September 2006
Environmental Technology
Verification Report
PHARMALEADS
EzyBot® A and EzyBot® B Test Kits
Prepared by
Batreiie
Tim Business .-/Innovation
Under a cooperative agreement with
SEPA
U.S. Environmental Protection Agency
ETV ETVETV
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THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
I'KUIjKWI >
ETV
X^FPA Baltelle
J/it? Business 0/Innovs
5. Environmental Protection Agency
ETV Joint Verification Statement
TECHNOLOGY TYPE: IMMUNOASSAY TEST KITS
APPLICATION: DETECTING BOTULINUM TOXIN
TECHNOLOGY NAME: EzyBot® A and EzyBot® B Test Kits
COMPANY: PharmaLeads
ADDRESS: 11/13 rue Watt
75013 Paris PHONE +33 1 44 85 62 21
FRANCE FAX: +33 1 44 85 62 27
WEB SITE: www.pharmaleads.com
E-MAIL: Jean-pierre.rogala@pharmaleads.com
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology Verification (ETV)
Program to facilitate the deployment of innovative or improved environmental technologies 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. Information and ETV
documents are available at www.epa.gov/etv.
ETV works in partnership with recognized standards and testing organizations, with stakeholder groups (consisting
of buyers, vendor organizations, and permitters), and with individual technology developers. The program evaluates
the performance of innovative technologies 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 Advanced Monitoring Systems (AMS) Center, one of six technology areas under ETV, is operated by Battelle
in cooperation with EPA's National Exposure Research Laboratory. The AMS Center evaluated the performance of
immunoassay test kits used to detect botulinum toxin in water. This verification statement provides a summary of
the test results for the PharmaLeads EzyBot® A and B test kits.
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VERIFICATION TEST DESCRIPTION
The verification test for the EzyBot® test kits was conducted at Battelle between November 2005 and January 2006
according to procedures specified in the Test/QA Plan for Verification of Immunoassay Test Kits for the following
parameters: contaminant presence/absence; false positive/false negative response to interferents, drinking water
(DW) matrix effects, and cross-reactivity; consistency; lowest detectable concentration; field portability; ease of
use; and sample throughput. The ability of the EzyBot® test kits to detect various concentrations of botulinum toxin
was evaluated by analyzing performance test (PT) and DW samples. PT samples included American Society for
Testing and Materials Type II deionized (DI) water fortified with the target contaminant, an interferent, both, or
only a cross-reactive species. Target analytes were added to DI water at lethal dose concentrations as well as at
several concentrations selected based on the vendor-stated limit of detection (LOD). The effect of interferents was
evaluated by analyzing two types of interferent solutions. The first type contained both humic and fiilvic acids in DI
water, and the second type contained magnesium (Mg) and calcium (Ca) in DI water. Both types of interferent
solutions were prepared with and without the addition of the contaminants at a single concentration level (10 times
the vendor-stated LOD). In addition, specificity was evaluated by exposing the EzyBot® test kits to
lipopolysaccharide, a potentially cross-reactive compound for botulinum toxin. PT samples were analyzed in
triplicate (with the exception of DI water fortified with target analytes at five times the vendor-stated LOD, for
which ten replicates were analyzed). DW samples were collected from four water utilities that use a variety of
treatment methods. DW samples, both unconcentrated and concentrated by a factor of 400, were analyzed in
triplicate both with and without the addition of botulinum toxin A and B at a concentration of 10 times the vendor-
stated LOD. The EzyBot® A test kit was specific to botulinum toxin A, and the EzyBot® B test kit was specific to
botulinum toxin B. In addition to the PT and DW samples analyzed, method blank (MB) samples consisting of DI
water 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.
QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a technical
systems audit and a data quality audit of 10% of the test data. This verification statement, the full report on which it
is based, and the test/QA plan for this verification are all available at www.epa.gov/etv/centers/centerl .html.
TECHNOLOGY DESCRIPTION
The following description of EzyBot® was provided by the vendor and was not verified in this test.
EzyBot® test kits provide a means for detecting botulinum toxins A (EzyBot® A) and B (EzyBot® B) in water. The
technology is based on the PharmaLeads internal collision fluorescence quenching technology. A fluorogenic
substance and a quenching substance in the substrate bracket an amino-acid sequence that, in the presence of
botulinum toxin A or B, is cleaved, generating an intense fluorescence. This fluorescence is measured using either a
laboratory or a field fluorimeter. Note that a laboratory fluorimeter is not provided by PharmaLeads with the
EzyBot® kit; however, a field fluorimeter is available for purchase as part of the field case. The type of fluorimeter
used for detection can affect the sensitivity of the analysis obtained with the EzyBot® test kit, therefore users may
want to contact the vendor for recommended fluorimeters in order to achieve optimal sensitivity with the EzyBot®
kits. The fluorescence generated by the EzyBot® test kit increases in intensity with time and with botulinum toxin
concentration. Data can be read from the fluorimeter display or for the PharmaLeads field portable fluorimeter can
be transferred to a computer through a cable provided with the fluorimeter. Note that a computer is not provided by
PharmaLeads.
EzyBot® A and B are available individually in kits of 50 ready-to-use cuvettes containing freeze-dried reagents,
which can be used in the laboratory or in the field. The PharmaLeads field case provides a field incubator which
can be plugged into the auxiliary power outlet of a car to perform the 1-hour incubation at 37°C in the field. The
field case also includes the PharmaLeads field portable fluorimeter. The price of an EzyBot® kit depends on the
quantity ordered. For large quantities, unit price is approximately $30 per ready-to-use cuvette. Cost for the field
case, including the field fluorimeter, the portable incubator, and 100 cuvettes, is less than $12,500.
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VERIFICATION OF PERFORMANCE
The tables below summarize the performance of the EzyBot®test kits in detecting botulinum toxins A and B.
EzyBot® A Summary Table
Parameter
Sample Information
Botulinum Toxin
A (mg/L)
Lab Bench Scale
Fluorimeter(a)
Field Portable
Fluorimeter(a)
30 min.
60 min.
30 min.
60 min.
0.01 (vendor-stated
limit of detection)
0
0
0
0
Contaminant-only
DI water
0.05
0
10
0
0
0.1
0
3
0
0
0.3 (lethal dose)
3
3
0
3
0.5
3
3
1
3
Interferent
0.5 and 2.5 mg/L each
humic/fulvic acids
0.1
0
50 and 250 mg/L each
Ca/Mg
0.1
3
DW-all locations
unconcentrated
0.1
NA
3
NA
DW-California
concentrated
0.1
3
DW-Florida
concentrated
0.1
3
DW-New York
concentrated
0.1
0
DW-Ohio
concentrated
0.1
3
Lowest Detectable Concentration® (mg/L)
0.3
0.05
ND
0.3
False positives
There were no false positive results from interferents including a preservative blank, humic and
fulvic acids, and Ca and Mg; DW from four locations using different water treatment
technologies; or the potentially cross-reactive lipopolysaccharide (0.1 mg/L).
False negatives
False negatives were obtained in the presence of both 0.5 and 2.5 mg/L each humic and fulvic
acids. A false negative was also obtained in New York water which was concentrated by a
factor of 400. A total of 3 false negative results were obtained out of the 12 solutions assessed
at 60 minutes. The vendor informed Battelle after testing that the lab bench scale fluorimeter
provided for testing may have had inconsistent functioning which could have caused the false
negative results that were obtained.
Consistency
Using the lab bench scale fluorimeter, results were consistent in 100% of the samples tested.
Using the field portable fluorimeter, results were consistent in 90% of the samples tested.
Other
Performance
Factors
Convenient ready-to use cuvettes. Easy to operate in the lab and easy to transport and operate in
the field. No formal scientific education would be required to use the kit; however, general lab
skills and training on fluorimeter use were helpful. Approximately 12-15 analyses were
completed in one hour in the laboratory. Only five samples could be processed in one hour in the
field due to size limitation of the field portable incubator. Each Ezybot ® kit contains 50 ready-
to-use cuvettes.
NA = Not tested. Testing concentration below detection in the contaminant only PT testing.
ND = not detectable at concentrations tested.
(a) Results out of 3 replicates except for the 0.05 mg/L contaminant only concentration for which results are out of 10 replicates
(b) The lowest concentration of contaminant-only PT samples to have at least two thirds of the measurements generate positive results.
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EzyBot®B Summary Table
Parameter
Sample Information
Botulinum Toxin
B (mg/L)
Lab Bench Scale
Fluorimeter(a)
Field Portable
Fluorimeter(a)
30 min.
60 min.
30 min.
60 min.
