iBaiteiie
. . . Putting Technology To Work
Environmental Technology
Verification Program
Advanced Monitoring
Systems Center
Test/QA Plan for
Verification of
Rapid Toxicity Technologies
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Test/QA Plan
Verification of
Rapid Toxicity Technologies
June 11, 2003
Prepared by
Battelle
505 King Avenue
Columbus, OH 43201-2693
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TABLE OF CONTENTS
Page
1.0 Introduction 1
1.1 Test Objective 1
1.2 Test Description 1
1.3 Organization and Responsibility 2
1.3.1 Battelle 2
1.3.2 Vendors 6
1.3.3 EPA 7
1.3.4 Analytical Laboratories Used for Stock Solution Confirmation 7
2.0 Verification Approach 9
2.1 Scope of Testing 9
2.2 Experimental Design 12
2.3 Test Samples 13
2.3.1 QC Samples 13
2.3.2 Drinking Water Fortified with Contaminants 14
2.3.3 Drinking Water Fortified with Interfering Compounds 16
3.0 Materials and Equipment 17
3.1 Laboratory Supplies 17
3.2 Field Supplies 18
3.3 Special Facilities 18
3.4 Technology Operators 19
4.0 Procedures 20
4.1 Test Sample Preparation and Storage 20
4.2 Sample Identification 21
4.3 Sample Analysis 21
4.3.1 Drinking Water Characterization 21
4.3.2 Stock Solution Confirmatory Methodologies 22
4.3.3 Technologies Undergoing Verification 22
4.4 Schedule 23
5.0 Data Handling and Reporting 24
5.1 Data Acquisition and Review 24
5.2 Data Evaluation 25
5.2.1 Endpoints 25
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TABLE OF CONTENTS (CONTINUED)
5.2.2 Precision 26
5.2.3 False Positive/Negative Responses 26
5.2.4 Field Portability 27
5.2.5 Other Performance Factors 27
5.3 Reporting 27
6.0 Quality Assurance/Quality Control 29
6.1 QC of Confirmatory Method 29
6.2 QC of Drinking Water QC Samples 29
6.3 Audits 30
6.3.1 Technical Systems 30
6.3.2 Performance Evaluation Audit 31
6.3.3 Audit of Data Quality 31
6.4 QA/QC Reporting 31
6.5 Corrective Action 32
7.0 Health and Safety 33
7.1 Handling of Contaminants 33
7.1.1 Stock Solution/Dechlorinated Drinking Water Sample Preparation 33
7.1.2 Field Handling 33
7.1.3 VX, Soman, Ricin, and Botulinum Toxin 34
8.0 References 35
APPENDIX Summary of Samples for Rapid Toxicity Technologies 37
LIST OF TABLES
Table 1. Categories, Contaminants, and Water Treatment Interferences 10
Table 2. Drinking Water Characterization Standard Methods 21
Table 3. Contaminant and Interfering Compounds Stock Solution Confirmatory Methods ... 22
Table 4. Summary of Data Recording Process for the Verification Test 24
LIST OF FIGURES
Figure 1. Organization Chart for the Verification Test 3
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ETV Advanced Monitoring Systems Center
Test/QA Plan for Verification of
Rapid Toxicity Technologies
Version 1
June 11, 2003
APPROVAL:
Name
Company
Date
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DISTRIBUTION LIST
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
Nirit Ulitzur
CheckLight Ltd
P.O. Box 72
Qiryat-Tiv'on 36000
Israel
Juha Lappalainen
Aboatox Oy
Lemminkaisenkatu 36
FI-20520 Turku, Finland
Risto Juvonen
Hidex Oy
Mustionkatu 2
FIN-20750 Turku, Finland
Karen Riggs
Amy Dindal
Ryan James
Zachary Willenberg
Battelle
505 King Ave.
Columbus, OH 43201
Robert Ferguson
Strategic Diagnostics Inc.
Ill Pencader Drive
Newark DE 19702-3322
Frank Kaiser
Severn Trent Services
3000 Advance Lane
Colmar, PA18915
Dan Kroll
Hach Company
5600 Lindbergh Drive
Loveland, CO 80538-8998
Ken Hayes
Aqua Survey, Inc.
499 Point Breeze Rd.,
Flemington, NJ 08822
Peter Perez
International Laboratory Supply
9200 New Trails Drive
The Woodlands, TX, 77381
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1.0 INTRODUCTION
1.1 Test Objective
This test/quality assurance (QA) plan provides procedures for a verification test of rapid
analysis technologies that measure toxicity in drinking water. The verification test will be
conducted under the auspices of the U.S. Environmental Protection Agency (EPA) through the
Environmental Technology Verification (ETV) program. The purpose of ETV is to provide
objective and quality assured performance data on environmental technologies, so that users,
developers, regulators, and consultants can make informed decisions about purchasing and
applying these technologies. The objective of this verification test of rapid toxicity technologies
is to evaluate their ability to detect certain toxins that are particularly toxic to humans and their
susceptibility to interfering chemicals in a controlled experimental matrix. Multiple water
quality matrices or mixtures of contaminants and interfering compounds will not be evaluated
because such is beyond the scope of this test and would more appropriately evaluated on a
case-by-case basis.
1.2 Test Description
The verification test will be performed by Battelle, of Columbus, Ohio, 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. In performing the
verification test, Battelle will follow the procedures specified in this test/QA plan, and will
comply with the data quality requirements in the "Quality Management Plan (QMP) for the ETV
Advanced Monitoring Systems Center"1. Various contaminants will be added to drinking water
at multiple concentration levels and analyzed by the participating technologies to assess their
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ability to detect the toxicity of these contaminants in drinking water. After analysis of several
concentrations of each contaminant, the lowest concentration of each contaminant that causes
inhibition greater than that of the negative control will be reported. The precision of the results
will be evaluated by making replicate measurements on each test sample and qualitative
characteristics of each technology such as ease of use and field portability will be assessed
through observations made by the test coordinator and operators throughout the verification test.
