June 2006
Environmental Technology
Verification Report
ENVIRONMENTAL BIO-DETECTION
PRODUCTS INC.
TOXI-CHROMOTEST
Prepared by
Battelle
Batreiie
//it? 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:
Toxi-Chromotest
Environmental Bio-Detection Products, Inc.
14 Abacus Road
Brampton, Ontario
CANADA L6T5B7
www.ebpi-kits.com
ebpi @ ebpi-kits.com
PHONE: (905) 794-3274
FAX: (905)794-2338
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 Environmental Bio-Detection Products, Inc. Toxi-Chromotest. 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 Toxi-Chromotest 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 Toxi-Chromotest 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 Toxi-Chromotest was evaluated by
• Endpoints and precision—color inhibition (as indicator of toxicity) with respect to that of the negative
control for all concentration levels of contaminants and potential interfering compounds and consistency of
the color change across 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
• False positive responses—chlorination and chloramination by-product inhibition with respect to
unspiked American Society for Testing and Materials Type II deionized water samples
• False negative responses—contaminants that, when present at lethal concentrations, did not produce any
color inhibition with respect to the negative control
• Other performance factors (sample throughput, ease of use, reliability).
The Toxi-Chromotest 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
1,000 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 bacteria within the Toxi-
Chromotest 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 Toxi-
Chromotest to detect toxicity, as well as to measure the precision of the Toxi-Chromotest results. The
response of the Toxi-Chromotest 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 (fortified with mercuric chloride); and
negative control samples, which consisted of the unspiked DDW.
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.
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TECHNOLOGY DESCRIPTION
The following description of the Toxi-Chromotest is based on information provided by the vendor. This
technology description was not verified in this test.
The Toxi-Chromotest detects toxic substances in water, chemicals, Pharmaceuticals, food, and body fluids.
The Toxi-Chromotest is a bacterial assay based on the ability of toxic materials and antibiotics to inhibit the
de novo synthesis of an inducible enzyme, B-galactosidase, in a strain of the bacteria, E. coli (K12 OR85). The
bacteria in the Toxi-Chromotest are exposed to stressing conditions and freeze dried. To test for toxicity, the
bacteria are mixed with a rehydration cocktail containing inducers of the enzyme B-galactosidase and factors
necessary for the recovery of the bacteria from their stressed condition. During the recovery phase, toxicants
present at sufficient concentrations penetrate the cell walls of the bacteria and inhibit the de novo synthesis of
the B-galactosidase. The rate of production of the induced enzyme is detected by a reaction of the excreted
enzyme with a chromogenic substrate in the bacterial suspension that was exposed to the potential toxicant.
Toxic materials above threshold levels interfere with the production of the enzyme and decrease color
formation.
The Toxi-Chromotest kit includes a reaction mixture (the cocktail containing an inducer for the enzyme
B-galactosidase and co-factors required for the recovery of the bacteria from their stressed condition),
lyophilized bacteria, rehydration solution, a positive control (4 micrograms per milliliter of mercuric chloride
in water), a chromogenic substrate (blue chromogen cocktail, ready for use), and diluent for the positive
control and test samples. In addition, the Toxi-Chromotest kit contains three 96-well microtiter plates and
biohazard bags. The user must supply a micropipette for adding the test samples, rehydrated bacteria, and
chromogenic substrates to the test wells and an incubator in which the plates containing the bacteria are
allowed to recover and begin to produce the enzyme that reacts with the added chromogenic substrate. The
incubator must maintain a constant temperature of 37 °C during the 90 minute incubation period.
The Toxi-Chromotest is supplied in a 25- by 13- by 8- centimeter (cm) Styrofoam box that contains the 96-
well plates, the biohazard bags for disposal of test materials, and all of the necessary reagents to carry out
three separate analytical test series. For field use, a 15-cm by 15-cm by 15-cm incubator can be supplied that
runs off a 12-volt battery or 120-volt alternating current.
The output from the Toxi-Chromotest can be measured in the laboratory by absorbance at 615 nanometers
using a plate reader. If the test is conducted in the field or a plate reader is not available (as during this test),
the results can be read by visually recording the intensity of blue color produced against an internally run set
of standards to obtain a relative toxicity reading. The standard Toxi-Chromotest kit, with reagent, bacteria,
and plates to run the tests in the three 96-well microtiter plates provided, sells for $375.
