September 2004
Environmental Technology
Verification Report


TETRACORE, INC.
BIOTHREAT ALERT® ANTHRAX, BOTULINUM
TOXIN, AND RlCIN IMMUNOASSAYTEST
STRIPS
               Prepared by
                Battelle


               Baltelle
             I he Business of Innovation
         Under a cooperative agreement with


           U.S. Environmental Protection Agency

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                                       September 2004
 Environmental Technology Verification
                  Report

    ETV Advanced Monitoring Systems Center

               Tetracore, Inc.
BioThreat Alert® Anthrax, Botulinum Toxin, and
        Ricin Immunoassay Test Strips
                      by
                   Ryan James
                   Amy Dindal
                 Zachary Willenberg
                   Karen Riggs
                    Battelle
                 Columbus, Ohio 43201

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                                       Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation by the EPA for use.
                                         11

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                                      Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
nation's air, water, and land resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, the EPA's  Office of Research and Development provides data and science support that
can be used to solve environmental problems and to build the scientific knowledge base needed
to manage our ecological resources wisely, to understand how pollutants affect our health, and to
prevent or reduce environmental risks.

The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols  developed with input from major stakeholders and customer groups
associated with the technology  area. ETV consists of six verification centers. Information about
each of these centers can be found on the Internet at http://www.epa.gov/etv/.

Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received funding to plan, coordinate,
and conduct such verification tests for "Advanced Monitoring Systems for Air, Water, and Soil"
and report the results to the community at large. Information concerning this specific
environmental technology area  can be found on the Internet at
http ://www. epa.gov/etv/centers/center 1 .html.
                                           in

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                                 Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. We sincerely appreciate the
contribution of drinking water samples from the New York City Department of Environmental
Protection (Paul Bennett), the City of Orlando (Terri Slifko), and the Metropolitan Water
District of Southern California (Paul Rochelle). Also, thank you to the Metropolitan Water
District of Southern California for concentrating each drinking water sample. We would also
like to thank Karen Bradham, U.S. EPA National Exposure Research Laboratory (NERL); Steve
Allgeier, U.S. EPA Office of Water; Ricardo DeLeon, Metropolitan Water District of Southern
California;  and Stanley States, Pittsburgh Water and Sewer Authority, for their careful review of
the test/QA plan and this verification report. Thanks go to Linda Sheldon, U.S. EPA NERL, for
her review of the verification reports and  statements.
                                           IV

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                                      Contents
                                                                                  Page


Notice	ii

Foreword  	 iii

Acknowledgments 	 iv

List of Abbreviations 	vii

1 Background  	 1

2 Technology Description  	2

3 Test Design and Procedures 	4
       3.1 Introduction	4
       3.2 Test Samples  	6
            3.2.1 Performance Test Samples	7
            3.2.2 Drinking Water Samples  	 8
            3.2.3 Quality Control Samples  	 8
       3.3 Test Procedure	9
            3.3.1 Laboratory Testing	9
            3.3.2 Non-Laboratory Testing	9
            3.3.3 Drinking Water Characterization	 10

4 Quality Assurance/Quality Control	 11
       4.1 Sample Chain-of-Custody Procedures	 11
       4.2 Equipment/Calibration  	 11
       4.3 Characterization of Contaminant Stock Solutions 	 11
            4.3.1 Characterization of Botulinum Toxin and Ricin  	 11
            4.3.2 Characterization of Anthrax Spores	 12
            4.3.3 Anthrax Enumeration Data  	 13
       4.4 Technical Systems Audit	 15
       4.5 Audit of Data Quality	 17
       4.6 QA/QC Reporting	 17
       4.7 Data Review	 17

5 Statistical Methods and Reported Parameters	 19
       5.1 Qualitative Contaminant Presence/Absence	 19
       5.2 False Positive/Negative Responses  	 19
       5.3 Consistency	 19
       5.4 Lowest Detectable Concentration  	 19
       5.5 Other Performance Factors  	20

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6 Test Results	21
      6.1  Qualitative Contaminant Presence/Absence 	21
            6.1.1 Anthrax  	21
            6.1.2 Botulinum Toxin  	24
            6.1.3 Ricin  	24
      6.2  False Positive/Negative Responses  	25
            6.2.1 Interfered PT Samples	25
            6.2.2 DW Samples 	26
            6.2.3 Cross-Reactivity PT Samples	27
      6.3  Consistency	28
      6.4  Lowest Detectable Concentration 	28
      6.5  Other Performance Factors  	29

7 Performance Summary	30

8 References  	34

                                       Figures

Figure 2-1.   Tetracore BioThreat Alert®	2


                                        Tables

Table 3-1.   Lethal Dose and Source of Contaminants 	 5
Table 3-2.   Performance Test Samples	6
Table 3-3.   Drinking Water Samples  	7
Table 3-4.   ATEL Water Quality Characterization of Drinking Water Samples	 10
Table 4-1.   Characterization Information for Battelle Preparation of Anthrax Spores	 12
Table 4-2.   Anthrax Enumeration Data for PT Samples	 14
Table 4-3.   Anthrax Enumeration Results for Fortified Interferent and
            Drinking Water Samples  	 16
Table 4-4.   Summary of Data Recording Process	 18
Table 6-la.   Anthrax Contaminant-Only PT Sample Results	22
Table 6-lb.   Botulinum Toxin Contaminant-Only PT Sample Results	24
Table 6-lc.   Ricin Contaminant-Only PT Sample Results	24
Table 6-2.   Interferent PT Sample Results 	25
Table 6-3.   DW Sample Results	27
Table 6-4.   Potentially Cross-Reactive PT Sample Results  	28
Table 7-1.   Anthrax Summary  Table  	30
Table 7-2.   Botulinum Toxin Summary Table  	32
Table 7-3.   Ricin Summary Table	33
                                          VI

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                              List of Abbreviations

AMS            Advanced Monitoring Systems
ATEL            Aqua Tech Environmental Laboratories, Inc.
Ca               calcium
CDC            Centers for Disease Control and Prevention
cfu              colony-forming units
COA            certificate of analysis
DI               deionized
DW             drinking water
EPA             U.S. Environmental Protection Agency
ETV            Environmental Technology Verification
HOPE           high-density polyethylene
L                liter
LOD            limit of detection
MB              method blank
Mg              magnesium
mg/L            milligram per liter
jiL               microliter
mL              milliliter
PT               performance test
QA              quality assurance
QC              quality control
QMP            quality management plan
RPD            relative percent difference
SOP             standard operating procedure
TSA            technical systems audit
                                         vn

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                                      Chapter 1
                                     Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental tech-
nologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.

ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative tech-
nologies by developing test plans that are responsive to the needs of stakeholders, conducting
field or laboratory tests (as appropriate), collecting and analyzing data, and preparing peer-
reviewed reports. All evaluations are conducted in accordance with rigorous quality  assurance
(QA) protocols to ensure that data of known and adequate quality are generated and  that the
results are defensible.

The EPA's National Exposure Research Laboratory and its verification organization  partner,
Battelle, operate the Advanced Monitoring Systems  (AMS) Center under ETV. The AMS Center
recently evaluated the performance  of the Tetracore,  Inc., BioThreat Alert® anthrax, botulinum
toxin, and ricin immunoassay test kits. Immunoassay test kits were identified as a priority
technology category for verification through the AMS Center stakeholder process.

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                                      Chapter 2
                               Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of environ-
mental monitoring technologies for air, water, and soil. This verification report provides results
for the verification testing of the Tetracore BioThreat Alert® test strips (Figure 2-1). The
following is a description of the Tetracore BioThreat Alert® test strips based on information
provided by the vendor. The information provided below was not subjected to verification in this
test.

The BioThreat Alert®test strip from Tetracore, Inc., is a lateral flow immunochromatographic
device that uses two antibodies in combination to specifically detect target antigen in solution.
One of the  specific antibodies is labeled with a colloidal
gold derivative. Samples applied to the BioThreat Alert® test
strips mix with the colloidal gold-labeled antibody and
move along the strip membrane by capillary action. The
second specific antibody captures the colloidal  gold-labeled
antibody and bound target. When  a sufficient amount of
target antigen is present, the colloidal gold label
accumulates in the sample
("S") window on the test strip,
forming a visible
reddish-brown colored line. As
an internal  control, a second
band in the control ("C")
window indicates that the test
strip functioned properly. Two
bands or colored lines (in the
"S" and "C" windows) are
required for a positive result
determination.
                             Figure 2-1. Tetracore BioThreat Alert8
Twenty-five individually
packaged BioThreat Alert® test strips (including a disposable pipette) are provided in a small
box. In addition to the test strips, the box contains several small plastic vials, 25 mL of sample
buffer, and step-by-step instructions. To complete a test on a liquid sample, the sample is mixed
with the provided buffer, and five or six drops are added to the sample well of the BioThreat
Alert® test strip. A positive result is indicated by the appearance of a colored line in the test
- ^ ,^- .„.