Contaminant-only
DI water
0.01 (vendor-stated
limit of detection)
0
3
0
0
0.05
7
10
0
0
0.1
3
3
0
0
0.3 (lethal dose)
3
3
3
3
0.5
3
3
0
3
Interferent
0.5 mg/L each
humic/fulvic acids
0.1
3
3
NA
2.5 mg/L each
humic/fulvic acids
0.1
1
3
50 and 250 mg/L each
Ca/Mg
0.1
0
0
DW- all but New York
unconcentrated
0.1
0
3
DW- New York
unconcentrated
0.1
3
3
DW-California
concentrated
0.1
0
3
DW-Florida
concentrated
0.1
3
3
DW-New York
concentrated
0.1
0
3
DW-Ohio
concentrated
0.1
3
3
Lowest Detectable Concentration(b) (mg/L)
0.05
0.01
ND 0.3
False positives
There were no false positive results from interferents including a preservative blank, humic and
fulvic acids, and Ca and Mg; DW from four locations using different water treatment technologies;
or the potentially cross-reactive lipopolysaccharide (0.1 mg/L).
False negatives
False negative results were obtained in the presence of both 50 and 250 mg/L Ca and Mg using
both a 30 minute and 60 minute incubation time. The 30 minute incubation time also generated
false negative results in unconcentrated water from California, Florida, and Ohio; and in
concentrated water from California and New York. A total of 8 false negative results were
obtained out of the 12 solutions assessed at 30 minutes. A total of 2 false negative results were
obtained out of the 12 solutions assessed at 60 minutes. The vendor informed Battelle after testing
that the lab bench scale fluorimeter provided for testing may have had inconsistent functioning
which could have caused the false negative results that were obtained.
Consistency
For the lab bench scale fluorimeter, results were consistent in 97% of the samples tested. With the
field portable fluorimeter, results were consistent in 100% of the samples tested.
Other Performance
Factors
Convenient ready-to use cuvettes. Easy to operate in the lab and easy to transport and operate in
the field. No formal scientific education would be required to use the kit; however, general lab
skills and training on fluorimeter use were helpful. Approximately 12-15 analyses were completed
in one hour in the laboratory. Only five samples could be processed in one hour in the field due to
size limitation of the field portable incubator. Each Ezybot ® kit contains 50 ready-to-use cuvettes.
NA = Not tested. Testing concentration below detection in the contaminant only PT testing.
ND = not detectable at concentrations tested.
(a) Results out of 3 replicates except for the 0.05 mg/L contaminant only concentration for which results are out of 10 replicates.
(b) The lowest concentration of contaminant-only PT samples to have at least two thirds of the measurements generate positive results.
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Original signed by Gregory A. Mack 10/26/2006
Gregory A. Mack Date
Vice President
Energy, Transportation, and Environment Division
Battelle
Original signed by Jonathan G. Herrmann 11/12/2006
Jonathan G. Herrmann Date
Director
National Homeland Security Research Center
U.S. Environmental Protection Agency
NOTICE: ETV verifications are based on an evaluation of technology performance under specific, predetermined
criteria and the appropriate quality assurance procedures. EPA and Battelle make no expressed or implied
warranties as to the performance of the technology and do not certify that a technology will always operate as
verified. The end user is solely responsible for complying with any and all applicable federal, state, and local
requirements. Mention of commercial product names does not imply endorsement.
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September 2006
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
PharmaLeads
EzyBot® A and EzyBot® B Test Kits
by
Mary Schrock
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 environmental technology 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 EPA 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.
111
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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 Metropolitan Water District of Southern
California (Paul Rochelle and Melinda Stalvey), the New York Department of Environmental
Protection (Virginia Murray), and Orange County Utilities, Orlando, Florida (Theresa Slifko and
Liza Robles). 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; 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.
iv
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Contents
Page
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
Chapter 1 Background 1
Chapter 2 Technology Description 2
Chapter 3 Test Design 4
3.1 Test Samples 5
3.1.1 Performance Test Samples 6
3.1.2 Drinking Water Samples 8
3.1.3 Quality Control Samples 8
3.2 Test Procedures 8
3.2.1 Laboratory Testing 8
3.2.2 Non-Laboratory Testing 9
3.2.3 Drinking Water Characterization 9
Chapter 4 Quality Assurance Quality Control 11
4.1 Quality Control of Stock Solution Confirmation Methods 11
4.2 Quality Control of Drinking Water Samples 11
4.3 Technical Systems Audit 11
4.4 Audit of Data Quality 12
4.5 QA/QC Reporting 12
4.6 Data Review 12
Chapter 5 Statistical Methods and Reported Parameters 14
5.1 Qualitative Contaminant Presence/Absence 14
5.2 False Positive/Negative Responses 14
5.3 Consistency 14
5.4 Lowest Detectable Concentration 14
Chapter 6 Test Results 16
6.1 Qualitative Contaminant Presence/Absence 16
6.2 False Positive/Negative Responses 22
6.2.1 Interferent PT Samples 22
6.2.2 DW Samples 23
6.2.3 Cross-Reactivity PT Samples 27
6.3 Consistency 27
6.4 Lowest Detectable Concentration 27
6.5 Other Performance Factors 28
6.5.1 Ease of Use 28
6.5.2 Field Portability 29
6.5.3 Throughput 30
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Chapter 7 Performance Summary 32
Chapter 8 References 35
Figures
Figure 2-1. PharmaLeads EzyBot® A Test Kit 2
Figure 6-1. Field Use of the PharmaLeads Fluorimeter 31
Figure 6-2. PharmaLeads' Portable Incubator Operates Using a Car Auxiliary Power Outlet and
Fits into a Cup Holder 31
Tables
Table 3-1. Lethal Dose and Source of Contaminants 5
Table 3-2. Performance Test Samples 7
Table 3-3. Drinking Water Samples 7
Table 3-4. Water Quality Characterization of Drinking Water Samples 10
Table 4-1. Summary of Data Recording Process 13
Table 6-1 a. Botulinum Toxin A Contaminant-Only PT Sample Results Using the Lab Bench
Scale Fluorimeter-Contaminant Presence/Absence Evaluation 18
Table 6-lb. Botulinum Toxin A Contaminant-Only PT Sample Results Using the Field Portable
Fluorimeter-Contaminant Presence/Absence Evaluation 19
Table 6-lc. Botulinum Toxin B Contaminant-Only PT Sample Results Using the Lab Bench
Scale Fluorimeter-Contaminant Presence/Absence Evaluation 20
Table 6-ld. Botulinum Toxin B Contaminant-Only PT Sample Results Using the Field Portable
Fluorimeter-Contaminant Presence/Absence Evaluation 21
Table 6-2. Botulinum Toxin Contaminant-Interferent Testing -False Positive/Negative
Evaluation 24
Table 6-3a. DW Sample Results Using EzyBot® A - False Positive/Negative Evaluation 25
Table 6-3b. DW Sample Results Using EzyBot® B - False Positive/Negative Evaluation 26
Table 6-4. Lowest Detectable Concentrations 28
Table 7-1. EzyBot® A Summary Table 32
Table 7-1. EzyBot® A Summary Table (Continued) 33
Table 7-2. EzyBot® B Summary Table 34
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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
COA
certificate of analysis
DI
deionized
DW
drinking water
EPA
U.S. Environmental Protection Agency
ETV
Environmental Technology Verification
L
liter
LD
lethal dose
LOD
limit of detection
MB
method blank
Mg
magnesium
mg/L
milligram per liter
mL
milliliter
mM
millimolar
PT
performance test
QA
quality assurance
QC
quality control
QMP
quality management plan
RPD
relative percent difference
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
technologies 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
technologies 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 PharmaLeads EzyBot® A and EzyBot® B test kits
which operate on principles of an enzymatic reaction between botulinum toxin and a specific
substrate. Immunoassay test kits and related technologies which determine the presence or
absence of botulinum toxin were identified as a priority technology category for verification
through the AMS Center stakeholder process.
1
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This verification report provides
results for the verification testing of EzyBot® A and EzyBot® B test kits. Following is a
description of the EzyBot" test kits, based on information provided by the vendor. The
information provided below was not verified in this test.
Botulinum toxins A and B cause paralysis by destroying neuronal compounds that are necessary
for muscle contraction. EzyBot8 test kits provide a means for detecting botulinum toxins A
(LzvBot" A) and B (EzyBotK B) in water. The technology is based on the PharmaLeads internal
collision fluorescence quenching technology. A fluorogenic substance and a quenching
substance in the substrate bracket an amino-acid sequence that, in the presence of botulinum
toxin A or B, is cleaved, generating an intense fluorescence. This fluorescence is measured using
either a laboratory or a field fluorimeter. Note that a laboratory fluorimeter is not provided by
PharmaLeads; however, a field fluorimeter is available for purchase as part of the field case. The
recorded intensity increases with time and with botulinum toxin concentration. The EzyBotlv A
test kit is shown in Figure 2-1.
