June 2006
Environmental Technology
Verification Report
AQUA SURVEY, INC.
CHEM-IQ Tox™ TEST KIT
Prepared by
Battelle
Batrene
/m? 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:
Chem-IQ Tox™
Aqua Survey, Inc.
469 Point Breeze Road
P.O. Box 72
Flemington, NJ 08822
www.aquasurvey.com
mail @ aquasurvey.com
PHONE: (908) 788-8700
FAX: (908) 788-9165
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 Aqua Survey, Inc. Chem-IQ Tox™ Test Kit. 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 Chem-IQ Tox™ Test Kit 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 Chem-IQ Tox™ Test Kit 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 Chem-IQ Tox™ Test Kit 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 Aqua
Survey, Inc. recommends that a 20% 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 20% inhibition threshold
• 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 were reported as producing inhibition less than 20% 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 Chem-IQ Tox™ Test Kit 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 can interfere
with the performance of the test and can degrade the contaminants during storage. Inhibition (endpoints) from
four replicates of each contaminant at each concentration level were evaluated to assess the ability of the
Chem-IQ Tox™ Test Kit to detect toxicity, as well as to measure the precision of the Chem-IQ Tox™ Test
Kit results. The response of the Chem-IQ Tox™ Test Kit 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 copper 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.
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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 Chem-IQ Tox™ Test Kit is based on information provided by the vendor.
This technology description was not verified in this test.
The Chem-IQ Tox™ Test Kit detects toxicants in drinking water using a chemical reaction that generates
fluorescence. The test can be conducted by a technician with basic laboratory skills. Sample analysis is
performed by adding two reagents to test and control water samples and measuring each sample's
fluorescence with a calibrated fluorometer. Percent inhibition values are calculated by comparing the light
production of the control with that of the test samples. If the average percent inhibition value of the replicate
test samples is greater than 20%, the test water sample is considered significantly impacted by a toxicant and
considered a positive response.
The Chem-IQ Tox™ Test Kit, which costs $250, contains 30 vials each of two reagents, 90 IQ Exposure
Chambers, disposal reagent pipettes, Chem-IQ Tox™ Test Kit score cards, and a Sharpie pen. Materials and
laboratory equipment required for the test include an Aquafluor™ hand-held fluorometer (Turner Design) or
equivalent and a supply of non-fluorescing 4 milliliter cuvettes (10 millimeter by 10 millimeter); an automatic
pipetter or equivalent with appropriate disposable tips for dispensing 10-milliliter, 250-microliter, and 50-
microliter volumes; a PCS liquid sonicator (L&R) or equivalent; a magnetic stir plate and stir bar (1/8 inch
diameter); a distilled or deionized water supply; and a digital timer that displays seconds.
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VERIFICATION RESULTS
Parameter
Contaminants 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
-16
-62
104
63
-55
50
-44
16
66
-44
LD/10
13
1
63
47
-30
84
-13
-8
16
-36
Average
Inhibition (%)
4
46
-26
11
34
LD/100
-33
4
17
-21
-38
71
10
19
-5
-14
Standard
0
LD/1,000
7
5
42
-18
-13
-3
-3
7
-25
-13
Deviation
9
3
20
9
2
Range of
Standard
Deviations
9-32
3-8
2-15
2-24
8-41
1-12
4-12
1-6
3-13
8-26
Toxicity
Thresh.
(mg/L)
ND
ND
24
25
ND
28
ND
ND
2,800
ND
Because DI water did not generate any measurable background light, the disinfection by-product
samples could not be compared with the inhibition due to DI water. Therefore only the absolute light
units produced by the chlorinated and chloraminated samples could be measured. Both of these
samples left adequate light for subsequent inhibition due to contamination and are thus not
considered to have generated false positive results.
False negative responses (inhibition less than 20%) were generated for aldicarb, botulinum toxin,
complex B, dicrotophos, ricin, soman, and VX when they were analyzed at the lethal dose
concentration.
The Chem-IQ Tox™ Test Kit instructions were clearly written; but a condensed summary with only
the necessary steps may be helpful. The contents of the Chem-IQ Tox™ Test Kit were well
identified. The test was not difficult to perform, but analyzing several samples simultaneously
required practice. No formal scientific education would be required to use the Test Kit.
