. Similar documentation will be recorded for shipping and
analysis of samples to the subcontract laboratory. Sample chain-of-custody procedures will be in
accordance with the Battelle SOP MDAS.I-009-Draft, Sample Chain of Custody.9 The chain-of-
custody form summarizes the samples collected and analyses requested. The chain-of-custody form
will track sample release from the DW utilities to the Battelle laboratory, and from the Battelle
laboratory to the subcontract laboratory. Each chain-of-custody form will be signed by the person
relinquishing samples once that person has verified that the chain-of-custody form is accurate. The
original sample chain-of-custody forms accompany the samples; the shipper will keep a copy. Upon
receipt at the sample destination, chain-of-custody forms will be signed by the person receiving the
samples once that person has verified that all samples identified on the chain-of-custody forms are
present in the shipping container. Any discrepancies will be noted on the form and the sample

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receiver will immediately contact the Verification Test Coordinator to report missing, broken, or
compromised samples. Reference samples shipped to the subcontract laboratory will be analyzed
within the holding time identified in the applicable reference method.
B4 LABORATORY REFERENCE METHODS
Table 4 provides the standard laboratory methods that will be used for the sample preparation
and reference analyses during this verification test. Table 5 provides the reference methods that will
be used for the analysis of the interferent solutions. Table 6 provides the methods that will be used
for determining the physio-chemical characteristics of the drinking water matrices. Instrumentation
used for each reference method is given in each table. All of the reference methods for the target
analytes will be performed by Battelle. Reference methods for the physio-chemical characterization
of the drinking water and the interferent solutions will be performed by a subcontract laboratory. In
all cases, laboratories will follow the QC requirements specified in B5. A subcontract laboratory will
be responsible for providing calibrated instrumentation, performing all method QA/QC, and
providing calibration records for any instrumentation used.

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Table 4. Reference Methods for Target Analytes
Target Analyte
Reference Method
Instrumentation®
VX
HMRC-IV-118-0510
GC/MS
GB (sarin)
HMRC-IV-118-0510
GC/MS
GD (soman)
HMRC-IV-118-0510
GC/MS
aldicarb
SOP for Analysis of Water Sample Extracts
For Type 1 Analytes by Liquid
Chromatography/ Mass Spectrometry11
LC/MS
dicrotophos
SOP for Extracting and Preparing Water
Samples For Analysis of Dicrotophos,
Mevinnhos. And Dichlorovos 12
GC/MS
a Gas chromatography (GC)/ mass spectrometry (MS)
Liquid chromatography (LC)/mass spectrometry (MS)
Table 5. Reference Methods for Interferents
Interferents
Reference Method
Instrumentation®
Ca and Mg
EPA 200.813
Simultaneous ICP
Humic and Fulvic
Acids
SM531014
Combustion infrared NDR
a Inductively coupled plasma (ICP)
Negative differential resistance (NDR)
Table 6. Reference Methods for the Physio-Chemical Characterization of Drinking Water
Parameter
Reference
Method
Instrumentation®
Turbidity
SM213014
Turbidimeter
Organic carbon
SM531014
Combustion infrared NDR
Specific
conductivity
SM251014
Conductivity meter
Alkalinity
SM 232014
pH meter
pH
EPA 150.115
pH meter
Hardness
EPA SM 234014
pH meter
Total organic
halides
SM 532014
Microcoulometric combustion analyzer

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Trihalomethanes
EPA 524.216
GC/MS
Haloacetic acids
EPA 552.216
GC/ECD
a Negative differential resistance (NDR)
Gas chromatography (GC)/ mass spectrometry (MS)
Gas chromatography (GC)/ Electron capture detection (ECD)
B5 QUALITY CONTROL AUDITS AND REQUIREMENTS
Steps will be taken to maintain the quality of the data collected during this verification test.
When confirmation analyses of the stock or reference solutions of both contaminant and potential
interfering compounds are performed, QC measures as noted in each laboratory's operating
procedures or the reference method will be followed. Similarly, appropriate QC measures as stated
in the reference method or the laboratory's operating procedures will be followed for the physio-
chemical characterization of the DW. The reference methods to be followed for this verification test
are listed in Tables 4 through 6. A summary of the QC samples and acceptance criteria associated
with each method is presented in Table 7. The QC measures for these reference methods will at
least include the analysis of a method blank sample with the analyses of the reference or stock
solution. Method blank samples will be analyzed to ensure that no sources of contamination are
present. If the analysis of a method blank sample indicates a concentration above the MDL for the
confirmatory instrument, contamination will be suspected. Any contamination source(s) will be
corrected, and proper blank readings will be achieved, before proceeding with the analyses. In
general, a matrix spike or laboratory fortified spike sample will also be analyzed. Average acceptable
recoveries for these samples are between 70-150%. Samples outside of the expected range will
generally be flagged and rerun once the QC acceptance criteria have been met. QC samples will be
run with every batch of 1-20 samples. Specific QC samples and acceptance criteria associated with
each method can be found in the appropriate reference (see Tables 4-6).
Quality control samples as provided with each enzymatic test kit will also be run per the
vendor's instructions (see Section B 1.1.3). Method blank samples will also be run as part of the
verification test (see Section B1.1.3).

