September 2004
Environmental Technology
Verification Report
ADVNT Biotechnologies
BADD™ Anthrax, Botulinum Toxin,
and Ricin Immunoassay Test Strips
Prepared by
Battelle
Battelle
The B usiness of Innovation
Under a cooperative agreement with
£EPA U.S. Environmental Protection Agency
ETV ElV ElV

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September 2004
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
ADVNT Biotechnologies
BADD™
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.
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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.
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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.
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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 	3
3.1	Introduction	3
3.2	Test Samples 	 5
3.2.1	Performance Test Samples	6
3.2.2	Drinking Water Samples 	7
3.2.3	Quality Control Samples 		8
3.3	Test Procedure		8
3.3.1	Laboratory Testing	 8
3.3.2	Non-Laboratory Testing	 8
3.3.3	Drinking Water Characterization	9
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 	25
6.2	False Positive/Negative Responses 	25
6.2.1	Interferent PT Samples	25
6.2.2	DW Samples 	27
6.2.3	Cross-Reactivity PT Samples	28
6.3	Consistency	28
6.4	Lowest Detectable Concentration 	29
6.5	Other Performance Factors 	29
7	Performance Summary	31
8	References 	35
Figures
Figure 2-1.	ADVNT BADD™ Immunoassay Test Strips	2
Tables
Table 3-1.	Lethal Dose and Source of Contaminants 	4
Table 3-2.	Performance Test Samples	 5
Table 3-3.	Drinking Water Samples 	6
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	25
Table 6-2.	Interferent PT Sample Results 	26
Table 6-3.	DW Sample Results	27
Table 6-4.	Potentially Cross-Reactive PT Sample Results 	28
Table 7-1.	Anthrax Summary Table 	31
Table 7-2.	Botulinum Toxin Summary Table 	33
Table 7-3.	Ricin Summary Table	34
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List of Abbreviations
AMS
Advanced Monitoring Systems
ATEL
Aqua Tech Environmental Laboratories, Inc
BADD™
Biowarfare Agent Detection Device
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
L
liter
LOD
limit of detection
MB
method blank
Mg
magnesium
mg/L
milligram per liter
|iL
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
vii

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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 ADVNT Biotechnologies Biowarfare Agent Detection
Devices (BADD™) 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 ADVNT Biotechnologies BADD™. BADD™ test strips
(Figure 2-1) are self-contained, qualitative assays for screening environmental samples for the
presence of anthrax, botulinum toxin, and ricin. These test strips work on similar principles, but
each is single use and can detect only one contaminant. The following is a description of the
BADD™ system based on information provided by the vendor. The information provided below
was not subjected to verification in this test.
The BADD™ test strips are stored in resealable packages, which include all the items necessary
to analyze each sample. Each individually packaged test includes approximately 250 microliters
(|iL) of buffer in a small plastic screw-top vial, a sample collection swab, a bulb syringe, the test
strip (within its own sealed package), and step-by-step instructions. This package is approxi-
mately 5 inches (12.7 centimeters) by 6 inches (15.2 centimeters) and weighs only a few ounces.
The vendor suggests that the
resealable package be used as a
sealed waste receptacle for all
testing materials.
The testing procedure involves
dipping the dry collection swab
into a solution suspected of
containing anthrax, botulinum
toxin, or ricin, followed by eluting
(extracting) the collected sample
into a collection tube containing a
sample diluent. After the sample is
collected, it is transferred onto the
BADD™ test strip where
dye-labeled antibodies detect trace amounts of the contaminant collected by the swab, as
indicated by the presence of two bands in the test result window. After 15 minutes, the results
are read visually.
BADD™ test strips are sold in boxes of 10 for approximately $250 per box.
Figure 2-1. ADVNT BADD™ Immunoassay Test Strips
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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 contaminants of interest. The
contaminants, or antigens, react with a selective antibody to produce a result that is indicated by
a color change. For the BADD™ test strips, the presence of contaminants is indicated by the
appearance of a colored line within approximately 20 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 BADD™ test strips were subjected to various concentrations of
anthrax spores, botulinum toxin, and ricin in American Society for Testing and Materials Type II
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 concentration, and the
source. The BADD™ 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)
and magnesium (Mg) and calcium (Ca) in DI water both with and without the addition of the
contaminants using the BADD™ test strips. In addition, specificity was evaluated by exposing
the BADD™ 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
Vendor-Stated
LOD
Lethal Dose
Concentration^
Source of Contaminant
Bacillus anthracis
Ames Strain (anthrax)
1 x 106
spores/mL
200 spores/mL(1)
Battelle and U.S. Army
Dugway Proving Ground
Botulinum toxin
Types A and B
0.4 mg/L
0.3 mg/L(2)
Metabiologics, Inc.
(Madison, Wisconsin)
Ricinus communis
Agglutinin II (ricin)
0.4 mg/L
15 mg/L(3)
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 BADD™ test kits was conducted from January 14 through April 23,
2004, according to procedures specified in the Test/QA Plan for Verification of Immunoassay
TestKits.(A) 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, trihalo-
methanes, and haloacetic acids. Battelle confirmed the presence of anthrax spores using plate
enumeration.
The BADD™ 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
BADD™ 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.
Table 3-2. Performance Test Samples
Type of PT
Sample
Sample Characteristics
Approximate Concentrations

