June 2006
Environmental Technology
Verification Report
STRATEGIC DIAGNOSTICS INC.
RAPIDTOXKIT
Prepared by
Battelle
Balteiie
The Business of Innovation
Under a cooperative agreement with
U.S. Environmental Protection Agency
ET1/ET1/ET1/
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THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
U.S. Environmental Protection Agency
Batreiie
Trtc Business o/ Innovation
ETV Joint Verification Statement
TECHNOLOGY TYPE: Rapid Toxicity Testing System
APPLICATION: Detecting Toxicity in Drinking Water
TECHNOLOGY
NAME:
COMPANY:
ADDRESS:
WEB SITE:
E-MAIL:
RAPIDTOXKIT
Strategic Diagnostics Inc.
Ill Pencader Drive
Newark, DE 19702
www.sdix.com
jambrosius@sdix.com
PHONE: (302) 456-6789
FAX: (302) 456-6782
The U.S. Environmental Protection Agency (EPA) has established 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 the Strategic Diagnostics Inc. RAPIDTOXKIT. This verification statement provides a
summary of the test results.
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VERIFICATION TEST DESCRIPTION
Rapid toxicity technologies use various biological organisms and chemical reactions to indicate the presence of
toxic contaminants. The toxic contaminants are indicated by a change or appearance of color or a change in
intensity. As part of this verification test, the RAPIDTOXKIT was subjected to various concentrations of
contaminants such as industrial chemicals, pesticides, rodenticides, Pharmaceuticals, nerve agents, and biological
toxins. Each contaminant was added to separate drinking water samples and analyzed. In addition to determining
whether the RAPIDTOXKIT could detect the toxicity caused by each contaminant, its response to interfering
compounds, such as water treatment chemicals and by-products in clean drinking water, was evaluated.
The RAPIDTOXKIT was evaluated by
• Endpoints and precision—percent inhibition for all concentration levels of contaminants and potential
interfering compounds and precision of replicate analyses
• Toxicity threshold for each contaminant—contaminant level at which higher concentrations generate
inhibition significantly greater than the negative control and lower concentrations do not. Note that
Strategic Diagnostics Inc. recommends that a 30% inhibition is required for a conclusive indication of
toxicity. During this test, a thorough evaluation of the toxicity threshold was performed. Therefore, the
toxicity threshold was determined with respect to the negative control rather than the 30% inhibition
threshold
• False positive responses—chlorination and chloramination by-product inhibition exceeding 30% with
respect to unspiked American Society for Testing and Materials (ASTM) Type II deionized (DI) water
samples
• False negative responses—contaminants that were reported as producing inhibition results less than 30%
when present at lethal concentrations (the concentration at which 250 milliliters of water would probably
cause the death of a 154-pound person) or negative background inhibition that caused falsely low
inhibition
• Other performance factors (sample throughput, ease of use, reliability).
The RAPIDTOXKIT was verified by analyzing a dechlorinated drinking water sample from Columbus, Ohio
(DDW), fortified with contaminants (at concentrations ranging from lethal levels to concentrations up to one
million times less than the lethal dose) and interferences (metals possibly present as a result of the water
treatment processes). Dechlorinated water was used because free chlorine kills the larval crustacean within the
RAPIDTOXKIT reagent and can degrade the contaminants during storage. Inhibition results (endpoints)
from four replicates of each contaminant at each concentration level were evaluated to assess the ability of the
RAPIDTOXKIT to detect toxicity, as well as to measure the precision of the RAPIDTOXKIT results. The
response of the RAPIDTOXKIT to possible interferents was evaluated by analyzing them at one-half of the
concentration limit recommended by the EPA's National Secondary Drinking Water Regulations guidance.
For analysis of by-products of the chlorination process, the unspiked DDW was analyzed because Columbus,
Ohio, uses chlorination as its disinfectant procedure. For the analysis of by-products of the chloramination
process, a separate drinking water sample was obtained from the Metropolitan Water District of Southern
California (LaVerne, California), which uses chloramination as its disinfection process. The samples were
analyzed after residual chlorine was removed using sodium thiosulfate. Sample throughput was measured
based on the number of samples analyzed per hour. Ease of use and reliability were determined based on
documented observations of the operators.
Quality control samples included method blank samples, which consisted of American Society for Testing
and Materials Type II deionized water; positive control samples (vendor-specified); and negative control
samples, which consisted of the unspiked DDW.
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QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a
technical systems audit, a performance evaluation 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 test
are all available at www.epa.gov/etv/centers/centerl.html.
TECHNOLOGY DESCRIPTION
The following description of the RAPIDTOXKIT is based on information provided by the vendor. This
technology description was not verified in this test.
The RAPIDTOXKIT uses larvae of the anostracan crustacean T. platyurus to detect freshwater (including
drinking water) contamination. The RAPIDTOXKIT bioassays are performed in disposable test tubes using
T. platyurus hatched from cysts. Cyst hatching must begin 30 to 45 hours prior to performing the test. The
T. platyurus are exposed to samples for 15 minutes to one hour, after which a suspension of red microspheres
is added. The organisms ingest the microspheres, resulting in a deep red color in their digestive tracts.
Stressed (intoxicated) organisms either fail to take up particles altogether or ingest at a much lower rate. The
presence or the absence of colored microspheres in the digestive tract of the larval crustaceans is observed
under a stereomicroscope, and data are recorded on a sheet supplied with the RAPIDTOXKIT. The total
number of T. platyurus in the control (standard freshwater) well(s), and the number of T. platyurus that have
taken up the red particles are counted, and the fraction of larval crustaceans affected by the contaminant is
defined as the percent inhibition. As a guideline, 30 percent inhibition of particle uptake is considered a
threshold for the presence of potentially toxic compounds in the water.
Each test kit includes three 1-milliliter test tubes containing cysts of T. platyurus, one bottle of standard
freshwater, three hatching vessels, six sub-sampling tubes, 48 test tubes, six test tube holders, one vial with
red microspheres, one vial with fixative, six observation plates, six transparent covers for observation plates, a
blue plastic sheet and grid designed to be placed under plates to aid in observing and scoring test organisms,
standard operating procedure booklet, bench protocol, six sheets for scoring test results and calculating mean
inhibition of particle uptake, and a specification sheet containing batch numbers and shelf lives of kit
components. Materials required but not provided as part of the kit include a 25°C incubator with 4,000-lux
constant illumination, a dissection microscope with minimum 10X magnification, and an overhead light
source for the microscope. The complete RAPIDTOXKIT, adequate for 7 to 15 water samples each,
depending on the sample size, measures 30 centimeters by 25 centimeters by 10 centimeters and costs $196.
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VERIFICATION RESULTS
Parameter
Contaminants in
DDW
Potential
interferences in
DDW
False positive
response
False negative
response
Ease of use
Field portability
Throughput
Compound
Aldicarb
Botulinum
toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium
sulfate
VX
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Lethal
Dose (LD)
Cone.
(mg/L)
260
0.3
240
250
1,400
2,800
15
1.4
2,800
2
Cone.
(mg/L)
0.5
0.6
0.15
0.25
2.5
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
100
4
56
100
100
100
27
100
100
99
LD/10
100
-51
13
100
100
100
14
99
100
10
Average
Inhibition (%)
29
100
20
-11
24
LD/100
100
-40
26
100
100
100
-2
100
79
4
LD/1,000
53
-32
28
51
-4
100
6
-2
29
22
Standard
Deviation (%)
6
0
4
11
9
Range of
Standard
Deviations
(%)
0-10
6-23
9-13
0-17
0-6
0
1-6
0-6
0-19
1-6
Toxicity
Thresh.
(mg/L)
0.26
ND
240
0.25
14
0.28
15
0.007
28
1.5
No results from the RAPIDTOXKIT were considered false positive because inhibition in the
chlorinated and chloraminated drinking water samples was always less than 30%.
Only botulinum toxin complex B exhibited inhibition less
dose concentration.
than 30% when analyzed at a lethal
The RAPIDTOXKIT contained clearly written instructions and illustrations. The contents of the
RAPIDTOXKIT were well identified. The only problem, other than the difficulty opening some
containers, was a slight difficulty getting the cysts out of the tubes with the recommended 1 mL
of water. Manually counting the number of red organisms under the microscope was tedious
when the results from many samples were determined one after the other over a few hours.
Overall, the RAPIDTOXKIT was easy to use, making it likely that a person with no formal
scientific training could conduct the tests.
The RAPIDTOXKIT was not evaluated for field portability.
Not including the 30 to 45-hour cyst-hatching period, approximately 25 analyses (including
method blanks and positive and negative controls) were completed in three hours. A maximum of
45 samples could be processed per kit.
ND = Significant inhibition was not detected.