The results from each technology will be reported individually. No direct comparison will be
made between technologies, but each technology will undergo similar testing so it is convenient
for end users to evaluate the ETV testing results.
1.3 Organization and Responsibility
The verification test will be performed by Battelle with the participation of the interested
vendors who will be having their technologies verified. The testing will occur at Battelle's
Columbus and West Jefferson, Ohio laboratories and at a field location in the Columbus area.
The organizational chart shown in Figure 1 shows the individuals from Battelle, the vendor
companies, and the EPA who will have responsibilities in the verification test.
1.3.1 Battelle
Dr. Ryan James is the AMS Center Verification Test Coordinator. In this role, Dr. James
will have overall responsibility for ensuring that the technical, schedule, and cost goals
established for the verification test are met. More specifically, he 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 QMP are followed.
• Prepare the draft test/QA plan, verification reports, and verification statements.
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Zachary Willenberg
Quality Manager
Gary Carlin
Safety Officer
Battelle
Management
Karen Riggs
AMS Center
Manager
Amy Dindal
Verification
Testing Leader
Ryan James
Verification
Test Coordinator
Battelle
Technical Staff
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Robert Fuerst
EPA AMS Center
Manager
Elizabeth Betz
EPA QA
Manager
Vendor
Representatives
Figure 1. Organization Chart for the Verification Test
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• Revise the draft test/QA plan, verification reports, and verification statements in response to
reviewers' comments.
• Coordinate distribution of the final test/QA plan, verification reports, and verification
statements.
• 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.
• Establish a budget for the verification test and monitor staff effort to ensure the budget is not
exceeded.
• Ensure that confidentiality of vendor information is maintained.
Ms. Amy Dindal is a Verification Testing Leader for the AMS Center. As such, Ms.
Dindal will provide technical guidance and oversee the various stages of verification testing.
She will:
• Support Dr. James in preparing the test/QA plan and organizing the testing.
• Assist Dr. James in anticipating and resolving potential technical problems with the
verification test.
• Review the draft test/QA plan.
• Review the draft verification reports and statements.
Ms. Karen Riggs is Battelle's manager for the AMS Center. As such, Ms. Riggs will:
• Review the draft test/QA plan.
• Review the draft verification reports and verification statements.
• Ensure that necessary Battelle resources, including staff and facilities, are committed to the
verification test.
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• Ensure that vendor confidentiality is maintained.
• Support Dr. James in responding to any issues raised in assessment reports and audits.
• Maintain communication with EPA's technical 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 technologies during the
verification test and associated experimental activities. The responsibilities of these technical
staff include:
• Assist in the preparation of samples.
• Analyze samples for the verification test as described in this test/QA plan.
• Make qualitative observations about the operation of the technologies.
Mr. Zachary Willenberg is Battelle's Quality Manager for the AMS Center. As such Mr.
Willenberg will:
• Review the draft test/QA plan.
Conduct quality review of the documentation exhibiting the capability of an outside
laboratory to perform solution confirmation analyses.
Conduct a technical systems audit during the verification test.
• Audit at least 10% of the verification data.
• Prepare and distribute an assessment report for each audit.
• Verify implementation of any necessary corrective action.
• Issue a stop work order if self audits indicate that data quality is being compromised; notify
Battelle's AMS Center Manager if stop work order is issued.
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• Provide a summary of the quality assurance/quality control (QA/QC) activities and results
for the verification reports.
• Review the draft verification reports and statements.
• Have an overall responsibility for ensuring that the test/QA plan is followed.
Mr. Gary Carlin is Battelle's Environmental Health and Safety representative for the
AMS Center. As such Mr. Carlin will:
• Review the safety issues related to handling the contaminants and provide input into the
test/QA plan.
• Advise staff working with the contaminants on personal protective equipment and training
needs.
1.3.2 Vendors
Vendor representatives will:
• Review the draft test/QA plan.
• Approve the test/QA plan.
• Provide one complete commercially-available rapid toxicity technology and associated
equipment/materials required for operation throughout the duration of the verification test.
• Provide all needed consumables for number of test samples included in the verification test.
• As desired, instruct Battelle personnel on how to operate the technology prior to testing.
• Review their respective draft verification report and statement.
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1.3.3 EPA
EPA's responsibilities in the AMS Center are based on the requirements stated in the
"Environmental Technology Verification Program Quality Management Plan"2. The roles of the
specific EPA staff are as follows:
Ms. Elizabeth Betz is EPA's Quality Assurance Manager. For the verification test, Ms.
Betzwill:
• Review the draft test/QA plan.
• Direct the performance, at the EPA's discretion, of external technical systems audit(s) during
the verification test.
• Notify the Battelle AMS Center Manager to facilitate a stop work order if the external audit
indicates that data quality is being compromised.
• Prepare and distribute an assessment report summarizing results of the external audit.
• Review draft verification reports and statements.
Mr. Robert Fuerst is EPA's manager for the AMS Center. As such, Mr. Fuerst will:
• Review the draft test/QA plan.
• Notify the Battelle AMS Center Manager to facilitate a stop work order if an external audit
indicates that data quality is being compromised.
• Review the draft verification statements.
• Review the final verification reports.
Coordinate submission of the verification reports and statements for final EPA approvals.
1.3.4 Analytical Laboratories Used for Stock Solution Confirmation
When methods are available, the contaminant concentration of the stock solutions will be
confirmed. When the analyses are within Battelle's capabilities, Battelle will perform the
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analyses, but if it is more cost effective, Battelle may collaborate with or establish a subcontract
with a commercial laboratory to perform the analyses.
In order to be selected to perform the confirmatory analyses during the verification test, a
commercial laboratory will need to provide Battelle documentation that demonstrates its
competence to perform the needed analysis, such documentation may include copies of: their
method/standard operating procedure, quality assurance manual, state government
certifications/approvals for analysis of the appropriate contaminant, and/or staff training records,
where available. If the prospective laboratory does not demonstrate their capability adequately,
another laboratory will be selected and their competence verified in a similar manner.