Anthrax spores
200 to 1010 sporcs/mL :"
Contaminant-only
Botulinum toxin Type A
0.5 to 25 mg/L
Botulinum toxin Type B
0.3 to 1,000 mg/L

Ricin
0.4 to 2,000 mg/L

Contaminants in 46 mg/L Ca and
18 mg/L Mg
Anthrax - 107 spores/mL
Botulinum toxin (Type B) - 4 mg/L
Ricin - 5 mg/L
Interferent
Contaminants in 230 mg/L Ca and
90 mg/L Mg
Anthrax - 107 and 10s spores/mL
Botulinum toxin (Type A) - 5 mg/L
Botulinum toxin (Type B) - 4 mg/L
Ricin - 5 mg/L

Contaminants in 0.5 mg/Lhumic
acid and 0.5 mg/L fulvie acid
Anthrax - 107 spores/mL
Botulinum toxin (Type B) - 4 mg/L
Ricin - 5 mg/L

Contaminants in 2.5 mg/L humic
acid and 2.5 mg/L ful vie acid
Anthrax - 107 and 10s spores/mL
Botulinum toxin (Type A) - 5 mg/L
Botulinum toxin (Type B) - 4 mg/L
Ricin - 5 mg/L

Bacillus thuringiensis (anthrax
analogue)
106 spores/mL
Potentially
Cross-reactive
Lipopolysaccharide
(botulinum toxin analogue)
5 mg/L

Lectin from soybean
(ricin analogue)
4 mg/L
w 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.
DW samples were analyzed using the BADD™ 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 BADD™ test strips.
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Table 3-3. Drinking Water Samples
Drinking Water Sample Description
Water Utility
Water
Treatment
Source
Type
Cone. /
Unconc.
Anthrax
(spores/mL)
Botulinum
Toxin
(mg/L)
Ricin
(mg/L)
Metropolitan
Water District of
California (CA)
filtered
chloraminated
surface
conc.
unspiked
107
10s
unspiked
4(Type B)
5(Type A)
unspiked
5
New York City,
New York (NY)
unfiltered
chlorinated
surface
conc.
unspiked
107
10s
unspiked
4(Type B)
5(Type A)
unspiked
5
Metropolitan
Water District of
California (CA)
filtered
chloraminated
surface
unconc.
unspiked
107
unspiked
4
unspiked
5
New York City,
New York (NY)
unfiltered
chlorinated
surface
unconc.
unspiked
107
unspiked
4
unspiked
5
Columbus, Ohio
(OH)
filtered
chlorinated
surface
both
unspiked
107
unspiked
4
unspiked
5
Orlando, Florida
(FL)
filtered
chlorinated
ground
both
unspiked
107
unspiked
4
unspiked
5
Approximate Contaminant
Concentrations
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 also were obtained from Dugway Proving Ground and 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 BADD™ test strips. Preliminary results indicated that only the
highest concentration PT sample produced positive results for ricin and anthrax, and there were
very few positive results for botulinum toxin Type B; therefore, the original test/QA plan was
amended to include preparing and analyzing higher concentration samples of anthrax and ricin
and testing a preparation of anthrax spores that were never preserved in phenol, a second source
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of anthrax spores, vegetative anthrax cells, and botulinum toxin Type A. 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
(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 BADD™ test strips to false negative results due to interferents, the test/QA
plan was amended to include the fortification of detectable types and concentrations of
contaminants 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 BADD™ 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 196 PT samples was analyzed by the BADD™ test strips for
this test. The results provided information about how well the BADD™ test strips detected the
presence of each contaminant at several concentration levels, the consistency of the responses,
and the susceptibility of the BADD™ 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 BADD™ 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 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. During the expanded DW testing for anthrax, a concentration level of
approximately 108 spores/mL was used to spike the DW samples. A total of 156 DW samples
was analyzed by the BADD™ test strips for this test.
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3.2.3 Quality Control Samples
In addition to the 352 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.
A control line in the result window on the BADD™ test strips appeared during the analysis of
each sample that indicated to the operator that the test kit was functioning properly. If this
control line did not appear, that test kit would be discarded and a new test strip used. There were
no such instances during testing. Because of this control line on the test strips, 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. In most cases, three
replicates of each test sample were analyzed. The testing procedure included the following steps
for analyzing liquid samples for the presence of anthrax spores, botulinum toxin, or ricin: (1) the
test strip was removed from its package; (2) the provided swab was submerged in the test sample
(minimum of 2 mL in a small test tube) for approximately 5 seconds to absorb the liquid sample;
(3) the swab was transferred into the buffer solution and pressed against the inside of the vial for
approximately 10 seconds to squeeze the sample out of the swab and mix the buffer/sample
solution; (4) the dropper was used to transfer 5 drops of the buffer/sample solution to the sample
well on the test strip (a sixth drop was added to spur the movement of the buffer/sample solution
across the adsorbent strip if the solution did not begin moving after 5 drops); and (5) for
anthrax, the strip was read after 15 minutes, for botulinum toxin and ricin, after 10 minutes. The
appearance of a colored line under the "C" on the test strip indicated that the proper procedure
had been followed and the test strip had functioned properly. Because the BADD™ test strips
did not require a reader to determine the positive or negative response, the result was read
visually. The vendor instructed Battelle to consider the appearance of a colored line under the
"T" on the test strip a positive response.
3.3.2	Non-Laboratory Testing
Because the toxic nature of these contaminants did not permit their use outside special
laboratory facilities, MB samples were analyzed at a non-laboratory location to evaluate the
BADD™ performance and ease of use outside of the laboratory. Both a trained technician and a
non-technical/untrained, first-time user performed analyses at a non-laboratory location. The
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purpose of these analyses was to test the performance of the BADD™ in a non-laboratory
setting, not to evaluate thoroughly the effect of changing conditions such as temperature and
humidity on the BADD™. Initially, the non-technical/untrained, first-time user was guided only
by the manual or by the vendor instructions. If the operators were about to complete the test
incorrectly, the Verification Test Coordinator prompted them to re-evaluate the 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.
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 BADD™ test strips.
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Table 3-4. ATEL Water Quality Characterization of Drinking Water Samples
Sources of Drinking Water Samples