.
       ftnthrax
   BioThreat Alert

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window of the test strip and can be read visually or with a reader. During this verification test, a
reader was used to make the determination of a positive or negative result.

One kit of 25 strips including sample buffer, instruction brochure, and vials needed for sampling
costs approximately $625. The Alexeter strip reader used during this verification test costs
approximately $4,000.

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                                      Chapter 3
                             Test Design and Procedures
3.1 Introduction
The objective of this verification test of immunoassay test kits was to evaluate their ability to
detect specific biological toxins and agents in water samples and to determine their
susceptibility to specific interferents added to pure water and to interferents inherently present in
several drinking water (DW) samples. The detection devices are based on immunological
interactions, where specific antibodies are used to detect antigens or contaminants of interest.
For the BioThreat Alert® test strips, the presence of contaminants is indicated by the appearance
of a colored line within 15 minutes of the application of a water sample. The single-use test
strips detect only one contaminant at a time.

During this verification test, the BioThreat Alert® test strips were subjected to various concen-
trations of anthrax spores, botulinum toxin (Types A and B), and ricin in American Society for
Testing and Materials Type n deionized (DI) water. Table 3-1 shows the contaminants and
information about their detection, including the vendor-stated limit of detection (LOD), the
lethal dose concentrations, and the source. The BioThreat Alert® test strips also were used to
analyze contaminant-fortified DW samples that were collected from four water utilities that use
a variety of treatment methods. The effect of interferents was evaluated by analyzing individual
solutions of organic acids (humic and fulvic), magnesium (Mg) and calcium (Ca) in DI water
both with and without the addition of the contaminants using the BioThreat Alert® test strips. In
addition, specificity was evaluated by exposing the BioThreat Alert® test strips to a potentially
cross-reactive compound or spore for each target contaminant.

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Table 3-1. Lethal Dose and Source of Contaminants
Contaminant
Bacillus anthracis
Ames Strain
(anthrax)
Botulinum toxin
Types A and B
Ricinus communis
Agglutinin n (ricin)
Vendor-Stated
LOD
1 x io5
spores/mL
0.01 mg/L
0.035 mg/L
Lethal Dose
Concentration'*0
200 spores/mL(1)
0.3 mg/L(2)
15mg/L(3)
Source of
Contaminant
BattelleandU.S.
Army Dugway
Proving Ground
Metabiologics, Inc.
(Madison,
Wisconsin)
Vector Laboratories,
Inc. (Burlingame,
California)
(a) The lethal dose of each contaminant was determined by calculating the concentration at which 250 mL of water
  would probably cause the death of a 154-pound person based on human mortality data.
mL = milliliter
mg/L = milligrams per liter

The verification test for the BioThreat Alert® test kits was conducted from January 14 through
April 23, 2004, according to procedures specified in the Test/QA Plan for Verification of
Immunoassay Test Kits.(4} This test was conducted at Battelle laboratories in Columbus and
West Jefferson, Ohio. Aqua Tech Environmental Laboratories, Inc. (ATEL) of Marion, Ohio,
performed physicochemical characterization for each DW sample to determine the following
parameters: turbidity; concentration of dissolved and total organic carbon; specific conductivity;
alkalinity; concentration of Mg and Ca; pH; hardness; and concentration of total organic halides,
trihalomethanes, and haloacetic acids. Battelle confirmed the presence of anthrax spores using
plate enumeration.

The BioThreat Alert® test strips were evaluated for the following parameters:

•   Qualitative contaminant presence/absence
•   False positive/false negative response
    - Interferents
    - DW matrix effects
    - Cross-reactivity
•   Consistency
•   Lowest detectable concentration
•   Other performance factors
    - Field portability
    -Ease of use
    - Sample throughput.

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3.2 Test Samples
Tables 3-2 and 3-3 summarize the samples analyzed for each contaminant. The ability of the
BioThreat Alert® test strips to individually detect various concentrations of anthrax spores,
botulinum toxin, and ricin was evaluated by analyzing performance test (PT) and DW samples.
PT samples included DI water fortified with either the target contaminant, an interferent, both,
or only a cross-reactive species. DW samples were analyzed using the BioThreat Alert® test
strips with and without the addition of each target contaminant. All the samples listed in the
test/QA plan were initially analyzed. As discussed below, additional concentration levels and
sample types were analyzed to more thoroughly evaluate the performance of the BioThreat
       test strips.
Alert®
Table 3-2. Performance Test Samples
Type of PT
Sample
Contaminant-only
Interferent
Potentially
Cross-reactive
Sample Characteristics
Anthrax spores
Botulinum toxin Type A
Botulinum toxin Type B
Ricin
Contaminants in 46 mg/L Ca
and 1 8 mg/L Mg
Contaminants in 230 mg/L Ca
and 90 mg/L Mg
Contaminants in 0.5 mg/Lhumic
acid and 0.5 mg/L fulvic acid
Contaminants in 2.5 mg/L humic
acid and 2.5 mg/L fulvic acids
Bacillus thuringiensis (anthrax
analogue)
Lipopolysaccharide
(botulinum toxin analogue)
Lectin from soybean
(ricin analogue)
Approximate Concentrations
200tol010spores/mL(a)
0.0 to 0.5 mg/L
0.01 to 0.5 mg/L
0.035 to 15 mg/L
Anthrax - 106 spore s/mL
Botulinum toxin (Type B only) - 0
Ricin -0.4 mg/L
Anthrax - 106 and 10s spores/mL
Botulinum toxin (Types A and B)
Ricin -0.4 mg/L
Anthrax - 106 spore s/mL
Botulinum toxin (Type B only) - 0
Ricin -0.4 mg/L
Anthrax - 106 and 10s spores/mL
Botulinum toxin (Types A and B)
Ricin -0.4 mg/L
.lmg/L
-0.1 mg/L
.lmg/L
-0.1 mg/L
105 spore s/mL
0.1 mg/L
0.4 mg/L
(a' This concentration range includes all samples analyzed, including spores preserved with and without phenol,
   spores prepared at Battelle and at Dugway Proving Ground, and vegetative anthrax cells.

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Table 3-3. Drinking Water Samples
Drinking Water Sample Description
Water Utility
Metropolitan Water
District of
California (CA)
New York City,
New York (NY)
Metropolitan Water
District of
California (CA)
New York City,
New York (NY)
Columbus, Ohio
(OH)
Orlando, Florida
(FL)
Water
Treatment
filtered
chloraminated
unfiltered
chlorinated
filtered
chloraminated
unfiltered
chlorinated
filtered
chlorinated
filtered
chlorinated
Source
Type
surface
surface
surface
surface
surface
ground
Cone. /
Unconc.
cone.
cone.
unconc.
unconc.
both
both
Approximate
Contaminant Concentrations
Anthrax
(spores/mL)
unspiked
106
10s
unspiked
106
10s
unspiked
106
unspiked
106
unspiked
106
unspiked
106
Botulinum
Toxin (mg/L)
unspiked
0.1 (Type B)
0.1 (Type A)
unspiked
0.1 (Type B)
0.1 (Type A)
unspiked
0.1 (Type B)
unspiked
0.1 (Type B)
unspiked
0.1 (Type B)
unspiked
0.1 (Type B)
Ricin
(mg/L)
unspiked
0.4
unspiked
0.4
unspiked
0.4
unspiked
0.4
unspiked
0.4
unspiked
0.4
3.2.1  Performance Test Samples

The contaminant-only PT samples were prepared in DI water using certified standards of ricin
and botulinum toxin. Reference methods were not available for quantitative confirmation of the
botulinum toxin and ricin test solutions so certificates of analysis (COA) and QA oversight of
solution preparation were used to confirm their concentrations. Anthrax PT samples also were
prepared in DI water using anthrax spores prepared and characterized by Battelle using standard
methods. All test samples were prepared from the standards or stock solutions on the day of
analysis. Spores obtained from Dugway Proving Ground were prepared there and then
enumerated by Battelle during this verification test.