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2.0 VERIFICATION APPROACH
2.1 Scope of Testing
The verification test of rapid toxicity technologies will focus on a broad range of
contaminants that have been prioritized by the ETV stakeholder process. These rapid toxicity
technologies do not provide identification or concentration of specific contaminants, but serve as
a rapid screening tool to determine if the water being tested is toxic. As part of this verification
test, the rapid toxicity technologies will be subjected to various concentrations of contaminants
representing several categories of chemicals such as industrial chemicals, pesticides,
rodenticides, pharmaceuticals, nerve agents, and biological toxins. Each contaminant will be
added to separate drinking water samples and analyzed by the rapid toxicity technologies. In
addition to determining whether or not these technologies can detect the toxicity caused by each
contaminant, the response of these technologies to interfering compounds likely to be present in
clean drinking water, such as chemicals used for water treatment and byproducts of water
treatment processes, will be evaluated. Table 1 shows the contaminants and the interfering
compounds that will be evaluated during this verification test.
This test/QA plan specifically addresses verification testing of technologies that rapidly
detect the toxicity of water samples. While there are many compounds that are toxic to the test
organisms that are not toxic to humans, this verification test will focus on contaminants that are
particularly toxic to humans. The technologies function by adding the water sample to a bacteria
(Vibrio fischeri\ enzyme (luciferase), or small crustacean (Daphnia magna) that either directly,
or in combination with reagents, produce a background level of light production or rate of
dissolved oxygen uptake (DOUR) in the absence of toxic contaminants. If toxic contaminants
are present in the water, depending on the vendor, their toxicity is indicated by a change in color
or intensity of the light production or by a decrease in the DOUR in proportion to the
concentration level of the contaminant. This indication of toxicity will be generically referred to
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as "inhibition" through the remainder of this test/QA plan. These technologies report the
inhibition of each drinking water sample with respect to the inhibition of either ASTM Type II
deionized (DI) water or non-toxic drinking water. The results are reported visually or via a
digital display or electronic output signal. The technologies that are designed
Table 1. Categories, Contaminants, and Water Treatment Interferences
Category
Industrial chemical
Carbamate pesticide
Organophosphate
pesticide
Rodenticide
Pharmaceutical
Nerve agents
Biological toxins
Contaminant
cyanide
aldicarb
dicrotophos
thallium sulfate
colchicine
VX, soman
ricin, botulinum toxin
Interfering Compounds
manganese, iron, aluminum,
copper sulfate, chloramination
byproducts, chlorination
byproducts, zinc sulfate
for use in a field location will be tested at a non-laboratory venue, and all of the technologies
will be tested in a laboratory.
The verification of the rapid toxicity technologies will be done through the analysis of
drinking water samples fortified with various concentrations of individual contaminants. ETV
verifications usually include a comparison of the results generated by the technologies being
verified with the results of analysis of the same samples using a standard reference method that
measures the same endpoint, usually concentration. In the case of this verification test, the most
common standard method for toxicity is the Whole Effluent Toxicity3 (WET) method. Most of
the rapid toxicity technologies employ organisms for the detection mechanism that are different
from the organisms suggested for used in the WET method. The sensitivity of different
organisms to contaminants will be different among different species. Therefore, this method
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would not provide an appropriate benchmark for direct comparison with the results produced by
all of the rapid toxicity technologies because of the lack of sameness of organism. It is well
documented that the contaminants used during this verification test are toxic and the objective of
the test is to determine each rapid toxicity technology's ability to detect this toxicity if the
contaminant is present, not to compare their response with the WET method. In lieu of a
traditional reference measurement of toxicity, the concentration of each contaminant or
interfering compound fortified into the drinking water sample will be confirmed independently
by standard confirmatory methods, when available. When a standard method is not available,
QA oversight (balance calibration, audits, and purity tracking of the standards) of the solution
preparation will ensure the accuracy of the sample concentration.
The rapid toxicity technologies provide a measure of toxicity, and will be evaluated by
reporting:
endpoint (% inhibition, EC50, EC30, toxicity threshold) for all concentration levels,
contaminants and potential interfering compounds
• precision
• false negative rate (frequency of inhibition similar to the negative control reported when a
contaminant is present at toxic concentrations)
• false positive rate (frequency of detectable inhibition reported in unspiked drinking water
samples)
• field portability
ease of use
• throughput.
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2.2 Experimental Design
The verification test will involve challenging the rapid toxicity technologies with
drinking water samples fortified separately with the contaminants and interfering compounds
listed in Table 1. Each technology will analyze samples containing the contaminants at
concentration levels that would be lethal if ingested. Subsequent dilutions (dilution factors as
per each vendor's protocol, otherwise tenfold) will be prepared and analyzed until there is no
longer inhibition as measured by each technology. From these data, the lowest concentration at
which the toxicity can be detected, or toxicity threshold, will be estimated for each technology
with respect to each contaminant. A common endpoint such as the effective concentration
causing 50% inhibition (EC50) will be reported for each technology along with data reflecting the
rate of false positive and negative results and the precision of the rapid toxicity technologies.
Inhibition results (endpoints) specific to each technology from four replicates of each
contaminant at each concentration level will be evaluated in order to evaluate the precision of the
technologies.
The response of the rapid toxicity technologies to compounds used during the water
treatment process (see Table 1) will be evaluated as potential interfering compounds by
analyzing separate drinking water samples fortified with the EPA's National Secondary Drinking
Water Regulations (NSDWR)4 level of each compound. For analysis of byproducts of the
chlorination process, the unspiked drinking water sample will be analyzed because it will be
from a water utility that uses chlorination as it disinfection process. For the analysis of
byproducts of the chloramination process, a separate water sample will be obtained from a water
system that uses chloramination as its disinfection process.
Sample throughput will be measured based on the number of samples analyzed per day.