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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
Visual Observance of Color
Inhibition at Concentrations
Relative to the LD Concentration
LD
-
-
-
+
-
+
-
-
+
-
LD/10
-
-
-
+
-
-
-
-
+
-
LD/100
-
-
-
-
-
-
-
-
-
-
LD/1,000
-
-
-
-
-
-
-
-
+
-
Visual Observance of Color
Inhibition
-
-
-
-
-
Toxicity Threshold
(mg/L)
ND
ND
ND
25
ND
2,800
ND
ND
280
ND
The Toxi-Chromotest did not generate any false positive results to water containing chlorination
or chloramination by-products.
Aldicarb, botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, and VX produced an
inhibition that was not visually distinguishable from the negative control at the lethal dose
concentrations.
The Toxi-Chromotest requires two 1.5-hour incubation periods. After bacteria rehydration, the
hydrated bacteria could be used only for one hour. In addition, the reaction of the Toxi-
Chromotest was observed visually, which was difficult when there were only slight variations in
color. No formal scientific training would be required to use the Toxi-Chromotest.
Overall the Toxi-Chromotest was easy to transport to the field and, with an incubator
warmed ahead of time, was deployed in a matter of minutes. Results were obtained within 3
to 4 hours of starting the test. Each Toxi-Chromotest kit contained materials to process three
96-well plates.
The number of samples that can be processed depends on the number of replicates per sample
and the number of dilutions per sample that are processed on each 96-well plate. One plate can be
taken through the procedure in 3 to 4 hours. Each Toxi-Chromotest kit contained materials to
process samples filling three 96-well plates.
+ = Visually distinguishable color inhibition from that of the negative control was observed.
- = Visually distinguishable color inhibition from that of the negative control was not observed
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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
Environmental Bio-Detection Products Inc.
Toxi-Chromotest
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 8
3.2 Test Procedure 8
3.2.1 Test Sample Preparation and Storage 8
3.2.2 Test Sample Analysis Procedure 8
3.2.3 Stock Solution Confirmation Analysis 9
Chapter 4 Quality Assurance/Quality Control 12
4.1 Quality Control of Stock Solution Confirmation Methods 12
4.2 Quality Control of Drinking Water Samples 12
4.3 Audits 13
4.3.1 Performance Evaluation Audit 13
4.3.2 Technical Systems Audit 13
4.3.3 Audit of Data Quality 14
4.4 QA/QC Reporting 14
4.5 Data Review 14
Chapter 5 Statistical Methods and Reported Parameters 16
5.1 Endpoints and Precision 16
5.2 Toxicity Threshold 16
5.3 False Positive/Negative Responses 16
5.4 Other Performance Factors 17
Chapter 6 Test Results 18
6.1 Endpoints 18
6.1.1 Contaminants and Potential Interferences 19
6.1.2 Precision 20
6.2 Toxicity Threshold 20
6.3 False Positive/Negative Responses 20
6.4 Other Performance Factors 21
6.4.1 Ease of Use 21
6.4.2 Field Portability 22
6.4.3 Throughput 22
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Chapter 7 Performance Summary 23
Chapters References 24
Figures
Figure 2-1. EBPI Toxi-Chromotest 2
Figure 6-1. Effect of Contaminant on Test Color 19
Tables
Table 3-1. Contaminants and Potential Interferences 5
Table 3-2. Summary of Quality Control and Contaminant Test Samples 7
Table 3-3. Stock Solution Confirmation Results 10
Table 3-4. Water Quality Parameters 11
Table 4-1. Summary of Performance Evaluation Audit 14
Table 4-2. Summary of Data Recording Process 15
Table 6-1. Inhibition of Bacterial Recovery in the Presence of Contaminants 18
Table 6-2. Toxicity Thresholds 21
Table 6-3. False Negative Responses 21
VI
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List of Abbreviations
AMS Advanced Monitoring Systems
ASTM American Society for Testing and Materials
ATEL Aqua Tech Environmental Laboratories
cm centimeter
DI deionized water
DDW dechlorinated drinking water from Columbus, Ohio
DPD n,n-diethyl-p-phenylenediamine
EBPI Environmental Bio-Detection Products Inc.