To perform an assay,
2 milliters (m l.) of the water
sample are introduced into a
cuvette containing freeze-dried
reagents. The cuvette is
incubated at 37°C for 15
minutes and an initial
fluorescence measurement is
taken as a baseline. The
cuvette is then incubated at
37°C with additional
fluorescence measurements
taken after a total incubation
time of 30 minutes and again
after a total incubation time of
one hour. If the difference
between the baseline
measurement and the 30 minute or 60 minute measurement reaches a pre-set threshold
(described in Section 3.2.1), the test is declared positive. Data can be read from the fluorimeter
REAGENT for
BOTULINUM TOXIN A
DETECTION
EzyBotr A
'00 ready-to uw cuvnUos X
= PHARMALEADS
Figure 2-1. PharmaLeads EzyBor A Test Kit
2
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display and/or transferred to a computer through a cable provided with the fluorimeter. Note that
a computer is not provided by PharmaLeads.
EzyBot® A and B are available individually in kits containing 50 ready-to-use cuvettes
containing freeze-dried reagents, which can be used in the laboratory or in the field. To perform
the 1-hour incubation at 37°C in the field, PharmaLeads provides a field incubator to be plugged
into the auxiliary power outlet of a car. The fluorescence level can be read on the PharmaLeads
field fluorimeter. Both incubator and field fluorimeter are included in the field case.
The price of an EzyBot® kit depends on the quantity ordered. For large quantities, unit price is
approximately $30 per ready-to-use cuvette. Cost for the field case, including the field
fluorimeter, the portable incubator, and 100 cuvettes, is less than $12,500.
3
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Chapter 3
Test Design
The objective of this verification test was to evaluate the ability of the EzyBot® test kits to detect
a specific biological toxin in water samples and to determine whether the test kits are susceptible
to interferents in drinking water (DW).
During this verification test, the EzyBot® test kits were subjected to various concentrations of
botulinum toxin in American Society for Testing and Materials Type II deionized (DI) water.
Note that the EzyBot® A test kits are specific to botulinum toxin A and were only tested with
botulinum toxin A. Similarly, EzyBot® B test kits were only tested with botulinum toxin B.
Table 3-1 shows the contaminants, the vendor-stated limit of detection (LOD), the lethal dose
(LD) concentrations, and the contaminant source. . It should be recognized that there is a wide
range of LD concentrations in the literature. In selecting an LD level for use in verification
testing, literature oral LD50 values were reviewed and included in the test/QA plan and
amendments.(1) In addition to reviewing the LD values in the literature, two factors were taken
into consideration in selecting the final LD concentration for use in testing:
1) Consistency with the LD concentrations used in the first round of ETV immunoassay
technology evaluations.
2) Applicability of the LD concentration level to the participating technologies'
expected limits of detection.
In some instances this resulted in an LD level being selected that was on the high end of the
literature values reported. Given the range of LD concentrations that are available in the
literature,it is recommended that all readers evaluate the LD concentrations used for verification
testing with respect to their particular LD requirements. The lethal dose concentration was
determined using a 250 mL ingestion volume.
The EzyBot® test kits 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 inter-
ferents was evaluated by analyzing two types of interferent solutions. The first type contained
both humic and fulvic acids in DI water and the second type contained magnesium (Mg) and
calcium (Ca) in DI water. Both types of interferent solutions were prepared with and without the
addition of the contaminants. In addition, specificity was evaluated by exposing the EzyBot® test
kits to lipopolysaccharide, a potentially cross-reactive compound for botulinum toxin.
4
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Table 3-1. Lethal Dose and Source of Contaminants
Contaminant
Vendor-Stated
LOD
Lethal Dose
Concentration^
Source of Contaminant
Botulinum toxin
A and B
0.01 milligrams/
liter (mg/L)
0.3 mg/L(a)
Metabiologics, Inc. (Madison,
Wisconsin)
/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 and as outlined in the
Test/QA Plan for Verification of Immunoassay Test Kits Amendment Number 5.(1)
The verification test for the EzyBot® test kits was conducted from November 2005 through
January 2006, according to procedures specified in the Test/QA Plan for Verification of
Immunoassay Test Kits including amendments 1-5.(1) This test was conducted at Battelle in
Columbus, 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. The EzyBot® test kits were evaluated for the following
parameters:
¦ 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.
3.1 Test Samples
Tables 3-2 and 3-3 summarize the samples analyzed for each contaminant. The ability of the
EzyBot® test kits to individually detect various concentrations of botulinum toxin 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 EzyBot® test kits with and without the addition of each target
contaminant. Note that the EzyBot® A test kit was tested with solutions containing only
botulinum toxin A and that the EzyBot® B test kit was tested with solutions containing only
botulinum toxin B.
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3.1.1 Performance Test Samples
The contaminant-only and method detection limit PT samples (shown in Table 3-2) were
prepared in DI water using certified standards of botulinum toxin. Reference methods were not
available for quantitative confirmation of the botulinum toxin test solutions so certificates of
analysis (COA) and QA oversight of solution preparation were used to confirm their
concentrations.
The interferent PT samples consisted of samples of humic and fulvic acids isolated from Elliott
Soil (obtained from the International Humic Substances Society) and Ca and Mg (prepared from
their chlorides with concentrations based on metals only), each spiked into DI water at two
concentration levels. These solutions were analyzed both with and without the target
contaminant. In addition, because the commercially available botulinum toxins contained a
preservative (sodium citrate), a preservative blank sample consisting of 0.025 millimolar (mM)
sodium citrate was prepared in DI water. This 0.025 mM sodium citrate solution represents the
concentration of the preservative that would be found in the most concentrated contaminant
solution. This preservative blank was analyzed along with the contaminant solutions to ensure
that the preservative would not cause false positive results during testing.
The last type of PT sample was a cross-reactivity check sample to determine whether the
EzyBot® test kits produced false positive results in response to similar analytes.
Lipopolysaccharide is biologically similar to botulinum toxin. Solutions of lipopolysaccharide
were prepared in DI water at concentrations ten times greater than the vendor-stated LOD for
botulinum toxin.
Three replicates of each PT sample were analyzed except for the sample concentration five times
greater than the vendor-stated LOD (0.05 mg/L) for which a total of ten replicates were
analyzed. The results provided information about how well the EzyBot® test kits detected the
presence of each contaminant at several concentration levels, the consistency of its responses,
and its susceptibility to interferents.
6
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Table 3-2. Performance Test Samples
Type of PT
Sample
Sample Characteristics
Botulinum toxin A or
B(a) Concentrations
Contaminant
Botulinum toxin A or B in DI water
0.01 to 0.5 mg/L
Botulinum toxin A or B in 50 mg/L Ca and 50 mg/L Mg
0.1 mg/L
Interferent
Botulinum toxin A or B in 250 mg/L Ca and 250 mg/L Mg
0.1 mg/L
Botulinum toxin A or B in 0.5 mg/L humic acid and 0.5
mg/L fulvic acid
0.1 mg/L
Botulinum toxin A or B in 2.5 mg/L humic acid and 2.5
mg/L fulvic acids
0.1 mg/L
Preservative Blank: 0.025 mM sodium citrate
NA
Cross-reactive
species
Lipopolysaccharide (botulinum toxin analogue):
0.1 mg/L in DI water
NA
NA = not applicable
tili = EzyBot® A test kits were tested with botulinum toxin A only, EzyBot® B test kits were tested with
botulinum toxin B only.
Table 3-3. Drinking Water Samples
Drinking Water Sample Description
Approximate Contaminant
Concentrations
Water Utility
Water
Treatment
Source
Type
Cone. /
Unconc.
Botulinum Toxin (mg/L)
Metropolitan Water
District of Southern
California (CA)
Filtered
chloraminated
surface
both
unspiked
0.1 (Type B)
0.1 (Type A)
New York City, New
York (NY)
Unfiltered
chlorinated
surface
both
Columbus, Ohio
(OH)
Filtered
chlorinated
surface
both
Orlando, Florida
(FL)
filtered
chlorinated
ground
both
7
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3.1.2 Drinking Water Samples
The DW samples were collected from four geographically distributed municipal sources
(Table 3-3). These samples were unique in terms of their source, treatment, and disinfection
process. All collected samples were finished DW either ready for the distribution system or from
within the distribution system.
Approximately 175 liters (L) of each of the DW samples were collected in pre-cleaned low-
density polyethylene containers. One hundred twenty-five liters of each DW sample were
shipped to the Metropolitan Water District of Southern California and dechlorinated with sodium
thiosulfate. Out of this, 100 L was concentrated using ultra-filtration techniques to a final volume
of 250 mL. This concentration factor was selected because it is the goal of an EPA on-site ultra-
filtration sample concentration method that is being developed to increase the concentration of
insoluble microbiological species in a water sample so they may be detected by available
detection technologies. Concentrated water samples were included in the test/QA plan due to
stakeholder interest in this technique and because the large concentration factor could affect the
amount of potential interferences in various types of water compared to testing only with
unconcentrated water. Twenty-five liters of each water sample was shipped to ATEL for water
quality analysis. The remaining 25 L of each sample was shipped to Battelle where the sample
was dechlorinated with sodium thiosulfate. Each DW sample (unconcentrated and concentrated)
was analyzed without adding any contaminant, as well as after fortification with individual
contaminants at a single concentration level.