Performance parameters, such as ease of use and reliability, will be based on documented
observations of the operators and test coordinator. Each technology will be used in a field
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environment, as well as in a laboratory setting, to assess the impact of field conditions on
performance.
2.3 Test Samples
Test samples to be used in this verification test will include drinking water (DW) and
quality control (QC) samples. Tables estimating the number of samples to be analyzed are
provided in the Appendix. The drinking water samples will be prepared from a single drinking
water sample collected from a tap in a system that uses chlorination as the disinfectant
procedure. The water will be dechlorinated (with sodium thiosulfate or as per vendor protocol)
and fortified with various concentrations of contaminants and interferences. Individual solutions
containing each contaminant and potential interfering compound separately will be prepared.
Subsequent dilutions of the contaminant samples with dechlorinated drinking water or ASTM
Type IIDI water (as per vendor protocol) will be analyzed by the rapid toxicity technologies
until there is no longer detectable inhibition. Mixtures of contaminants and interfering
compounds will not be analyzed. The QC samples will include method blank samples and
positive and negative control samples.
2.3.1 QC Samples
The QC samples will include method blank samples, which will consist of ASTM Type II
DI water; positive control samples, which will consist of ASTM Type II DI water fortified with a
contaminant and concentration selected by each vendor; and negative control samples, which
will consist of the unspiked dechlorinated drinking water sample. The method blank samples
will be used to help ensure that no sources of contamination are introduced in the sample
handling and analysis procedures. The positive control samples will provide an indication to the
operator whether or not the rapid toxicity technology is functioning properly. The vendor will
provide the approximate inhibition endpoint that should result upon analysis of the positive
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control by their technology. While there will not be required performance limits placed on the
result of the positive control sample, if the result is not in the range of anticipated results
specified by the vendor, it will indicate to the operator that the technology may have been
operated incorrectly. The negative control sample will be used to set a background toxicity of
the water sample. Any change in inhibition from the negative control sample can be considered
to be due to the presence of a contaminant. At least one method blank and positive control
sample will be analyzed for every 20 samples (more often if required by vendor's protocol)
analyzed by each rapid toxicity technology. Negative control samples will be analyzed as often
as required to accommodate the vendor's analytical protocol.
2.3.2 Drinking Water Fortified with Contaminants
Approximately 50-L of a single drinking water sample, drinking water from a municipal
supply that uses chlorination as its disinfectant procedure, will be collected in a high density
polyethylene (HDPE) container as part of this verification test. The sample container will be
pre-cleaned by the manufacturer and certified to be contaminant free. After sample collection,
aliquots of the water sample will be analyzed for several water quality parameters such as: the
concentration of trihalomethanes, haloacetic acids, total organic halides, copper, aluminum, iron,
manganese, and zinc; turbidity; dissolved organic carbon content; pH; alkalinity; specific
conductivity; and hardness. Prior to analysis, these aliquots will have been preserved as per the
standard methods for these analyses. The remaining drinking water sample will be dechlorinated
with approximately 1 mg of sodium thiosulfate pentahydrate for every liter of drinking water
remaining. A scoop that holds approximately 1 mg will be used to measure the sodium
thiosulfate pentahydrate into the water sample. It is acceptable for the vendors to use different
dechlorination procedures as part of their protocol. All subsequent test samples will be prepared
from this dechlorinated drinking water (DW) and stored in glass containers to avoid the leaching
of chlorine from HDPE plastics.
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A stock solution of each contaminant at the lethal dose concentration level will be
prepared in the DW. Table A-l in the Appendix lists the concentration level of the lethal dose
sample of each contaminant and the number of samples to be analyzed. Those stock solutions
may be diluted by factors of 10, 100, 1,000, and 10,000 to generate a dilution series for each
technology to analyzed All the technologies will analyze at least the lethal dose concentration
and the first two dilutions, but the technologies will only continue on and analyze the rest of the
dilutions as long as they are detecting inhibition with respect to the negative control sample. For
example, if the 100-fold dilution sample produced no detectable inhibition, the technology would
not be required to analyze the more dilute samples. However, if the 100-fold dilution sample
produced detectable inhibition, the technology would be required to analyze additional dilutions
until the inhibition is similar to the negative control sample, even if samples with dilution factors
greater than 10,000 need to be prepared. If a technology detects inhibition at the lethal dose
level, but not at the 10-fold dilution factor, additional dilutions should be performed in order to
obtain detectable results at a minimum of two concentration levels. Each dilution set will be
analyzed four times by each technology. The endpoints (% inhibition, EC50, etc.) reported by
each technology will be recorded for each dilution sample or dilution series, whichever is
appropriate for the specific endpoint. Each of the participating technologies will be tested as
described above in Battelle laboratories.
Testing the operation of the rapid toxicity technologies in a field setting is a key
component of the verification test. Evaluating the performance of each field portable technology
while being used outside the laboratory without the availability of miscellaneous laboratory
supplies is important to the buyers and users of these technologies. Technologies will only be
evaluated in a field setting if the vendor states that the technology has that capability. At a non-
laboratory field location, each field portable technology will analyze a single water sample (four
aThe scenario of multiple ten-fold dilutions is suggested for each vendor's technology to alleviate the practical
difficulties of preparing different test solutions for each vendor. However, if the vendor's protocol includes dilutions (not
necessarily ten-fold) to non-detectable levels, using their protocol would be acceptable as long as the appropriate endpoint data
are obtained. Data forms will be provided to the operators to aid the documentation of the dilutions.
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replicates) fortified with one of the contaminants (from Table 1) known only to the Verification
Test Coordinator. The technologies will report its toxicity as prescribed by each vendor's
protocol. The operators will follow the vendor's protocol exactly, as no guidance on
concentration level or dilutions will be provided. All data obtained and dilutions performed
while performing these analyses will be documented and the endpoints reported by each
technology will be compared with those obtained in the laboratory setting.