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Original signed by Gregory A. Mack
Gregory A. Mack Date
Vice President
Energy, Transportation, and Environment Division
Battelle
6/22/06 Original signed by Andrew P. Avel
Andrew P. Avel
Acting Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
8/7/06
Date
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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June 2006
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Strategic Diagnostics Inc.
RAPIDTOXKIT
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 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; Yves
Mikol, New York City Department of Environmental Protection; and Stanley States, Pittsburgh
Water and Sewer Authority, for their careful review of the test/quality assurance plan and/or 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 6
3.1.1 Quality Control Samples 6
3.1.2 Drinking Water Fortified with Contaminants 6
3.1.3 Drinking Water Fortified with Potential Interferences 7
3.2 Test Procedure 7
3.2.1 Test Sample Preparation and Storage 7
3.2.2 Test Sample Analysis Procedure 7
3.2.3 Stock Solution Confirmation Analysis 9
Chapter 4 Quality Assurance/Quality Control 13
4.1 Quality Control of Stock Solution Confirmation Methods 13
4.2 Quality Control of Drinking Water Samples 13
4.3 Audits 14
4.3.1 Performance Evaluation Audit 14
4.3.2 Technical Systems Audit 15
4.3.3 Audit of Data Quality 15
4.4 QA/QC Reporting 15
4.5 Data Review 16
Chapter 5 Statistical Methods and Reported Parameters 17
5.1 Endpoints and Precision 17
5.2 Toxicity Threshold 18
5.3 False Positive/Negative Responses 18
5.4 Other Performance Factors 19
Chapter 6 Test Results 20
6.1 Endpoints and Precision 20
6.1.1 Contaminants 20
6.1.2 Potential Interferences 32
6.1.3 Precision 33
6.2 Toxicity Threshold 34
6.3 False Positive/Negative Responses 35
6.4 Other Performance Factors 35
6.4.1 Ease of Use 35
6.4.2 Field Portability 36
6.4.3 Throughput 36
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Chapter 7 Performance Summary 37
Chapters References 38
Figures
Figure 2-1. Strategic Diagnostics Inc. RAPIDTOXKJT 2
Figure 3-1. Magnification of T. platyurus on Observation Plate 10
Tables
Table 3-1. Contaminants and Potential Interferences 5
Table 3-2. Summary of Quality Control and Contaminant Test Samples 8
Table 3-3. Stock Solution Confirmation Results 11
Table 3-4. Water Quality Parameters 12
Table 4-1. Summary of Performance Evaluation Audit 15
Table 4-2. Summary of Data Recording Process 16
Table 6-la. Aldicarb Percent Inhibition Results 21
Table 6-lb. Aldicarb Percent Inhibition Results—Additional Dilutions 21
Table 6-lc. Botulinum Toxin Complex B Percent Inhibition Results 22
Table 6-ld. Colchicine Percent Inhibition Results 22
Table 6-le. Cyanide Percent Inhibition Results 23
Table 6-lf. Cyanide Percent Inhibition Results—Additional Dilutions 23
Table 6-lg. Dicrotophos Percent Inhibition Results 24
Table 6-lh. Dicrotophos Percent Inhibition Results—Additional Dilutions 24
Table 6-li. Nicotine Percent Inhibition Results 25
Table 6-lj. Nicotine Percent Inhibition Results—Additional Dilutions 25
Table 6-lk. Ricin Percent Inhibition Results 26
Table 6-11. Soman Percent Inhibition Results 27
Table 6-lm. Soman Percent Inhibition Results—Additional Dilutions 28
Table 6-ln. Thallium Sulfate Percent Inhibition Results 29
Table 6-lo. VX Percent Inhibition Results 29
Table 6-lp. VX Percent Inhibition Results—Additional Dilutions 30
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results 31
Table 6-3. Potential Interferences Results 33
Table 6-4. Toxicity Thresholds 34
Table 6-5. False Negative Responses 35
VI
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List of Abbreviations
AMS Advanced Monitoring Systems
ASTM American Society for Testing and Materials
ATEL Aqua Tech Environmental Laboratories
DI deionized water
DDW dechlorinated drinking water from Columbus, Ohio
DPD n,n-diethyl-p-phenylenediamine
EPA U.S. Environmental Protection Agency
ETV Environmental Technology Verification
HDPE high-density polyethylene
LD lethal dose
mM millimolar
[iL microliter
mg/L milligram per liter
mL milliliter
mm millimeter
NSDWR National Secondary Drinking Water Regulations
%D percent difference
PE performance evaluation
QA quality assurance
QC quality control
QMP quality management plan
SOP standard operating procedure
TSA technical systems audit
vn
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental
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 Strategic Diagnostics Inc. RAPIDTOXKIT. Rapid
toxicity technologies were identified as a priority verification category 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
environmental monitoring technologies for air, water, and soil. This verification report provides
results for the verification testing of the RAPIDTOXKIT. Following is a description of the
RAPIDTOXKIT, based on information provided by the vendor. The information provided below
was not verified in this test.
The RAPIDTOXKIT (Figure 2-1) uses
larvae of the anostracan crustacean
T. platyurus to detect freshwater
(including drinking water) contamination.
The RAPIDTOXKIT bioassays are
performed in disposable test tubes using
T. platyurus hatched from cysts. Cyst
hatching must begin 30 to 45 hours prior to
performing the test. The T. platyurus are
exposed to samples for 15 minutes to one
hour, after which a suspension of red
microspheres is added. The organisms
ingest the microspheres, resulting in a deep
red color in their digestive tracts. Stressed
(intoxicated) organisms either fail to take up
particles altogether or ingest at a much
lower rate. The presence or the absence of
colored microspheres in the digestive tract
of the larval crustaceans is observed under a
Figure 2-1. Strategic Diagnostics Inc.
RAPIDTOXKIT
stereomicroscope, and data are recorded on a sheet supplied with the RAPIDTOXKIT. The total
number of T. platyurus in the control (standard freshwater) well(s), and the number of T. platyurus
that have taken up the red particles are counted, and the fraction of larval crustaceans affected by the
contaminant is defined as the percent inhibition. As a guideline, 30 percent inhibition of particle
uptake is considered a threshold for the presence of potentially toxic compounds in the water.
Each test kit includes three 1 -milliliter test tubes containing cysts of T. platyurus, one bottle of
standard freshwater, three hatching vessels, six sub-sampling tubes, 48 test tubes, six test tube
holders, one vial with red microspheres, one vial with fixative, six observation plates, six transparent
covers for observation plates, a blue plastic sheet and grid designed to be placed under plates to aid in
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observing and scoring test organisms, standard operating procedure booklet, bench protocol, six
sheets for scoring test results and calculating mean inhibition of particle uptake, and a specification
sheet containing batch numbers and shelf lives of kit components. Materials required but not
provided as part of the kit include a 25°C incubator with 4,000-lux constant illumination, a dissection
microscope with minimum 10X magnification, and an overhead light source for the microscope. The
complete RAPIDTOXKIT, adequate for 7 to 15 water samples each, depending on the sample size,
measures 30 centimeters by 25 centimeters by 10 centimeters and costs $196.
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Chapter 3
Test Design
The objective of this verification test of rapid toxicity technologies was to evaluate their ability
to detect certain toxins and to determine their susceptibility to interfering chemicals in a
controlled experimental matrix. Rapid toxicity technologies do not identify or determine the
concentration of specific contaminants, but serve as a screening tool to quickly determine
whether water is potentially toxic.
As part of this verification test, the RAPIDTOXKIT was subjected to various concentrations of
contaminants such as industrial chemicals, pesticides, rodenticides, Pharmaceuticals, nerve
agents, and biological toxins. Each contaminant was added to separate drinking water samples
and analyzed. In addition to determining whether the RAPIDTOXKIT could detect the toxicity
caused by each contaminant, its response to interfering compounds such as water treatment
chemicals and by-products in clean drinking water, was evaluated. Table 3-1 shows the
contaminants and potential interferences that were evaluated during this verification test.
This verification test was conducted from August to December 2005 according to procedures
specified in the Test/QA Plan for Verification of Rapid Toxicity Technologies including
Amendments 1 and 2.(1) The RAPIDTOXKIT was verified by analyzing a dechlorinated drinking
water sample from Columbus, Ohio (hereafter in this report referred to as DDW), fortified with
various concentrations of the contaminants and interferences shown in Table 3-1. Where
possible, the concentration of each contaminant or potential interference was confirmed
independently by Aqua Tech Environmental Laboratories (ATEL), Marion, Ohio, or by Battelle,
depending on the analyte.