Columbus,
Orlando,
New York City,
MWD,



Ohio
Florida
New York
California
Parameter
Unit
Method
(OH DW)
(FL DW)
(NY DW)
(CA DW)
Turbidity
NTU
EPA 180.1(5)
0.2
0.5
1.3
0.1
Dissolved
organic carbon
mg/L
SM 5310(6)
2
2
2
2
Total organic
carbon
mg/L
SM 5310(6)
2
2
2
2
Specific
conductivity
|iS/cm2
SM 2510(6)
357
325
85
740
Alkalinity
mg/L
SM 2320(6)
55
124
4
90
pH

EPA 150.1(7)
7.33
7.93
6.80
7.91
Calcium
mg/L
EPA 200.8®
42
41
5.7
35
Magnesium
mg/L
EPA 200.8(8)
5.9
8.4
19
1.5
Hardness
mg/L
EPA 130.2(7)
125
137
28
161
Total organic
halides
\ig/L
SM 5320(6)
360
370
310
370
Trihalomethanes
|ig/L/
analyte
EPA 524.2(9)
26.9
80.9
38.4
79.7
Haloacetic acids
l-ig/L/
analyte
EPA 552.2(10)
23.2
41.1
40.3
17.6
NTU = nephelometric turbidity unit
MWD = Metropolitan Water District
|_iS/cm2 = microSiemen per square centimeter
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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 11-007, Standard Operating
Procedure for Chain of Custody for Dioxin/Furan 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 BADD™ 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 characterization and con-
firmation of the possible interferents, analytical equipment was calibrated by ATEL according to
the procedures specified in the appropriate standard methods. Pipettes used during the verifica-
tion 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 supernatant
consisting of the phenol/water solution was decanted from the spores, 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 5.26 xlO9 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 and had not been characterized like the previously described lot. The second
lot had been subjected only to enumeration in order 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 BADD™ 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. Cells from an enumeration of the Battelle-prepared, phenol-preserved spores were collected,
placed in solution, and then enumerated to determine the concentration. No further characteriza-
tion was performed on these cells. Solutions of these cells were used to determine the sensitivity
of the BADD™ 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 and BL-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 |j,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 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 (RPD)
between the two are given in the table. 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 = 1 E ' x 100%
For the Battelle-prepared, phenol-preserved spores, only one enumeration resulted in a concen-
tration 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

Expected
Actual

Description (units)
Date
Concentration
Concentration^
RPD

January 28
53
58
9

January 28
58
53
9
Battelle-prepared,
January 30
53
61
15
phenol-preserved
February 2
61
53
14
stock solution
February 10
61
82
55
(108 spores/mL)
February 26
82
63
23

March 1
63
67
5

March 23
67
57
14
Battelle-prepared,
January 28
10
7.8
22
phenol-preserved serial
January 30
40
32
20
dilution validations



(104 spores/mL)
March 2
10
7.7
24

March 23
1,000
992
1
Battelle-prepared, non-
February 5
Unknown
14
NA
phenol-preserved



(108 spores/mL)
February 12
14
106
657
Vegetative anthrax
March 23
Unknown
26
NA
(104 cfu/mL)
March 24
260
350
35