2.3.3 Drinking Water Fortified with Interfering Compounds
Individual aliquots of the drinking water sample described above (DW) will be fortified
with one half the NSDWR guidance level of each potential interfering compound. Table A-2 in
the appendix lists the interfering compounds along with the concentration at which they will be
tested. Four replicates of each of these samples will be analyzed.
For the byproducts of the chlorination process, the unspiked drinking water sample (same
as the negative control) will be analyzed in replicate since the water sample will be from a
system disinfected with the chlorination process. For the byproducts of the chloramination
process, a separate drinking water sample will be collected from a water utility that uses
chloramination as the disinfecting process. This water sample will be characterized by
measuring trihalomethanes, haloacetic acids, total organic halides, turbidity, dissolved organic
carbon content, pH, alkalinity, specific conductivity, and hardness. Upon receipt, this sample
will be dechlorinated as per vendor protocols to quench the chloramination process and, as for
the other possible interfering compounds and chlorination byproducts, analyzed in replicates of
four.
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3.0 MATERIALS AND EQUIPMENT
In general, this verification test relies on the materials and equipment provided by the
vendors. Battelle will provide the following equipment and materials.
3.1 Laboratory Supplies
The following supplies will be needed for the preparation of the DW and QC samples:
• ASTM Type IIDI water
• various laboratory supplies necessary for accurate preparation of the test samples and
subsequent dilutions (ie. volumetric pipets, pipet bulbs, Eppendorf micro pipettes/pipette
tips, volumetric flasks, disposable pipets, etc.)
• reference standards with a known level of purity for target analytes (NIST traceable or
equivalent)
• glass sample containers
sodium thiosulfate pentahydrate
• sodium hypochlorite
• n, n-diethly-p-phenylenediamine (DPD) tablet
• personal protective equipment.
3.2 Field Supplies
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For the analysis of samples in the field setting, Battelle will provide only the water
sample for analysis, the DW sample to be used by the operators for any necessary dilutions, and
the QC samples. The operators will depend on only supplies provided by the vendor to analyze
the samples.
3.3 Special Facilities
Four of the contaminants to be evaluated in this verification test (VX, soman, ricin, and
botulinum toxin) can only be handled in laboratories that are specially designed and certified for
the use of chemical and biological agents. Battelle's Medical Research and Evaluation Facility
(MREF), which is a Department of Defense laboratory-scale facility conducting research with
chemical and biological agents will be used for verification testing of the specified contaminants.
The MREF is licensed to ship, receive, and handle select agents, as defined by the Centers for
Disease Control and Prevention. The facility maintains state-of-the-art equipment and
professional and technical staffing expertise to safely conduct testing and evaluation of
hazardous chemical and biological materials.
The MREF and its personnel have the demonstrated capability for storing and safely
handling VX, soman, ricin, and botulinum toxin. Battelle's agent stocks will be analyzed prior to
testing to verify the purity of the agent used to make the test samples. Only chemical agents
(CA) with purity greater than 85 percent will be used in this program. Handling of CA at the
MREF are detailed in the following standard operating procedures (SOP): MREF SOP 1-002
Storage, Dilution, and Transfer of GA, GB, GD, GF, TGD, VX, HD, HL, HN and L when CA
Concentration/Quantity is Greater than Research Dilute Solution (RDS), MREF SOP 1-003
Receipt, Transfer, Storage, and Use of Research Dilute Solution (RDS), and MREF SOP 1-003
Disposal of Chemical Agent. These SOPs can be reviewed at the MREF facility, but copies
cannot be made for external review. Biological agent use will be according to the CDC Select
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Agents Program (32 CFR 626 and 627)5'6 administered through the Biological Defense Research
Program and the Battelle MREF Facility Safety Plan.
3.4 Technology Operators
Given the agent facility restrictions, vendors will not be able to operate their technologies
during this verification test. Each rapid toxicity technology will be tested independently while
being operated by a Battelle staff member. All operators will have a bachelor's level degree in
the sciences or equivalent work experience. The vendor will provide training to the operators by
means of a visit to Battelle or a conference call prior to the start of the verification test and then
will be asked to sign a consent form stating the names of the Battelle staff they have trained.
Each operator will manipulate the water samples and reagents to generate solutions that can be
analyzed by the rapid toxicity technologies. More than one operator may be used by Battelle,
but operators will be restricted to only operating technologies on which they have been trained.
Battelle staff from the MREF will operate the technologies while analyzing samples fortified
with VX, soman, ricin, and botulinum toxin.
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4.0 PROCEDURES
4.1 Test Sample Preparation and Storage
The drinking water sample will be collected as described in Section 2.3.2 and because
free chlorine will degrade many of the contaminants and interferences during storage, the sample
will be immediately dechlorinated with sodium thiosulfate (or other dechlorination reagent as per
vendor's protocol). The dechlorination of the drinking water will be qualitatively confirmed by
adding a DPD tablet to an aliquot of the DW. If the water does not turn pink, the dechlorination
process will be determined to be successful, if it does turn pink an additional 0.5 mg of sodium
thiosulfate pentahydrate (or other dechlorinating reagent as per vendor's protocol) will be added
to the sample for every liter of water and the dechlorination confirmation procedure will be
repeated. Once dechlorination is confirmed, all the DW samples and the negative control QC
samples will be made from this sample, while the method blank and positive control QC samples
will be prepared from ASTM Type IIDI water. The positive control samples will be diluted to
appropriate concentrations using ASTM Type II DI water in Class A volumetric glassware. All
QC samples will be prepared prior to the start of the testing and stored at room temperature for a
maximum of 60 days. The DW samples will be prepared within 7 days of testing and stored in
the dark at room temperature. Once dechlorinated and fortified with the contaminants, the DW
samples will only be preserved chemically if absolutely necessary, as this verification test is
designed to evaluate the ability of rapid toxicity technologies to measure toxicity in drinking
water. Therefore, minimal adaptation of the water sample from between the time it comes out of
the tap to the analysis time is preferred. Sample treatment as required by the vendors (such as
pH adjustment) may need to be performed in order for the technologies to operate properly.