Initially, the test/QA plan called for the analysis of PT samples with concentrations including the
lethal dose; the vendor-stated LOD; and approximately 5, 10, and 50 times the LOD. These
samples were analyzed using the BioThreat Alert® test strips. Preliminary results indicated that
anthrax was not detectable; therefore, the original test/QA plan was amended to include the
analysis of higher concentration levels of anthrax, as well as anthrax spores that were never
preserved in phenol, a second source of anthrax spores, and vegetative anthrax cells. This testing
and the subsequent results are fully described in Section  6.1.
The interferent PT samples consisted of samples of humic and fulvic acids isolated from the
Elliott River (obtained from the International Humic Substances Society) and Ca and Mg

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(prepared from their chlorides), each spiked into DI water at two concentration levels. These
solutions were analyzed both with the addition of each target contaminant at one concentration
level and without the addition of any target contaminant. To be able to evaluate the
susceptibility of the BioThreat Alert® test strip to false negative results due to interferents, the
test/QA plan was amended to include the fortification of detectable concentrations of anthrax
spores into interferent solutions.

The last type of PT sample was a cross-reactivity check sample to determine whether the test
strips produce false positive results in response to similar analytes. Bacillus thuringiensis (for
anthrax), lectin from soybean (for ricin), and lipopolysaccharide (for botulinum toxin) are
chemically or biologically similar to the specified targets. Solutions of these were prepared in DI
water at concentrations similar to the vendor-stated LOD of the test kits for the specified targets
and analyzed using the appropriate BioThreat Alert® test strip.

In most cases,  three replicates of each PT sample were analyzed. In some instances, the anthrax
test samples were analyzed less than three times, depending on the number of test strips
available for the analysis. A total of 186 PT samples was analyzed by the BioThreat Alert® test
strips for this test.  The results provided information about how well the BioThreat Alert® test
strips detected the presence of each contaminant at several concentration levels, the consistency
of the responses, and the susceptibility of the BioThreat Alert® test strips to some selected
interferents and possibly cross-reactive species.

3.2.2 Drinking Water Samples

Table 3-3 lists the DW samples collected from four geographically distributed municipal sources
to evaluate the performance  of the BioThreat Alert® with various sample matrices. These
samples were unique in terms of their source and treatment and disinfection process.  All
collected samples were finished DW either ready for the distribution system or from within the
distribution system.

Approximately 120 L of each of the DW samples were collected in pre-cleaned high-density
polyethylene (HOPE) containers. All but 20 L of the DW samples were shipped to the
Metropolitan Water District  of Southern California, dechlorinated with sodium thiosulfate, and
then concentrated through ultra-filtration techniques to a final volume of 250 mL. This
concentration factor was selected because it is the goal of an EPA onsite ultra-filtration method
which is currently being developed.  The remaining 20 L of each DW sample was shipped to
ATEL for water quality analysis. Each DW sample (non-concentrated and concentrated) was
analyzed without adding any contaminant, as well  as after fortification with individual
contaminants at a single concentration level. A total  of 156 DW samples was analyzed by the
BioThreat Alert® test strips for this test.

3.2.3 Quality Control Samples

In addition to the 342 PT and DW samples analyzed, 41 method blank (MB) samples consisting
of DI water also were analyzed to confirm negative responses in the absence of any contaminant
and to ensure that no sources of contamination were introduced during the analysis procedures.

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The Tetracore reader produced an error message if a control line in the result window on the
BioThreat Alert® test strips did not appear, according to specifications, during the analysis of
each sample. If the Tetracore reader produced an error message instead of a result, that test strip
was discarded and a new test strip was used. Such an error message occurred just two times
during the verification test. Because of this control feature, other positive control samples were
not analyzed.
3.3 Test Procedure

3.3.1 Laboratory Testing

The scope of this verification test required that most of the test samples be analyzed within
Battelle laboratories staffed with technicians trained to safely handle anthrax, botulinum toxin,
and ricin. Each day, fresh samples were prepared from standards or stock solutions in either DI
water, an interferent matrix, or a DW matrix. Each sample was prepared in its own container and
labeled only with a sample identification number that also was recorded in a laboratory record
book along with details of the sample preparation. Prior to the analysis of each sample, the
verification staff recorded the sample identification number on a sample data sheet; then, after
the analysis was complete, the result was recorded on the sample data sheet.  Three replicates of
each test sample were analyzed. The BioThreat Alert® test strip testing procedure included the
following steps for analyzing liquid samples for the presence of anthrax  spores, botulinum toxin,
or ricin: (1) sample buffer was added to the  0.5-mL mark on the sample vial; (2) the liquid
sample was added to the buffer until the solution reached the  1-mL mark on the sample vial; (3)
the cap was closed, and the vial was shaken vigorously for approximately 10 seconds;  (4) the
disposable pipette was used to remove liquid from the sample vial; (5) 5 or 6 drops were placed
in the sample port of the BioThreat Alert® test strip;  and (6) after 15 minutes, the test strip was
placed in the reader and the reader's instructions were followed to obtain the result. The reader
was operated by turning it on, entering the sample identification, and pressing the "enter"
button. The test strip was then taken into the reader,  and a positive or negative result was
generated within approximately 2 minutes. Each result, along with the time,  date,  and  sample
identification was printed using a printer provided by Tetracore.

3.3.2 Non-Laboratory Testing

Because the toxic nature of the contaminants did not permit their use outside special laboratory
facilities, MB samples were analyzed at a non-laboratory location to evaluate the BioThreat
Alert® test strip performance and ease of use outside of the laboratory. Both a trained technician
and a non-technical/untrained, first-time user performed analyses at the non-laboratory location.
The purpose of these analyses was to test the performance of the BioThreat Alert® test  strips in a
non-laboratory  setting, not to evaluate thoroughly the effect of changing conditions such as
temperature and humidity on the BioThreat Alert® test strips.  The non-technical/untrained, first-
time user was guided by only the manual or by vendor instructions. The operators for the rest of
the verification test had undergraduate degrees in the sciences or equivalent work experience
and either participated in a training session provided by the vendor prior to the verification test
or were trained by a vendor-trained operator.

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3.3.3 Drinking Water Characterization

An aliquot of each DW sample, collected as described in Section 3.2.2, was sent to ATEL prior
to concentration to determine the following water quality parameters: turbidity; concentration of
dissolved and total organic carbon; conductivity; alkalinity; pH; concentration of Ca and Mg;
hardness; and concentration of total organic halides, trihalomethanes, and haloacetic acids.

Table 3-4 lists the methods used to characterize the DW samples, as well as the characterization
data from the four water samples collected as part of this verification test. Water samples were
collected and water quality parameters were measured by ATEL in January. Samples were then
transported and test strips were analyzed from January through March. Because of this, some of
the water quality parameters may have changed from the time of analysis by ATEL until testing
with the BioThreat Alert® test strips.

Table 3-4.  ATEL Water Quality Characterization of Drinking Water Samples
Sources of Drinking Water Samples
Parameter
Turbidity
Dissolved

organic carbon
Total organic
carbon
Specific
conductivity
Alkalinity
pH
Calcium
Magnesium
Hardness
Total organic
halides
Trihalomethanes

Haloacetic acids

Unit
NTU

mg/L
O
mg/L
|iS/cm2
mg/L

mg/L
mg/L
mg/L
Hg/L
^g/L/
analyte
VgfU
analvte
Method
EPA180.1(5)

SM5310(6)

SM5310(6)
SM2510(6)
SM 2320(6)
EPA150.1(7)
EPA200.8(8)
EPA200.8(8)
EPA 130.2(7)
SM 5320(6)
EPA 524.2(9)

EPA 552.2(10)

Columbus,
Ohio
(OH DW)
0.2

2

2
357
55
7.33
42
5.9
125
360
26.9

23.2

Orlando,
Florida
(FL DW)
0.5

2

2
325
124
7.93
41
8.4
137
370
80.9

41.1

New York City,
New York
(NY DW)
1.3

2

2
85
4
6.80
5.7
19
28
310
38.4

40.3

MWD,
California
(CA DW)
0.1

2

2
740
90
7.91
35
1.5
161
370
79.7

17.6

NTU = nephelometric turbidity unit
MWD = Metropolitan Water District
I^S/cm2 = micro Siemens per square centimeter
                                            10

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                                     Chapter 4
                        Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the
quality management plan (QMP) for the AMS Center(11) and the test/QA plan(4)  for this
verification test.
4.1 Sample Chain-of-Custody Procedures

Sample custody was documented throughout collection, shipping, and analysis of the samples.
Sample chain-of-custody procedures were in accordance with ASAT n-007, Standard Operating
Procedure for Chain of Custody for Dioxin/Fur an Analysis. The chain-of-custody forms
summarized the samples collected and analyses requested and were signed by the person
relinquishing samples once that person had verified that the custody forms were accurate. The
original sample custody forms accompanied the samples; the shipper kept a copy. Upon receipt
at the sample destination,  sample custody forms were signed by the person receiving the samples
once that person had verified that all samples identified on the custody forms were present in the
shipping container.
4.2 Equipment/Calibration

The BioThreat Alert® test strips and all appropriate reagents and supplies specific for the
detection of anthrax, botulinum toxin, and ricin were provided to Battelle by the vendor. These
test kits, each containing an internal control line, required no calibration. For DW characteriza-
tion and confirmation of the possible interferents, analytical equipment was calibrated by ATEL
according to the procedures specified in the appropriate standard methods.  Pipettes used during
the verification test were calibrated according to Battelle Standard Operating Procedure (SOP)
VI-025, Operation, Calibration, and Maintaining Fixed and Adjustable Volume Pipettes.
4.3 Characterization of Contaminant Stock Solutions

4.3.1  Characterization of Botulinum Toxin and Ricin

Certificates of analysis  for botulinum toxin and ricin were provided by the supplier. Because
standard reference methods do not exist, the concentration of botulinum toxin and ricin were not
                                           11

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independently confirmed. The COAs stated that the ricin standard (Vector Laboratories, Inc.,
Burlingame, California) had a concentration of 1,000 mg/L and the botulinum toxin standards
(Metabiologics, Inc., Madison, Wisconsin) had concentrations of 2,000 mg/L for Type B and
1,000 mg/L for Type A. Test samples containing these contaminants were prepared by diluting
aliquots of these stock solutions with DI water.