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 Environmental Bio-Detection Products Inc. (EBPI)
Toxi-Chromotest. 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 Toxi-Chromotest. Following is a description of the Toxi-
Chromotest, based on information provided by the vendor. The information provided below was
not verified in this test.
The Toxi-Chromotest (Figure 2-1) detects toxic substances in water, chemicals, Pharmaceuticals,
food, and body fluids. The Toxi-Chromotest is a bacterial assay based on the ability of toxic
materials and antibiotics to inhibit the de novo synthesis of an inducible enzyme, B-galactosidase,
in a strain of the bacteria, E. coli (K12 OR85). The bacteria in the Toxi-Chromotest are exposed
to stressing conditions and freeze dried. To test for
toxicity, the bacteria are mixed with a rehydration
cocktail containing inducers of the enzyme
B-galactosidase and factors necessary for the
recovery of the bacteria from their stressed
iw»
. ^ S" £»^ condition. During the recovery phase, toxicants
11 --* ^^. present at sufficient concentrations penetrate the
cell walls of the bacteria and inhibit the de novo
synthesis of the B-galactosidase. The rate of
production of the induced enzyme is detected by a
reaction of the excreted enzyme with a chromo-
genic substrate in the bacterial suspension that was
exposed to the potential toxicant. Toxic materials
above threshold levels interfere with the
production of the enzyme and decrease color
formation.
Figure 2-1. EBPI Toxi-Chromotest
The Toxi-Chromotest kit includes a reaction
mixture (the cocktail containing an inducer for the
enzyme B-galactosidase and co-factors required for the recovery of the bacteria from their
stressed condition), lyophilized bacteria, rehydration solution, a positive control (4 micrograms
per milliliter of mercuric chloride in water), a chromogenic substrate (blue chromogen cocktail,
ready for use), and diluent for the positive control and test samples. In addition, the Toxi-
Chromotest kit contains three 96-well microtiter plates and biohazard bags. The user must supply
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a micropipette for adding the test samples, rehydrated bacteria, and chromogenic substrates to
the test wells and an incubator in which the plates containing the bacteria are allowed to recover
and begin to produce the enzyme that reacts with the added chromogenic substrate. The
incubator must maintain a constant temperature of 37 °C during the 90-minute incubation period.
The Toxi-Chromotest standard kit is supplied in a 25- by 13- by 8- centimeter (cm) Styrofoam
box that contains three 96-well plates, biohazard bags for disposal of test materials, and all of the
necessary reagents to carry out three separate analytical test series. For field use, a 15-cm by 15-
cm by 15-cm incubator can be supplied that runs off a 12-volt battery or 120-volt alternating
current.
The output from the Toxi-Chromotest can be measured in the laboratory by absorbance at
615 nanometers using a plate reader. If the test is conducted in the field or a plate reader is not
available (as during this test), the results can be read by visually recording the intensity of blue
color produced against an internally run set of standards to obtain a relative toxicity reading. The
standard Toxi-Chromotest kit with reagent, bacteria, and plates to run the tests in the three 96-
well microtiter plates provided, sells for $375.
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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 Toxi-Chromotest 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 Toxi-Chromotest 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 Toxi-Chromotest 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 Toxi-Chromotest was evaluated by
• Endpoints and precision—color inhibition (as indicator of toxicity) with respect to that of the
negative control for all concentration levels of contaminants and potential interfering
compounds and consistency of the color change across 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
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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 with respect
to unspiked American Society for Testing and Materials (ASTM) Type II deionized (DI)
water samples
• False negative responses—contaminants that, when present at lethal concentrations, did not
produce any color inhibition with respect to the negative control
• Other performance factors (sample throughput, ease of use, reliability).
The Toxi-Chromotest was used to analyze the DDW samples fortified with contaminants at
concentrations ranging from lethal levels to concentrations up to 1,000 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) The decrease in color intensity from four
replicates of each contaminant at each concentration level was evaluated to assess the ability of
the Toxi-Chromotest to detect toxicity at various concentrations of contaminants, as well as to
evaluate the repeatability of the Toxi-Chromotest results.