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Table 7. Quality Control Measures for Reference Methods
Reference Method
QC Samples
Frequency
Acceptance Criteria3
HMRC-IV-118-05
Duplicate
Once in every 10 samples
±10% RPD

Blank
Once in every 10 samples
< MDL

Matrix spike
Once in every 10 samples
Depends on matrix
SOP for Analysis of Water
Sample Extracts For Type 1
Analytes by Liquid
Chromatography/ Mass
Spectrometry
Duplicate
Once in every 10 samples
±20% RPD

Fortified blank
Once in every 10 samples
50-100% recovery

Method blank
Once in every 10 samples
<1 ng/mL

Spike
Once in every 10 samples
80-120% recovery
SOP for Extracting and
Preparing Water Samples For
Analysis of Dicrotophos,
Mevinphos, And Dichlorovos
Duplicate
Once in every 10 samples
±50% RPD

Method blank
Once in every 10 samples
<50 ppb

Spike
Once in every 10 samples
70-150% recovery
EPA 200.8
Initial performance
check
First, before anything else
is run
95-105% recovery

Continuing
performance check
After every 10 samples
90-110% recovery

Quality check
standards
Once in every 10 samples
95-105% recovery

Blank
At the start of a run and
after every 10 samples
± the PQL

Matrix spike
Once in every 10 samples
85-115% recovery

Duplicate
Once in every 10 samples
±20% RPD if >5x the
PQL
SM 5310
Blank
At the start of a run and
after every 10 samples
< RDL

RDL
Once per run
70-130% recovery

Matrix spike
Once in every 10 samples
75-125% recovery

Duplicate
Once in every 10 samples
±20% RPD
SM 2130
Check standards
First, before anything else
90-110% recovery

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Duplicate
Once in every 10 samples
±10% RPD
SM 2510
Check standards
At the start of a run and
after every 10 samples
±10% of the true value

Blank
At the start of a run and
after every 10 samples
< 2 UHMO

Duplicate
At the start of a run and
after every 10 samples
±20% RPD
EPA 150.1
Buffer
At the start of a run and
after every 10 samples
±1% of the true value

Duplicate
Once in every 10 samples
±10% RPD
SM 2320
Check standards
At the start of a run and
after every 10 samples
±10% of the true value

Blank
At the start of a run and
after every 10 samples
< RDL

Duplicate
Once in every 10 samples
±10% RPD

Matrix spike
Once in every 10 samples
±10% of the true value
SM 2340
Check standards
At the start of a run and
after every 10 samples
±10% of the true value

Blank
At the start of a run and
after every 10 samples
< RDL

Duplicate
Once in every 10 samples
±10% RPD

Matrix spike
Once in every 10 samples
±10% of the true value
EPA 524.2
Check standards
Every 12 hours
70-130% recovery

LCS
Every 24 hours
70-130% recovery

Blank
Every 12 hours
70-130% recovery

Duplicate
Every 24 hours
70-130% recovery
EPA 552.2
Blank
Once in every 10 samples
70-130% recovery

Matrix spike
Once in every 10 samples
70-130% recovery

Duplicate
Once in every 10 samples
70-130% recovery
SM 5320
Blank
Once per batch not to
exceed 20 samples
< RDL