4.3.2 Characterization of A nthrax Spores

Multiple sources and forms of the Ames strain of Bacillus anthracis (anthrax) were evaluated
during this verification test. The primary source was a lot of spores prepared by Battelle and
stored in a 1% stock solution of phenol in water as a means to prevent vegetative cell growth.
This lot of spores is referred to in this report as Battelle-prepared, phenol-preserved. Prior to
testing, an aliquot of the stock solution described above was centrifuged, the phenol/water
solution was removed, and the  spores were reconstituted with DI water. This process was
repeated two times to ensure that the spores were suspended only in DI water. This lot of spores
was characterized with an 11-step characterization process prior to use in the verification test.
For confidentiality reasons,  Table 4-1  gives the outcome of only five of the characterization
parameters, as well as the location at which each step was performed. These characterization
steps were performed when  this lot of spores was prepared  in September 2003. It should be
noted that, once a stock solution of spores is characterized, less concentrated solutions of spores
can be prepared from the stock solution without questioning the integrity of the spores. This lot
of spores met all 11 acceptance criteria. Two parts of the characterization process—DNA
sequencing and gene identification—were performed by Dr. Alex Hoffmaster at the
Epidemiologic Investigations Laboratory, Meningitis and Special Pathogens Branch of the
Centers for Disease Control and Prevention (CDC). The CDC analyses confirmed that the spores
were Ames strain anthrax spores, and the guinea pig LD50 study confirmed their virulence. The
stock solution of spores was enumerated after preparation to determine its original
concentration. In addition, a vegetative cell analysis showed that the stock solution was 99.94%
anthrax spores. Because at least one spore is needed to spur the growth of a colony during an
enumeration,  the concentrations determined represented a minimum concentration of spores.
Care was taken to spread the samples to avoid clumping; but, if clumping occurred, the spore
concentrations would only be higher than shown in the data tables.

Table 4-1. Characterization Information for Battelle Preparation of Anthrax Spores

          Characterization                 Outcome           Analysis Performed By

 % vegetative cells                  0.06%                             Battelle

 Viable spore count                  5.26xio9                           Battelle

 Guinea pig 10 day LD50             10 spores                           Battelle

 DNA fingerprinting                 MLVA Genotype 62                  CDC

 PA gene  sequencing	Protective Antigen  Type I	CDC	
                                           12

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Another lot of anthrax spores prepared by Battelle was used during the verification test. This lot
had been prepared in the same way as the other, but it had never been stored in phenol or any
other preservative. The second lot had been subjected only to enumeration to determine the
concentration. Test solutions were made from this stock solution to investigate whether the
phenol preservation was affecting the sensitivity of the test strips.

Similarly, a lot of anthrax spores from Dugway Proving Ground was obtained and used to
investigate the sensitivity of BioThreat Alert® test strips to a different spore preparation (referred
to as Dugway-prepared in this report). Again, enumeration was the only characterization step
performed on this lot of spores.

A stock solution of vegetative anthrax cells also was prepared and used during this verification
test. This solution was prepared by  harvesting vegetative cells from an enumeration of the
Battelle-prepared, phenol-preserved spores, placing them into solution, and then enumerating to
determine the solution's concentration. No further characterization was performed on these
vegetative cells.  Solutions of these cells were used to determine the sensitivity of the BioThreat
Alert® test strips to vegetative cells.

Regardless of the source and type of anthrax stock solution used to make test samples, its
concentration was confirmed by a plate enumeration method. This was done within 24 hours of
any stock solution being used for test sample preparation and is described in Battelle SOP
MREF X-054, Enumeration ofBL-2 andBL-3 Bacteria Samples Via the Spread Plate
Technique. In addition, four times during the verification test the serial dilution method was
validated by enumerating the PT samples. For example, for a 109 spores/mL sample to be
enumerated, the  method requires that it be diluted to at least 103 spores/mL so 100 |^L of sample
will provide a countable number of spores on a culture plate. Therefore, if 100 |j,L of the 103
spores/mL solution provided the correct number of spores to the plate, the concentration of every
serial dilution made to obtain that concentration was confirmed.

4.3.3 Anthrax Enumeration Data

Table 4-2 gives the results of all plate enumerations performed throughout the verification test
on anthrax solutions prepared in DI water. The data from  enumerations to validate the serial
dilution method  are also given in Table 4-2. The expected concentration, as determined from a
previous enumeration (if available), the actual  concentration, and the relative percent difference
between the two are given in the table.  Relative percent difference (RPD) is determined using
the following equation, where E is the expected concentration and A is the actual concentration
as determined by the enumeration.

                                      E-A\
                              RPD= —j^x 100%
                                        E

For the Battelle-prepared, phenol-preserved spores, only one enumeration resulted in a
concentration that was more than 25% different from the expected concentration. The average
                                           13

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Table 4-2. Anthrax Enumeration Data for PT Samples
     Spore Solution
   Description (units)
Date
   Expected          Actual
Concentration   Concentration^
RPD


Battelle-prepared,
phenol -preserved
stock solution
(108 spores/mL)


Battelle-prepared,
phenol-preserved serial
dilution validations
(104 spores/mL)

Battelle-prepared, non-
phenol-preserved
(108 spores/mL)
Vegetative anthrax
(104 cfu/mL)

Dugway-prepared
(106 spores/mL)

January 28
January 28
January 30
February 2
February 10
February 26
March 1
March 23
January 28
January 30
J
March 2
March 23
February 5

February 12
March 23
March 24
March 22
March 23
March 24
53
58
53
61
61
82
63
67
10
40

10
1,000
Unknown

14
Unknown
260
Unknown
0.010
10
58
53
61
53
82
63
67
57
7.8
32

7.7
992
14

106
26
350
666
0.0081
8.0
9
9
15
14
55
23
5
14
22
20

24
1
NA

657
NA
35
NA
19
20
(a) Each enumeration involved the development of three to five plates. The average, standard deviation, and relative
  standard deviation for each set of Battelle-prepared, phenol-preserved enumeration data were determined, and the
  average relative standard deviation of all enumerations was calculated to estimate the variability in the enumeration
  process, which was 15%.
NA = not applicable.

concentration of the Battelle stock solution was 6 x 109 spores/mL (ranging from 5.3 x  109 to
8.2 x io9 spores). Over the two-month period that the stocks were used and the enumerations
performed, the relative standard deviation of the eight results was 15%. The accuracy and
precision of these enumerations indicate that the concentration of the spore stock solution was
consistent over several months and was usually close to the expected concentration. The serial
dilution validation data confirm that the PT samples containing the Battelle-prepared, phenol-
preserved spores were prepared accurately at various concentration levels. Also shown in
Table 4-2 are the enumerations performed to determine the concentration of the alternate
Battelle preparation of spores (Battelle-prepared, non-phenol-preserved), vegetative anthrax
cells, and a stock solution of spores obtained from Dugway Proving Ground. Notable among
these results was the significant increase in concentration of the alternative Battelle-prepared
stock solution from February 5 to February 12, 2004. Because this lot of spores was used only to
                                             14

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determine the effect of phenol preservation on the sensitivity of the BioThreat Alert® test strips,
this observation was not fully investigated. For enumerations with unknown expected
concentrations, the concentration of that particular solution or the stock from which it had been
prepared had not previously been determined.