The RAPIDTOXKIT was evaluated by
• Endpoints and precision—percent inhibition for all concentration levels of contaminants and
potential interfering compounds and precision of replicate analyses
• Toxicity threshold for each contaminant—contaminant level at which higher concentrations
generate inhibition significantly greater than the negative control and lower concentrations
do not. Note that Strategic Diagnostics Inc. recommends that a 30% inhibition is required for
a conclusive indication of toxicity. During this test, a thorough evaluation of the toxicity
threshold was performed. Therefore, the toxicity threshold was determined with respect to
the negative control rather than the 30% inhibition threshold
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Table 3-1. Contaminants and Potential Interferences
Category
Biological toxins
Botanical pesticide
Carbamate pesticide
Industrial chemical
Nerve agents
Organophosphate pesticide
Pharmaceutical
Potential interferences
Rodenticide
Contaminant
Botulinum toxin complex B, ricin
Nicotine
Aldicarb
Cyanide
Soman, VX
Dicrotophos
Colchicine
Aluminum, copper, iron, manganese, zinc, chloramination
by-products, and chlorination by-products
Thallium sulfate
• False positive responses—chlorination and chloramination by-product inhibition exceeding
30% with respect to unspiked American Society for Testing and Materials (ASTM) Type II
deionized (DI) water samples
• False negative responses—contaminants that were reported as producing inhibition less than
30% when present at lethal concentrations or negative background inhibition that caused
falsely low inhibition results or negative background inhibition that caused falsely low
inhibition
• Other performance factors (sample throughput, ease of use, reliability).
The RAPIDTOXKIT was used to analyze the DDW samples fortified with contaminants at
concentrations ranging from lethal levels to concentrations up to one million times less than the
lethal dose. The lethal dose of each contaminant was determined by calculating the concentration
at which 250 milliliters (mL) of water would probably cause the death of a 154-pound person.
These calculations were based on toxicological data available for each contaminant that are
presented in Amendment 2 of the test/QA plan.(1) Inhibition (endpoints) from four replicates of
each contaminant at each concentration level were evaluated to assess the ability of the
RAPIDTOXKIT to detect toxicity at various concentrations of contaminants, as well as to
measure the precision of the RAPIDTOXKIT results.
The response of the RAPIDTOXKIT to compounds used during the water treatment process
(identified as potential interferences in Table 3-1) was evaluated by analyzing separate aliquots
of DDW fortified with each potential interference at one-half of the concentration limit
recommended by the EPA's National Secondary Drinking Water Regulations (NSDWR)(2)
guidance. For analysis of by-products of the chlorination process, the unspiked DDW was
analyzed because Columbus, Ohio, uses chlorination as its disinfectant procedure. For the
analysis of by-products of the chloramination process, a separate drinking water sample was
obtained from the Metropolitan Water District of Southern California (LaVerne, California),
which uses chloramination as its disinfection process. The samples were analyzed after residual
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chlorine was removed using sodium thiosulfate. Sample throughput was measured based on the
number of samples analyzed per hour. Ease of use and reliability were determined based on
documented observations of the operators.
3.1 Test Samples
Test samples used in the verification test included drinking water and quality control (QC)
samples. Table 3-2 shows the number and type of samples analyzed. QC samples included
method blanks and positive and negative control samples. The fortified drinking water samples
were prepared from a single drinking water sample collected from the Columbus, Ohio, system.
The water was dechlorinated using sodium thiosulfate and then fortified with various concen-
trations of contaminants and interferences. The DDW containing the potential interferences was
analyzed at a single concentration level, while at least four dilutions were analyzed for each
contaminant using the RAPIDTOXKIT and additional dilutions of some contaminants were
analyzed to better determine the toxicity threshold. Mixtures of contaminants and possible
interfering compounds were not analyzed.
3.1.1 Quality Control Samples
QC samples included method blanks, positive controls, negative controls, and preservative
blanks. The method blank samples consisted of ASTM Type IIDI water and were used to help
ensure that no sources of contamination were introduced in the sample handling and analysis
procedures. A positive control sample was included in the RAPIDTOXKIT and was used as
provided from the vendor. While performance limits were not placed on the results, significant
inhibition for the positive control sample indicated to the operator that the RAPIDTOXKIT was
functioning properly. Two negative control samples were included. One was provided by the
vendor. The second consisted of unspiked DDW and was used to set a background inhibition of
the DDW, the matrix in which each test sample was prepared. To ensure that the preservatives in
the contaminant solutions did not have an inhibitory effect, preservative blank samples were
prepared. These preservative blanks consisted of DDW fortified with a concentration of
preservative equivalent to that in the test solutions of botulinum toxin complex B, ricin, soman,
and VX.
3.1.2 Drinking Water Fortified with Contaminants
Approximately 50 liters of Columbus, Ohio, tap water were collected in a low-density
polyethylene container. The water was dechlorinated with sodium thiosulfate. Dechlorination
was confirmed by adding an n,n-diethyl-p-phenylenediamine (DPD) tablet to a 10-mL aliquot of
the water. Lack of color development in the presence of DPD indicated that the water was
dechlorinated. All subsequent test samples were prepared from this water sample.
A stock solution of each contaminant was prepared in DDW at concentrations at or above the
lethal dose level. The stock solution was further diluted to obtain one sample containing the
lethal dose concentration for each contaminant and three additional samples with concentrations
10, 100, and 1,000 times less than the lethal dose. Additional concentrations of some contam-
inants were prepared and analyzed for two reasons: one was because of the large difference in
response between two concentration levels. For example, if only one dilution level was almost
completely inhibitory and the next dilution level was non-inhibitory, several intermediate
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concentrations were analyzed to better determine the toxicity threshold of that contaminant. The
other reason was because sometimes the lowest concentration analyzed was mostly inhibitory,
thus, not providing even an estimate of the toxicity threshold. For these contaminants, additional
tenfold dilutions were analyzed to more accurately determine the toxicity threshold. Table 3-2
lists each concentration level and the number of samples analyzed at each level.
3.1.3 Drinking Water Fortified with Potential Interferences
Individual aliquots of the DDW were fortified with one-half the concentration specified by the
EPA's NSDWR for each potential interference. Table 3-2 lists the interferences, along with the
concentrations at which they were tested. Four replicates of each of these samples were analyzed.
To test the sensitivity of the RAPIDTOXKIT to by-products of the chlorination process as
potential interferences, the unspiked DDW (same as the negative control) was used since the
water sample originated from a utility that uses chlorination as its disinfectant procedure. In a
similar manner, by-products of the chloramination process were evaluated using a water sample
from the Metropolitan Water District of Southern California. The residual chlorine in both of
these samples was removed using sodium thiosulfate, and then the samples were analyzed in
replicate with no additional fortification of contaminants.
3.2 Test Procedure
The procedures for preparing, storing, and analyzing test samples and confirming stock solutions
are provided below.
3.2.1 Test Sample Preparation and Storage
A drinking water sample was collected as described in Section 3.1.2 and, because free chlorine
kills the larval crustaceans within the RAPIDTOXKIT reagent and can degrade the contaminants
during storage, was immediately dechlorinated with sodium thiosulfate. Dechlorination of the
water sample was qualitatively confirmed by adding a DPD tablet to a 10-mL aliquot of the
DDW. All the contaminant samples, potential interference samples, preservative blanks, and
negative control QC samples were made from this water sample, while the method blank sample
was prepared from ASTM Type IIDI water. The positive control samples were made by adding
the vendor-specified positive control solution to ASTM Type II DI water using calibrated auto-
pipettes. All QC samples were prepared prior to the start of the testing and stored at room
temperature. The stability of each contaminant for which analytical methods are available was
confirmed by analyzing it three times over a two-week period. Throughout this time, each
contaminant maintained its original concentration to within approximately 25%. Therefore, the
aliquots of DDW containing the contaminants were prepared within two weeks of testing and
were stored at room temperature without chemical preservation. The contaminants without
analytical methods were analyzed within 48 hours of their preparation. To maintain the integrity
of the test, test samples provided to the operators were labeled only with sample identification
numbers so that the operators did not know their content.