3.1.3 Quality Control Samples
In addition to the PT and DW samples analyzed, method blank (MB) samples consisting of DI
water 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. Method
blanks were to be analyzed at a frequency of at least 10% of all samples. A positive control
consisting of a metabolite solution provided by PharmaLeads was analyzed to ensure that the
fluorimeter was operating properly. The positive control was to be analyzed at a frequency of at
least 5% of all samples.
3.2 Test Procedures
3.2.1 Laboratory Testing
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
with a sample identification number that was recorded in a laboratory record book with details of
the sample preparation. PharmaLeads provided two fluorimeters for use in this test. One was a
laboratory bench scale fluorimeter (Jenway Model 6200), the second was an early model of a
field portable fluorimeter under development by PharmaLeads. The EzyBot® kits can be used in
both a "quick" screen mode where fluorescence is measured after incubating for 30 minutes or in
a higher sensitivity mode where fluorescence is measured after incubating for 60 minutes. The
8
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60 minute incubation can also be used to confirm the "quick" screen mode result. Tests were
conducted using both the 30 minute and 60 minute incubation times.
To test a liquid sample for the presence of botulinum toxin using the EzyBot® kit and the lab
bench scale fluorimeter, the following procedure was used. Two milliliters of sample were
placed in an EzyBot® ready-to-use cuvette which contained freeze-dried reagents. The lid was
replaced on the cuvette and the cuvette was shaken until the freeze-dried reagent was completely
dissolved. The cuvette was incubated at 37°C for 15 minutes and then a baseline fluorescence
reading was taken. The cuvette was incubated at 37°C for an additional 15 minutes and a
fluorescence reading was taken, in total, 30 minutes from the time the sample and reagents were
first mixed. Per instructions from PharmaLeads, if the difference between the fluorescence
reading at 30 minutes and the baseline fluorescence reading was greater than 100, the result was
considered positive. The cuvette was incubated for an additional 30 minutes at 37°C and the
fluorescence was recorded again at 60 minutes from the time the sample and reagents were first
mixed. Similarly, if the difference between the fluorescence reading at 60 minutes and the
baseline fluorescence reading was greater than 100, the result was considered positive. For
testing with the field portable fluorimeter, samples were prepared in the cuvettes, incubated, and
fluorescence readings taken at the same intervals as described above; however, positive results
were determined by subtracting the baseline fluorescence reading (at 15 minutes) from the
fluorescence reading at 30 minutes and dividing that quantity by the baseline fluorescence
reading. A fluorescence measurement that was 20% higher than the baseline was considered
positive. The same calculation was carried out for the 60 minute fluorescence measurement;
however, a 40% increase in fluorescence at the 60 minute interval was considered a positive
result. Data generated using the laboratory bench scale fluorimeter were manually recorded on
data sheets and calculations between the 30 or 60 minute measurement and the baseline
measurement were hand calculated. Data generated with the field portable fluorimeter were
collected on a laptop computer and manually transferred into an electronic spreadsheet for data
calculations.
3.2.2 Non-Laboratory Testing
Because of the toxic nature of botulinum toxin, only MB samples and the vendor supplied
positive control were analyzed at a non-laboratory location. Because the field portable incubator
supplied by PharmaLeads was designed to operate using a car auxiliary power outlet, the non-
laboratory testing took place in a parked car. Only the field portable incubator and field portable
fluorimeter were tested in this setting. Because the PharmaLeads field fluorimeter did not have a
finalized instruction manual at the time of testing and because PharmaLeads intends to provide a
customized training session to kit purchasers, this technology was only tested by operators that
had received training from PharmaLeads.
3.2.3 Drinking Water Characterization
An aliquot of each DW sample, collected as described in Section 3.1.2, was sent to ATEL to
characterize the water samples based on the water quality parameters shown in Table 3-4.
The table lists the methods used as well as the characterization data for the four water samples
collected as part of this verification test. Water quality parameters were characterized upon
sampling in June 2005, while the EzyBot® kits were tested with DW in November 2005. The
9
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time delay between collection and testing was due to the fact that the water samples were
collected for use during a separate ETV test conducted prior to this one. Because of this, an
aliquot of each DW was tested by ATEL again in January 2006 to verify some of the parameters
with the most potential to change over time. Note that dissolved organic carbon was not retested
as this result was verified by the total organic carbon results, additionally the total organic
halides and calcium and magnesium were not verified as there was no reason to expect a change
in these parameters. The concentrations of most water quality parameters were similar; however,
there was a decrease in levels of volatile compounds such as trihalomethanes and haloacetic
acids over this time-period.
Table 3-4. Water Quality Characterization of Drinking Water Samples
Columbus,
Ohio
Metropolitan
Water District
of Southern
California
New York City,
New York
Orlando,
Florida
Parameter
Method
2005
2006
2005
2006
2005
2006
2005
2006
Alkalinity (mg/L)
SM 2320 B(2)
40
44
71
97
14
12
142
125
Specific conductivity
(|imho)
SM 2510 B(2)
572
602
807
812
84
78
322
325
Hardness (mg/L)
EPA 130.2(3)
118
107
192
182
20
26
143
130
pH
EPA 15 0.1(3)
7.6
7.4
8.0
7.9
6.9
6.8
8.5
7.6
Total haloacetic acids
(Mg/L)
EPA 552.2(5)
32.8
<6.0
17.4
<6.0
39.0
<6.0
34.6
<6.0
Total organic carbon
(mg/L)
SM 5310 B(2)
2.1
2.3
2.5
2.7
1.6
4.1
1.7
2.1
Dissolved organic
carbon (mg/L)
SM 5310 B(2)
2.1
NA
2.9
NA
1.1
NA
1.6
NA
Total organic halides
(l-Lg/L)
SM 5320B(2)
220
NA
170
NA
82
NA
300
NA
Total trihalomethanes
(l-Lg/L)
EPA 524.2(4)
74.9
16.6
39.2
24.1
39.0
23.1
56.4
41.8
Turbidity (NTU)
SM 2130 B(7)
0.1
0.6
0.1
0.2
1.1
1.3
0.5
0.1
Calcium (mg/L)
EPA 200.7 (6)
33
NA
45
NA
5.6
NA
8.8
NA
Magnesium (mg/L)
EPA 200.7 (6)
7.7
NA
20
NA
1.3
NA
43
NA
NTU = nephelometric turbidity unit
NA = not retested
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(8) and the test/QA plan(1) for this
verification test.
4.1 Quality Control of Stock Solution Confirmation Methods
The COA for botulinum toxin was provided by the supplier of those contaminants. Because
standard reference methods do not exist, the concentration of botulinum toxin was not
independently confirmed. The COA stated that both botulinum toxin A and B standards
(Metabiologics, Inc., Madison, Wisconsin) had concentrations of 1000 mg/L. Each toxin stock
solution was in 50 mM sodium citrate buffer at a pH of 5.5 and had passed Metabiologics' tests
for activity, identity and purity. Test samples containing these contaminants were prepared by
diluting aliquots of these stock solutions. All records pertaining to stock solution dilutions were
reviewed as part of the technical systems audit review. For the interferent samples, the
concentration of calcium and magnesium was confirmed by EPA Method 200.7.(6)
4.2 Quality Control of Drinking Water Samples
A method blank sample consisting of DI water was analyzed once for approximately every 7
water samples analyzed for a frequency of approximately 15% of all samples. A positive control
sample was analyzed once for approximately every 13 water samples for a frequency of
approximately 8% of all samples. While performance limits were not placed on the results of the
positive control sample, the vendor informed Battelle that for the lab bench scale fluorimeter
(Jenway Model 6200) the positive control should read near the instrument maximum reading of
1999 and for the field portable fluorimeter the positive control should have a significantly higher
fluorescence reading than the method blank to indicate that the instrument was functioning
properly.
4.3 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 and amendments'^ and the
AMS Center QMP.(8) 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 and
11
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amendments,(1) and reviewed data acquisition and handling procedures. Observations and
findings from this audit were documented 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.
4.4 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.
4.5 QA/QC Reporting
Both the technical systems and data quality audits were documented in accordance with Sections
3.3.4 and 3.3.5 of the QMP for the ETV AMS Center/8-* Once an assessment report was prepared
for each audit, the Battelle Verification Test Coordinator responded to any findings 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. During
this test, all quality assurance findings were minor and had minimal impact on the overall test
results.