March 22
Unknown
666
NA
Dugway-prepared
March 23
0.010
0.0081
19
(106 spores/mL)
March 24
10
8.0
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 109 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 BADD™, 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
ADVNT had claimed, additional PT 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


Expected
Actual

Sample
Date
Concentration
Concentration^

Description
(2004)
(105 spores/mL)
(105 spores/mL)
RPD
Cone. CA DW
January 28
10
0.38
96
Cone. CA DW
January 30
100
8.7
91
Unconc. CA DW
January 30
40
8
80
i) 5 mu 1. OC
I'chruaiy 2
15
10
i)
1 J
' J\
'ic
w
f-s
I'chruaiy 3
15
10
i)
23o mu 1. C"a
l>o mu 1. Mu
1 'chinai\ 3
15
5 o
03
4o mu 1. ("a
1 S mu 1. Mu
1 'chiliai\ 3
15
S 3
45
Cone. CA DW
February 3
15
6.9
54
Unconc. CA DW
February 3
15
6.5
57
Cone. OH DW
February 3
15
5.7
62
Unconc. OH DW
February 3
15
6.9
54
Cone. NY DW
February 3
15
13
17
Unconc. NY DW
February 3
15
12
21
Cone. FL DW
February 3
15
9.1
39
Unconc. FL DW
February 3
15
7.5
50
Cone. NY DW
March 3
1,000
933
7
Cone. CA DW
March 3
1,000
1,100
10
1 J
' J\
'ic
w
f-s
March 3
1 .ODD
<.)<.)}
1
23o mu 1. Ca
l>o mu 1. Mu
March 3
1 .ODD
1 .01)1)
0
2 5 mu 1. OC
March 23
1 .odd
l>02
4
Cone (A l)\V
March 23
1 .1)1)1)
44S
5 5
23o mu 1. ("a
l>o mu 1. Mu
March 24
1 .()()()
7SS
21
Cone. NY DW
March 24
1,000
486
51
OC = Organic carbon (humic and fulvic acids).
Shading on table distinguishes the interferent and cross-reactivity PT samples from the DW samples.
(a)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
Responsible
Partv
Where Recorded
How Often
Recorded
Disposition
of Data
Dates and times of test
events
Battelle
ETV data sheets
Start/end of test,
and at each
change of a test
parameter
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
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
procedures and sample
results
Battelle
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
procedures and sample
results
ATEL
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 BADD™ 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 BADD™ 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 BADD™ performance such as ease of use, field portability, and sample through-
put are discussed in Section 6. Also addressed are qualitative observations of the verification
staff pertaining to the performance of the BADD™.
20

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Chapter 6
Test Results
6.1 Qualitative Contaminant Presence/Absence
The responses for the BADD™ test strips using the contaminant-only PT samples containing
anthrax, botulinum toxin, and ricin are discussed in the following sections. The BADD™ test
strips provide indication of only a positive or negative response based on whether or not a red
line appears under the "T" (for test) after a liquid test sample is applied to the adsorbent strip. A
red line appears under the "C" (for control) after every test sample regardless of whether or not
the target contaminant is present.
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 only the 4 x 107 concentration level (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. The results of this enumeration confirms the concentration
of spores in these samples. After discussions with ADVNT Biotechnologies, 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 BADD™ to the affected spores.)
2.	The sensitivity of the BADD™ 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.
3.	The BADD™ test strips are more sensitive to vegetative anthrax cells than spores. (This
hypothesis stemmed from the analysis of one sample that was prepared by collecting a single
vegetative anthrax colony from an enumeration plate and placing it into DI water and mixing
well. This sample produced one out of two positive results using the BADD™ test strips;
however, the solution was not enumerated so the concentration was not known.)
21

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Table 6-1 a. Anthrax Contaminant-Only PT Sample Results
Purpose of
Analysis
Actual Fortified
Concentration'*0
Anthrax
Description
Prep
Location
Phenol-
Preserved
Positive
Results Out
of Total
Replicates

200 spores/mL(b)
Spores
Battelle
Yes
0/3
Original test/QA
plan PT samples
8 x 105 spores/mL(c)
3 x 106spores/mL
8 x 106 spores/mL
Spores
Spores
Spores
Battelle
Battelle
Battelle
Yes
Yes
Yes
0/3
0/3
0/3

4 x 107 spores/mL
Spores
Battelle
Yes
2/3

5 x 107 spores/mL
Spores
Battelle
No
2/3
Investigation of
phenol storage of
1 x 109 spores/mL
8 x 108 spores/mL
Spores
Spores
Battelle
Battelle
No
Yes
2/2
2/2
spores
1 x 1010 spores/mL
Spores
Battelle
No
2/2

8 x 109 spores/mL
Spores
Battelle
Yes
2/2

8 x 108 spores/mL
Spores
Battelle
Yes
3/3
Sensitivity
determination
8 x 107 spores/mL
8 x 106 spores/mL
Spores
Spores
Battelle
Battelle
Yes
Yes
3/3
0/3