3.2.2 Test Sample Analysis Procedure
Prior to test sample analysis, the larval T. platyurus was required to be hatched from cysts. This
was done by hydrating the cysts for 1 hour in 1 mL of fresh water and then transferring them to
7
-------
Table 3-2. Summary of Quality Control and Contaminant Test Samples
Type of Sample
Quality control
DDW fortified
with contaminants
DDW tortitied
with potential
interferences
Disinfectant by-
products
Sample Characteristics
Method blank
(ASTM Type II water)
Positive control
(potassium iodide/iodine solution)
Negative control
(standard freshwater: moderately
hard EPA medium)
Negative control
(unspiked DDW)
Preservative blank: botulinum
toxin complex B
Preservative blank: VX and soman
Preservative blank: ricin
Aldicarb
Botulinum toxin complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Aluminum
Copper
Iron
Manganese
Zinc
Chloramination by-products
Chlorination by-products
Concentration Levels
NA
Used as provided in kit
Used as provided in kit
NA
0.015 millimolar (mM) sodium
citrate
0.21% isopropyl alcohol
0.00024% NaN3, 0.00045 M
NaCl, 0.03mM phosphate
260; 26; 2.6; 0.26; 0.026;
0.0026; 0.00026
milligrams/liter (mg/L)
0.30; 0.030; 0.0030; 0.00030
mg/L
240; 24; 2.4; 0.24 mg/L
250; 25; 2.5; 0.25; 0.1875;
0.1250; 0.0625 mg/L
1,400; 140; 14; 1.4; 0.140;
0.014; 0.0014 mg/L
2,800; 280; 28; 2.8; 0.28;
0.028; 0.0028 mg/L
15; 1.5; 0.15; 0.015 mg/L
1.4; 0.14; 0.014; 0.0105;
0.0035; 0.0014; 0.007 mg/L
2,800; 280; 28; 2.8 mg/L
2.0; 1.5; 1.0; 0.5; 0.2; 0.02;
0.002; mg/L
0.5 mg/L
0.6 mg/L
0.15 mg/L
0.25 mg/L
2.5 mg/L
NA
NA
No. of Sample
Analyses
20
20
76
76
4
4 with VX, 4 with soman
4
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4
4
4
4
4
4
76
NA = not applicable, samples not fortified with any preservative, contaminant, or potential interference.
-------
the hatching vessel with 8 mL of fresh water for an incubation time of 30 to 45 hours at 25°C
with continuous illumination. Afterward, the test organisms were transferred into the sub-
sampling tube until use. The test organisms must be used within 30 and 45 hours after the start of
incubation.
Once the organisms were hatched, the test tubes were filled up to the horizontal mark with both
control (positive and negative) and test sample solutions. The larvae were distributed evenly
throughout the suspension in the sub-sampling tube by repeated aspiration and dispensation of
the water/larvae suspension from a pipette, and 0.5 mL of larval suspension was transferred into
each control and sample test tube. The samples were then incubated for 1 hour at 25°C. After an
hour, 0.2 mL of the microsphere bead suspension (mixed well) was added to each sample test
tube. The test tubes were then shaken gently to homogenize the contents. The tubes were
incubated for 15 to 30 minutes at 25°C. Then, three drops of fixative (which kills the larvae)
were added to each test tube, and the samples were mixed again.
After a 5-minute wait to allow the dead larvae to settle to the bottom of the test tubes, the micro-
pipette was set to 0.2 mL; and all the larvae from the bottom of each test tube were transferred
into wells on the observation plate. The observation plate was covered with a transparent cover
and placed underneath the microscope. A magnification was selected that allowed a complete
view of the well surface (example shown in Figure 3-1). The total number of larvae in the well
(colored and not colored digestive tracts) was counted, as well as the number of larvae with
distinct colored digestive tracts. In the figure, an example of a colored digestive tract is labeled
"P" for positive microparticle uptake, indicating that the contaminant was not inhibiting the
organism; and an example of a non-colored digestive tract that did not experience microparticle
uptake is labeled "N" for negative. The uptake of colored particles may vary among larvae.
Lightly colored larvae were still counted as positive. Two examples of these are labeled "LC" on
the figure. In addition, because not all of the cysts hatch completely, among the larvae collected
for the analysis, there will still be some at an early stage of development (smaller, orange in
color, and not transparent). These opaque larvae were completely excluded from the scoring. In
the figure, two larvae with these characteristics are labeled "NC" for not counted. The
observation plate was placed on a grid to make counting the larvae easier. In general, fully
developed larvae with colored digestive tracts have not been inhibited by a contaminant, while
those without have. Two operators performed all the analyses using the RAPIDTOXKIT. One
operator performed testing with contaminants that did not require special chemical and biological
agent training and one performed testing with those that did. Both held bachelor's degrees in the
sciences and were trained by the vendor to operate the RAPIDTOXKIT.
3.2.3 Stock Solution Confirmation Analysis
The concentrations of the contaminant and interfering compound stock solutions were confirmed
with standard analytical methods, with the exception of colchicine, ricin, and botulinum toxin
complex B—contaminants without standard analytical methods. Aliquots to be analyzed by
standard methods were preserved as prescribed by the method. In addition, the same standard
methods were used to measure the concentration of each contaminant/potential interference in
the unspiked DDW so that background concentrations of contaminants or potential interferences
were accounted for within the displayed concentration of each contaminant/potential interference
sample. Table 3-3 lists the standard methods used to measure each analyte; the results from the
stock solution confirmation analyses (obtained by analyzing the lethal dose concentration for the
-------
^ a 3 ^ I
Figure 3-1. Magnification of T. platyurus on Observation Plate. P = positive, LC = lightly
colored (still positive), N = negative, and NC = not counted.
contaminants and the single concentration that was analyzed for the potential interferences); and
the background levels of the contaminants and potential interferences measured in the DDW
sample, which were all non-detect or negligible.
Standard methods were also used to characterize several water quality parameters such as
alkalinity; dissolved organic carbon content; specific conductivity; hardness; pH; concentration
of haloacetic acids, total organic carbon, total organic halides, and trihalomethanes; and
turbidity. Table 3-4 lists these measured water quality parameters for both the water sample
collected in Columbus, Ohio, representing a water system using chlorination as the disinfecting
process, and the water sample collected at the Metropolitan Water District of Southern
California, representing a water system using chloramination for disinfection.
10
-------
Table 3-3. Stock Solution Confirmation Results
Contaminant
Aldicarb
Botulinum toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Potential
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Method
Battelle
method
(a)
(a)
EPA335.3(3)
Battelle
method
Battelle
method
(a)
Battelle
method
EPA 200.8(4)
(a) Battelle
method
EPA 200.7(5)
EPA 200.7(5)
EPA 200.7(5)
EPA 200.7(5)
EPA 200.7(5)
Average Concentration ± Standard
Deviation N = 4 (mg/L)(b)
260 ±7
NA
NA
249 ±4
296 ± 26 (field portability)
1,168 ±18
2,837 ± 27
NA
1.3 ±0.1 (10/1 8/05)
1.16 ±0.06 (10/21/05)
2,469 ±31
1.89 ±0.08 (10/17/05)
1.77 ±0.03 (10/20/05)
0.50 ± 0.02
0.60 ± 0.03
0.155 ±0.006
0.281 ±0.008
2.63 ± 0.05
Background in
DDW (mg/L)
<0.005
NA
NA
0.006
<3.0
<0.01
NA
<0.025
<0.001
<0.0005
<0.2
<0.02
<0.04
<0.01
0.27
NA = Not applicable.
(a) No standard method available. QA audits and balance calibration assured accurately prepared
(b) Target concentration was highest concentration for each contaminant or interference on Table
solutions.
3-2.
11
-------
Table 3-4. Water Quality Parameters
Parameter
Alkalinity (mg/L)
Specific conductivity
(umho)
Hardness (mg/L)
pH
Total haloacetic acids
(Hg/L)
Dissolved organic
carbon (mg/L)
Total organic carbon
(mg/L)
Total organic halides
(Hg/L)
Total trihalomethanes
(Hg/L)
Turbidity (NTU)
Method
SM 2320 B(6)
SM2510B(6)
EPA 130.2(7)
EPA 150.1(7)
EPA 552.2(8)
SM5310B(6)
SM5310B(6)
SM 5320B(6)
EPA 524.2(9)
SM2130(10)
Dechlorinated Columbus,
Ohio, Tap Water
(disinfected by
chlorination)
40
572
118
7.6
32.8
2.1
2.1
220
74.9
0.1
Dechlorinated Southern
California Tap Water
(disinfected by
chloramination)
71
807
192
8.0
17.4
2.9
2.5
170
39.2
0.1
NTU = nephelometric turbidity unit.
12
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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center*11} and the test/QA plan for this verification test.(1)
4.1 Quality Control of Stock Solution Confirmation Methods
The stock solutions for the contaminants cyanide and thallium sulfate and for the potential
interferences aluminum, magnesium, zinc, iron, and copper were analyzed at ATEL using
standard reference methods. As part of ATEL's standard operating procedures (SOPs), various
QC samples were analyzed with each sample set. These included matrix spike, laboratory control
spike, and method blank samples. According to the standard methods used for the analyses,
recoveries of the QC spike samples analyzed with samples from this verification test were within
acceptable limits of 75% to 125%, and the method blank samples were below the detectable
levels for each analyte. For VX, soman, aldicarb, nicotine, and dicrotophos, the confirmation
analyses were performed at Battelle using a Battelle SOP or method. Calibration standard
recoveries of VX and soman were always between 62% and 141%, and most of the time were
between 90% and 120%. Dicrotophos standard recoveries ranged from 89% to 122%. Aldicarb
standard recoveries ranged from 95% to 120%. Nicotine standard recoveries ranged from 96% to
99%. Standard analytical methods for colchicine, ricin, and botulinum toxin complex B were not
available and, therefore, not performed. QA audits and balance calibrations assured that solutions
for these compounds were accurately prepared.