The Chem-IQ Tox™ Test Kit was transported from a laboratory to a storage room to simulate a non-
laboratory location. All materials were easily transported by one person in a small cardboard box.
The Test Kit was set up in less than 10 minutes, except that Reagent Two took approximately
20 minutes to thaw. A source of electricity was required for the sonicator, while the fluorometer ran
on batteries. A cooler to transport and store reagents, pipettes and tips, the sonicator and a power
source, the fluorometer, and a waste container were needed for field use. Results were obtained
within 10 minutes of starting the test.
Approximately 30 analyses were completed in one hour. The 30 analyses included method blanks,
positive controls, and test samples. Approximately 130 samples could be processed per pair of
Reagent One and Reagent Two vials.
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
Aqua Survey, Inc.
Chem-IQ Tox™Test Kit
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 3
3.1 Test Samples 5
3.1.1 Quality Control Samples 5
3.1.2 Drinking Water Fortified with Contaminants 5
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 8
Chapter 4 Quality Assurance/Quality Control 11
4.1 Quality Control of Stock Solution Confirmation Methods 11
4.2 Quality Control of Drinking Water Samples 11
4.3 Audits 12
4.3.1 Performance Evaluation Audit 12
4.3.2 Technical Systems Audit 12
4.3.3 Audit of Data Quality 13
4.4 QA/QC Reporting 13
4.5 Data Review 13
Chapter 5 Statistical Methods and Reported Parameters 15
5.1 Endpoints and Precision 15
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 and Precision 18
6.1.1 Contaminants 18
6.1.2 Potential Interferences 27
6.1.3 Precision 29
6.2 Toxicity Threshold 29
6.3 False Positive/Negative Responses 29
6.4 Other Performance Factors 31
6.4.1 Ease of Use 31
6.4.2 Field Portability 31
6.4.3 Throughput 32
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Chapter 7 Performance Summary 33
Chapters References 34
Figures
Figure 2-1. Aqua Survey, Inc. Chem-IQ Tox™ Test Kit 2
Tables
Table 3-1. Contaminants and Potential Interferences 4
Table 3-2. Summary of Quality Control and Contaminant Test Samples 6
Table 3-3. Stock Solution Confirmation Results 9
Table 3-4. Water Quality Parameters 10
Table 4-1. Summary of Performance Evaluation Audit 13
Table 4-2. Summary of Data Recording Process 14
Table 6-la. Aldicarb Percent Inhibition Results 19
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results 19
Table 6-lc. Colchicine Percent Inhibition Results 20
Table 6-ld. Cyanide Percent Inhibition Results 21
Table 6-le. Dicrotophos Percent Inhibition Results 22
Table 6-lf. Nicotine Percent Inhibition Results 22
Table 6-lg. Ricin Percent Inhibition Results 23
Table 6-lh. Soman Percent Inhibition Results 24
Table 6-li. Thallium Sulfate Percent Inhibition Results 24
Table 6-lj. VX Percent Inhibition Results 25
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results 26
Table 6-3. Potential Interferences Results 28
Table 6-4. Disinfection By-Product Background Light Production 29
Table 6-5. Toxicity Thresholds 30
Table 6-6. False Negative Responses 30
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 Aqua Survey, Inc. Chem-IQ Tox™ Test Kit. 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 Chem-IQ Tox™ Test Kit. Following is a description of
Chem-IQ Tox™ Test Kit, based on information provided by the vendor. The information
provided below was not verified in this test.
The Chem-IQ Tox™ Test Kit (Figure 2-1) detects toxicants in drinking water using a chemical
reaction that generates fluorescence. The test can be conducted by a technician with basic
laboratory skills. Sample analysis is
performed by adding two reagents to test
and control water samples and measuring
each sample's fluorescence with a
calibrated fluorometer.
Percent inhibition values are calculated
by comparing the light production of the
control with that of the test samples. If
the average percent inhibition value of
the replicate test samples is greater than
20%, the test water sample is considered
significantly impacted by a toxicant and
considered a positive response.