8 x 10s spores/mL
Spores
Battelle
Yes
0/3
Alternate spore
preparation
8 x 107 spores/mL
8 x 106 spores/mL
Spores
Spores
Dugway
Dugway
No
No
3/3
0/1

Unknown Cone.
Vegetative
Battelle
NA
1/2
Vegetative cell
sensitivity
4 x 106cfu/mL
3 x 105cfu/mL
Vegetative
Vegetative
Battelle
Battelle
NA
NA
1/1
2/3

3 x 104cfu/mL
Vegetative
Battelle
NA
0/1
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 uncertainty of the enumeration technique is approximately 15%.
(b:i Lethal dose concentration.
^ This concentration is very close to the vendor-stated LOD.
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 BADD™ 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 BADD™ test strips;
increasing the concentration of spores beyond what was required by the test/QA plan; 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; and testing the response of the
test strips to vegetative anthrax cells at various concentrations.
To address the possibility that storing spores in phenol affected the sensitivity of the BADD™, 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 Analyses, Investigation of phenol storage of spores." Both solutions had been
prepared at Battelle using the same preparation method. The 5 x 107 spores/mL sample made
with spores not stored in phenol produced positive results in two out of three samples, as did the
4 x 107 spores/mL sample made with spores that had been 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. All four samples were detectable in each of two
replicates analyzed. These results suggested that the effect of phenol storage was probably
inconsequential to the sensitivity of the BADD™ 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
BADD™ 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 by tenfold factors several times.
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 deter-
mined in this manner, the lowest detectable concentration of Dugway spores was 108 spores/mL,
a level similar to that determined for the Battelle-prepared spores.
The third explanation of the results 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
detectable concentration of vegetative cells was 10s colony-forming units (cfu)/mL, an order of
magnitude lower than what the vendor claimed to be able to attain for anthrax spores. While
23

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ADVNT Biotechnologies has not provided information with regard to the BADD™ test kit's
vegetative cell sensitivity, these results suggest that BADD™ test strips are much more sensitive
to vegetative cells than to spores.
6.1.2 Botulinum Toxin
The results obtained for the PT samples containing botulinum toxin are given in Table 6-lb.
Upon analyzing the first five concentration levels listed in the test/QA plan, the only positive
results were produced from one replicate out of three at the 2 and 4 mg/L concentration levels,
with no positive responses at concentrations above or below those levels. In response to these
results, ADVNT Biotechnologies informed us that their test strips were only sensitive to
botulinum toxin Type A. Since botulinum toxin Type B was described for use in the test/QA
plan, samples were not initially analyzed using botulinum toxin Type A. To more completely
verify the BADD™ test strips, a limited amount of expanded testing was conducted by
analyzing four PT samples containing a range of concentration levels (0.5, 2, 5, and 25 mg/L) of
botulinum toxin Type A and two higher concentration levels (200 and 1,000 mg/L) of botulinum
toxin Type B. The results showed that the BADD™ test strips were sensitive to botulinum toxin
Type A at approximately 5 mg/L, but were not able to reproducibly detect botulinum toxin
Type B at concentration levels up to 1,000 mg/L.
Table 6-1 b. Botulinum Toxin Contaminant-Only PT Sample Results
Purpose
Concentration
Type of Botulinum
Positive Results Out of Total
of Analysis
(mg/L)
Toxin
Replicates

0.3(a)
B
0/3

0.4®
B
0/3
Original test/QA plan
2
B
B
1/3
1/3
PT samples
4

20
B
0/3

0.5
A
1/3

2
A
0/3
Expanded testing
5
25
A
A
3/3
3/3

200
B
0/3

o
O
O
B
0/3
^ Lethal dose concentration.
(b) Vendor-stated LOD.
24

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6.1.3 Ricin
The results obtained for the PT samples containing ricin are given in Table 6-lc. Upon
analyzing the first five concentration levels listed, the only positive results were produced at the
20 mg/L concentration level. Two additional concentration levels (200 and 2,000 mg/L) were
analyzed to better define the performance of B ADD™ for the detection of ricin. Positive results
were obtained at each of these additional concentration levels.
Table 6-lc. Ricin Contaminant-Only PT Sample Results
Purpose	Concentration	Positive Results Out of
of Analysis (mg/L)	Total Replicates
0.4(a)	0/3
2	0/3
Original test/QA plan	.
PT samples
15(b)	0/3
20	3/3
200	3/3
Expanded testing
	2,000	3/3	
(a)	Vendor-stated LOD.
(b)	Lethal dose concentration.
6.2 False Positive/Negative Responses
Three types of samples were analyzed to evaluate the susceptibility of BADD™ 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 contaminant; 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 and botulinum toxin,
25

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Table 6-2. Interferent PT Sample Results
Positive Results Out of Total Replicates