Table 4-3 gives the enumeration data for all of the interferent PT (shaded) and DW samples that
were spiked with anthrax spores. For possible interferent samples and samples prepared in DW,
the addition of spores was confirmed by enumeration for at least one sample representing each
matrix. The results of the DW samples enumerated in late January and early February indicated
that the relative difference between the expected concentration and the actual concentration
ranged from 17 to 96%. The larger percent differences for the DW samples as compared with the
PT samples were not a surprise,  considering that DW is presumably an interferent-prone matrix.
These data suggest that spore health is dependent on whether the solution is in DI water or DW.
However, the effect of DW on spore health seemed to be less significant when the concentration
of spores was higher. For example, in March, when the DW and interferent samples were spiked
with higher concentrations of anthrax spores, the difference between the expected concentration
and the actual  concentration for the interferent samples was between 0 and 21% and for the DW
samples between 7 and 55%. Enumerations were performed to characterize the concentration of
spores in each sample matrix. For each test matrix, spores were enumerated within a day of
testing. In the Chapter 6 tables, the actual concentrations of the test samples have been corrected
for the result of the appropriate enumeration for that sample. Because not every test sample was
enumerated and some of the test samples were the result of dilutions of enumerated samples, not
every actual concentration will be represented directly in Table 4-2 or Table 4-3.

The concentrations of the possible cross-reactive interferents of soybean lectin (analogue of
ricin) and lipopolysaccharide (analogue of botulinum toxin) were not confirmed independent of
the COA received from the supplier because of the lack of available analytical methodologies for
these analytes. Samples containing Bacillus thuringiensis (analogue of anthrax) were confirmed
by the same enumeration method used for anthrax and were approximately an order of
magnitude less than expected because some spores were lost during washing with water.
Because the lowest detectable concentration of anthrax was much more concentrated than
Tetracore, Inc., had claimed, additional samples containing higher concentration levels of
anthrax were prepared and analyzed. Additional resources were not expended to determine the
cross-reactivity of Bacillus thuringiensis at comparable concentration levels.
4.4 Technical Systems Audit

The Battelle Quality Manager conducted a technical systems audit (TSA) to ensure that the
verification test was performed in accordance with the test/QA plan(4) and the AMS Center
QMP(11) As part of the audit, the Battelle Quality Manager reviewed the standards and methods
used, compared actual test procedures with those specified in the test/QA plan,(4) and reviewed
data acquisition and handling procedures. Observations and findings from this audit were docu-
mented and submitted to the Battelle Verification Test Coordinator for response. No findings
were documented that required any significant action. The records concerning the TSA are
permanently stored with the Battelle Quality Manager.

                                           15

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Table 4-3. Anthrax Enumeration Results for Fortified Interferent and Drinking Water
Samples
        Sample
      Description
              Expected         Actual
  Date     Concentration   Concentration'"'
  (2004)   (105 spores/mL)  (105 spores/mL)
                                 RPD
 Cone. CA DW
 Cone. CA DW
 Unconc. CA DW
January 28
January 30
January 30
 10
 100
 40
0.38
 8.7
96
91
80
 0.5mg/LOC
 2.5mg/LOC
 230 mg/L Ca
 90 mg/L Mg
 46 mg/L Ca
 18 mg/L Mg
 Cone. CA DW
 Unconc. CA DW
 Cone. OH DW
 Unconc. OH DW
 Cone. NY DW
 Unconc. NY DW
 Cone. FL DW
 Unconc. FL DW
February 2
February 3
February 3

February 3
February 3
February 3
February 3
February 3
February 3
February 3
February 3
February 3
 15
 15
 15
 15
 15
 15
 15
 15
 6.9
 6.5
 5.7
 6.9
 13
 12
 9.1
 7.5
54
57
62
54
17
21
39
50
 Cone. NY DW
 Cone. CA DW
 2.5 mg/L OC
 230 mg/L Ca
 90 mg/L Mg
 March 3
 March 3
 March 3
 March 3
1,000
1,000
1,000
1,000
1,000
 2.5 mg/L OC
 Cone. CA DW
March 23
March 23
1,000
1,000
 962
 448
 4
55
 230 mg/L Ca
 90 mg/L Mg
 Cone. NY DW
March 24
March 24
1,000
1,000
 788
 486
OC = Organic carbon (humic and fulvic acids)
Shading on table distinguishes the interferent and cross-reactivity PT samples from the DW samples.
 The uncertainty of the enumeration technique is approximately 15%.
                                          16

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4.5 Audit of Data Quality

At least 10% of the data acquired during the verification test was audited. Battelle's Quality
Manager or designee traced 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 were checked.
4.6 QA/QC Reporting

Each internal assessment and audit was documented in accordance with Sections 3.3.4 and 3.3.5
of the QMP for the ETV AMS Center.(11) Once the assessment report was prepared, the Battelle
Verification Test Coordinator responded to each potential problem and implemented any
necessary follow-up corrective action. The Battelle Quality Manager ensured that follow-up
corrective action was taken. The results of the TSA were sent to the EPA.
4.7 Data Review

Records generated in the verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-4 summarizes the types of data recorded. The
review was performed by a technical staff member involved in the verification test, but not the
staff member who originally generated the record. The person performing the review added
his/her initials and the date to a hard copy of the record being reviewed.
                                           17

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Table 4-4.  Summary of Data Recording Process
Data to Be Recorded
Dates and times of test
events
Responsible
Party
Battelle
How Often
Where Recorded Recorded
ETV data sheets Start/end of test,
and at each
change of a test
parameter
Disposition
of Data
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
  Sample collection and
  preparation information,
  including chain-of-
  custody
Battelle
ETV data sheets
and chain-of-
custody forms
At time of sample
collection and
preparation
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
 Detection device           Battelle
 procedures and sample
 results
                 ETV data sheets
                   Throughout test
                   duration
                   Manually incorporated
                   in data spreadsheets
 Anthrax enumeration
 data
Battelle
Enumeration data
forms
With every
enumeration
Manually incorporated
in data spreadsheets
 Reference method          ATEL
 procedures and sample
 results
                 Data acquisition
                 system, as
                 appropriate
                   Throughout
                   sample analysis
                   process
                   Transferred to
                   spreadsheets and
                   reported to Battelle
                                                   18

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                                     Chapter 5
                  Statistical Methods and Reported Parameters

The methods presented in this chapter were used to verify the performance parameters listed in
Section 3.1. The BioThreat Alert® test strips produce qualitative results; i.e., they indicate only
the presence or absence of a contaminant, not a measure of the concentration present. Therefore,
the data evaluation methods were used in that context.
5.1  Qualitative Contaminant Presence/Absence

Accuracy was assessed by reporting the number of positive results out of the total number of
samples tested for the BioThreat Alert® test strips at each concentration level of contaminant-
only PT sample tested for anthrax spores, botulinum toxin, and ricin.
5.2 False Positive/Negative Responses

A false positive response was defined as a positive response when the DI water or DW sample
was spiked with a potential interferent, a cross-reactive compound, or not spiked at all. A false
negative response was defined as a negative response when any sample was spiked with a
contaminant at a concentration greater than the lowest detectable concentration of the test strip
for each analyte in DI water. Interferent PT samples, cross-reactivity PT samples, and DW
samples were included in the analysis. The number of false positive and negative results is
reported.
5.3 Consistency

The reproducibility of the results was assessed by calculating the percentage of individual test
samples that produced positive or negative results without variation within replicates.
5.4  Lowest Detectable Concentration

The lowest detectable concentration for each contaminant was determined to be the
concentration level at which at least two out of the three replicates generated positive responses.
These concentration levels are determined for each target contaminant in solutions of DI water.
                                           19

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5.5 Other Performance Factors

Aspects of the instrument performance such as ease of use, field portability, and sample
throughput are discussed in Section 6. Also addressed are qualitative observations of the
verification staff pertaining to the performance of the BioThreat Alert® test strips.
                                           20

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                                      Chapter 6
                                      Test Results
6.1 Qualitative Contaminant Presence/Absence

The responses for the BioThreat Alert® test strips using the contaminant-only PT samples
containing anthrax, botulinum toxin, and ricin are discussed in the following sections. The
BioThreat Alert® test strips provide indication of only a positive or negative response based on
whether or not a line appears in the left half (sample) of the absorbent strip window after a liquid
test sample is applied. A line appears in the right half (control) after every test sample regardless
of whether or not the target contaminant is present. For this verification test, Tetracore, Inc.,
instructed Battelle to use an electronic reader to determine whether or not that line appeared.

6.1.1 Anthrax

The results obtained for the performance test samples containing anthrax spores are given in
Table 6-1 a. The first five concentration levels listed were initially analyzed, and the results
indicated that none of those samples (up to 50 times the vendor-stated LOD) produced
detectable results. The Battelle-prepared, phenol-preserved serial dilution validation
enumeration on January 30 (1  x 10s spores/mL expected) was a part of the serial dilution process
to make all five of these PT samples, so the results of this enumeration confirm the
concentration of spores in these samples. After discussions with Tetracore, Inc., the following
speculative explanations for these results were considered:

1.      The target proteins on the spore's surface may have been stripped off or chemically
       altered by phenol in the storage solution. (The absence or alteration of these proteins
       would probably decrease the sensitivity of the BioThreat Alert® test strips to the affected
       spores.)