The concentration of the contaminant and interfering compound stock solutions will be
verified with standard confirmatory methods, when available. Four aliquots of each stock
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solution will be analyzed. Aliquots to be analyzed by confirmatory methods will be preserved
as prescribed by the standard method to be used.
4.2 Sample Identification
Aliquots to be analyzed by each technology will be drawn from the QC or drinking water
samples and placed in uniquely identified sample containers. The sample containers will be
identified by a unique identification (ID) number. A master log of the samples and sample ID
numbers for each technology will be kept by Battelle. The ID number, date, person collecting,
sample location, and time of collection will be recorded on a chain-of-custody form for all field
samples.
4.3 Sample Analysis
4.3.1 Drinking Water Characterization
Table 2 lists the methods to be used to characterize the drinking water sample used as the
water source throughout this verification test.
Table 2. Drinking Water Characterization Standard Methods
Chemical
Turbidity
Organic Carbon
Specific
Conductivity
Alkalinity
pH
Hardness
Method
EPA 180. 17
SM53108
SM25109
SM 23209
EPA 150. 19
EPA 130.29
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4.3.2 Stock Solution Confirmatory Methodologies
Table 3 lists the methods to be used to verify the concentration of the stock solutions of
the contaminants and metal interfering compounds analyzed during this verification test.
Table 3. Contaminant and Interfering Compounds Stock Solution Confirmatory Methods
Chemical
Cyanide
Aldicarb
Dicrotophos
Thallium, Manganesea, Iron3,
Aluminum3, Copper3, and Zinc3
Total Organic Halides
Trihalomethanes
Haloacetic Acids
Colchicine
VX, Soman, Botulinum Toxin, Ricin
Method
EPA 335. 110
EPA 531. 111
EPASW-846(8141A)12
EPA 200. 813
SM 5320B12
EPA 524.214
EPA 552.215
No standard method available. QA audits and
balance calibration will assure accurate solutions.
MREF facility will perform purity analyses on
chemical and biological agent materials using
internal standard operating procedures
3Potential interfering compounds
4.3.3 Technologies Undergoing Verification
Each vendor will be required to provide one complete unit of their rapid toxicity
technology and associated equipment and consumables required for the duration of the
verification test. Each of the technologies being verified will be used to analyze the full set of
samples. As has been described, the sample set will include replicates of each of the QC and
drinking water samples. The analyses will be performed according to the vendor's recommended
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procedures as described in the user's instructions or manual, or during training provided to the
Battelle staff. Similarly, calibration and maintenance of the technologies will be performed as
specified by the vendor.
Results from the technologies being verified will be recorded manually by the operator
on appropriate data sheets. In addition to the analytical results, the data sheets will include
records of the time required for sample analysis and operator observations concerning the use of
the technologies (i.e., frequency of calibration, ease of use, maintenance, etc.).
4.4 Schedule
The verification test described here will take place throughout July and August 2003 at
Battelle's laboratories in and near Columbus, Ohio and at a nearby field location. It will be
necessary for participating vendors to provide their technologies to Battelle by a specified date
so testing staff may become familiar with operating the units before testing begins. Vendor staff
should provide training in operating the technologies either in person or by teleconference.
Rapid toxicity technologies and associated equipment (but not consumables) will be returned to
the vendors at the completion of report writing. Technologies will be decontaminated by
washing with an aqueous solution of bleach before being returned to vendor.
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5.0 DATA HANDLING AND REPORTING
5.1 Data Acquisition and Review
A variety of data will be acquired and recorded electronically or manually by Battelle
staff in this verification test. Operation, maintenance, and results from the rapid toxicity
technologies and sampling procedures, will generally be documented on data sheets or in
laboratory record books. Results from the confirmatory method instruments used will be
compiled in electronic format. Table 4 summarizes the types of data to be recorded.
Table 4. Summary of Data Recording Process for the Verification Test
Data to be Recorded
Dates, times of test
events
Sample preparation
(dates, concentrations,
preservation)
Test parameters
(contaminant
concentrations,
location, etc.)
Stock solution
confirmatory method
sample analysis, chain
of custody, and results
Responsible
Party
Battelle
Battelle
Battelle
Battelle or
contracted
laboratory
Where Recorded
Laboratory record
books
Laboratory record
books
Laboratory record
books
Laboratory record
books, data sheets,
or data acquisition
system, as
appropriate
How Often Recorded
Start/end of test, and at
each change of a test
parameter.
When each solution is
prepared
When set or changed,
or as needed to
document stability.
Throughout sample
handling and analysis
process
Disposition of Data00
Used to organize/check test
results; manually
incorporated in data
spreadsheets as necessary.
Used to confirm the
concentration and integrity
of the samples analyzed
Used to organize/check test
results, manually
incorporated in data
spreadsheets as necessary.
Transferred to
spreadsheets/agreed upon
report
(a) All activities subsequent to data recording are carried out by Battelle.
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Records received by or generated by any Battelle staff during the verification test will be
reviewed by a more senior Battelle staff member within two weeks of data collection, before
these records are used to calculate, evaluate, or report verification results. These records may
include electronic records; laboratory record books; field sampling records; or equipment
calibration records. 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 received, generated, or will be storing the
record. In addition, data calculations performed by Battelle will be spot-checked by a more
senior Battelle technical staff member to ensure that calculations are performed correctly.
Calculations to be checked include confirmation analysis results and statistical calculations
described in this test/QA plan.
The data obtained from this verification test will be compiled and reported independently
for each rapid toxicity technology being verified. No intercomparison of the results from one
vendor's technology to another's will be made.
5.2 Data Evaluation
5.2.1 Endpoints
Each of the technologies produce their own unique endpoints that are all derived from the
inhibition data gathered when analyzing various concentrations of contaminants in drinking
water samples. Some of these endpoints include EC50, effective concentration causing 30%
inhibition (EC30), and toxicity thresholds (estimate of lowest detectable concentration). For each
technology, these data will be documented and presented with respect to each contaminant and
concentration level. Where possible, the data from each technology will be converted to
common endpoints to ease comparison among the verification reports.