4.6 Data Review
Data generated during this verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-1 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.
12
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Table 4-1. Summary of Data Recording Process
Data to Be Recorded
Responsible
Party
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 and
Water
Utilities
providing
DW samples
Laboratory
record books
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
Fluorimeter
procedures and
sample results
Battelle
ETV data
sheets or
captured in data
acquisition
system
Throughout test
duration
Manually
incorporated in ETV
data sheets or
transferred to
electronic
spreadsheets
Reference method
procedures and
sample results
ATEL
Data
acquisition
system, as
appropriate
Throughout
sample analysis
process
Transferred to
spreadsheets and
reported to Battelle
13
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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
Chapter 3. The EzyBot® kits produce qualitative results; i.e., the kits indicate only the presence
or absence of a contaminant and do not measure the concentration present. Therefore, the data
evaluation methods were applied in that context.
5.1 Qualitative Contaminant Presence/Absence
Accuracy of the EzyBot® A Kit for botulinum toxin A and EzyBot® B Kit for botulinum toxin B
was assessed by reporting the number of positive results out of the total number of contaminant-
only PT samples tested at each concentration level. A result was considered positive based on
the vendor's specifications for a positive result which are described in Section 3.2.1.
5.2 False Positive/Negative Responses
A result was considered a false positive when a DI water or DW sample was spiked with a
potential interferent, a cross-reactive compound, or not spiked at all and a positive response was
obtained. A result was considered a false negative when any DW or interferent sample was
spiked with botulinum toxin at a concentration greater than lowest detectable concentration (as
determined during DI water contaminant-only testing) and produced a negative response.
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 within a set (i.e., within a single concentration level or type of interferent) 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-thirds of the replicates generated positive responses. In
addition, all concentrations greater than that lowest detected level were required to have a
14
-------
positive responses in at least two-thirds of the replicates. These concentration levels were
determined for botulinum toxin A and B in solutions of DI water.
5.5 Other Performance Factors
Aspects of the EzyBot test kit 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 EzyBot® test kits.
15
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Chapter 6
Test Results
6.1 Qualitative Contaminant Presence/Absence
The results obtained for the PT samples containing botulinum toxin are given in Tables 6-la
through 6-ld. The EzyBot® kits can be used in both a "quick" screen mode where fluorescence
is measured after incubating for 30 minutes or in a higher sensitivity mode where fluorescence is
measured after incubating for 60 minutes. The 60 minute incubation can also be used to confirm
the "quick" screen mode result. During this test, results were obtained using both the 30 and 60
minute incubation times with both a lab bench scale fluorimeter (Jenway Model 6200) and an
early model of a small, field portable fluorimeter. It should be noted that the quality of the
fluorimeter used for analysis can affect the sensitivity of the results obtained with the EzyBot®
kits. At the time of verification testing, the vendor provided Battelle with the Jenway Model
6200 for use in testing. Subsequently, the vendor was informed by the Jenway manufacturer that
this fluorimeter model is no longer in production due to some discontinuities in its functioning.
Therefore, it is important to note that use of this particular fluorimeter may have affected the lab
bench scale fluorimeter results determined during verification testing. Similarly, the sensitivity
of the field portable fluorimeter used for analysis will affect the sensitivity of results obtained
with the EzyBot® kits. After the verification test, the vendor informed Battelle that the field
portable fluorimeter available from PharmaLeads has undergone modification for improved
sensitivity and stability since this verification testing took place and is available as the EzyBot
2®. Any subsequent changes to the field portable fluorimeter were not verified as part of this
report. Because of the variation in EzyBot® test kit sensitivity that can be attributed to the type of
fluorimeter used, users may want to contact the vendor for recommendations on fluorimeters to
use in order to achieve optimal sensitivity with the EzyBot® kits.
The interpretation of results from the lab bench scale and field portable fluorimeters used for this
verification test is described in Section 3.2.1, and briefly summarized as follows. Using the lab
bench scale fluorimeter, the difference between the fluorescence reading at 30 or 60 minutes and
the baseline fluorescence reading needed to be greater than 100 to be considered positive. For the
field portable fluorimeter the 30 minute fluorescence reading needed to be 20% higher than the
baseline reading and the 60 minute reading needed to be 40% higher than the baseline reading to
be considered positive.
As shown in Table 6-la, with the lab bench scale fluorimeter and the 30 minute incubation time,
EzyBot® A generated positive results to botulinum toxin A in all replicates at the lethal dose
16
-------
concentration (0.3 mg/L) and higher. Note that while the 30 minute results for the 0.1 mg/L
concentration (10 x LOD) had consistently elevated responses, these responses did not meet the
criteria for a positive result. With the lab bench scale fluorimeter and the 60 minute incubation
time, EzyBot® A generated positive results in all replicates at concentrations five times the
vendor-stated LOD (0.05 mg/L) and higher. Table 6-lb shows EzyBot® A results using the field
portable fluorimeter. With this fluorimeter and the 30 minute incubation, only one of three
replicates generated a positive response at the highest concentration tested (0.5 mg/L). With the
60 minute incubation time and the field portable fluorimeter, EzyBot® A generated positive
results in all replicates at the lethal dose concentration (0.3 mg/L) and higher.
Table 6-1 c presents results using EzyBot B and the lab bench scale fluorimeter. With this
fluorimeter and the 30 minute incubation time, EzyBot® B generated positive results to
botulinum toxin B in all replicates at ten times the vendor-stated LOD (0.1 mg/L) and higher and
in seven of ten replicates at five times the vendor-stated LOD (0.05 mg/L). A total of ten
replicates were analyzed at this concentration level because three replicates were contaminant-
only PT samples and seven were included in the test/QA plan as a method detection limit study.
Because of the qualitative nature of the EzyBot® test kits, the results of all ten analyses are
reported as additional contaminant-only PT replicates because a method detection limit cannot be
calculated for a technology that reports a presence/absence result. With the lab bench scale
fluorimeter and the 60 minute incubation time, EzyBot® B generated positive results in all
replicates at the vendor-stated LOD (0.01 mg/L) and higher. Table 6-ld shows EzyBot® B
results using the field portable fluorimeter. With this fluorimeter and the 30 minute incubation,
positive results were generated only for the three replicates at the lethal dose concentration (0.3
mg/L). Why the highest concentration (50 times the vendor-stated LOD, 0.5 mg/L) was not
detected with the field portable fluorimeter when the 0.3 mg/L concentration was detected is
uncertain. With the 60 minute incubation time and the field portable fluorimeter, EzyBot® B
generated positive results to all replicates at the lethal dose level (0.3 mg/L) and higher.
17
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Table 6-la. Botulinum Toxin A Contaminant-Only PT Sample Results Using the Lab
Bench Scale Fluorimeter-Contaminant Presence/Absence Evaluation
Analyte
Concentration
(mg/L)
Testing
Level
30 Minute
Results(a)
No. of
Positives
(30 min.)
60 Minute
Results(a)
No. of
Positives
(60 min.)
9
-7
0.01
LOD
-29
0
-18
0
3
30
61
4(i(i
-18
172
1
2M
6
0.05
5 x LOD
0
0
75 X
10
3
742
-1
7X3
Botulinum
4
754
Toxin A
3
747
31
Xh2
55
5<)X
0.1
10 x LOD
61
0
4fifi
3
mi
4i IX
:m
1374
0.3
LD
157
h>x
3
13X4
135fi
3
25n
1342
0.5
50 x LOD
2^2
247
3
1374
1321
3
LOD = vendor-stated limit of detection.
LD = lethal dose
> indicates that the 60 minute fluorescence reading exceeded the fluorimeter maximum.
i :" Difference between fluorescence at 30 or 60 minutes and baseline fluorescence at 15 minutes. Result is
considered positive if the difference is greater than 100.
Shaded areas highlight positive results.
18
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Table 6-lb. Botulinum Toxin A Contaminant-Only PT Sample Results Using the Field
Portable Fluorimeter-Contaminant Presence/Absence Evaluation
Analyte
Concentration
(mg/L)
Testing
Level
30 Minute
Results(a)
No. of
Positives
(30 min.)
60 Minute
Results(a)
No. of
Positives
(60 min.)
16
21.1
0.01
LOD
8.7
0
2.5
0
2.8
-5.1
5.3
15.6
-4.5
0.9
3.6
28.4
-3.4
-0.1
0.05
5 x LOD
-1.2
0
1.9
0
-3.9
-1.6
-1.6
-0.6
Botulinum
0.9
2.8
Toxin A
-3.1
0.1
-0.6
7.9
8.2
9.5
0.1
10 x LOD
-19.2
0
-6.4
0
10.3
14 5
9.5
0
ft 7 1
0.3
LD
5.0
52 ft
3
8.4
ft 5 7
9.5
71 3
0.5
50 x LOD
15.8
1
Xft (I
3
2i).5
l>3 8
LOD = vendor-stated limit of detection.