Matrix spike
Once per batch not to
exceed 20 samples
55-139% recovery

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Duplicate
Once per batch not to
90-131% recovery


exceed 20 samples

a Relative percent difference (RPD)
Method detection limit (MDL)
Practical quantitation limit (PQL)
Reporting detection limit (RDL)
B6 INSTRUMENT/EQUIPMENT TESTING, INSPECTION, AND MAINTENANCE
The instalments used for the reference analyses (see Tables 4-6) will be tested and inspected
as per the standard operating procedures of Battelle or the subcontract laboratory or per the standard
methods being used to make each measurement. Instruments to be used in the reference analyses for
this test include (but are not limited to) gas chromatography (GC)/mass spectrometry (MS), liquid
chromatography/MS, pH electrodes, inductively-coupled plasma/MS, and GC/electron capture
detection. Any discovered deficiencies with a particular instrument will be resolved per the protocol
of the laboratory in a timely manner. Any maintenance required on components of the enzymatic test
kits will be the responsibility of the vendor.
B7 INSTRUMENT/EQUIPMENT CALIBRATION AND FREQUENCY
The instruments used for the reference analyses will be calibrated per the standard reference
methods being used to make each measurement or the standard operating procedures of the analysis
laboratory. Instruments to be used in the reference analyses for this test include (but are not limited
to) gas chromatography (GC)/mass spectrometry (MS), liquid chromatography/MS, pH electrodes,
inductively-coupled plasma/MS, and GC/electron capture detection. If any component of an
enzymatic test kit requires calibration, the vendor will provide Battelle technical staff with
instructions on how to properly maintain such components. All calibrations performed will be
documented by Battelle in the project laboratory record book. Calibration of spectrometry
instruments will generally involve a 4-8 point calibration curve covering the range of concentrations
of the reference solutions to be analyzed. Calibration of each reference instrument will be performed
as frequently as required in the reference method guidelines. Calibration procedures for each method

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are summarized in Table 8.
Calibration of the applicable enzymatic test kit components will be done as often as suggested
by the vendor. This calibration will use National Institute of Standards and Technology (NIST)-
traceable standards if applicable or calibration solutions and/or devices supplied by the vendor.
Pipettes used during solution preparation will be maintained and calibrated as required by Battelle
standard operating procedures (i.e., minimum of every 6 months). Pipettes will be checked and either
recalibrated or replaced if they are dropped accidentally over the course of testing.
Table 8. Calibration Procedures for Reference Methods
Reference Method
Calibration Procedures
HMRC-IV-118-05
5 point curve performed once with each set of samples.
Calibration check also performed.
SOP for Analysis of Water Sample Extracts For Type 1
Analytes by Liquid Chromatography/ Mass
Spectrometry
At least a 4 point curve performed once with each set
of samples. Continuing calibration blanks and
verification standards run also.
SOP for Extracting and Preparing Water Samples For
Analysis of Dicrotophos, Mevinphos, And Dichlorovos
4 point curve performed once with each set of samples
EPA 200.8
3 point curve performed once with each set of samples.
Performance check standards also run.
SM 5310
6 pint curve performed once with each set of samples
or once every 24 hours
SM 2103
5 point curve performed quarterly each year.
Continuing calibration checks and check standards
also performed.
SM 2510
N/A. Performance check standards run.
EPA 150.1
Calibration performed once before each set of samples
SM 2320
Calibration performed once before each set of samples.
Performance check standards also run.
SM 2340
Calibration performed once before each set of samples.
Performance check standards also run.
EPA 524.2
7 point curve performed once before each set of
samples. Performance check standards also run.
EPA 552.2
5-6 point curve performed once before each ste of
samples.

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SM 5320
5 point curve performed once before each set of

samples. Recalibrated if the performance check

standards fail.
B8 INSPECTION/ACCEPTANCE OF SUPPLIES AND CONSUMABLES
All materials, supplies, and consumables will be ordered by the Verification Test Coordinator
or designee. Where possible, Battelle will rely on sources of materials and consumables that have
been used previously as part of ETV verification testing without problems. Battelle will also rely on
previous experience or recommendations from EPA advisors, host facility staff, or enzymatic test kit
vendors. NIST-traceable materials will be used whenever possible. All items will be compared with
their Certificates of Analysis, or similar documentation. Items that don't match will be sent back to
their supplier.
B9 NON-DIRECT MEASUREMENTS
Data published previously in the scientific literature relating to enzymatic test kits'
performance will not be used during this verification test.
BIO DATA MANAGEMENT
Various types of data will be acquired and recorded electronically or manually by Battelle
technical staff during this verification test. Table 9 summarizes the type of data to be recorded.
All data and observations for the operation of the test kits will be documented by Battelle
technical staff on data sheets or in laboratory record books. If a subcontract laboratory is used,
results from the subcontract laboratory reference instruments will be compiled by the
subcontractor's staff in electronic format and submitted to Battelle upon obtaining the results.
Results from any Battelle-performed reference methods will be similarly compiled and submitted.