2.      The sensitivity of the BioThreat Alert® test strips to anthrax spores is dependent on the
       method used to prepare the spores;  therefore, the spores prepared  at Battelle may result in
       decreased responsiveness compared with spores prepared elsewhere.
                                            21

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Table 6-1 a. Anthrax Contaminant-Only
Purpose of
Analysis


Original test/QA
plan PT samples



Investigation of
phenol storage of
spores



Sensitivity
determination

Alternate spore
preparation


Vegetative cell
sensitivity


Actual Fortified
Concentration^'
200 spores/mL(b)
8 x 104spores/mL
4 x 105spores/mL
8 x 10s spores/mL
4 x 106spores/mL
5 x 106spores/mL
1 x 109spores/mL
8 x 108spores/mL

1 x 1010spores/mL
8 x 109spores/mL
8 x 108spores/mL
8 x 107spores/mL
8 x 106spores/mL
8 x 105spores/mL
7 x 108 spores/mL
8 x 107 spores/mL
Unknown Cone.
4 x 106cfu/mL
3 x 105cfu/mL
3 x 104cfu/mL
3 x 103cfu/mL
PT Sample Results
Anthrax
Description
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Spores

Spores
Spores
Spores
Spores
Spores
Spores
Spores
Spores
Vegetative
Vegetative
Vegetative
Vegetative
Vegetative
Prep
Location
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Battelle

Battelle
Battelle
Battelle
Battelle
Battelle
Battelle
Dugway
Dugway
Battelle
Battelle
Battelle
Battelle
Battelle
Phenol-
Preserved
Yes
Yes
Yes
Yes
Yes
No
No
Yes

No
Yes
Yes
Yes
Yes
Yes
No
No
NA
NA
NA
NA
NA
Positive
Results Out
of Total
Replicates
0/3
0/3
0/3
0/3
0/3
0/3
0/3
3/3

3/3
3/3
3/3
3/3
0/3
0/1
2/2
0/1
2/2
1/1
1/1
2/3
0/1
Vendor-stated LOD was 1 x 105 spores/mL.
NA = not applicable. Vegetative cells were not prepared from any stock solution; they were grown and placed in
solution.
®  Actual concentrations were corrected for the enumeration of the stock solution from which each sample was
   prepared. The uncertainly of the enumeration technique is approximately 15%.
(b:i Lethal dose concentration.
                                                    22

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Additional testing beyond that described in the test/QA plan was performed to explore these
possible explanations and to gain more information about the performance of the BioThreat
Alert® test strips. It included evaluating whether Battelle's storage of the stock solution of
anthrax spores in a 1% solution of phenol had any impact on the performance of the BioThreat
Alert® test strips; increasing the concentration of spores beyond what was required by the
test/QA plan; and subjecting the test strips to Ames strain anthrax spores prepared by Dugway
Proving Ground using a preparation method that is different from the one Battelle uses.

To address the possibility that storing spores in phenol affected the sensitivity of the BioThreat
Alert® test strips, a series of samples was prepared and analyzed using one anthrax spore stock
solution that had been stored in a phenol solution and one that had not. The data are given in
Table 6-1 a under "Purpose of Analysis, Investigation of phenol storage of spores." Both
solutions had been prepared at Battelle using the same preparation method. The 5 x 106
spores/mL sample made with spores not stored in phenol  was not detectable, as was the case for
the 4  x 106 spores/mL solution made from a stock that was stored in phenol. In addition,
samples containing concentrations of approximately 1010  and 109 spores/mL of spores from both
phenol and non-phenol stock solutions were analyzed. The approximately 109 spore/mL
solutions were not detectable, but the 1010 spore/mL solutions were detectable. These results
suggested that the effect of phenol storage was probably inconsequential to the sensitivity of the
BioThreat Alert® test strips to anthrax spores.

The second explanation of the results at the first five concentration levels was investigated by
preparing and analyzing samples containing approximately 109, 108, 107, and 106 spores/mL
from the original stock solution that had been stored in phenol, but washed with water prior to
testing. Since phenol storage apparently did not affect the sensitivity of the technologies to
spores, this series of samples was analyzed to determine the approximate sensitivity of the
BioThreat Alert® test strips to the Battelle-prepared  spores. Only the two highest concentration
levels were detectable; therefore, the lowest detectable concentration was approximately 108
spores/mL. Solutions of spores that were prepared at Dugway Proving Ground and received at
Battelle in 2001 were then analyzed. Since 2001, the Dugway stock solution had been
refrigerated as a solution of spores in spent media. The solution was washed in DI water as
described for the phenol storage solution above and diluted to make several solutions with
concentrations separated by factors often. Both the stock solution concentration and the dilution
methodology were confirmed by plate enumeration as shown in Table 4-2.  These samples were
analyzed one concentration level at a time by decreasing concentration to determine the
approximate sensitivity to these spores. Three replicate analyses were performed on the lowest
detectable individual replicate. When determined in this manner, the lowest detectable
concentration of Dugway spores was approximately 109 spores/mL, a concentration higher than
the lowest detectable concentration of the Battelle-prepared spores.

Tetracore  informed Battelle that the BioThreat Alert® test strips are more sensitive to vegetative
anthrax than spores. This was investigated by preparing a solution of vegetative cells as
described above. This solution was diluted by a factor of  10 four times, and then the stock and
two diluted samples were enumerated to determine the concentration of vegetative cells in each
sample. These samples were analyzed one concentration level at a time by decreasing
concentration to determine the approximate sensitivity to these vegetative cells.  The lowest

                                           23

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detectable concentration of vegetative cells was 3 x 104 colony-forming units (cfu)/mL,
approximately an order of magnitude lower than Tetracore claimed to be able to attain for
anthrax spores.

6.1.2 Botulinum Toxin

The results obtained for the PT samples containing botulinum toxin Types A and B are given in
Table 6-lb. The results showed that the BioThreat Alert® test strips were reproducibly sensitive
to botulinum toxin Type A at 0.01 mg/L and Type B at 0.05 mg/L.

Table 6-lb. Botulinum Toxin Contaminant-Only PT Sample Results
Purpose
of Analysis

Botulinum toxin
PT samples


Concentration
(mg/L)
0.01(a)
0.05
0.1
0.300
0.5
Positive Results Out of
Total Replicates (Type A)
3/3
3/3
3/3
NA(c)
3/3
Positive Results Out of Total
Replicates (Type B)
1/3
3/3
3/3
3/3
3/3
^a' Vendor-stated LOD for botulinum toxin.
(b:i Lethal dose concentration.
(G:I This concentration level was not analyzed using Type A botulinum toxin.
6.1.3 Ricin

The results obtained for the PT samples containing ricin are given in Table 6-1 c. When
analyzing concentrations ranging from 0.035 to 15 mg/L, all replicate samples generated
positive results.

Table 6-1 c. Ricin Contaminant-Only PT Sample Results
Purpose
of Analysis


Ricin PT samples


Concentration
(mg/L)
0.035(a)
0.2
0.4
2
15<">
Positive Results Out of
Total Replicates
3/3
3/3
3/3
3/3
3/3
^Vendor-stated LOD.
(b:i Lethal dose concentration.
                                            24

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6.2 False Positive/Negative Responses

Three types of samples were analyzed to evaluate the susceptibility of BioThreat Alert® test
strips to false positive and negative results. These included interferent PT samples, made up of
DI water fortified with Ca and Mg and samples fortified with humic and fulvic acids with and
without the addition of target contaminants; cross-reactivity PT samples, made up of DI water
fortified with a contaminant similar biologically or chemically with each specific target con-
taminant; and DW samples both concentrated and unconcentrated and both with and without the
addition of target contaminants. A false positive result was defined as a positive result in the
absence of the target contaminant, and a false negative result was defined as a negative result
from a sample containing detectable levels of each target contaminant.

6.2.1 Interferent PT Samples

The results from the interferent PT samples are given in  Table 6-2. For test strips specific to
each contaminant, the number of positive results out of the number of replicates is given for PT
samples containing only the possible interferents and those possible interferents in the presence
of the listed concentration of target contaminant. For anthrax, expanded testing included
additional interferent PT samples with a higher concentration of anthrax. Results for botulinum
toxin Types A and B and ricin are also presented.