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5.2.2 Precision
The standard deviation (S) of the results for the replicate samples will be calculated and
used as a measure of technology precision at each concentration.
n 1/2
s =
-X2
(3)
where n is the number of replicate samples, Ik is the inhibition measured for the A* sample, and
/ is the average inhibition of the replicate samples. The rapid toxicity technology precision at
each concentration level will be reported in terms of the relative standard deviation (RSD), e.g.,
S
RSD =
x 100 (4)
Also, the relative standard deviation (RSD) of the results for endpoints such as EC50 determined
from analysis of replicate dilution series will be calculated and used as a measure of rapid
toxicity technology precision with respect to each contaminant.
5.2.3 False Positive/Negative Responses
Results will be considered false positive only when an unspiked drinking water sample,
administered blindly to the operators, produces inhibition greater than that of the negative
control.
Results will be considered false negative only when a rapid toxicity technology is
exposed to a lethal concentration of some contaminant in the drinking water sample and the
rapid toxicity technology does not indicate inhibition greater than the negative control. The rate
of false positives/false negatives will be expressed as a percentage of total samples analyzed for
each contaminant.
5.2.4 Field Portability
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The results obtained from the measurements made on drinking water samples in the
laboratory and field setting will be compiled independently for each rapid toxicity technology
and compared to assess the accuracy of the measurements under the different analysis
conditions. Means and standard deviations of the endpoints generated in both locations will be
used to make the comparison. Also, qualitative observations of each technology's performance
in a non-laboratory setting will be made by the Verification Test Coordinator and operators.
Factors such as the ease of transport and set-up, demand for electrical power, and space
requirement will be documented and discussed in the report.
5.2.5 Other Performance Factors
Ease of use will be qualitatively assessed throughout the verification test through
observations of the operators and Verification Test Coordinator. Clarity of the vendors
instruction manual, user-friendliness of any needed software, overall convenience of the
technologies and accessories/consumables, and any unique qualitative observations are among
the observations that will be documented and discussed in the verification report.
Sample throughput will be evaluated quantitatively based on the number of samples that
can be analyzed per hour.
5.3 Reporting
The data obtained in the verification test will be compiled separately for each vendor's
technology, and the data evaluations will be applied to each technology's data set without
reference to any other. At no time will data from different vendor's rapid toxicity technology be
intercompared or ranked. Following completion of the data evaluations, a draft verification
report will be prepared for each vendor's rapid toxicity technology, stating the verification test
procedures and documenting the performance observed. The draft verification reports will each
be submitted to the respective vendors for review and comment. Battelle will consider the
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comments provided by each vendor when revising the verification reports, but does not
guarantee that revisions made to the final verification reports will reflect those comments. A
written response to all substantive vendor comments not included in the body of the report will
be prepared and submitted to the vendor. After vendor review, the revised reports will be
submitted to EPA and AMS Center stakeholders for peer review. The reports will then be
revised again to address the peer review comments and submitted for final EPA approval.
A verification statement for each technology will be prepared in parallel with the
verification reports. The verification statement is a two- to three-page summary of the
technology, the test procedures, and the test results. Upon approval by EPA, each verification
statement will be signed by a senior manager of Battelle and by an EPA laboratory director.
Battelle will reserve the right to post the final verification reports and statements on the ETV
website (http://www.epa.gov/etv). Original signed verification statements will be provided to the
respective vendors for use in marketing their products.
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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, and data recording and 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 QC of Stock Solution Confirmatory Method
When confirmation analyses of the stock solutions of both contaminant and potential
interfering compounds are performed, both method blank and negative control samples will also
be performed. 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
verification test. The negative control sample will indicate the background level of contaminant
or potential interfering compound in the DW sample. If this concentration is more than 15% of
the fortified concentration of contaminant or potential interfering compound, a correction for the
recovery of the confirmation analyses will be performed to account for the amount present in the
background.
6.2 QC of Drinking Water QC Samples
The method blank samples analyzed with the DW samples are expected to produce very
little or no inhibition. If the inhibition is different from what the vendor suggests for a method
blank sample, the analysis will be repeated once. For this re-analysis, a new method blank
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sample will be prepared and clean vials/containers and fresh reagents will be used. If similar
results persist, the vendor will be notified, but the verification test will proceed.
While there will not be required performance limits placed on the result of the positive
control sample, if the result is not in the range of anticipated results specified by the vendor, it
will indicate to the operator that the technology may have been operated incorrectly and the
analysis of that sample will be repeated once using clean vials/containers and fresh reagents. If
the unexpected result persists, the vendor will be notified, but the verification test will proceed.
6.3 Audits
6.3.1 Technical Systems Audit
The Battelle Quality Manager 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 test/QA plan and the AMS Center QMP1, and that all
procedures described in this test/QA plan are being followed. This audit will review the
contaminant standard and stock solution confirmatory methods used, compare actual test
procedures to those specified in this test/QA plan, and review data acquisition and handling
procedures. An independent technical systems audit may also be performed by EPA Quality
Management staff during the verification test, at EPA's discretion.
Before using an outside laboratory to perform stock solution confirmation analyses, the
Battelle Quality Manager will conduct an audit of the laboratory's quality documents. If there are
areas of concern with the quality documents, the commercial laboratory will be notified, and if they
are willing to adapt their procedures, the laboratory will still be used. If not, another laboratory will
be selected.
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6.3.2 Performance Evaluation (PE) Audits
The concentration of the standards used to prepare the DW samples fortified with
contaminants and potential interfering compounds will be confirmed by analyzing standards
prepared in Type IIDI Water from two separate commercial vendors using the confirmatory
methods. 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% is required
for the measurements to be considered as acceptable. Failure to achieve this agreement will
trigger a repeat of the performance evaluation comparison. Failure in the second comparison
requires obtaining another set of standards, and repeating the performance audit.