The response of the Toxi-Chromotest 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
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.
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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 Toxi-Chromotest kit. 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 ensure
that no sources of contamination were introduced in the sample handling and analysis
procedures. A positive control sample was included in the Toxi-Chromotest and was used as
provided from the vendor. While performance limits were not placed on the results, a steadily
increasing color intensity across a serial dilution indicated to the operator that the Toxi-
Chromotest was functioning properly. The negative control consisted of unspiked DDW and was
used to set a background color intensity 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 DDW.
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. Table 3-2 lists each concentration level and the
number of samples analyzed at each level.
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Table 3-2. Summary of Quality Control and Contaminant Test Samples
Type of Sample
Quality control
DDW fortified with
contaminants
DDW fortified with
potential interferences
Disinfectant
by-products
Sample
Characteristics
Method blank
(ASTM Type II water)
Positive control
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
4 mg/L mercury chloride (used as
provided in kit)
NA
0.015 millimolar (mM) sodium citrate
0.21% isopropyl alcohol
0.00024% NaN3, 0.00045 molar
NaCl, 0.03mM phosphate
260; 26; 2.6; 0.26 milligrams/liter
(mg/L)
0.3; 0.03; 0.003; 0.0003 mg/L
240; 24; 2.4; 0.24 mg/L
250; 25; 2.5; 0.25 mg/L
1,400; 140; 14; 1.4; mg/L
2,800; 280; 28; 2.8 mg/L
15; 1.5; 0.15; 0.015 mg/L
1.4; 0.14; 0.014; 0.0014 mg/L
2,800; 280; 28; 2.8 mg/L
2; 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
10
10 serial dilutions
52
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
52
NA = not applicable, samples not fortified with any preservative, contaminant, or potential interference.
-------
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 Toxi-Chromotest 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 bacteria within the Toxi-Chromotest 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 that so that the operators did not know their content.
3.2.2 Test Sample Analysis Procedure
The first step in analyzing the test samples was to reconstitute the lyophilized bacteria by adding
the solution in Bottle C to Bottle B, inverting several times, and allowing it to sit for 15 minutes.
In the meantime, 200 ^iL of each test sample were added to a well within a 96-well plate.
Typically, four wells of each concentration level were analyzed. After the bacteria sat for
15 minutes, 1 mL was transferred into Bottle A, the reaction mixture. This solution was capped
and inverted to mix, and 100 (^L of the mixture were added to all the positive control and test
samples. The 96-well plate was placed in an incubator at 37°C for 90 minutes, and then 100 (^L
8
-------
of blue chromogen were added to each of the wells. The samples were returned to the incubator
for an additional 90 minutes to allow a period of time for color development. Afterward, each
well containing a test sample was compared with the negative control; and, if the color was
visibly less intense than the negative control, it was considered detectable. For the purpose of
data collection, each detectable solution was compared to the column of positive control samples
and assigned the dilution level that its color most closely matched.
For each contaminant, a minimum of the lethal dose concentration and three additional
concentration levels were analyzed four times using the Toxi-Chromotest. Only one
concentration of each potential interference was analyzed four times. Two operators performed
all the analyses using the Toxi-Chromotest. 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 Toxi-Chromotest.
3.2.3 Stock Solution Confirmation Analysis
The concentrations of the contaminant and interfering compound stock solutions were verified
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
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.
-------
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)
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 (10/18/05)
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 solutions.
(b) Target concentration was highest concentration for each contaminant or interference on Table 3-2.
10
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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.
11
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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 Toxi-
Chromotest for every 96-well plate of samples that was analyzed. A positive control sample also
was analyzed on every plate of samples. Specifically, one column on each plate was dedicated to
a serial twofold dilution (consisting of eight concentration levels) of a 4-mg/L mercuric chloride
sample provided by the vendor. On every plate analyzed, this increasing color intensity (propor-
tional to the decrease in positive control concentration) was observed in the positive control
column, indicating that the proper procedure for the analysis of samples was being followed and
that the reagents were performing as expected. A negative control sample (unspiked DDW) was
analyzed with approximately every four samples. The color of these samples was compared with
that of the test samples to determine the toxic effect of the test sample.
12
-------
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
concentration of the PE sample, and the nominal concentration of that sample was calculated
using the following equation:
M
%D=—xlOO% m
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.