4.2 Quality Control of Drinking Water Samples
A method blank sample consisting of ASTM Type IIDI water was analyzed once by the
RAPIDTOXKIT for approximately every 20 drinking water samples that were analyzed.
Because inhibition has to be calculated with respect to a control sample, none were calculated for
the method blank samples. The method blanks were used as the control for calculating the
background inhibition of the DDW for the disinfection by-product evaluation. A positive control
solution of potassium iodide and iodine also was analyzed once for approximately every
20 drinking water samples. While performance limits were not placed on the results of the
positive control sample, the vendor informed Battelle that, if the positive control samples did not
cause significant inhibition, it would indicate to the operator that the RAPIDTOXKIT was not
functioning properly. For 20 positive control samples, inhibition was complete (100%) in each
13
-------
case. A negative control sample (unspiked DDW) was analyzed with approximately every four
samples. The percent inhibition calculation for each sample incorporated the average inhibition
of the negative control samples analyzed with that particular sample set; therefore, by definition,
the average inhibition of four negative control samples was 0%. The negative control included in
the kit was analyzed once for approximately every 20 drinking water samples. This freshwater
control sample was analyzed to confirm the viability of the RAPIDTOXKIT organisms. Strategic
Diagnostics Inc. recommended that in the freshwater control the organisms have a minimum
survival rate of 50% before any samples were analyzed. This requirement was met for each
sample set and, on average, the survival rate (ratio of surviving to total organisms) in the
freshwater negative control was 70% ± 10% for the 76 freshwater samples that were analyzed.
Results for this negative control were similar to those obtained for the method blank and the
DDW negative control, which had average survival rates of 67% ±11% (N=20) and 57% ± 13%
(N=76), respectively.
4.3 Audits
A performance evaluation (PE) audit, a technical systems audit (TSA), and an audit of data
quality were performed for this verification test.
4.3.1 Performance Evaluation Audit
The accuracy of the reference method used to confirm the concentrations of the stock solutions
of the contaminants and potential interferences was confirmed by analyzing solutions of each
analyte from two separate commercial vendors. The standards from one source were used to
prepare the stock solutions during the verification test, while the standards from a second source
were analyzed as the PE sample. The percent difference (%D) between the measured concentra-
tion of the PE sample, and the nominal concentration of that sample was calculated using the
following equation:
%D=— xlOO% (!
A
where M is the absolute value of the difference between the measured and the nominal concen-
tration, and A is the nominal concentration. The %D between the measured concentration of the
PE standard and the nominal concentration had to be less than 25% for the measurements to be
considered acceptable. Table 4-1 shows the results of the PE audit for each compound. All %D
values were less than 25.
PE audits were performed when more than one source of the contaminant or potential
interference was commercially available and when methods were available to perform the
confirmation; therefore, PE audits were not performed for all of the contaminants. To assure the
purity of the other standards, documentation, such as certificates of analysis, was obtained for
colchicine, botulinum toxin complex B, and ricin. In the cases of VX and soman, which were
obtained from the U.S. Army, the reputation of the source, combined with the confirmation
analysis data, provided assurance of the concentration analyzed.
14
-------
Table 4-1. Summary of Performance Evaluation Audit
Contaminant
Potential
interference
Aldicarb
Cyanide
Dicrotophos
Nicotine
Thallium
Aluminum
Copper
Iron
Manganese
Zinc
Measured
Concentration
(mg/L)
0.057
1,025
1.10
0.120
1,010
960
1,000
960
922
1,100
Nominal
Concentration
(mg/L)
0.050
1,000
1.00
0.100
1,000
1,000
1,000
1,000
1,000
1,000
%D
14
3
10
20
1
4
0
4
8
10
4.3.2 Technical Systems Audit
The Battelle Quality Manager conducted a TSA to ensure that the verification test was performed
in accordance with the test/QA plan(1) and the AMS Center QMP.(11) As part of the audit, the
Battelle Quality Manager reviewed the contaminant standard and stock solution confirmation
methods, compared actual test procedures with those specified in the test/QA plan, 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.
4.3.3 Audit of Data Quality
At least 10% of the data acquired during the verification test were audited. Battelle's Quality
Manager 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.
4.4 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 ensured that a response was provided for each adverse finding or
15
-------
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.
4.5 Data Review
Records generated in the verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-2 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 signature or initials and the date to a hard copy of the record being reviewed.
Table 4-2. Summary of Data Recording Process
Data to be
Recorded
Dates, times of test
events
Sample
preparation (dates,
procedures,
concentrations)
Test parameters
(contaminant
concentrations,
location, etc.)
Stock solution
confirmation
analysis, sample
analysis, chain of
custody, and
results
Responsible
Party
Battelle
Battelle
Battelle
Battelle or
contracted
laboratory
Where
Recorded
Laboratory
record books
Laboratory
record books
Laboratory
record books
Laboratory
record books,
data sheets, or
data acquisition
system, as
appropriate
How Often
Recorded
Start/end of test,
and at each change
of a test parameter
When each sample
was prepared
When set or
changed
Throughout sample
handling and
analysis process
Disposition of Data(a)
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
Used to confirm the
concentration and
integrity of the samples
analyzed; procedures
entered into laboratory
record books
Used to organize/check
test results, manually
incorporated in data
spreadsheets as
necessary
Transferred to
spreadsheets/agreed
upon report
All activities subsequent to data recording were carried out by Battelle.
16
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Chapter 5
Statistical Methods and Reported Parameters
The statistical methods presented in this chapter were used to verify the performance parameters
listed in Section 3.
5.1 Endpoints and Precision
The raw data for the RAPIDTOXKIT was collected by observing the test organisms underneath
a microscope. The organisms affected by the toxicant did not take up the colored particles and,
therefore, appeared completely colorless. As described in Section 3.2.2, organisms that were
lightly colored were considered positive. The total number of organisms present and the number
of affected organisms were counted. Each test sample containing contaminants was compared
with a negative control sample that, for this verification test, was unspiked DDW. The negative
control supplied with the kit (freshwater) was analyzed for QC purposes, but was not used in the
percent inhibition calculation. This comparison was made by accounting for the inhibition of the
negative control in the calculation of the percent inhibition. Therefore, the percent inhibition of
the four negative control samples within each sample set always averaged zero. The percent
uptake (%Ł/) and percent inhibition (%/) for each sample was calculated using the following
equations:
„ , colored organisms .nnm
% uptake = x 100% n,
total organisms \L>
% inhibition = f 1 - J^sample ] x 100% (3)
%U
negative control J
Where %[/sampie is the percent uptake for each test sample and %U negative control is the average %U
of the four negative control samples analyzed in the same sample set as the subject test sample.
The negative control sample was always DDW, except when the inhibition of the disinfectant
by-products was being determined, in that case, ASTM Type IIDI water served as the control
sample.
The standard deviation (SD) of the results for the replicate samples was calculated, as follows,
and used as a measure of technology precision at each concentration. The standard deviation
around the average negative control results represented the variability of the inhibition caused by
the negative control water. Similarly, the standard deviation of the rest of the contaminant
17
-------
concentrations represented the precision of the inhibition caused by the background water
combined with the contaminant.
SD =
(4)
where n is the number of replicate samples, h is the percent inhibition measured for the &th
sample, and / is the average percent inhibition of the replicate samples. Because the average
inhibition was frequently near zero for this data set, relative standard deviations often would
have greatly exceeded 100%, making the results difficult to interpret. Therefore, the precision
results were left in the form of standard deviations of the percent inhibition so the reader could
easily view the uncertainty around the average percent inhibition for results that were both near
zero and significantly larger than zero.
5.2 Toxicity Threshold
The toxicity threshold was defined as the lowest concentration of contaminant to exhibit a
percent inhibition significantly greater than the negative control. Also, each concentration level
higher than the toxicity threshold had to be significantly greater than the negative control, and
the inhibition produced by each lower concentration analyzed had to be significantly less than
that produced by the toxicity threshold concentration. Since the inhibition of the test samples was
calculated with respect to the inhibition of each negative control sample, the percent inhibition of
the negative control was always zero. A significant difference in the inhibition at two
concentration levels required that average inhibition at each concentration level, plus or minus its
respective standard deviation, did not overlap.