Table 6-2.  Interferent PT Sample Results

Interferent
Sample
46 mg/L Ca
18mg/LMg
230 mg/L Ca
90 mg/L Mg
0.5 mg/L
humic and
fulvic acid
2.5 mg/L
humic and
fulvic acid


Positive Results Out
Anthrax (spores/mL)
Blank
0/3
0/3
0/3
0/3
lxl06(a)
0/3
5xl05(b)
0/3
5xl05(b)
0/3
Ixl06(b)
0/3
Ixl06(b)
lxl08(a)
NA
0/3
Ixl08(b)
NA
3/3
Ixio8(b>
of Total
Replicates
Botulinum Toxin (mg/L)
Blank
0/3
0/3
0/3
1/3
TypeB
0.1
3/3
3/3
3/3
3/3
Type A
0.1
NA
3/3
NA
3/3
Ricin
Blank
0/3
0/3
0/3
0/3

(mg/L)
0.4
3/3
3/3
3/3
3/3
NA = not applicable. Sample not analyzed during expanded testing.
(^ Expected concentration.
(b:i Actual concentration.

One replicate of the botulinum toxin Type B test strips subjected to 2.5 mg/L humic and fulvic
acids generated a false positive result. With that exception, when the unspiked interferent
solutions were analyzed, there were no false positive results for the test strips specific for any of
                                            25

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the three target contaminants. The lack of detectable results for several DW samples spiked at
1 x 106 spores/mL for anthrax indicated false negative responses with respect to the vendor-
stated LOD; however, because those tested concentration levels were not detectable when
analyzed in DI water (see Section 6.1.1), the lack of sensitivity within this testing scenario
cannot be attributed to the presence of the possible interferents. Expanded testing was performed
by analyzing samples prepared using concentration levels of anthrax detectable when prepared
in DI water only. Of these samples, three out of three replicates of 230-mg/L Ca and 90-mg/L
Mg fortified with 1 x 108 spores/mL anthrax generated false negative responses. For botulinum
toxin and ricin, no false negative results were generated from interferent solutions spiked with
target contaminants. The lower concentration interferent matrix was not analyzed during the
expanded testing of anthrax samples or the botulinum toxin Type A samples.

6.2.2 DW Samples

The results from the DW samples are given in Table 6-3. For test strips specific to each
contaminant, the number of positive results out of the number of replicates is given for the DW
samples containing no target contaminants and also the DW samples in the presence of the listed
concentration of each target contaminant. For anthrax, expanded testing included additional DW
samples containing a higher concentration of anthrax. Results for botulinum toxin Types A and
B and ricin are also given.

There were several false positive results for the test strips when used to analyze the  unspiked
DW samples. For the anthrax test strips, one out of three replicates was falsely positive when
used to analyze FL and concentrated NY DW. For the botulinum toxin test strips, one out of
three replicates was falsely positive when subjected to concentrated OH DW; for the ricin test
strips, two out of three replicates generated positive results when used to analyze the
concentrated FL DW.

The second column of results under anthrax shows false negative responses with respect to the
vendor-stated LOD (not as defined in Section 5.2). But, because  those tested concentration
levels were not detectable when analyzed in DI water (see Section 6.1.1), the negative results
cannot necessarily be attributed to the presence of the DW matrix. For anthrax spores, expanded
testing was performed using a concentration level of anthrax that was detectable when prepared
in DI water only. Only two DW samples, concentrated CA and concentrated NY DW, were
analyzed during the expanded testing of anthrax samples or the botulinum toxin Type A
samples. For anthrax, two out of three samples of 1 x 108 spores/mL in concentrated  DW from
NY and one out of three samples of the same concentration in concentrated CA DW produced
false negative results; for botulinum toxin, there were no false negative responses; and for ricin,
one replicate out of three was falsely negative when 0.4 mg/L of  ricin was spiked into NY DW.
                                           26

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Table 6-3. DW Sample Results
DW Sample
Unconcentrated
CADW
Concentrated
CADW
Unconcentrated
FLOW
Concentrated
FLOW
Unconcentrated
NYDW
Concentrated
NYDW
Unconcentrated
OHDW
Concentrated
OHDW
Positive Results Out
Anthrax (spores/mL)
Blank Ixl06« Ixl08«
0/3
0/3
1/3
0/3
0/3
1/3
0/3
0/3
0/3
2xio5(b)
0/3
4xl04(b)
0/3
5xl05(b)
0/3
6xl05(b)
0/3
8xl05(b)
0/3
8xl05(b)
0/3
5xl05(b)
0/3
4xl05(b)
NA
2/3
Ixio8(b>
NA
NA
NA
1/3
Ixio8(b>
NA
NA
of Total Replicates
Botulinum Toxin
TypeB
Blank 0.1
0/3
0/3
0/3
0/3
0/3
0/3
0/3
1/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
(mg/L)
Type A
0.1
NA
3/3
NA
NA
NA
3/3
NA
NA
Ricin (mg/L)
Blank 0.4
0/3
0/3
0/3
2/3
0/3
0/3
0/3
0/3
3/3
3/3
3/3
3/3
2/3
3/3
3/3
3/3
(a) Expected concentration.
(b) Actual concentration.
NA = not applicable. Sample not analyzed during expanded testing.
6.2.3 Cross-Reactivity PT Samples

The results from the cross-reactivity PT samples are given in Table 6-4. For test strips specific to
each target contaminant, a PT sample fortified with a spore or chemical similar to each target
contaminant was analyzed in the absence of any target contaminant. The number of positive
results out of the number of replicates is given for each sample. The only false positive result in
this evaluation of cross-reactivity was for lipopolysaccharide, a compound chemically similar to
botulinum toxin. The rest of the results were correctly reported as negative.
                                            27

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Table 6-4. Potentially Cross-Reactive PT Sample Results
                                                  Positive Results Out of Total
                                                           Replicates

                                                            Botulinum
                                              Anthrax        Toxin        Ricin
 Bacillus thuringiensis (1 x  105 spores/mL)(a)

 Lipopolysaccharide (0.1 mg/L)

 Lectin from soybean (0.4 mg/L)	
(a)  Concentration was determined after the fact to be below the lowest detectable concentration. Therefore, the non-
   detectable results may not indicate a lack of cross-reactivity.


6.3  Consistency

For the anthrax testing, at times the number of replicate analyses was reduced to conserve time
or available supplies. However, the available replicate data for anthrax suggests that
performance of the test strips was reproducible for the contaminant and interferent PT samples
since 96% of the test strips generated results that were either all negative or all positive.
However, the results were less consistent for DW samples (unspiked or spiked with the target
contaminant), where 22% of the sets generated mixed results. For the botulinum toxin test strips,
there was only one mixed result for the contaminant PT samples, one for the interferent PT
samples, and one for the DW samples (overall 92% consistency for botulinum toxin). They were
generated from the lowest concentration of botulinum toxin Type B sample, the unspiked 2.5
mg/L humic and fulvic acid sample, and the unspiked concentrated OH DW sample,
respectively. For ricin, the results were consistent 100% of the time for the contaminant and
interferent PT samples. For the ricin DW samples, two sample sets, the unspiked concentrated
FL DW, and the spiked NY DW generated mixed results. Overall, 95% of all the contaminant
and interferent PT sample results and 87% of all of the DW results were obtained in sets of two
or three in which all the individual replicates had the same result, whether positive or negative.


6.4  Lowest Detectable  Concentration

The lowest detectable concentration of each target contaminant was defined as the lowest
concentration of contaminant-only PT sample to have at least two out of three positive results.
For anthrax, that concentration was 8 x 107  spores/mL (Battelle spores), 7 x 108 spores/mL
(Dugway spores), and 3 x 104 cfu/mL (vegetative cells); for botulinum toxin, 0.01 mg/L (Type
A); 0.05 mg/L (Type B); and for ricin, 0.035 mg/L.
                                           28

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6.5 Other Performance Factors

Battelle technicians, who had been trained by Tetracore to perform testing using the BioThreat
Alert® test strips, performed all of the required laboratory testing. The technicians had no
problem performing the tests as they were trained. The BioThreat Alert® test strips do not require
the use of a reader because the zone of the test strip that changes color is exposed and therefore
the results can be read visually. However, because Tetracore recommended using the reader to
improve the sensitivity of the test strips and remove human bias from the results, a reader was
used to determine positive or negative results during the verification test. The reader comes in a
rugged carrying case that weighs approximately 20 pounds and is about the size of a
medium-sized suitcase.

To test the ability of the BioThreat Alert® test strips to be used outside a laboratory environment
and by a non-trained user, both a trained operator and person without any training in the
sciences or in the operation of the BioThreat Alert® test strips were given a liquid sample (DI
water) and told to analyze the sample three times. The non-technical person was guided only by
the instructions provided with each test strip. The experienced operator analyzed this sample in
the correct way. The non-technical operator followed the instructions properly and tested all
three samples without error. Each of the six DI water samples correctly produced negative
results. The Verification Test Coordinator observed both operators and made this assessment.