Anthrax (spores/mL)
Botulinum Toxin (mg/L)
Ricin (mg/L)
Interferent




Type B Type A

Sample
Blank
1 x I07(a)
1 x 108
Blank
4
5
Blank 5
46 mg/L Ca
18 mg/L Mg
0/3
0/3(b)
6 x 106
NA
0/3
0/3
NA
0/3 0/3
230 mg/L Ca
90 mg/L Mg
0/3
0/3(b)
4 x 106
3/3(h)
1 x io8
0/3
0/3
3/3
0/3 0/3
0.5 mg/L
humic and
0/3
0/3(b)
1 x io7
NA
0/3
0/3
NA
0/3 0/3
fulvic acids






2.5 mg/L
humic and
0/3
0/3(b)
1 x io7
3/3(h)
1 x io8
0/3
0/3
3/3
0/3 0/3
fulvic acids





NA = not applicable. Sample not analyzed during expanded testing.
Expected concentration.
(b) Actual concentration.
expanded testing included additional interferent PT samples (a higher concentration in the case
of anthrax and a different type in the case of botulinum toxin). No expanded testing involving
interferent PT samples was done for the ricin test strips.
When interferent solutions not fortified with target contaminants were analyzed, no false
positive results occurred for the test strips specific for any of the three target contaminants. The
lack of detectable results at 1 x 107 spores/mL for anthrax, 4 mg/L for botulinum toxin Type B,
and 5 mg/L for ricin indicated false negative responses with respect to the vendor-stated LOD;
however, because those tested concentration levels for anthrax were not detectable when
analyzed in DI water and the test strips were not sensitive to botulinum toxin Type B (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 and
botulinum toxin Type A. For both anthrax spores and botulinum toxin, there were no false
negative responses for the expanded testing. The lower concentration interferent matrix was not
analyzed during the expanded testing of anthrax and botulinum toxin samples.
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6.2.2 DWSamples
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 and botulinum toxin, expanded testing
included additional DW samples (a higher concentration in the case of anthrax and a different
type in the case of botulinum toxin) fortified with those two target contaminants. No expanded
testing involving DW samples was performed for the ricin test strips.
Table 6-3. DW Sample Results
Positive Results Out of Total Replicates

Anthrax (spores/mL)
Botulinum Toxin (mg/L)
Ricin (mg/L)
DW Sample
Blank
lxl07(a)
lxlO8
Blank
Type B
4
Type A
5
Blank
5
Unconcentrated
CA DW
0/3
4
0/3
x io6
(b)
NA
0/3
0/3
NA
0/3
0/3
Concentrated CA
DW
0/3
4
0/3
x io6
(b)
OO
£2 o
m 1—1
X
0/3
1/3
3/3
0/3
0/3
Unconcentrated
FL DW
0/3
5
0/3
x io6
(b)
NA
0/3
1/3
NA
0/3
0/3
Concentrated FL
DW
0/3
6
0/3
x io6
(b)
NA
0/3
0/3
NA
0/3
0/3
Unconcentrated
NYDW
0/3
8
0/3
x io6
(b)
NA
0/3
0/3
NA
0/3
0/3
Concentrated
NYDW
0/3
9
0/3
x io6
(b)
2/3
1 X 108(b)
0/3
0/3
3/3
0/3
0/3
Unconcentrated
OH DW
0/3
5
0/3
x io6
(b)
NA
0/3
0/3
NA
0/3
0/3
Concentrated
OH DW
0/3
4
0/3
x io6
(b)
NA
0/3
0/3
NA
0/3
0/3
NA = not applicable. Sample not analyzed during expanded testing.
^ Expected concentration.
® Actual concentration.
Table 6-3 shows that there were no false positive results for the test strips specific for any of the
three target contaminants when the unspiked DW samples were analyzed. Two false positives
occurred when the BADD™ test strips were used to analyze DW fortified with botulinum toxin
Type B, which, according to the vendor and as noted in Section 6.1.2, the test strips cannot
27

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detect. These occurred in one unconcentrated FL DW sample and one concentrated CA DW
sample, each generating one out of three positive results. The second column of results under
anthrax and botulinum toxin and all results under ricin are false negative responses with respect
to the vendor-stated LOD; but for the reasons detailed in Section 6.2.1, the negative responses
may not be the result of the DW matrix. Only two DW samples, concentrated CA and
concentrated NY DW, were analyzed during the expanded testing of anthrax and botulinum
toxin samples. For anthrax spores, one out of three samples of 1 x 108 spores/mL in concentrated
DW from NY produced a false negative result; for botulinum toxin Type A, there were no false
negative responses.
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.
Table 6-4. Potentially Cross-Reactive PT Sample Results
Bacillus thuringiensis (5 x 10s spores/mL)(a)
Lipopolysaccharide (5 mg/L)
Lectin from soybean (4 mg/L)	
Positive Results Out of Total
Replicates
Botulinum
Anthrax	Toxin	Ricin
(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 the
consistency of the positive (or negative) results depended on how close the concentration level
was to the lowest detectable concentration for the spores being analyzed. The contaminant PT
sample data using the Battelle-prepared spores showed that the lowest detectable concentration
for the BADD™ was between 107 and 108 spores/mL. At approximately 107 spores/mL (8 x
106 spores/mL of the original test/QA plan PT samples and of the sensitivity determination
samples), there were no positive responses out of six replicates; at concentrations above
108 spores/mL, there were 100% positive responses (14 out of 14 replicates). When analyzing
28