Figure 2-1 Aqua Survey, Inc. Chem-IQ Tox™
Test Kit
The Chem-IQ Tox™ Test Kit, which
costs $250, contains 30 vials each of two
reagents, 90 IQ Exposure Chambers, disposal reagent pipettes, Chem-IQ Tox™ Test Kit score
cards, and a Sharpie pen. Materials and laboratory equipment required for the test include an
Aquafluor™ hand-held fluorometer (Turner Design) or equivalent and a supply of non-
flu orescing 4-milliliter cuvettes (10 millimeter by 10 millimeter); an automatic pipetter or
equivalent with appropriate disposable tips for dispensing 10-milliliter, 250-microliter, and 50-
microliter volumes; a PCS liquid sonicator (L&R) or equivalent; a magnetic stir plate and stir bar
(1/8 inch diameter); a distilled or deionized water supply; and a digital timer that displays
seconds.
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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, Chem-IQ Tox™ Test Kit 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 Chem-IQ Tox™ Test Kit can 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) Chem-IQ Tox™ Test Kit 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.
Chem-IQ Tox™ Test Kit 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 Aqua Survey, Inc. recommends that a 20% 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 20% 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
20% 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
20% when present at lethal concentrations or negative background inhibition that could cause
falsely low inhibition
• Other performance factors (sample throughput, ease of use, reliability).
Chem-IQ Tox™ Test Kit 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) Inhibition (endpoints) from four replicates of
each contaminant at each concentration level were evaluated to assess the ability of Chem-IQ
Tox™ Test Kit to detect toxicity at various concentrations of contaminants, as well as to measure
the precision of Chem-IQ Tox™ Test Kit results.
The response of Chem-IQ Tox™ Test Kit 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
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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 Chem-IQ Tox™ Test 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 solution consisting of 600 mg/L copper was used as a positive control. While
performance limits were not placed on the results, significant inhibition for the positive control
sample indicated to the operator that Chem-IQ Tox™ Test Kit was functioning properly. The
negative control sample 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 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
600 mg/L copper
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; 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
15
15
60
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
60
NA = not applicable, samples not fortified with any preservative, contaminant, or potential interference.
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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 Chem-IQ Tox™ Test Kit 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, the 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
can interfere with the performance of the test 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. 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
To analyze the test samples, kit reagents were prepared as specified in the instructions. This
involved diluting Reagent One with ASTM Type II water and sonicating for 60 seconds. Reagent
Two was removed from freezer storage and allowed to thaw. The fluorometer was calibrated
using ASTM Type II water as the blank and DDW as the control water sample prepared as a
"standard sample." Five milliliters of each test sample, as well as the negative and positive
control samples were placed in an individual cell in the exposure chamber. Reagent One, 125|iL,
was added to each cell. Then, 25 |iL of Reagent Two were added to each cell, and the time was
recorded. The contents of the cell were gently mixed by aspirating and dispensing using a
7
-------
disposable pipette. A 2- to 3-mL aliquot from each cell was transferred to an individual cuvette.
Fluorescence was measured exactly 10 minutes after the addition of Reagent Two. Aqua Survey
recommends analyzing at least three replicate samples to conclusively determine inhibition.
For each contaminant, a minimum of the lethal dose concentration and three additional
concentration levels were analyzed four times using Chem-IQ Tox™ Test Kit. Only one
concentration of each potential interference was analyzed four times. The fluorescence was
recorded, and the percent inhibition was calculated for each sample. Two operators performed all
the analyses using Chem-IQ Tox™ Test Kit. 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 Chem-IQ Tox™ Test Kit.
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 (10/2 1/05
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.
-------
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.
10
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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 a
standard reference method. 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 Chem-IQ
Tox™ Test Kit for approximately every 10 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 inhibition
of the DDW for the disinfecting by product evaluation. A positive control sample of 600 mg/L
copper also was analyzed once for approximately every 10 drinking water samples. While
performance limits were not placed on the results of the positive control sample, if the positive
control samples did not cause nearly complete inhibition, it would indicate to the operator that
Chem-IQ Tox™ Test Kit was not functioning properly. For 15 positive control samples, an
inhibition of 88% ± 12% was measured. This inhibition indicated that Chem-IQ Tox™ was
11
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functioning properly. 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%.
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:
%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.