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.
13
-------
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
4.3.3 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
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.
14
-------
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
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.
15
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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 results from the Toxi-Chromotest were interpreted visually, in a qualitative manner. Overall,
the negative control sample made up of DDW and the very dilute positive control samples
generated an intense bluish color. Toxic contaminants inhibit the color development; therefore,
shades of blue that were less intense than the negative control were detectable. If three out of
four replicates exhibited detectable color inhibition, the overall sample was considered to have a
positive result (indicated by a "+" in Chapter 6). If two or more of the replicate samples were not
detectable, the overall sample was considered a negative result (indicated by a "-" in Chapter 6).
The color of the detectable sample wells was compared with the colors of the positive control
sample. The dilution level that it matched most was recorded as raw data. If the color was similar
to or darker than the negative control, the sample was considered non-detectable (indicated by a
"-" in Chapter 6).
Because of the qualitative nature of the results, there was no quantitative measure of
reproducibility. Reproducibility was evaluated simply by noting the similarity of the colors that
developed in the sample wells. The fraction of sample sets that produced the same color was
reported. Examples of how the color development was interpreted are presented in Section 6.1.1.
5.2 Toxicity Threshold
The toxicity threshold was defined as the lowest concentration of contaminant to exhibit
inhibited color development with respect to the negative control. Also, each concentration level
higher than the toxicity threshold had to have a less intense color than the negative control.
5.3 False Positive/Negative Responses
A response would be considered false positive if an unspiked drinking water sample produced a
color visibly less intense than the negative control. To test for this possibility, unspiked drinking
16
-------
water from a water utility using chlorination, as well as from a utility using chloramination, was
analyzed. A response was considered false negative when the Toxi-Chromotest was subjected to
a lethal concentration of some contaminant in the DDW, and the color intensity was not
decreased with respect to the negative control.
5.4 Other Performance Factors
Ease of use (including clarity of the instruction manual 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.
17
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Chapter 6
Test Results
6.1 Endpoints
The Toxi-Chromotest was evaluated by visually comparing a negative control and the test
sample data. A positive control that consisted of a serial dilution of a 4-mg/L mercuric chloride
solution was analyzed with each sample set to confirm the performance of the Toxi-Chromotest.
Contaminant test samples in which the bacteria were not inhibited, such as the negative control,
were bright blue; while samples containing contaminants that inhibited bacterial recovery
exhibited decreased color production. Data collected during the test are summarized in
Table 6-1. Contaminants and potential interferences are in the left column, and the concentra-
tions of contaminants with respect to the lethal dose are across the top of the table. The lethal
dose concentrations can be found in Table 3-2. A less intense blue than the negative control
(indicating a toxic effect) in the majority (defined as three out of four) of four sample replicates
is shown with a positive sign, and a color similar to or a more intense blue than the negative
control (indicating a non-toxic effect) in the majority of the replicates is shown with a negative
sign.
Table 6-1. Inhibition of Bacterial Recovery in the Presence of Contaminants
Contaminant
Lethal Dose
(LD)
LD/10
LD/100
LD/1,000
Aldicarb
Botulinum toxin complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
All interferences
a) Only one concentration of possible interferences was analyzed (see Table 3-2).
18
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6.1.1 Contaminants and Potential Interferences
As shown in Figure 6-1, the Toxi-Chromotest detected toxic effects in three of the contaminants
analyzed: cyanide, thallium sulfate, and nicotine. Cyanide was detected at the lethal dose concen-
tration of 250 mg/L as well as the 25 mg/L level. Thallium sulfate was detected at concentrations
of 2,800 mg/L, 280 mg/L, and 2.8 mg/L. The color of the 28-mg/L concentration was not
significantly different from the negative control. There was no clear reason why this was the
case. Nicotine was only detectable at the highest concentration analyzed. Figure 6-1 shows a
fully developed 96-well plate that contains each of these contaminants.