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Table 9. Summary of Data Recording Process
Data to Be
Recorded
Where Recorded
How Often
Recorded
By Whom
Disposition of Data
Dates, times,
and details of
test events
ETV laboratory
record books or
data recording
forms
Start/end of test
procedure, and at
each change of a
test parameter
Battelle
Used to organize
and check test
results; manually
incorporated in data
spreadsheets as
necessary
Sample
preparation
(dates,
concentrations,
etc.)
ETV laboratory
record books
When each
solution is
prepared
Battelle
Used to confirm the
concentration and
integrity of the
samples analyzed
Enzymatic test
kit procedures
and sample
results
ETV data sheets
and laboratory
record book
Throughout test
duration
Battelle
Manually
incorporated into
data spreadsheets
for statistical
analysis and
comparisons
Reference
method sample
preparation
ETV laboratory
record book
Throughout
sample
preparation
Battelle
Used to demonstrate
validity of samples
submitted for
reference
measurements
Reference
method
procedures,
calibrations,
QA, etc.
Laboratory record
books or data
recording forms
Throughout
sampling and
analysis processes
Battelle or
subcontract
laboratory
Retained as
documentation of
reference method
performance
Reference
method analysis
results
Electronically from
reference analytical
method
Every sample
analysis
Battelle or
subcontract
laboratory
Converted to
spreadsheets for
calculations
Records received by or generated by any of the Battelle technical staff during the verification
test will be reviewed by a Battelle staff member within two weeks of receipt or generation,
respectively, before the records are used to calculate, evaluate, or report verification results. If a
Battelle staff member generated the record, this review will be performed by a Battelle technical

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staff member involved in the verification test, but not the staff member who originally generated
the record. The review will be documented by the person performing the review by adding
his/her initials and date to the hard copy of the record being reviewed. In addition, data
calculations performed by Battelle technical staff will be spot-checked by Battelle technical staff
to ensure that calculations are performed correctly. Calculations to be checked include any
statistical calculations described in this test/QA plan. The data obtained from this verification test
will be compiled and reported independently for each enzymatic test kit. Results for the
enzymatic test kits from different vendors will not be compared with each other.
Among the QA activities conducted by Battelle QA staff will be an audit of data quality. This
audit will consist of a review by the Battelle Quality Manager of at least 10% of the test data.
During the course of any such audit, the Battelle Quality Manager will inform the technical staff
of any findings and any immediate corrective action that should be taken. If serious data quality
problems exist, the Battelle Quality Manager will request that Battelle's AMS Center Manager
issue 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.

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SECTION C
ASSESSMENT AND OVERSIGHT
CI ASSESSMENTS AND RESPONSE ACTIONS
Every effort will be made in this verification test to anticipate and resolve potential
problems before the quality of performance is compromised. One of the major objectives of this
test/QA plan is to establish mechanisms necessary to ensure this. Internal QC measures described
in this test/QA plan, which is peer reviewed by a panel of outside experts, implemented by the
technical staff and monitored by the Verification Test Coordinator, will give information on data
quality on a day-to-day basis. The responsibility for interpreting the results of these checks and
resolving any potential problems resides with the Verification Test Coordinator. Technical staff
have the responsibility to identify problems that could affect data quality or the ability to use the
data. Any problems that are identified will be reported to the Verification Test Coordinator, who
will work with the Battelle Quality Manager to resolve any issues. Action will be taken to control
the problem, identify a solution to the problem, and minimize losses and correct data, where
possible. Independent of any EPA QA activities, Battelle will be responsible for ensuring that the
following audits are conducted as part of this verification test.
Cl.l Performance Evaluation Audits
When possible, the concentration of the standards used to prepare the samples fortified
with contaminants and potential interfering compounds will be confirmed by analyzing standards
prepared in ASTM Type IIDI water from two separate commercial vendors using the reference
methods noted in Tables 4 and 5. The standards from one vendor will be used during the
verification test, while the standards from the second vendor will be used exclusively to confirm
the accuracy of the displayed concentration of the first vendor. Agreement of the standards within
25% (percent difference) is required for the measurements to be considered as acceptable. Failure
to achieve this agreement will trigger a repeat of the performance evaluation (PE) comparison.