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.
12
-------
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.
13
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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.
14
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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 fluorometer provided with the Chem-IQ Tox™ Test Kit reported the fluorescence for each
sample analyzed. Each test sample was compared with a negative control sample that, for this
verification test, was unspiked DDW. 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 inhibition for each sample was calculated using the following equation:
% inhibition = 1 - =—^ x 100% (2)
I J-/negative control j
Where Lsampie is the fluorescence produced for the test samples and Lnegative control is the average
fluorescence of the replicate negative control sample analyzed with each sample set. For each
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 SD of the rest of the contaminant concentrations
represented the precision of the inhibition caused by the background water combined with the
contaminant.
k=i
15
-------
where n is the number of replicate samples, h is the percent inhibition measured for the kih
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 differences in the inhibition at two
concentration levels required that the average inhibition at each concentration level, plus or
minus its respective standard deviation, did not overlap.
Aqua Survey, Inc. suggests that a 20% 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 20% 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 20% when determined with respect to DI water. Depending on the degree of
inhibition in the 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 20%, was not significantly different from the
negative control, and was not significantly different from the other concentration levels analyzed
(for a lethal dose inhibition less than 100%). The inhibition of the lethal dose sample was
required to be significantly greater than the other concentration levels because it more
thoroughly incorporated the uncertainty of all the measurements made by the Chem-IQ Tox™
Test Kit in determining false negative results. 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.
16
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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.
17
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Chapter 6
Test Results
6.1 Endpoints and Precision
Tables 6-la-j 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.
Contaminant test samples that produced negative percent inhibition values indicated an increase
in light production by the Chem-IQ Tox reagents and were considered non-toxic.
6.1.1 Contaminants
The contaminants that generated inhibition significantly greater than the negative control
included colchicine, cyanide, nicotine, soman, and thallium sulfate. Colchicine and cyanide
generated detectable inhibition at the two highest concentration levels analyzed, nicotine at the
three highest concentration levels, and thallium sulfate at only the highest concentration level.
Alternatively, dicrotophos produced an average negative inhibition at all four concentration
levels and aldicarb resulted in some negative and some positive average inhibition, depending on
the concentration level; but no concentration level of aldicarb produced an average inhibition
that differed significantly from the negative control.
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
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
18
-------
Table 6-la. Aldicarb Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.26
2.6
26
260
(Lethal Dose)
Inhibition
(%)
4
-23
9
10
32
30
1
-35
-22
-24
-24
-65
4
21
19
7
-1
-18
-34
-10
Average
(%)
0
7
-33
13
-16
Standard
Deviation
(%)
15
32
21
9
14
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.0003
0.003
0.03
0.3
(Lethal Dose)
Lethal Dose
Preservative
Blank
Inhibition
(%)
2
9
-17
6
5
9
-7
11
5
7
1
2
5
-2
1
0
-58
-64
-59
-66
-25
-29
-25
-25
Average
(%)
0
5
4
1
-62
-26
Standard
Deviation
(%)
12
8
3
3
4
2
19
-------
Table 6-lc. Colchicine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.24
2.4
24
240
(Lethal Dose)
Inhibition
(%)
43
-46
-5
8
43
47
43
35
33
-2
17
19
59
61
66
64
106
101
104
106
Average
(%)
0
42
17
63
104
Standard
Deviation
(%)
37
5
15
3
2
20
-------
Table 6-ld. Cyanide Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.25
2.5
25
250
(Lethal Dose)
Field
Portability
Negative
Control
Field
Portability
250
Inhibition
(%)
-29
9
11
9
-14
-18
-22
-18
-56
-17
-6
-7
41
49
44
53
64
61
62
64
-6
2
1
3
-38
-36
-35
-37
Average
(%)
0
-18
-21
47
63
0
-37
Standard
Deviation
(%)
19
4
24
5
2
4
1
(a) Results for cyanide at the field location were much different than in the laboratory.
Results did not seem to be correlated with non-laboratory analysis.