LD = lethal dose concentration.
(a) ((Fluorescence at 30 or 60 minutes - baseline fluorescence at 15 minutes) x 100)/(baseline fluorescence at 15
minutes). Result is considered positive if >20% for 30 minute measurements and >40% for 60 minute
measurements. Shaded areas highlight positive results.
19
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Table 6-lc. Botulinum Toxin B Contaminant-Only PT Sample Results Using the Lab
Bench Scale Fluorimeter-Contaminant Presence/Absence Evaluation
Analyte
Concentration
(mg/L)
Testing
Level
30 Minute
Results(a)
No. of
Positives
(30 min.)
60 Minute
Results(a)
No. of
Positives
(60 min.)
11
172
0.01
LOD
16
0
I Wi
3
9
ISS
84
h7S
67
5fi3
85
731
|w(i
14^2
0.05
5 x LOD
i 3i>
127
133
7
l"l 3
11)41
1 <)7(i
10
Botulinum
Toxin B
154
121
131
1113
gi)2
1237
253
1 fo5
0.1
10 x LOD
ISS
I (ill
3
I4()3
1 15"
3
1
13l>3
0.3
LD
1 ()7o
|t)4h
3
I42w
1425
3
123d
1430
0.5
50 x LOD
1>3S
11)75
3
1442
I4WS
3
LOD = vendor-stated limit of detection.
LD = lethal dose concentration.
> indicates that the 60 minute fluorescence reading exceeded the fluorimeter maximum.
(a) Difference between fluorescence at 30 or 60 minutes and baseline fluorescence at 15 minutes. Result is
considered positive if the difference is greater than 100. Shaded areas highlight positive results.
20
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Table 6-ld. Botulinum Toxin B Contaminant-Only PT Sample Results Using the Field
Portable Fluorimeter-Contaminant Presence/Absence Evaluation
Analyte
Concentration
(mg/L)
Testing
Level
30 Minute
Results(a)
No. of
Positives
(30 min.)
60 Minute
Results(a)
No. of
Positives
(60 min.)
-14.6
-25.9
0.01
LOD
-17.9
0
-22.8
0
-20.5
-26.6
-9.0
-9.9
-10.9
-10.3
0.1
-0.2
-13.3
14.7
0.05
5 x LOD
-10.0
0
-4.7
0
-17.0
-5.0
-9.6
16.3
Botulinum
-14.1
-4.2
Toxin B
16.6
9.5
-16.2
-0.7
-8.9
38.4
0.1
10 x LOD
-14.1
0
-2.4
0
-4.3
-1.2
> 7
21)^.3
0.3
LD
32 v
23.4
3
2(i(i 2
1 n ) 5
3
S.2
I Mi 3
0.5
50 x LOD
14.1
0
154 w
3
9.7
121 h
LOD = vendor-stated limit of detection.
LD= lethal dose concentration.
(a) ((Fluorescence at 30 or 60 minutes - baseline fluorescence at 15 minutes) x 100)/(baseline fluorescence at 15
minutes). Result is considered positive if >20% for 30 minute measurements and >40% for 60 minute
measurements. Shaded areas highlight positive results.
21
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6.2 False Positive/Negative Responses
Three types of samples were analyzed to evaluate the susceptibility of the EzyBot® kits to false
positive and negative results. These included interferent PT samples, made up of DI water
fortified with Ca and Mg or with humic and fulvic acids, both with and without the addition of
target contaminants; cross-reactivity PT samples made up of DI water fortified with a
contaminant biologically similar to botulinum toxin; and DW samples both concentrated and
unconcentrated and both with and without the addition of botulinum toxin. In addition, a
preservative blank containing sodium citrate, which is used as a preservative in commercially
available botulinum toxin, was analyzed to evaluate the potential for false positive results from
the preservative. A false positive result was defined as a positive result in the absence of
botulinum toxin and a false negative result was defined as a negative result from a sample
containing botulinum toxin at ten times the vendor-stated LOD, if that concentration level was
detectable in the PT contaminant-only testing. Note that only the lab bench scale fluorimeter
was used for the false positive and false negative assessments due to lack of sensitivity of the
field portable fluorimeter to the botulinum toxin concentrations used for assessing false negative
results.
6.2.1 Interferent PT Samples
The results from the interferent PT samples are given in Table 6-2. Neither EzyBot® A nor
EzyBot® B had false positive results from the PT samples containing possible interferences, but
no contaminant. Based on the EzyBot® A contaminant-only PT testing described in Section 6.1,
the 0.1 mg/L botulinum toxin A solutions were detectable for 60 minute incubation/lab bench
scale fluorimeter readings, while the 30 minute incubation time was below the detection limit.
Therefore for EzyBot® A, only the 60 minute incubation/lab bench scale fluorimeter results were
assessed for false negatives in the inteferent testing. While the 0.1 mg/L botulinum toxin A
solution was detected in the presence of calcium and magnesium, false negative results were
obtained in the presence of both 0.5 mg/L each and 2.5 mg/L each humic and fulvic acids.
As described in Section 6.1, the 0.1 mg/L botulinum toxin B solutions were detectable with
EzyBot® B for both the 30 and 60 minute incubation times with the lab bench scale fluorimeter.
Therefore for interferent testing, both the 30 and 60 minute incubation/lab bench scale
fluorimeter results were assessed for false negatives. False negative results were obtained for
two of three replicates of 0.1 mg/L botulinum toxin B in the presence of 5 mg/L humic and
fulvic acids with the 30 minute incubation time. False negative results were also obtained in the
presence of both concentrations of calcium and magnesium at both the 30 and 60 minute
incubation times. There were no false negative results for 0.5 mg/L each humic and fulvic acids
during the 30 or 60 minute incubation and no false negative results for 2.5 mg/L each humic and
fulvic acids during the 60 minute incubation.
The false negative results obtained while analyzing interferent PT samples using the 60 minute
incubation time with both the EzyBot® A and EzyBot® B kits were unexpected given the
performance of the EzyBot® kits while testing drinking water samples which are presented in
Section 6.2.2. Because similar concentrations of total organic carbon, calcium, and magnesium
were in the various drinking waters (Table 3-4) as were added to interferent PT samples, similar
22
-------
results were expected. No definitive reason could be found which would explain this unexpected
performance. Possible reasons are that the sources of interferents added to DI water to create the
interferent PT samples are not identical to the interferents in the drinking waters and that matrix
effects from the various drinking waters enhance the detection capability of the EzyBot® kits;
however, these possible reasons for differences in performance were not further evaluated during
verification testing. Additionally, the vendor informed Battelle after testing that the lab bench
scale fluorimeter provided for testing may have had inconsistent functioning which could have
affected the results obtained.
6.2.2 DWSamples
The results from the DW samples are given in Tables 6-3a for EzyBot® A and 6-3b for EzyBot®
B. For both EzyBot® A and EzyBot® B there were no false positive results generated by DW
samples without contaminant. EzyBot® A was evaluated with DW fortified with 0.1 mg/L
botulinum toxin A using only the 60 minute incubation time because no positive results were
obtained with the 30 minute incubation when analyzing the contaminant-only PT samples at the
0.1 mg/L concentration. With the 60 minute incubation, positive results were generated for 0.1
mg/L botulinum toxin A in all geographic types of water except the concentrated water from
New York, for which a false negative result was obtained. The reason for the false negative
result for concentrated New York water is not clear; however, the New York water is from the
only source that was not filtered and had the highest turbidity reading; therefore, the high particle
content of the New York water may have interfered with EzyBot® A. Additionally, as mentioned
previously, the vendor informed Battelle after testing that the lab bench scale fluorimeter
provided for testing may have had inconsistent functioning which could have affected the results
obtained.
EzyBot® B results are reported for both the 30 minute and 60 minute incubation times. With the
60 minute incubation, there were no false negative results as botulinum toxin B (0.1 mg/L) was
detected in all geographic types of water, both unconcentrated and concentrated. The 30 minute
incubation results were not easily interpreted. All of the unconcentrated drinking water samples,
except for New York, generated false negative results, while two out of four concentrated water
samples (New York and California) generated false negative results.
23
-------
Table 6-2. Botulinum Toxin Contaminant-Interferent Testing -False Positive/Negative
Evaluation
Botulinum
Lab Bench Scale Fluorimeter
Toxin
30
No. of
60
No. of
Cone.
Interferent
Minute
Positives
Minute
Positives
Results(a)
(30 min.)
Results(a)
(60 min.)