Only ASTM Type II DI water was added to the first column of wells (method blank). No
chromogen was added to any of these wells, and no color development was expected. The
second column (PC) is the serial dilution of the mercury chloride positive control. The highest
concentration of mercury chloride (4 ppm) is at the top of the column, with twofold dilutions in
each well down to the bottom (0.031 mg/L). It is clear that color is almost completely depleted in
the top two wells, in the third well it is moderately depleted, and, thereafter, less than 0.5 mg/L,
the colors in the wells are virtually indistinguishable. The top four rows of the next five columns
contain the cyanide solutions. The color is almost completely depleted in the 250 mg/L sample,
while the 25 mg/L sample produced a light blue color. For the 25 mg/L sample, the lower three
replicates were visually clearly distinguishable from the negative control sample, but the top
sample was very similar to the top two negative control samples, causing it to be categorized as
negative. Nonetheless, because three out of four results for that concentration level were positive,
the result for the 25 -mg/L concentration was considered positive. The other two cyanide
Cyanide (mg/L)
Thallium Sulfate (mg/L)
Blank PC 25 0.25
PC = positive control
NC = negative control
28 2,800 280 NC 2.8
Nicotine (mg/L)
Figure 6-1. Effect of Contaminant on Test Color
19
-------
concentrations generated colors that were darker blue than the DDW negative control sample,
indicating the lack of a toxic effect at these two concentrations. The top four rows of the next
five columns contained the thallium sulfate solutions. The highest concentration of thallium
sulfate inhibited all color production in the sample, while the 280-mg/L and 2.8-mg/L
concentrations produced a light blue easily distinguishable from the DDW negative control. As
mentioned previously, the 28-mg/L concentration, in the wells in the second column from the
right, did not produce a change in color distinguishable from the negative control. The bottom
four rows in columns three through seven contained the nicotine samples. The only concentration
that produced any depleted color was 2,800 mg/L, which appeared to be just slightly less intense
than the negative control and the other nicotine samples.
None of the other contaminants inhibited color production in any of the samples. In addition,
none of the preservatives used in the stock solutions of ricin, botulinum toxin complex B, soman,
and VX; or possible chemical interferences inhibited color production. However, the serial
dilution of the mercury chloride positive control produced a gradual color gradient proportional
to the concentration on each of the plates used to analyze these samples, indicating proper
functioning of the test and reagents.
6.1.2 Precision
Because of the qualitative nature of the Toxi-Chromotest data, no numerical calculation for
repeatability was possible. The only measure of repeatability that could be evaluated was
whether individual replicates of each sample represented positive or negative responses. All of
the sample wells within four replicates of the same concentration had the same result with
respect to the negative control except for 25 mg/L cyanide, which exhibited one negative and
three positive responses (see discussion in Section 6.1).
6.2 Toxicity Threshold
Table 6-2 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 color inhibition with respect to 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
cyanide at 25 mg/L. Thallium sulfate was also detected at 2.8 mg/L; but the 28-mg/L sample was
not detectable, causing the toxicity threshold to become 280 mg/L.
6.3 False Positive/Negative Responses
The Toxi-Chromotest did not generate any results that could be considered false positive. Neither
the chlorination nor chloramination by-product samples generated detectable toxicity.
20
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6.4 Other Performance Factors
6.4.1 Ease of Use
The Toxi-Chromotest step-by-step instructions were easy to read; however, the letters with
which the solutions were identified did not correspond to the solution names. For example, the
diluent was labeled "G," while mercury chloride was labeled "D," which was confusing because
there was a tendency to think of diluent as "D." Storage conditions were marked on the vial
labels. The reaction of the Toxi-Chromotest could be observed visually or with a plate reader.
For this evaluation, color intensity was determined by visually comparing the samples with the
Table 6-2. Toxicity Thresholds
Contaminant
Aldicarb
Botulinum toxin complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Concentration (mg/L)
ND
ND
ND
25
ND
2,800
ND
ND
280
ND
ND = Significant color depletion was not observed.
Table 6-3. 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
yes
yes
yes
no
yes
no
yes
yes
no
yes
21
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positive control. Visual observation of reactions was difficult when there were only slight
variations in color intensity.
The Toxi-Chromotest requires two 1.5-hour incubation periods; and, after the 15-minute
bacterial rehydration, the hydrated bacteria could be used for only one hour. Because all reagents
started out colorless, it was somewhat difficult to tell whether reagents had been added and
difficult to observe the level of reagents in the wells. All reagents were stored in a refrigerator.