Strategic Diagnostics Inc. suggests that a 30% inhibition be attained for a conclusive indication
of toxicity; however, for this test, a more thorough evaluation of sensitivity was performed.
Therefore, the toxicity threshold was determined as described here, and the 30% inhibition
threshold was used for the false negative/false positive evaluation.
5.3 False Positive/Negative Responses
A response was considered false positive if an unspiked drinking water sample produced an
inhibition exceeding 30% when determined with respect to DI water. Depending on the degree of
background inhibition in a sample, toxicity from subsequent contamination of that sample may
not be detectable or could be exaggerated as a result of the baseline inhibition. Drinking water
samples collected from water systems using chlorination and chloramination as the disinfecting
process were analyzed in this manner.
A response was considered false negative if, when a lethal concentration of some contaminant
was analyzed, the average inhibition did not exceed 30%, was not significantly different from the
negative control, or was not significantly different from the other concentration levels analyzed
(for lethal dose inhibition less than 100%). The inhibition of the lethal dose sample was required
to be significantly greater than other concentration levels because it more thoroughly
18
-------
incorporated the uncertainty of all the measurements made by the RAPIDTOXKIT in
determining a false negative result. A difference was considered significant if the average
inhibition plus or minus the standard deviation did not encompass the value or range of values
that were being compared.
5.4 Other Performance Factors
Ease of use (including clarity of the instruction manual, user-friendliness of software, and overall
convenience) was qualitatively assessed throughout the verification test through documented
observations of the operators and Verification Test Coordinator. Sample throughput was
evaluated quantitatively based on the number of samples that could be analyzed per hour.
19
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Chapter 6
Test Results
6.1 Endpoints and Precision
Tables 6-la-p present the percent inhibition data for 10 contaminants; and Table 6-2 gives the
percent inhibition for preservatives with concentrations similar to what would be contained in a
lethal dose of botulinum toxin complex B, ricin, soman, and VX. Given in each table are the
concentrations analyzed, the percent inhibition for each replicate at each concentration, and the
average and standard deviation of the inhibition of the four replicates at each concentration.
6.1.1 Contaminants
The RAPIDTOXKIT produced a detectable inhibition for all the contaminants tested, with the
exception of botulinum toxin complex B. Aldicarb, cyanide, dicrotophos, nicotine, soman, and
VX all exhibited complete inhibition at some concentration level and required additional
dilutions to more closely determine the toxicity threshold for each of those contaminants (Tables
6-lb, 6-lf, 6-lh, 6-lj, 6-lm, and 6-lp). As shown in the data tables throughout this chapter,
aldicarb, cyanide, and nicotine were detectable in the top four concentrations analyzed;
dicrotophos, thallium sulfate, and soman in the top three concentrations analyzed; and ricin and
VX in the top one or two concentrations analyzed. The only contaminant that generated an
inhibition that was not completely intuitive was colchicine, for which the highest concentration
sample (240 mg/L) generated a 56% ± 13% inhibition, the next highest concentration (24 mg/L)
generated an inhibition that was not significantly different from the negative control, and the
lowest two concentrations generated an inhibition that was both significantly greater than the
negative control. Because the 24 mg/L sample was not detectable, the lowest concentration of
colchicine considered detectable was the lethal dose concentration.
It is important to note that the botulinum toxin complex B, ricin, soman, and VX stock solutions
used to prepare the test samples were stored in various preservatives that included sodium azide,
sodium chloride, and sodium phosphate for ricin; sodium citrate only for botulinum toxin
complex B; and isopropyl alcohol for soman and VX. During the previous ETV test of this
technology category, the preservatives were not accounted for in the negative control; therefore,
the results from each test should be interpreted accordingly. The results for this test are more
thorough because they show the sensitivity (or lack thereof) to both the preservative and the
20
-------
Table 6-la. Aldicarb Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.26
2.6
26
260
(Lethal Dose)
Inhibition
(%)
3
21
-23
5
57
48
65
44
100
100
100
100
100
100
100
100
100
100
100
100
Average
(%)
0
53
100
100
100
Standard
Deviation
(%)
18
10
0
0
0
Table 6-lb. Aldicarb Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
0.00026
0.0026
0.026
0.26
Inhibition
(%)
-14
16
19
21
12
4
-41
4
-29
-38
-15
-24
-17
-18
-11
-1
35
62
63
56
Average
(%)
0
-5
-26
-12
54
Standard
Deviation
(%)
21
24
9
8
13
21
-------
Table 6-lc. Botulinum Toxin Complex B Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.0003
0.003
0.03
0.3
(Lethal Dose)
Preservative
Blank
Inhibition
(%)
0
-12
3
9
-28
-32
-40
-26
-47
-60
-7
-47
-61
-33
-41
-68
-2
-4
8
14
14
27
-10
20
Average
(%)
0
-32
-40
-51
4
12
Standard
Deviation
(%)
9
6
23
16
9
16
Table 6-ld. Colchicine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.24
2.4
24
240
(Lethal Dose)
Inhibition
(%)
-17
1
13
2
43
24
15
31
22
28
17
38
5
19
26
2
48
42
69
64
Average
(%)
0
28
26
13
56
Standard
Deviation
(%)
12
12
9
11
13
22
-------
Table 6-le. Cyanide Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.25
2.5
25
250
(Lethal Dose)
Inhibition
(%)
8
-12
5
-1
53
31
73
46
100
100
100
100
100
100
100
100
100
100
100
100
Average
(%)
0
51
100
100
100
Standard
Deviation
(%)
9
17
0
0
0
Table 6-If. Cyanide Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
0.0625
0.125
0.1875
0.25
Inhibition
(%)
7
-19
19
-7
0
31
25
-4
-23
4
2
11
-6
6
12
1
49
43
45
74
Average
(%)
0
13
-2
3
53
Standard
Deviation
(%)
16
18
15
8
14
23
-------
Table 6-lg. Dicrotophos Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
1.4
14
140
1,400
(Lethal Dose)
Inhibition
(%)
-12
27
-17
2
-3
-8
-9
4
100
100
100
100
100
100
100
100
100
100
100
100
Average
(%)
0
-4
100
100
100
Standard
Deviation
(%)
20
6
0
0
0
Table 6-lh. Dicrotophos Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
0.0014
0.014
0.14
1.4
Inhibition
(%)
17
-4
-3
-9
11
17
16
13
12
19
12
27
21
-3
14
26
20
-7
1
11
Average
(%)
0
14
17
14
6
Standard
Deviation
(%)
12
3
7
13
12
24
-------
Table 6-li. Nicotine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Inhibition
(%)
-21
5
11
6
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Average
(%)
0
100
100
100
100
Standard
Deviation
(%)
14
0
0
0
0
Table 6-lj. Nicotine Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
0.0028
0.028
0.28
2.8
Inhibition
(%)
-25
31
15
-22
-42
-30
-16
-98
-7
29
-18
-6
75
48
69
32
100
100
100
100
Average
(%)
0
-47
-1
56
100
Standard
Deviation
(%)
28
36
20
20
0
25
-------
Table 6-Ik. Ricin Percent Inhibition Results
Concentration
(ms/L)
Negative
Control
0.015
Lethal
Dose/1,000
Preservative
Blank
0.15
Lethal
Dose/100
Preservative
Blank
1.5
Lethal Dose/10
Preservative
Blank
15
(Lethal Dose)
Lethal Dose
Preservative
Blank
Inhibition
(%)
l
-6
3
2
10
4
2
11
3
8
-10
3
-3
0
-2
-1
13
17
-11
-6
18
12
21
7
22
2
-9
1
26
29
30
22
3
2
-9
1
Average
(%)
0
6
1
-2
3
14
4
27
3
Standard
Deviation
(%)
4
4
8
1
14
6
13
4
3
26
-------
Table 6-11. Soman Percent Inhibition Results
Concentration
(ms/L)
Negative
Control
0.0014
0.014
0.14
1.4
(Lethal Dose)
Lethal Dose
Preservative
Blank
Inhibition
(%)
9
3
-5
-7
-2
-6
-7
6
100
100
100
100
100
95
100
100
100
100
100
100
16
20
31
37
Average
(%)
0
-2
100
99
100
26
Standard
Deviation
(%)
7
6
0
3
0
10
27
-------
Table 6-1m. Soman Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
0.0014
0.0035
0.007
0.0105
0.014
Lethal Dose
Preservative
Blank
Inhibition
(%)
8
-4
-7
2
4
-27
-16
-32
5
-1
7
12
100
100
94
100
100
100
100
100
100
100
100
100
-5
-1
-4
-2
Average
(%)
0
-18
6
98
100
100
-3
Standard
Deviation
(%)
7
16
5
3
0
0
2
28
-------
Table 6-ln. Thallium Sulfate Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Inhibition
(%)
13
-18
6
-1
48
7
20
40
74
91
70
80
100
100
100
100
100
100
100
100
Average
(%)
0
29
79
100
100
Standard
Deviation
(%)
13
19
9
0
0
Table 6-lo. VX Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.002
0.02
0.2
2
(Lethal Dose)
Lethal Dose
Preservative
Blank
Inhibition
(%)
-9
-2
8
3
20
25
22
20
3
-3
12
4
5
14
12
9
100
97
100
100
25
21
31
5
Average
(%)
0
22
4
10
99
21
Standard
Deviation
(%)
8
2
6
4
1
11
29
-------
Table 6-lp. VX Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
0.2
0.5
1.0
1.5
2
Lethal Dose
Preservative
Blank
Inhibition
(%)
-18
-23
-6
47
-19
-38
-28
-19
2
10
11
0
25
6
8
2
100
100
100
100
100
100
100
100
-21
-27
-3
-28
Average
(%)
0
-26
6
10
100
100
-20
Standard
Deviation
(%)
32
9
5
10
0
0
12
contaminant. In the earlier verification test, toxicity could have been the result of either.