Over 400 BioThreat Alert® test strips were tested during the verification test; all except five
functioned properly. In two of those instances, the identification microchip inside the test strip
was not recognized  by the reader, so the type of test (ricin) had to be manually selected using the
reader's arrow buttons, and the test strip was then read by the reader.  Additionally, the following
three problems caused a test strip to be discarded and the sample re-analyzed using a new test
strip: (1) lack of sample flow after the application of the required 6 drops of sample; (2) after a
sample had flowed across the strip, no control line was visible; and (3) the reader generated a
"control line out of  range" that results when the reader does not detect an adequately intense
control line. Overall, 99% of the test strips functioned properly. The verification staff were able
to test approximately 20 samples per hour.
                                           29

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                              Chapter 7
                        Performance Summary
Table 7-1. Anthrax Summary Table
Parameter
Qualitative
contaminant
results
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Positive
Actual Results Out
Fortified Anthrax of Total
Sample Information Concentration'3' Replicates
8 x 108 spores/mL 3/3
Battelle -prepared, 8 x 1 07 spores/mL 3/3
phenol-preserved spores 8 x 106 spores/mL 0/3
8 x 105 spores/mL 0/1
4xl06cfu/mL 1/1
3xl05cfu/mL 1/1
Vegetative cells _ , «4 ,, , T _ „
5 3 x 104cfu/mL 2/3
3xl03cfu/mL 0/1
7x 108spores/mL 2/2
Dugway -prepared spores _ . ,7 , , _ n
5 }i F v 8 x 107spores/mL 0/1
230 mg/L Ca and 90 mg/L Mg 1 x 108 spores/mL00 0/3
2.5 mg/L humic acid and 2.5 mg/L , , n8 / T rw
,, . e . e 1 x 10s spores/mL™
ml vie acid 3/3
Concentrated CA 1 x 108 spores/mL00 2/3
Concentrated NY 1 x 108 spores/mL® 1/3
Unconcentrated DW 1 x 106 spores/mL 0/24
1 x 105 spores/mL ... „,,
n .„ , . . . unspiked 0/3
Bacillus thurmgiensis
Two false positives resulted from the analysis of the DW samples. One out of
three replicates for each of the FL D W and concentrated NY D W falsely generated
positive results. Bacillus thuringiensis was prepared at concentrations much lower
than the lowest detectable concentration of Bacillus anthracis. Therefore, negative
results with these samples do not necessarily indicate a lack of cross-reactivity.
None of three results was positive for the 230-mg/L Ca and 90-mg/L Mg spiked
with a detectable concentration of anthrax. In addition, one and two false negative
results were generated for the concentrated CA and concentrated NY DW samples,
respectively. Bio Threat Alert® test strips were not able to detect anthrax spores at
the vendor-stated LOD. All of the unconcentrated DW samples were spiked at
concentrations less than detectable by the test strips and, therefore, were, as
expected, negative.
96% (25 of 26 replicates) of the contaminant and interferent PT sample results
were obtained in replicate sets in which all the individual replicates had the same
result, whether positive or negative. This was the case for 78% of the DW
samples.
                                  30

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Table 7-1.  Anthrax Summary Table (continued)
Parameter
Lowest detectable concentration
Other performance factors
Sample Information
8 x 107 spores/mL - Battelle prep; 7x 108 spores/mL - Dugway prep (vendor-stated
LOD: 1 x 105 spores/mL); 3 x 104 cfu/mL - vegetative anthrax (no vendor-stated
LOD)
All components for testing were provided in a box of 25 test strips; the strip reader
used during the verification test was powered using electricity or batteries, was
easy to operate, and was contained in a rugged carrying case; test strips and reader
were used easily inside and outside a laboratory with trained operator;
non-technical operator performed tests as well as a trained operator; and sample
throughput was 20 samples per hour.
(a)  The uncertainly of the enumeration technique was approximately 15%.
*• '  Battelle-prepared, phenol-preserved spores.
                                               31

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Table 7-2. Botulinum Toxin Summary Table
Parameter
Qualitative
contaminant
positive
results
Contaminant-
only PT
samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Other performance factors
Botulinum Toxin Positive Results Out
Sample Information Concentration (mg/L) of Total Replicates
0.01 3/3
0.05 3/3
Type A
0.1 3/3
0.5 3/3
0.01 1/3
0.05 3/3
Type B 0. 1 3/3
0.3 3/3
0.5 3/3
n AM m 3/3 Type A
CaandMg 0.1 Jr
& 6/6 Type B
Humic acid and fulvic „ , 3/3 Type A
acid ' 6/6 Type B
Concentrated DW 0.1 n/io?*6 ™
12/12 TypeB
Unconcentrated DW 0.1 12/12 Type B
0.1 mg/L
, . . . . , unspiked 1/3
Lipopolysaccharide
There was one false positive replicate out of three for the unspiked 2.5-mg/L
humic and fulvic acid interferent PT sample; the unspiked concentrated OH
DW sample and the lipopolysaccharide each generated one false positive result
out of three replicates.
No false negatives resulted from the analysis of the interferent and DW samples
spiked with detectable levels of Types A and B botulinum toxin.
92% of the results were obtained in replicate sets in which all the individual
replicates had the same result, whether positive or negative.
0.01 mg/L (Type A); 0.05 mg/L (Type B) (vendor- stated LOD for both Types
A and B: 0.01 mg/L)
All components for testing were provided in a box of 25 test strips; the strip
reader used during the verification test was powered using electricity or
batteries, was easy to operate, and was contained in a rugged carrying case; test
strips and reader were used easily inside and outside a laboratory with trained
operator; non-technical operator performed tests as well as a trained operator;
and sample throughput was 20 samples per hour.
                                       32

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Table 7-3. Ricin Summary Table
Parameter
Qualitative
contaminant
positive
results
Contaminant-only
PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Other performance factors
Ricin Concentration Positive Results Out of
(mg/L) Total Replicates
0.035 3/3
0.2 3/3
0.4 3/3
2 3/3
15 3/3
Ca and Mg 0.4 6/6
Humic acid and _ . , , ,
f . . ., 0.4 6/6
tulvic acid
Concentrated DW 0.4 12/12
Unconcentrated DW 0.4 11/12
T°-4mg^ K unspiked 0/3
Lectm irom soybean
The unspiked concentrated FL DW generated two false positives out of
three. All other DW and cross-reactivity samples resulted in correctly
negative responses.
There was one false negative out of three for the NY D W sample spiked
with a detectable concentration of ricin. The other spiked interferent and
DW samples were correctly determined to be positive.
100% of the contaminant and interferent PT results were obtained in
replicate sets in which all the individual replicates had the same result,
whether positive or negative. That was the case 88% (14 out of 16) of the
time for the DW samples.
0.035 mg/L (Vendor-stated LOD: 0.035 mg/L)
All components for testing were provided in a box of 25 test strips; the strip
reader used during the verification test was powered using electricity or
batteries, was easy to operate, and was contained in a rugged carrying case;
test strips and reader were used easily inside and outside a laboratory with
trained operator; non-technical operator performed tests as well as a trained
operator; and sample throughput was 20 samples per hour.
                                         33

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                                    Chapter 8
                                    References
1.   Personal communication with Dick Burrows, U.S. Army Center for Health Promotion and
    Preventive Medicine.

2.   U.S. EPA threat prioritization study provided by Steve Allgeier, U.S. EPA Office of Water.

3.   Center for Defense Information Fact Sheet: Ricin, www.cdi.org/terrorism;ricin-pr.cfm.

4.   Test/QA Plan for Verification of Immunoassay Test Kits, Battelle, Columbus, Ohio,
    January 2004.

5.   U.S. EPA Method 180.1, "Turbidity (Nephelometric)," Methods for the Determination of
    Inorganic Substances in Environmental Samples, EPA/600/R-93/100, August 1993.

6.   American Public Health Association, et al. Standard Methods for the Examination of Water
    and Wastewater.  19th Edition, Washington, D.C., 1997.

7.   U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020,
    March 1983.

8.   U.S. EPA Method 200.8, "Determination of Trace Elements in Waters and Wastes by
    Inductively-Coupled Plasma Mass Spectrometry," in Methods for the Determination of
    Organic Compounds in Drinking Water, Supplement I, EPA/600/R-94/111, October 1994.

9.   U.S. EPA Method 524.2, "Permeable 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, August 1995.

10. U.S. 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,
    August 1995.
                                         34

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11.  Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
    Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle,
    Columbus, Ohio, March 2004.
                                         35

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