An expiration date was listed on the vial labels, but there was no indication how long they were
good once they were opened.
All the necessary supplies were provided with the Toxi-Chromotest except for pipettes with tips.
No formal scientific training would be required to use the Toxi-Chromotest. However, good
laboratory skills, especially in pipetting, would be helpful. Verification testing staff were able to
use the Toxi-Chromotest after a 30-minute training session.
The Toxi-Chromotest generated a small amount of aqueous waste, pipette tips, and 96-well
plates as waste. It was not clear whether the bacteria or other reagents should be considered
hazardous waste. Providing this information in the instructions or on reagent vials would be
helpful.
6.4.2 Field Portability
The Toxi-Chromotest was transported from a laboratory setting to a storage room for the field
portability evaluation. The storage room contained several tables and light and power sources,
but no other laboratory facilities. The Styrofoam container that reagents were shipped in was
used to carry the Toxi-Chromotest and related supplies. The incubator was carried separately.
One person could carry the kit and incubator; but, when laboratory peripherals, such as pipettes,
were added, it became more convenient to use a cart. The Toxi-Chromotest was set up easily in
only a few minutes. A source of electricity for the incubator and a flat surface of approximately
45 by 60 cm on which to fill the plate wells were required for analysis. The non-rehydrated
reagents are good at room temperature for up to one week. A plastic bag in the Toxi-Chromotest
was used to collect solid and liquid waste. Because the bacteria must be used within one hour of
rehydration, reagents are best prepared in the field. Overall the Toxi-Chromotest was easy to
transport to the field and, with an incubator warmed ahead of time, was deployed in a matter of
minutes. Results were obtained within 3 to 4 hours of starting the test. At this location, the Toxi-
Chromotest was tested using one concentration of cyanide at 250 mg/L. Apparently, there was a
problem with the reagents or the procedure because very little color development took place;
however, there is no reason to think that this had anything to do with the location where the test
was performed.
6.4.3 Throughput
The number of samples that can be processed depends on the number of replicates per sample
and the number of dilutions per sample that are processed on each 96-well plate. One plate can
be taken through the procedure in 3 to 4 hours. Each Toxi-Chromotest kit contained materials to
process samples filling three 96-well plates.
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Chapter 7
Performance Summary
Parameter
Contaminants in
DDW
Potential
interferences in
DDW
False positive
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
Visual Observance of Color
Inhibition by Contaminants at
Concentrations Relative to the LD
Concentration
LD
-
-
-
+
-
+
-
-
+
-
LD/10
-
-
-
+
-
-
-
-
+
-
LD/100
-
-
-
-
-
-
-
-
-
-
LD/1,000
-
-
-
-
-
-
-
-
+
-
Visual Observance of Color
Inhibition
-
-
-
-
-
Toxicity Threshold
(mg/L)
ND
ND
ND
25
ND
2,800
ND
ND
280
ND
The Toxi-Chromotest did not generate any false positive results to water containing chlorination
or chloramination by-products.
False negative
response
Aldicarb, botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, and VX produced an
inhibition that was not visually distinguishable from the negative control at the lethal dose
concentrations.
Ease of use
The Toxi-Chromotest requires two 1.5-hour incubation periods. After bacteria rehydration, the
hydrated bacteria could be used only for one hour. In addition, the reaction of the Toxi-
Chromotest was observed visually, which was difficult when there were only slight variations in
color. No formal scientific training would be required to use the Toxi-Chromotest.
Field portability
Overall the Toxi-Chromotest was easy to transport to the field and, with an incubator
warmed ahead of time, was deployed in a matter of minutes. Results were obtained within
3 to 4 hours of starting the test. Each Toxi-Chromotest kit contained materials to process
three 96-well plates.
Throughput
The number of samples that can be processed depends on the number of replicates per sample
and the number of dilutions per sample that are processed on each 96-well plate. One plate can be
taken through the procedure in 3 to 4 hours. Each Toxi-Chromotest kit contained materials to
process samples filling three 96-well plates.
+ = Visually distinguishable color inhibition from that of the negative control was observed.
- = Visually distinguishable color inhibition from that of the negative control was not observed
23
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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.
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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.
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