Table 3-2 details the concentrations of preservatives in the lethal dose samples of each
contaminant. These data could be evaluated in two ways to determine the sensitivity of the
RAPIDTOXKIT to contaminants stored in preservatives. The first approach would be to
determine the inhibition of the test samples containing preservatives with respect to the
background negative control, as was the case for the contaminants not stored in preservatives.
This technique, however, could indicate that the RAPIDTOXKIT was sensitive to the
contaminant when, in fact, it was sensitive to one of the preservatives. Since these contaminants
are only available (either commercially or from the government) in aqueous formulations with
the preservatives, this may be appropriate. The second approach would be to fortify negative
control samples with the same concentrations of preservative contained in all the samples so that
the inhibition resulting from the preservatives could be subtracted from the inhibition caused by
the contaminant. This approach would greatly increase the number of samples required for
analysis. Therefore, for this test, aspects of both approaches were incorporated without
substantially increasing the number of samples. Negative control samples fortified with a
concentration of each preservative equivalent to the concentration in the lethal dose test samples
(preservative blanks) were analyzed prior to and with every set of test samples. For those sets of
test samples for which it was especially difficult to determine whether inhibitory effects were
30
-------
from the contaminant or the preservative, the preservative blank was diluted identically to all the
contaminant samples and analyzed so a background subtraction could take place if necessary.
Interestingly, when the preservative blanks were analyzed prior to the contaminant analysis, all
of the preservatives generated detectable inhibition (Table 6-2). The contaminants were analyzed
along with only the lethal dose equivalent preservative blank before moving forward with
additional dilution levels of the preservative (except for ricin, where dilutions of the preservative
blank were performed for each contaminant dilution level). In all four cases during contaminant
testing, the inhibition caused by the lethal dose of the preservative blank was less than 30%, the
minimum considered to indicate toxicity, according to Strategic Diagnostics Inc.; and, for ricin
and botulinum toxin complex B, the inhibition of the lethal dose preservative blank was not
significantly different from the negative control. The ricin test samples were analyzed initially
along with preservative dilutions; but, for the other three contaminants, further analysis of the
preservative blanks was unnecessary because of the lack of toxic effect. It was not clear why the
preservative blanks exhibited a toxic effect initially but did not when analyzed with the test
samples.
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results
Preservative
Blank
Negative
Control
Ricin
Soman/VX
Botulinum
Toxin
Complex B
Inhibition
(%)
0
-18
4
13
61
70
72
61
49
40
59
58
14
16
24
20
Average
(%)
0
66
52
19
Standard
Deviation
(%)
13
6
9
5
For botulinum toxin complex B, the lethal dose preservative blank was not significantly different
from the negative control, and all the test samples were either negative or not significantly
greater than the negative control. Therefore, botulinum toxin complex B apparently does not
generate toxic effects on the RAPIDTOXKIT organisms.
The lethal dose concentration of ricin (27% ± 4%) resulted in an inhibition that was significantly
greater than the negative control (0% ± 4%) and the preservative blank (3% ± 3%), indicating a
slight toxic effect. However, Strategic Diagnostics Inc. suggests that 30% inhibition is the lowest
detectable inhibition, so the result is borderline detectable. As previously mentioned, preserva-
tive blanks diluted identically to concentrations of each of the other ricin test samples were
analyzed. None of these ricin test samples generated an inhibition significantly different from
their respective preservative blanks.
31
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VX and soman were similar in that both of their lethal dose preservative blanks analyzed with
the contaminant samples generated an inhibition that was greater than 20% and significantly
greater than the negative control. However, in neither case were additional dilutions of the
preservative blank analyzed because, in the case of VX, the lethal dose contaminant sample
generated an inhibition of nearly 100% and, for soman, the top three concentration levels
generated an inhibition of nearly 100%. Therefore, it was clear that almost all of the inhibition
exhibited by the test samples was caused by the contaminant and not the preservative. Because of
the very strong inhibition, additional dilutions of the test samples were analyzed to more
accurately determine the toxicity threshold of each contaminant. The lethal dose preservative
blanks were analyzed along with the additional dilutions of VX and soman to confirm whether
the preservatives cause inhibitory effects. In neither case did the preservative blanks generate an
inhibition greater than the negative control. Again, it is not clear why the preservative blanks
caused inhibition upon initial analysis and did not when analyzed with the contaminants. For
VX, two of the additional dilutions generated complete inhibition; and, for soman, three of the
additional concentration levels also generated complete inhibition.
6.1.2 Potential Interferences
All of the potential interference samples were prepared in DDW and compared with the negative
control to determine the level of inhibition. This determination is crucial because the ability of
the RAPIDTOXKIT to detect toxicity is dependent on the organism's ability to take up particles
in whatever drinking water matrix is being used. If the background drinking water sample com-
pletely inhibits the uptake of particles, inhibition caused by contaminants could not be detected.
Table 6-3 presents the results from the samples analyzed to test the effect of potential inter-
ferences on the RAPIDTOXKIT organisms. Of the five metal solutions evaluated as possible
interferences with the RAPIDTOXKIT, four of them, zinc (24% ± 9%), copper (100% ± 0%),
iron (20% ± 4%), and aluminum (29% ± 6%), exhibited an inhibition that was significantly
different from the DDW negative control (0% ± 3%). Because zinc, iron, and aluminum
exhibited an inhibition less than that considered a minimum detectable inhibition by Strategic
Diagnostics Inc., they should be considered very slight interferences. Therefore, water samples
containing similar concentration of metals could be used as a representative negative control
sample because there was still enough particle uptake in the presence of these metals to detect
any additional inhibition of particle uptake caused by contaminants. Copper, on the other hand,
should be considered a possible interference because the organisms' particle uptake would be
completely inhibited by the matrix if a similar copper concentration was present, leaving no
residual particle uptake to be inhibited by contamination.
To investigate whether the RAPIDTOXKIT is sensitive to by-products of disinfecting processes,
DDW samples from water systems that use chlorination and chloramination were analyzed and
compared with ASTM Type n DI water as the control sample. In the absence of a background
water sample, it seems likely that DI water may be used as a "clean water" control; therefore, it
would be helpful to know what the results would be if this is done. The sample from the water
supply disinfected by chlorination (N=76) exhibited an average inhibition of 12 ± 18%, while the
sample from the water supply disinfected by chloramination exhibited an inhibition of 3% ± 8%
on four replicates. The difference in the number of replicates is because the dechlorinated water
was used as the negative control with each sample set; therefore, much more data were collected
32
-------
Table 6-3. Potential Interferences Results
Potential
Interferences
Negative control
(Metals)
Aluminum
Copper
Iron
Manganese
Zinc
Negative control
(By-products)
Chlorination
by-products
Chloramination
by-products
Concen-
tration
(mg/L)
NA
0.5
0.6
0.15
0.25
2.5
NA
NA
NA
Inhibition
(%)
-2
-3
4
1
30
30
20
35
100
100
100
100
19
22
24
14
-27
-6
-10
-1
35
15
18
26
4
-4
-1
1
(a)
-5
2
14
0
Average
(%)
0
29
100
20
-11
24
0
12
3
Standard
Deviation
(%)
3
6
0
4
11
9
3
18
8
NA = Not applicable.
(a) Average inhibition across all DDW negative control samples (N=76).
on that water. These inhibition data suggest that samples disinfected by either process are not
likely to interfere with the RAPIDTOXKIT results because the inhibition caused by the two
disinfected drinking water matrices left most of the organisms able to take up particles to
potentially be inhibited by subsequent contamination.