21
-------
Table 6-le. Dicrotophos Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
1.4
14
140
1,400
(Lethal Dose)
Inhibition
(%)
-3
-10
14
0
17
-33
-14
-21
-14
-5
-96
-37
-50
-55
-15
0
-60
-49
-64
-47
Average
(%)
0
-13
-38
-30
-55
Standard
Deviation
(%)
10
21
41
27
8
Table 6-If. Nicotine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Inhibition
(%)
-l
ll
12
-22
5
-20
-4
7
72
72
70
72
84
83
84
84
50
49
49
51
Average
(%)
0
-3
71
84
50
Standard
Deviation
(%)
16
12
1
1
1
22
-------
Table 6-lg. Ricin Percent Inhibition Results
Concentration
(mg/L)
Negative Control
0.015
0.15
1.5
15
(Lethal Dose)
Lethal Dose
Preservative Blank
Inhibition
(%)
10
-2
-8
1
-1
-10
-1
0
19
0
6
15
-28
-2
-15
-6
-48
-38
-54
-37
16
7
17
8
Average
(%)
0
-3
10
-13
-44
12
Standard
Deviation
(%)
7
4
8
12
8
5
23
-------
Table 6-lh. Soman Percent Inhibition Results
Concentration
(mg/L)
Negative Control
0.0014
0.014
0.14
1.4
(Lethal Dose)
Lethal Dose Level
Preservative Blank
Inhibition
(%)
-l
0
3
-2
11
4
9
5
23
24
17
12
-9
-8
-10
-6
17
15
15
17
1
-6
-2
4
Average
(%)
0
7
19
-8
16
-1
Standard
Deviation
(%)
2
3
6
2
1
5
Table 6-li. Thallium Sulfate Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Inhibition
(%)
4
7
6
-17
-15
-17
-44
-25
-2
-6
-2
-11
29
1
20
12
62
69
67
68
Average
(%)
0
-25
-5
16
66
Standard
Deviation
(%)
12
13
5
12
3
24
-------
Table 6-lj. VX Percent Inhibition Results
Concentration
(ms/L)
Negative Control
0.002
0.02
0.2
2
(Lethal Dose)
Lethal Dose Level
Preservative Blank
Inhibition
(%)
l
-2
3
-2
-1
-17
-21
-13
-28
-13
-15
0
-26
-38
-26
-53
-31
-33
-82
-28
-23
-55
-30
-26
Average
(%)
0
-13
-14
-36
-44
-34
Standard
Deviation
(%)
3
8
11
13
26
14
Chem-IQ Tox™ Test Kit 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 that were not stored in
preservatives. This technique, however, could indicate that Chem-IQ Tox™ Test Kit 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 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.
25
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During the initial analysis of the preservative blanks (Table 6-2), none of the samples generated
an inhibition significantly greater than the negative control or greater than 20% (Aqua Survey's
suggested benchmark for significant toxicity). Because the preservatives apparently did not have
toxic effects at the lethal dose concentration, no additional dilutions of preservative blanks were
required to determine whether there were toxic effects from each individual concentration level;
and each contaminant concentration level was evaluated and compared with the negative control
to determine any toxic effects. The inhibition of the lethal dose preservative blank was deter-
mined with each contaminant sample set and is shown with each table of the contaminant
inhibition.
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results
Preservative
Blank
Negative
Control
Ricin
Soman/VX
Botulinum
Toxin
Complex B
Inhibition
(%)
-4
8
-9
4
24
1
4
22
11
11
7
11
-39
-47
-54
-36
Average
(%)
0
13
10
-44
Standard
Deviation
(%)
8
12
2
8
ta) Soman and VX use the same preservative.
Samples from three of the four botulinum toxin complex B concentrations produced an average
inhibition that was not significantly different from the negative control and was less than 20%
(the Aqua Survey criterion), indicating no toxic effect. The other sample, at the lethal dose
concentration, was significantly different from the negative control, but in the negative direction
(-62%). In addition, the lethal dose preservative blank analyzed with this sample set also
produced a negative inhibition (-26%). The inhibition of the lethal dose botulinum toxin complex
B sample was more negative than the lethal dose preservative blank and the negative control,
indicating no toxic effect.