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Failure in the second comparison requires obtaining another set of standards, and repeating the PE
audit.
Given the security requirements and lack of alternate sources for some of the chemical
agents used in this verification test, PE audits will not be performed for these contaminants. PE
audits will be done for all remaining compounds when more than one source of the contaminant
or potential interfering compounds are available. PE audits will only be performed on
compounds used to prepare test samples and will not be performed on any solutions supplied as
part of an enzymatic test kit.
C1.2 Technical Systems Audits
The Battelle Quality Manager will perform a technical systems audit (TSA) at least once
during this verification test. The purpose of this audit is to ensure that the verification test is being
performed in accordance with the AMS Center QMP1, this test/QA plan, published reference
methods, and any SOPs used by Battelle. In the TSA, the Battelle Quality Manager, or designee,
may review the reference methods used, compare actual test procedures to those specified or
referenced in this plan, and review data acquisition and handling procedures. In the TSA, the
Battelle Quality Manager will observe testing in progress, observe the reference method sample
preparation and analysis (when available), inspect documentation, and review technology-specific
record books. He will also check standard certifications and may confer with other Battelle staff.
A TSA report will be prepared, including a statement of findings and the actions taken to address
any adverse findings. The EPA AMS Center Quality Manager will receive a copy of Battelle's
TSA report. At EPA's discretion, EPA QA staff may also conduct an independent on-site TSA
during the verification test. The TSA findings will be communicated to technical staff at the time
of the audit and documented in a TSA report.
C1.3 Data Quality Audits
The Battelle Quality Manager will audit at least 10% of the verification data acquired in
the verification test. The Battelle Quality Manager will trace the data from initial acquisition,
through reduction and statistical comparisons, to final reporting. All calculations performed on

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the data undergoing the audit will be checked.
CI.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 TSA 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.
C2 REPORTS TO MANAGEMENT
The Battelle Quality Manager, during the course of any assessment or audit, will identify
to the technical staff performing experimental activities any immediate corrective action that
should be taken. If serious quality problems exist, the Battelle Quality Manager will notify the
AMS Center Manager to authorize 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.
The test/QA plan and final report are reviewed by EPA AMS Center QA staff and the EPA AMS
Center program management staff. Upon final review and approval, both documents will then be
posted on the ETV website (www.epa.gov/etv).

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SECTION D
DATA VALIDATION AND USABILITY
D1 DATA REVIEW, VALIDATION, AND VERIFICATION REQUIREMENTS
The key data review, validation, and verification requirements for the verification test are
the collection of QC samples as outlined in the test/QA plan, a comparison of field data sheet
comments against final data to flag any suspect data, and a review of final data (as described in
Section BIO). The QA audits, as described in Section C, are designed to assure the quality of this
data.
D2 VALIDATION AND VERIFICATION METHODS
Data verification is conducted as part of the data review as described in Section BIO and
CI.2 of this test/QA plan. This verification will include a visual inspection of handwritten data to
ensure that all entries were properly recorded or transcribed, and that any erroneous entries were
properly noted (i.e, single line through the entry, with an error code and the initials of the recorder
and data of entry). Data manually incorporated into spreadsheets for use in calculations will be
checked against the handwritten data to ensure no transcription errors occurred. Calculations will
be spot-checked to ensure the accuracy and the appropriateness of the calculations. Calculations
performed manually will be reviewed and repeated using a handheld calculator or commercial
software (i.e., Excel). Calculations performed using standard commercial software (i.e., Excel)
will be reviewed by inspection of the equations used for the calculations and verification of
selected calculations by handheld calculator.
A number of data validation procedures will also be performed. Section C of this test/QA
plan provides a description of the validation safeguards employed for this verification test. Data
validation efforts include the completion of QC activities, and the performance of TSA and PE
audits as described in Section C.
An audit of data quality will be conducted by the Battelle Quality Manager to ensure that