33
-------
6.1.3 Precision
Across all the contaminants and potential interferences, the standard deviation (not relative
standard deviation) was measured and reported for each set of four replicates to evaluate the
precision of the RAPIDTOXKIT. Out of 105 opportunities, the standard deviation of the four
replicate inhibition measurements was less than 10% inhibition 68 times (65% of the time),
between 10% and 20% inhibition 29 times (27% of the time), and greater than 20% inhibition
just 8 times (8%). As described in Section 3.2.2, the analysis procedure required that each
replicate undergo the entire analysis process; therefore, the measurement of precision represents
the precision of the analysis method performed on a single water sample on a given day. The
precision does not reflect the repeatability of the method across more than one day or more than
one preparation of reagents or more than one operator.
6.2 Toxicity Threshold
Table 6-4 gives the toxicity thresholds, as defined in Section 5.2, for each contaminant. Note the
difference between detectability with respect to the negative control and the toxicity threshold
with respect to the other concentration levels analyzed. A contaminant concentration level can
have an inhibition significantly different from the negative control (thus detectable), but if its
inhibition is not significantly different from the concentration levels below it, it would not be
considered the toxicity threshold because in the context of this test, its inhibition would not be
distinguishable from that of the lower concentrations. The lowest toxicity threshold
concentration was for soman at 0.007 mg/L. Only botulinum toxin complex B did not generate
detectable inhibition at some concentration level.
Table 6-4. Toxicity Thresholds
Contaminant
Aldicarb
Botulinum toxin complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Concentration (mg/L)
0.26
ND
240
0.25
14
0.28
15
0.007
28
1.5
ND = Significant inhibition was not detected.
34
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6.3 False Positive/Negative Responses
No results from the RAPIDTOXKIT were considered false positive because the chlorination and
chloramination by-product samples did not inhibit particle uptake in 30% of the exposed
organisms. Since the background inhibition is not complete, it can be accounted for by using
negative control samples that are very similar to the water being analyzed. If samples are
analyzed daily, a good practice would be to archive a negative control sample each day in case of
contamination the next day.
Table 6-5 shows the RAPIDTOXKIT false negative responses, which are described in
Section 5.3. Only botulinum toxin complex B did not exhibit a detectable inhibition at the lethal
concentration. Ricin's average inhibition at the lethal dose was 27% ± 4%, not meeting the
minimum requirement set by the vendor. It was not, however, considered false negative because
the relatively small uncertainty around the average encompassed 30%.
Table 6-5. False Negative Responses
Contaminant
Aldicarb
Botulinum toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Lethal Dose
Concentration (mg/L)
260
0.30
240
250
1,400
2,800
15
1.4
2,800
2.0
False Negative
No
Yes
No
No
No
No
No(a)
No
No
No
Inhibition was 27% ± 4%; vendor suggests 30% as minimum indicator of
toxicity, but it was not considered false negative because relatively small
uncertainty encompassed 30%.
6.4 Other Performance Factors
6.4.1 Ease of Use
RAPIDTOXKIT contained clearly written instructions and illustrations. The detailed information
on which organisms to count and which to disregard was especially useful. Microscope slides
with a grid facilitated the process of counting the organisms. Contents of the RAPIDTOXKIT
were well identified. The tubes in which the cysts were stored before hatching were somewhat
difficult to open, while the test tubes for the test waters were extremely difficult to open. The
vendor indicated that a design change for these tubes was underway. The only problem, other
35
-------
than the difficulty opening some containers, was a slight difficulty getting the cysts out of the
tubes with the recommended 1 mL of water. The cysts tended to sink to the bottom of the tube
and get stuck, often requiring more than 1 mL to transfer. Additionally, the test organisms could
be used only 30 to 45 hours after starting the hatching process. This also may make spontaneous
testing problematic. According to Strategic Diagnostics Inc., a programmable incubator is now
available to pre-program cyst hatching to make the use of living test organisms more convenient.
Reagents were easy to prepare. Storage conditions were not indicated on the reagent containers,
but were noted on a warning label on the RAPIDTOXKIT and also in the manual. The
RAPIDTOXKIT warning label indicated that the cysts, microspheres, and fixative needed to be
refrigerated, while the manual indicated that only the cysts needed to be refrigerated.
All the necessary supplies were provided with the kit except for pipettes with tips, an incubator
used to hatch the cysts, and a microscope. Manually counting the number of red organisms under
the microscope was tedious when the results from many samples were determined one after the
other over a few hours. Highly colored organisms were easy to identify; but, if only a small
amount of red spheres had been ingested, identification was more difficult. The microscope was
easily wiped clean and did not require significant routine maintenance.
No formal scientific education would be required to use the RAPIDTOXKIT. However, good
laboratory skills, especially pipetting, would be beneficial. Basic math skills are required for
interpreting results. Verification testing staff were able to use the RAPIDTOXKIT after a two-
hour training session. Test tubes, observation plates, and pipette tips were generated as solid
waste. It was not stated whether the organisms or the fixative solution should be considered
hazardous waste.
6.4.2 Field Portability
The RAPIDTOXKIT was not evaluated for field portability because the vendor indicated that it
was not intended to be used in the field at this time.
6.4.3 Throughput
Approximately 25 analyses were completed in three hours. The 25 analyses included method
blanks and positive and negative controls, as well as test samples. Note that additional lead time
(30 to 45 hours) is required to hatch the cysts. The hatching process took approximately one hour
of labor prior to the extended incubation. Throughput evaluations assumed that the cysts were
already hatched. A maximum of 45 samples, without replicates, could be processed per kit.
36
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Chapter 7
Performance Summary
Parameter
Contaminants in
DDW
Potential
interferences in
DDW
False positive
response
False negative
response
Compound
Aldicarb
Botulinum
toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium
sulfate
VX
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Lethal
Dose (LD)
Cone.
(mg/L)
260
0.3
240
250
1,400
2,800
15
1.4
2,800
2
Cone.
(mg/L)
0.5
0.6
0.15
0.25
2.5
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
100
4
56
100
100
100
27
100
100
99
LD/10
100
-51
13
100
100
100
14
99
100
10
LD/100
100
-40
26
100
100
100
_2
100
79
4
LD/1,000
53
-32
28
51
-4
100
6
_2
29
22
Average Standard
Inhibition (%) Deviation (%)
29 6
100 0
20 4
-11 11
24 9
Range of
Standard
Deviations
(%)
0-10
6-23
9-13
0-17
0-6
0
1-6
0-6
0-19
1-6
Toxicity
Thresh.
(mg/L)
0.26
ND
240
0.25
14
0.28
15
0.007
28
1.5
No results from the RAPIDTOXKIT were considered false positive because inhibition in the
chlorinated and chloraminated drinking water samples was always less than 30%.
Only botulinum toxin complex B exhibited inhibition less than 30% when analyzed at a lethal
dose concentration.
Ease of use
The RAPIDTOXKIT contained clearly written instructions and illustrations. The contents of the
RAPIDTOXKIT were well identified. The only problem, other than the difficulty opening some
containers, was a slight difficulty getting the cysts out of the tubes with the recommended 1 mL
of water. Manually counting the number of red organisms under the microscope was tedious
when the results from many samples were determined one after the other over a few hours.
Overall, the RAPIDTOXKIT was easy to use, making it likely that a person with no formal
scientific training could conduct the tests.
Field portability
Throughput
The RAPIDTOXKIT was not evaluated for field portability.
Not including the 30 to 45-hour cyst-hatching period, approximately 25 analyses (including
method blanks and positive and negative controls) were completed in three hours. A maximum of
45 samples could be processed per kit.
ND = Significant inhibition was not detected.
37
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Chapter 8
References
1. Test/QA Plan for Verification of Rapid Toxicity Technologies, Battelle, Columbus, Ohio,
June 2003; Amendment 1: June 9, 2005; Amendment 2: August 19, 2005.
2. United States Environmental Protection Agency, National Secondary Drinking Water
Regulations: Guidance for Nuisance Chemicals, EPA/810/K-92/001, July 1992.
3. U.S. EPA Method 335.3, "Cyanide, Total—Colorimetric, Automated UV" in Methods for
the Chemical Analysis of Water and Wastes, EPA/600/4-79/020, March 1983.
4. 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
Metals in Environmental Samples, Supplement I, EPA/600/R-94/111, 1994.
5. 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.
6. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, 1997. Washington, DC.
7. U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020.
8. 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.
9. U.S. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
Spectrometry," Methods for the Determination of Organic Compounds in Drinking Water—
Supplement III, EPA/600/R-95/131.
10. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater, 20th edition, 1998, Washington, DC.
38
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11. Quality Management Plan (QMP)for the ETV Advanced Monitoring Systems Center,
Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, December 2004.
39
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