Given the security requirements and lack of confirmatory methodology for some of the
contaminants in this verification test, PE audits will not be performed for all of the contaminants.
They will be done when more than one source of the contaminant or potential interfering
compounds are available and there are available confirmatory methods.
6.3.3 Audit of Data Quality
At least 10% percent of the data generated during the verification test will be audited
during the verification test. Battelle's Quality Manager will trace the data from the 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.
6.4 QA/QC Reporting
Each assessment and audit will be documented in accordance with Section 3.3.4 of the
QMP for the AMS Center. The results of the technical systems audit will be sent to the EPA.
Assessment reports will include the following:
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• 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
The Battelle or EPA Quality Managers 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 is
authorized to stop work. 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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7.0 HEALTH AND SAFETY
7.1 Handling of Contaminants
7.1.1 Stock Solution / DW 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. (The same gloves can be used for sodium hydroxide)
• All contaminated waste shall be handled as hazardous waste and sent out through Battelle
Waste Operations.
7.1.2 Field Handling
Field handling of the contaminant and interfering compound solutions will be accomplished
by taking the following precautions:
• All containers shall be stored and transported in double containment.
• Safety goggles, nitrile gloves with long cuffs, and a chemical resistant disposable lab coat
shall be worn when handling chemicals. Gloves shall be immediately changed if they
become contaminated.
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7.1.3 VX, Soman, Ricin, and Botulinum Toxin
These contaminants will be handled following the safety procedures required at the MREF
facility as listed in Section 3.3.
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8.0 REFERENCES
1. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center, U.S.
EPA Environmental Technology Verification Program, prepared by Battelle, Columbus,
Ohio, Version 4.0 December 2002
2. "Environmental Technology Verification Program Quality Management Plan" (QMP),
December 2002, EPA/600/R-03/021
3. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater
and Marine Organisms, U.S. EPA Document, 5th Ed., EPA-821-R-02-012 October 2002.
4. Secondary Drinking Water Regulations: Guidance for Nuisance Chemicals, U.S. EPA
Document, EPA-810-K-92-001, July 1992.
5. Code of Federal Regulations, Title 32, Chapter 5, Part 626, Biological Defense Safety
Program.
6. Code of Federal Regulations, Title 32, Chapter 5, Part 626, Biological Defense Safety
Program, Technical Safety Requirements.
7. EPA Method 180.1, "Turbidity (Nephelometric)", Methods for the Determination of
Inorganic Substances in Environmental Samples EPA-600-R-93-100 1993.
8. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, 1997. Washington, DC.
9. EPA Methods for Chemical Analysis of Water and Wastes EPA-600-4-79-020.
10. EPA Method 335.1, Cyanides Amenable to Chlorination, 1974, in "Methods for Chemical
Analysis of Water and Wastes, " EPA-600-4-79-020, March 1983.
11. EPA Method 531.1, Measurement of n-Methylcarbamoyloximes and n-methylcarbamates in
Water by Direct Aqueous Injection HPLC with Post Column Derivatization, Revision 3.1,
1995.
12. SW846 Method 8141A, "Organophosphorous Compounds by Gas
Chromatography:Capillary Column Technique", Revision 1, September 1994.
13. EPA Method 200.8, "Determination of Trace Elements in Waters and Wastes by
Inductively-Coupled Plasma Mass Spectrometry", Revision 5.4, 1994.
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14. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
Spectrometry", Methods for the Determination of Organic Compounds in Drinking
Water-Supplement III EPA/600/R-95-131.
15. EPA Method 552.2, "Haloacetic Acids and Dalapon by Liquid-Liquid Extraction,
Derivatization and GC with Electron Capture Detector", Methods for the Determination of
Organic Compounds in Drinking Water-Supplement III EPA/600/R-95-131.
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APPENDIX
SUMMARY OF SAMPLES FOR
RAPID TOXICITY TECHNOLOGIES
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Table A-l. Summary of Contaminant Test Samples
Type of Sample
Quality Control
Drinking Water
Field Location
Sample Characteristics
Method Blank
Positive Control
(contaminant and
concentration determined by
each vendor)
Negative Control
(unfortified drinking water)
Unfortified drinking water
Cyanide
Aldicarb
Dicrotophos
Thallium Sulfate
Colchicine
VX
Soman
Botulinum Toxin
Ricin
One drinking water sample
fortified with any one of the
above contaminants
Lethal Dose
Concentration
NA
NA
NA
NA
250 mg/L
140 mg/L
1.4g/L
2.8 g/L
240 mg/L
0.2 mg/L
0.30 mg/L
0.3 mg/L
15 mg/L
One of the
above
concentrations.
No. of Samplesa
At least 5% of all
samples
At least 5% of all
samples
One per dilution series
At least 5% of all
samples
12-20
12-20
12-20
12-20
12-20
12-20
12-20
12-20
12-20
12-20
a Each lethal dose concentration will be analyzed along with 2 to 4 dilutions. Four replicates of each dilution series will be
analyzed. Therefore, the number of samples listed in the table is an approximate range.
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Table A-2. Summary of the Interfering Compound Test Samples
Type of Sample
Quality Control
Drinking Water
Sample Characteristics
Method Blank
Positive Control
(contaminant and
concentration determined by
each vendor)
Negative Control
(unfortified drinking water)
Unfortified drinking water
Manganese
Iron
Aluminum
Copper
Zinc
Chloramination byproducts
Chlorination byproducts
Concentration
NA
NA
NA
NA
0.25 mg/L
0.15mg/La
0.5 mg/La
0.6 mg/La
2.5 mg/La
NAb
NA
No. of Samples
At least 5% of all
samples
At least 5% of all
samples
One per interfering
compound
At least 5% of all
samples
4
4
4
4
4
4
4
National Secondary Drinking Water Regulations that set non-mandatory water quality standards
b A separate water sample will be collected from a system using chloramination as its disinfection process for these analyses.
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