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two samples containing 5 x 107 spores/mL, the results in both cases were two positive responses
out of three replicates.
For botulinum toxin, the 5- and 25-mg/L samples of botulinum toxin Type A produced positive
results in three out of three replicates, while the rest of the samples produced negative results
except for three individual replicates at separate concentration levels. Two of these replicates
were generated from botulinum toxin Type B, which was not detectable, and one was from a
concentration of botulinum toxin Type A that was below the lowest detectable concentration
(false positive results). For ricin, the results were 100% consistent for all the samples analyzed.
Either all replicates within a sample were positive or all were negative. Overall, 90% of the
anthrax results were obtained in sets of two or three in which all the individual replicates had the
same result, whether positive or negative. For botulinum toxin, this statistic was 84%; for ricin,
it was 100%).
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 4 x 107 spores/mL (Battelle-prepared), 8 x 107 spores/mL
(Dugway-prepared) and 3 x 10s cfu/mL (vegetative cells); for botulinum toxin Type A, 5 mg/L;
and for ricin, 20 mg/L.
6.5	Other Performance Factors
Battelle technicians, who had been trained by ADVNT Biotechnologies to operate the BADD™,
performed all of the testing in a laboratory setting. The technicians had no problem performing
the tests as they were trained. To test the ability of the BADD™ test strips to be used outside a
laboratory environment and by a non-trained user, both a trained operator and a person without
any training in the sciences or in the operation of the BADD™ were given a liquid sample (DI
water) and told to analyze the sample three times. Initially, the non-technical person was guided
only by the instructions provided with each test strip. However, if they were about to complete
the test incorrectly, the Verification Test Coordinator prompted them to re-evaluate the
instructions. The experienced operator analyzed the sample in the correct way. The non-
technical operator followed the instructions properly until it was time to drop the sample onto
the test strip. The operator had to be stopped from dropping the sample onto the rectangular
adsorbent rather than the round sample well. A reason for this may have been that the operator
had been using the side of the instruction insert that did not have a diagram of the test strip. The
Verification Test Coordinator directed the operator to the instructions with the diagram, and the
operator dispensed the sample in the correct place. However, the operator initially held the
dropper too close to the sample well, creating many bubbles during dispensing. The Verification
Test Coordinator emphasized that the operator was to apply the sample dropwise, and the
operator corrected the technique. After those two simple directions, the non-technical operator
tested two additional replicates flawlessly. More than 400 BADD™ test strips were used during
the verification test, all except one functioned properly. In that instance, upon application of the
29

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sample, the sample did not move across the adsorbent strip. That test strip was discarded and the
analysis was repeated.
As described in Section 3.3.1, using the BADD™ test strips required the visual determination of
the result. The appearance of even a faint line with a red color under the "T" on the test strip
indicated a positive result. For many of the results that were determined to be positive, the lines
indicating that result were very faint. For anthrax, 38 results were determined to be positive;
and, of those, the technician performing the tests described 17, or 44%, of those results to be
"faintly" or "extremely faintly" positive. The technician, who has normal vision, made this
comment when the indicator line was present, but not obvious at a glance (i.e., the strip had to
be studied closely to determine whether or not the line had appeared). For botulinum toxin, 29%
of the 24 positive results were described that way; for ricin, all 9 of the positive responses were
described in that manner. The verification staff was able to test 20 to 30 samples per hour using
the BADD™ test strips.
30

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Chapter 7
Performance Summary
Table 7-1. Anthrax Summary Table
Parameter
Sample Information
Actual Fortified
Anthrax
Concentration'3'
Positive Results Out
of Total Replicates



8 x 108 spores/mL
3/3

Contaminant-
only PT
samples
Battelle-prepared,
phenol-preserved spores
8 x 107 spores/mL
4xl07 spores/mL
8 x 106 spores/mL
8 x 105 spores/mL
3/3
2/3
0/3
0/3

Vegetative cells
4 x 106 cfu/mL
3 x 105 cfu/mL
1/1
2/3
Qualitative
contaminant
positive


3 x 104 cfu/mL
0/1

Dugway-prepared spores
8 x 107 spores/mL
8 x 106 spores/mL
3/3
0/1
results
Interferent
PT samples
230 mg/L Ca
90 mg/L Mg
2.5 mg/L humic acid
2.5 mg/L fulvic acid
1 x 10s spores/mL®
1 x 108 spores/mL®
3/3
3/3