0.025 mM sodium citrate
0
0
EzyBot® A
and
EzyBot® B
0.5 mg/L each
humic/fulvic acids
Individual
0
Individual
results not
listed
0
None
2.5 mg/L each
humic/fulvic acids
results not
listed
0
0
50 mg/L each Ca/Mg
0
0
250 mg/L each Ca/Mg
0
0
0.5 mg/L each
humic/fulvic acids
26
NA
NA
28
0
62
2.5 mg/L each
humic/fulvic acids
-18
NA
NA
-103
0
EzyBot® A
0.1 mg/L
-Si.
Type A
'til
50 mg/L each Ca/Mg
NA
NA
:s'«
:'X
3
250 mg/L each Ca/Mg
NA
NA
|()'J
1 'X
3
0.5 mg/L each
humic/fulvic acids
I'JS
">
:s(.
3
mx
!(¦(¦()
It.'l
3
2.5 mg/L each
humic/fulvic acids
It >5
1
XX4
3
EzyBot® B
0.1 mg/L
83
_ 1 _
Type B
0
42
50 mg/L each Ca/Mg
1
0
62
0
2
62
-4
-2
250 mg/L each Ca/Mg
-23
0
-17
0
-3
-3
NA = EzyBot® A was not sensitive to the 0.1 mg/L contaminant concentration after 30 minutes in the contaminant-
only PT testing and was therefore not included in the interferent testing.
> indicates that the 60 minute fluorescence reading exceeded the fluorimeter maximum.
(a) Difference between fluorescence at 30 or 60 minutes and baseline fluorescence at 15 minutes. Result is
considered positive if the difference is greater than 100. Shaded areas highlight positive results.
24
-------
Table 6-3a. DW Sample Results Using EzyBot® A - False Positive/Negative Evaluation
Botulinum
Lab Bench Scale Fluorimeter
Toxin
60 Minute
No. of Positives
Cone.
DW
Results(a)
(60 min.)
California unconcentrated
0
and concentrated
Florida unconcentrated and
0
concentrated
Individual
None
New York unconcentrated
results not listed
0
and concentrated
Ohio unconcentrated and
0
concentrated
California unconcentrated
4~<>
4<>5
535
3
California concentrated
45(1
:i(.
:i5
3
Florida unconcentrated
5SI
:
3
Florida concentrated
::i
^r
3
0.1 mg/L
4:1
Type A
45(1
New York unconcentrated
4'(>
4^X
3
-12
New York concentrated
-30
0
-50
"5'J
Ohio unconcentrated
445
544
3
241
Ohio concentrated
4" 1
U4
3
Note that EzyBot" A was not sensitive to the 0.1 mg/L contaminant concentration after 30 minutes in the
contaminant-only PT testing. Therefore the 30 minute incubation was not included in the interferent testing.
"" Difference between fluorescence at 60 minutes and baseline fluorescence at 15 minutes. Result is considered
positive if the difference is greater than 100. Shaded areas highlight positive results.
25
-------
Table 6-3b. DW Sample Results Using EzyBot®B - False Positive/Negative Evaluation
Botulinum
Lab Bench Scale Fluorimeter
Toxin
30
No. of
60
No. of
Cone.
DW
Minute
Positives
Minute
Positives
Results(a)
(30 min.)
Results(a)
(60 min.)
California unconcentrated
0
0
and concentrated
Florida unconcentrated and
concentrated
Individual
results not
listed
0
Individual
results not
listed
0
None
New York unconcentrated
and concentrated
0
0
Ohio unconcentrated and
0
0
concentrated
California unconcentrated
19
0
i:i
3
30
155
41
California concentrated
99
0
4'5
3
60
'52
77
'<>()
25
l(.'
Florida unconcentrated
18
0
144
3
22
I-
I'W
~4<>
Florida concentrated
3
-<>:
3
0.1 mg/L
:i5
"14
Type B
:i><.
New York unconcentrated
M5
n.
3
')<)<)
\:m,
3
29
1 15
New York concentrated
28
0
i id
3
23
1 15
46
241
Ohio unconcentrated
32
0
:i:
3
28
141
4SS
14'4
Ohio concentrated
4^(.
4~<>
3
12 'S
141"
3
(a) Difference between fluorescence at 30 or 60 minutes and baseline fluorescence at 15 minutes. Result is
considered positive if the difference is greater than 100. Shaded areas highlight positive results.
> indicates that the 60 minute fluorescence reading exceeded the fluorimeter maximum.
26
-------
6.2.3 Cross-Reactivity PT Samples
DI water fortified with a chemical similar to the target contaminant was analyzed in the absence
of the target contaminant. Lipopolysaccharide at a concentration of 0.1 mg/L (equivalent to ten
times the vendor-stated LOD for the target contaminant) was used as the cross-reactivity analyte
for botulinum toxin and was analyzed in triplicate with each EzyBot® kit. Neither EzyBot® A
nor EzyBot® B had any positive response to the lipopolysaccharide using either the lab bench
scale fluorimeter or the field portable fluorimeter with both 30 and 60 minute incubation times.
6.3 Consistency
Using the lab bench scale fluorimeter, EzyBot® A results were consistent between replicates in
100% of the samples tested. With the field portable fluorimeter, EzyBot® A results were
consistent between replicates in four out of the five sets of samples analyzed (90%). The 30
minute reading of 0.5 mg/L botulinum toxin A had a positive response in only one of three
replicates with the field portable fluorimeter.
For EzyBot B, using the lab bench scale fluorimeter, results were consistent between replicates
for all but 0.1 mg/L botulinum toxin B in the presence of 2.5 mg/L each of humic and fulvic
acids (for which only one of three replicates had a positive response) and 0.05 mg/L botulinum
toxin B (for which seven out of ten replicates had a positive response). This resulted in a
consistent response in 58 of the 60 sample sets analyzed (97%). Using the field portable
fluorimeter, EzyBot® B results were consistent between replicates in 100%) of the samples tested.
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 thirds of the measurements
generate positive results. In addition, all concentrations greater than the lowest detectable
concentration were required to have at least two thirds of the measurements generate positive
results. The concentrations in Table 6-4 summarize the results presented in Table 6-la through 6-
ld. Note that while the field portable fluorimeter results for the 30 minute incubation with the
EzyBot® B kit had 3 positive detects of the 0.3 mg/L botulinum toxin B solution, because there
were no detects of the 0.5 mg/L solution, no detectable concentration is reported. The highest
sensitivity results for the both the EzyBot® A and EzyBot® B kits were obtained using the 60
minute incubation time and the lab bench scale fluorimeter resulting in detection of 0.05 mg/L
botulinum toxin A with EzyBot® A and 0.01 mg/L botulinum toxin B with EzyBot® B.
27
-------
Table 6-4. Lowest Detectable Concentrations
Lab Bench Scale Fluorimeter
Field Portable Fluorimeter
30 minutes
60 minutes
30 minutes
60 minutes
EzyBot® A
0.3 mg/L
0.05 mg/L
ND
0.3 mg/L
EzyBot® B
0.05 mg/L
0.01 mg/L
ND
0.3 mg/L
ND = Not detectable at concentrations tested.
6.5 Other Performance Factors
6.5.1 Ease of Use
Both kits contained clearly written and informative instructions for use with the lab bench scale
fluorimeter. At the time of testing, instructions for the field portable fluorimeter were still being
finalized so testing staff relied on the training provided by PharmaLeads. Contents of the kit
were clearly labeled. Storage requirements were marked on the outer container and in the
instruction manual. Overall, all packaging was easy to open. Ready-to-use cuvettes required no
reagent preparation. The sample was added to the cuvettes, mixed and incubated at 37°C.
Readings were taken at 15, 30, and 60 minutes. Prior to use, cuvettes were required to be stored
at 4° C. Expiration dates were clearly printed on the outer container.
All equipment was supplied with the kit except for pipettes with tips needed to dispense 2 mL of
sample into the cuvettes, the incubator, and a fluorimeter. Laboratory scale fluorimeters and
incubators are available commercially. A field portable incubator and fluorimeter are available
from PharmaLeads in a field case. The field case also contains pipettes necessary for dispensing
sample into the cuvettes. A laptop computer (not included with the kit or field case) was needed
to acquire data using the field portable fluorimeter. The lab bench scale fluorimeter used for this
test required manually recording a 3-4 digit fluorescence measurement. The field portable
fluorimeter acquired multiple readings of a 6-7 digit fluorescence measurement on a laptop
computer. These readings were averaged and transferred into a spreadsheet for further
calculations. With the lab bench scale fluorimeter, because relatively few digits were involved in
each measurement and because a positive response only involved assessing whether the 30 or 60
minute fluorescence measurement differed by 100 units from the baseline 15 minute
measurement, a positive result was easily determined. With the field portable fluorimeter, a
positive or negative result required more extensive data manipulation (determining the
percentage change in fluorescence between the baseline measurement and the 30 or 60 minute
measurement) with each measurement involving more digits. The vendor states that since the
verification testing took place, the field portable fluorimeter has been revised so that its results
are determined by measuring a difference of 100 units, similar to the measurement with the lab
bench scale fluorimeter and now also includes a warning sound emitted when a positive result is
obtained. The revised field portable fluorimeter (EzyBot® 2) was not evaluated during this ETV
test. The lab bench scale fluorimeter required calibration prior to use using a standard provided
in the EzyBot® kit. The field portable fluorimeter had fluorimeter and software settings that
28
-------
needed to be set prior to use, but did not require calibration. The surfaces of both fluorimeters
were easily wiped clean.