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data review, verification, and validation procedures were completed, and to assure the overall
quality of the data.
D3 RECONCILIATION WITH USER REQUIREMENTS
This purpose of this verification test is to evaluate the performance of enzymatic test kits.
To meet the requirements of the user community, the data obtained in this verification test should
include thorough documentation of the performance of each enzymatic test kit during the
verification test. The data review, verification, and validation procedures described in previous
sections will assure that data meet these requirements, are accurately presented in the verification
reports generated from this test, and that data not meeting these requirements are appropriately
flagged and discussed in the verification reports. The QC acceptance criteria described in Section
B4 will be used to ensure that the reference methods provide accurate measurements of the
reference or stock solutions and that the levels of contaminants tested are accurately presented in
the verification reports.
The data from this verification test will be compiled into an ETV verification report. The
report will be submitted to EPA in Word Perfect and Adobe pdf format and subsequently posted
on the ETV website. This test/QA plan and the resulting ETV verification report(s) will be
subjected to review by the enzymatic test kit vendors, Battelle staff, EPA, and expert peer
reviewers. The reviews of this test/QA plan will assure that this verification test and the resulting
reports meet the needs of potential users of the enzymatic test kits.

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SECTION E
HEALTH AND SAFETY
El STANDARD/TEST SAMPLE PREPARATION
All handling of solid and highly concentrated aqueous solutions of contaminants and
possible interferences will be done inside of a laboratory hood with hood sash set to the lowest
height that still allows for safe manipulation of materials. The following guidelines should be
adhered to:
Personal protective equipment shall include safety glasses with side shields, a
laboratory coat, and nitrile lab gloves. Gloves shall be immediately changed if they
become contaminated.
All contaminated waste shall be handled as hazardous waste and disposed of according
to facility regulations.
E2	SAMPLE HANDLING DURING VERIFICATION TESTING
Laboratory handling of any solutions used during the verification test will be
accomplished by taking the following precautions:
All containers shall be stored and transported in double containment.
Safety goggles or glasses, nitrile gloves with long cuffs, and a chemical resistant
disposable lab coat, or other safety equipment specifically called for in facility
guidelines, shall be worn when handling all chemicals. Gloves shall be immediately
changed if they become contaminated.
E3	TESTING OF VX, GB, AND GD
Use of these contaminants in the verification of enzymatic test kits will be done following
the safety procedures required at the HMRC facility.

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SECTION F
REFERENCES
1.	Quality Management Plan for the ETV Advanced Monitoring Systems Center, Version 5.0,
U.S. EPA Environmental Technology Verification Program, Battelle, Columbus, Ohio, March
2004.
2.	"Environmental Technology Verification Program Quality Management Plan", December
2002, EPA/600/R-03/021.
3.	Army Regulation 50-6, Chemical Surety, June 2001.
4.	Battelle, Chemical Safety Information Program, SIH-PP-005, September, 2004.
5.	Battelle SOP HMRC-III-007, Standard Operating Procedure 3X and 4X Proof of Decon.
6.	U.S. Army Center for Health Promotion and Preventative Medicine, USACHPPM Technical
Guide 230, Chemical Exposure Guidelines for Deployed Military Personnel, January 2002.
7.	Gosselin et al., Clinical Toxicology of Commercial Products. 5th edition, Baltimore, MD,
1984.
8.	World Health Organization, The WHO Recommended Classification of Pesticides by Hazard
and Guidelines to Classifiction: 2004, 2005.
9.	Battelle SOP MDAS.I-009-Draft, Sample Chain of Custody, June 2004.
10.	Battelle SOP HMRC-IV-118, Standard Operating Procedure for the Determination of CA in
Wastewater.
11.	Battelle, Standard Operating Procedure for Analysis of Water Extracts for Type I Analytes
by Liquid Chromatography/Mass Spectrometry, Version 1, January 2004.
12. Battelle, Standard Operating Procedure for Extracting and Preparing Water Samples for
Analysis of Dicrotophos, Mevinphos, andDichlorovos, Version 3, March 2005.

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13.	U.S. EPA, EPA Method 200.8, Determination of Trace Elements in Waters and Wastes by
Inductively Coupled Plasma-Mass Spectrometry, Revision 5.4, May 1994.
14.	American Public Health Association, et al., Standard Methods for the Examination of Water
and Wastewater. 20th Edition, Washington, D.C., 1999.
15.	U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020, March
1983.
16. U.S. EPA, Methods for Determination of Organic Compounds in Drinking Water,
Supplement III, EPA/600/R-95-131, August 1995.

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