Concentrated CA
1 x 108 spores/mL®
3/3

DW samples
Concentrated NY
1 x 108 spores/mL®
2/3


Unconcentrated DW
1 x 107 spores/mL®
0/24

Cross-reactivity
5 x 105 spores/mL
Bacillus thuringiensis
unspiked
0/3
False positives
No false positives resulted from the analysis of the interferent, DW, or cross-
reactivity samples. 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.
F alse negatives
One false negative replicate resulted from the analysis of the interferent and
DW samples spiked with detectable levels of anthrax spores (concentrated NY
DW); the BADD™ test strips were not able to detect anthrax spores at the
vendor-stated LOD, but they were able to detect much higher concentration
levels. All of the unconcentrated DW samples were spiked at concentrations
less than detectable by the test strips and, therefore, were, as expected, negative.
Consistency
90% of the results were obtained in replicate sets in which all the individual
replicates had the same result, whether positive or negative.
31

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Table 7-1. Anthrax Summary Table (continued)
Parameter
Sample Information
Lowest detectable concentration
4 x 107 spores/mL - Battelle prep; 8 x 107spores/mL - Dugway prep (vendor-
stated limit of detection [LOD]: 1 x 106 spores/mL);
3 x 105 cfu/mL - vegetative anthrax (no vendor-stated LOD)
Other performance factors
All components for testing were provided in a resealable package weighing just
a few ounces; strips used easily inside and outside a laboratory with trained
operator; non-technical operator needed minor direction from a trained
operator; indicator line color change for the anthrax test strips was very faint
44% of the time, increasing the likelihood of false negative results; and sample
throughput was 20 to 30 samples per hour.
(a) The uncertainty of the enumeration technique was approximately 15%.
® Battelle-prepared, phenol-preserved spores.
32

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Table 7-2. Botulinum Toxin Summary Table
Parameter
Sample Information
Botulinum Toxin
Concentration (mg/L)
Positive Results Out
of Total Replicates



0.5
1/3


Type A
2
5
25
0/3
3/3
3/3
Qualitative
contaminant
positive results
Contaminant-
only PT samples
Type B
0.3
0.4
2
4
20
200
1,000
0/3
0/3
1/3
1/3
0/3
0/3
0/3

Interferent
PT samples
230 mg/L Ca
90 mg/L Mg
2.5 mg/L humic acid
2.5 mg/L fulvic acid
5(a)
5(a)
3/3
3/3


Concentrated CA
5(a)
3/3

DW samples
Concentrated NY
5(a)
3/3


Unconcentrated DW
4(b)
2/24

Cross-reactivity
5mg/L
Lipopoly saccharide
unspiked
1/3
False positives
No false positives resulted from the analysis of the interferent or unspiked
DW samples. There was one false positive replicate out of three when
lipopoly saccharide was analyzed as a possible cross-reactive compound.
F alse negatives
No false negatives resulted from the analysis of the interferent and DW
samples spiked with detectable levels of Type A botulinum toxin; however,
the BADD™ test strips were not able to reproducibly detect Type B
botulinum toxin when spiked into DW or interferent samples at 4 mg/L or
DI water up to 1,000 mg/L.
Consistency
84% of the results were obtained in replicate sets in which all the individual
replicates had the same result, whether positive or negative.
Lowest detectable concentration
5 mg/L (Type A), Type B was not reproducibly detectable, (vendor-stated
LOD for botulinum toxin [non-specific]: 0.4 mg/L)
Other performance factors
All necessary components for testing were provided in a resealable package
weighing just a few ounces; strips used easily inside and outside a laboratory
with trained operator; non-technical operator needed minor direction from a
trained operator; indicator line color change for the botulinum toxin test
strips was very faint 29% of the time, increasing the likelihood of false
negative results; and sample throughput was 20 to 30 samples per hour.
Type A botulinum toxin.
33

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Table 7-3. Ricin Summary Table
Parameter
Ricin
Concentration Positive Results Out
(mg/L) of Total Replicates
Qualitative
contaminant positive
results
Contaminant-only
PT samples
0.4 0/3
2 0/3
5 0/3
15 0/3
20 3/3
200 3/3
2,000 3/3
Interferent PT and
DW Samples
5 0/36
Cross-reactivity
4 unspiked 0/3
Lectin irom soybean
False positives
No false positives resulted from the analysis of the interferent, DW,
or cross-reactivity samples.
F alse negatives
Ricin was not reproducibly detectable when spiked into DW and
interferent samples at 5 mg/L. No expanded testing was done
involving the interferent or DW samples.
Consistency
100% of the results were obtained in replicate sets in which all the
individual replicates had the same result, whether positive or
negative.
Lowest detectable concentration
20 mg/L (vendor-stated LOD: 0.4 mg/L)
Other performance factors
All necessary components for testing were provided in a resealable
package weighing just a few ounces; strips used easily inside and
outside a laboratory with trained operator; non-technical operator
needed minor direction from a trained operator; indicator line color
change for the ricin test strips was very faint for every positive
response, increasing the likelihood of false negative results; and
sample throughput was 20 to 30 samples per hour.
34

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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.
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.
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