No formal scientific education would be required for using the kits, but general good laboratory
skills are needed. Verification testing staff were able to conduct tests with the kit after a training
session which lasted several hours. In particular the training related to operating the fluorimeters
was helpful. The vendor's address and website are included in the instruction manual providing
easy access to the vendor's contact information. One cuvette per sample and pipette tips were
generated as solid waste. It was not stated in the kit or product literature whether the ready-to-
use cuvettes should be considered hazardous waste due to any of the reagents contained within.
6.5.2 Field Portability
Because the field portable incubator supplied by PharmaLeads was designed to operate using a
car auxiliary power outlet and because the fluorimeter and laptop computer were battery
powered allowing easy portability, the non-laboratory testing took place in a parked car. The
incubator only needed to be plugged into the car auxiliary outlet, the car did not need to be
turned on and running in order for the incubator to work. Only the field portable incubator and
field portable fluorimeter were tested in this setting (and not the laboratory bench scale
fluorimeter). The technology was tested with a method blank and the vendor-provided positive
control. The fluorimeter, incubator, and necessary supplies were transported in several small
boxes, and the laptop for use with the fluorimeter was carried in its case. Two people easily
transported all equipment. Once at the field testing location, the equipment was set up and the
incubator was warmed to temperature within five minutes. The small size of all the equipment
allowed all analyses for this test to be carried out in the back seat of a car (see Figures 6-1 and 6-
2). The field portable incubator only holds 5 cuvettes which limits the number of samples that
can be processed during the 30 minute or 60 minute incubation time. The vendor stated that the
reagent in the cuvettes is stable for 24 hours at 25°C. Therefore, within that timeframe, field
deployment could be carried out without concern for reagent storage. Longer-term deployment
would require a means of keeping the cuvettes at 4°C. The following items in addition to the kit,
incubator, computer, and fluorimeter were needed for field use: a timer, pipettes and tips, and a
waste container. Note that with purchase of the PharmaLeads field case, the field portable
incubator and fluorimeter, as well as pipettes are included. As mentioned in Section 6.5.1, the
field portable fluorimeter generates raw data which needs to be taken through calculations in
order to assess whether the result was positive. Such an assessment could not be made by
looking at the raw data alone and required additional time to arrive at a result beyond the field
analysis time. The vendor states that since the verification testing took place, the field portable
fluorimeter has been revised so that its results are determined by measuring a difference of 100
units, similar to the measurement with the lab bench scale fluorimeter and now also includes a
warning sound emitted when a positive result is obtained. The revised field portable fluorimeter
(EzyBot® 2) was not evaluated during this ETV test.
29
-------
6.5.3 Throughput
Approximately 12-15 sample analyses plus method blanks and controls were completed in one
hour in the laboratory where a larger incubator was available. Note that the field portable
incubator only held 5 cuvettes so unless multiple portable incubators or a different type of
incubator were used only 5 samples could be processed in one hour in the field. Each EzyBot®
kit contains 50 ready-to-use cuvettes.
30
-------
Figure 6-1. Field Use of the PharmaLeads Fluorimeter
Figure 6-2. PharmaLeads' Portable Incubator Operates Using a Car Auxiliary Power
Outlet and Fits into a Cup Holder
31
-------
Chapter 7
Performance Summary
Table 7-1. EzyBot® A Summary Table
Parameter
Sample Information
Botulinum Toxin
A (mg/L)
Lab Bench Scale
Fluorimeter
-------
Table 7-1. EzyBot® A Summary Table (Continued)
False positives
There were no false positive results from interferents including a preservative blank, humic and
fulvic acids, and Ca and Mg; DW from four locations using different water treatment
technologies; or the potentially cross-reactive lipopolysaccharide (0.1 mg/L).
False negatives
False negatives were obtained in the presence of both 0.5 and 2.5 mg/L each humic and fulvic
acids. A false negative was also obtained in New York water which was concentrated by a
factor of 400. A total of 3 false negative results were obtained out of the 12 solutions assessed
at 60 minutes. The vendor informed Battelle after testing that the lab bench scale fluorimeter
provided for testing may have had inconsistent functioning which could have caused the false
negative results that were obtained.
Consistency
Using the lab bench scale fluorimeter, results were consistent in 100% of the samples tested.
Using the field portable fluorimeter, results were consistent in 90% of the samples tested.
Other
Performance
Factors
Convenient ready-to use cuvettes. Easy to operate in the lab and easy to transport and operate in
the field. No formal scientific education would be required to use the kit; however, general lab
skills and training on fluorimeter use were helpful. Approximately 12-15 analyses were
completed in one hour in the laboratory. Only five samples could be processed in one hour in the
field due to size limitation of the field portable incubator. Each EzyBot ® kit contains 50 ready-
to-use cuvettes.
33
-------
Table 7-2. EzyBot®B Summary Table
Parameter
Sample
Information
Botulinum Toxin B
(mg/L)
Lab Bench Scale
Fluorimeter00
Field Portable
Fluorimeter00
30 min.
60 min.
30 min.
60 min.
Contaminant-only
DI Water
0.01 (vendor-stated
limit of detection)
0
3
0
0
0.05
7
10
0
0
0.1
3
3
0
0
0.3 (lethal dose)
3
3
3
3
0.5
3
3
0
3
Interfere nt
0.5 mg/L each
humic/fulvic acids
0.1
3
3
NA
2.5 mg/L each
humic/fulvic acids
0.1
1
3
50 and 250 mg/L each
Ca/Mg
0.1
0
0
DW- all but New York
unconcentrated
0.1
0
3
DW- New York
unconcentrated
0.1
3
3
DW-California
concentrated
0.1
0
3
DW-Florida
concentrated
0.1
3
3
DW-New York
concentrated
0.1
0
3
DW-Ohio
concentrated
0.1
3
3
Lowest Detectable Concentration® (mg/L)
0.05
0.01
ND 0.3
False positives
There were no false positive results from interferents including a preservative blank, humic and
fulvic acids, and Ca and Mg; DW from four locations using different water treatment
technologies; or the potentially cross-reactive lipopolysaccharide (0.1 mg/L).
False negatives
False negative results were obtained in the presence of both 50 and 250 mg/L Ca and Mg using
both a 30 minute and 60 minute incubation time. The 30 minute incubation time also generated
false negative results in unconcentrated water from California, Florida, and Ohio; and in
concentrated water from California and New York. A total of 8 false negative results were
obtained out of the 12 solutions assessed at 30 minutes. A total of 2 false negative results were
obtained out of the 12 solutions assessed at 60 minutes. The vendor informed Battelle after
testing that the lab bench scale fluorimeter provided for testing may have had inconsistent
functioning which could have caused the false negative results that were obtained.
Consistency
For the lab bench scale fluorimeter, results were consistent in 97% of the samples tested. With
the field portable fluorimeter, results were consistent in 100% of the samples tested.
Other Performance
Factors
Convenient ready-to use cuvettes. Easy to operate in the lab and easy to transport and operate in
the field. No formal scientific education would be required to use the kit; however, general lab
skills and training on fluorimeter use were helpful. Approximately 12-15 analyses were
completed in one hour in the laboratory. Only five samples could be processed in one hour in the
field due to size limitation of the field portable incubator. Each EzyBot ® kit contains 50 ready-
to-use cuvettes.
NA = Not tested. Testing concentration below detection in the contaminant only PT testing.
ND = not detectable at concentrations tested.
(a) Results out of 3 replicates except for the 0.05 mg/L contaminant only concentration for which results are out of 10 replicates.
(b) The lowest concentration of contaminant-only PT samples to have at least two thirds of the measurements generate positive results.
34
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Chapter 8
References
1. Test/QA Plan for Verification of Immunoassay Test Kits, Battelle, Columbus, Ohio,
January 2004.
2. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, Washington, D.C., 1997.
3. U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020,
March 1983.
4. 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.
5. 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.
6. U.S. EPA Method 200.7, "Trace Elements in Water, Solids, and Biosolids by Inductively
Coupled Plasma—Atomic Emission Spectrometry," EPA-821-R-01-010, January 2001.
7. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 20th Edition, Washington, D.C., 1998.
8. 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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