The ricin preservative blank analyzed prior to the contaminant analysis did not generate a
detectable inhibition; therefore, as with botulinum toxin complex B, additional dilutions of the
preservative blank were not required. In addition, inhibition of the lethal dose preservative blank
during contaminant testing did not differ significantly from the negative control; therefore, the
inhibition of each ricin sample could be compared directly to the negative control. The average
inhibition of each of the three lower concentrations of ricin was not significantly different from
the negative control. The lethal dose sample was significantly different, but in the negative
direction, indicating the lack of a toxic effect.
26
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For soman, the average inhibition of the preservative blank analyzed prior to the contaminant
samples was 10% ± 2%, not significantly different from that of the negative control and not
above the 20% threshold of toxicity suggested by the vendor. Again, dilutions of the preservative
blank were not required, and the contaminant inhibition was calculated only with respect to the
negative control. Each of the four soman test concentrations generated an average inhibition that
was significantly different from the negative control (three in a positive direction and one in a
negative direction). On average, none of them exceeded 20%, indicating no toxic effect.
For VX, the inhibition of the preservative blank analyzed prior to the contaminant samples was
not significantly different from that of the negative control and not above the 20% threshold of
toxicity suggested by the vendor. Thus, dilutions of the preservative blank were not analyzed
with the contaminant samples. When the set of contaminant samples was analyzed with a lethal
dose preservative blank, the preservative blank inhibition was -34% ± 14%, indicating an
enhancement of luminescence from the preservative. The reason for the difference between this
result and the result obtained prior to the contaminant analysis was not clear. However, while
this preservative blank result was significantly different from the negative control, it did not
differ significantly from any test sample inhibition, indicating that VX had no significant toxic
effect.
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 Chem-IQ Tox™ Test Kit to detect toxicity is dependent on the background light production
in whatever drinking water matrix is being used. If the background drinking water sample
completely inhibits background light, 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 Chem IQ Tox™ Test Kit. Of the five metal solutions evaluated as possible
interferences with the Chem IQ Tox™ Test, three of them, copper (46% ± 3%), iron (-26% ±
20%), and zinc (34% ± 2%), exhibited an average inhibition that was significantly different from
the DDW negative control (0% ± 4%).
The iron inhibition was negative; therefore, drinking water with similar concentrations of iron
would likely be amenable to the Chem-IQ Tox™ Test Kit because there would actually be an
increase in background light that could potentially be inhibited by contaminants. Zinc and copper
generated inhibition greater than 20% but less than 50%. While these levels of inhibition are
greater than the vendor's toxicity threshold, enough background light remains that toxicity from
contaminants could likely be detected provided water containing these metals were analyzed
with respect to negative controls with similar background toxicity. Aluminum and manganese
generated inhibition not significantly different from the negative control. These results
underscore the need for negative control samples that are extremely similar to the water matrices
that are suspected of having been contaminated. Small differences in the water composition can
cause the appearance of toxicity.
To investigate whether the Chem-IQ Tox™ Test Kit is sensitive to by-products of disinfecting
processes, DDW samples from water systems that use chlorination and chloramination were
analyzed and would have been compared with ASTM Type IIDI water as the control sample.
27
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Table 6-3. Potential Interferences Results
Potential
Interferences
Negative control
(Metals)
Aluminum
Copper
Iron
Manganese
Zinc
Concen-
tration
(ms/L)
NA
0.5
0.6
0.15
0.25
2.5
Inhibition
(%)
-2
-5
2
5
-8
3
11
9
44
51
46
44
-23
-55
-15
-9
13
20
-1
12
32
34
33
36
Average
(%)
0
4
46
-26
11
34
Standard
Deviation
(%)
4
9
3
20
9
2
NA = Not applicable.
However, when ASTM Type IIDI water was analyzed with the Chem-IQ Tox™ Test Kit, almost
all of the light was inhibited; prohibiting the calculation of inhibition with respect to the DI
water. Therefore, instead of calculating inhibition, the background light generated when the two
water samples were analyzed was compared (see Table 6-4). The background light units
produced for the 60 DDW (chlorination by-product) samples analyzed throughout the
verification test was 1,043 ± 233. This seemed to be adequate background light for subsequent
inhibition to occur. In addition, the average number of light units produced in the sample
containing chloraminated water was 2,817 ± 201 (N=4); thus, it seems that Chem-IQ Tox™ Test
Kit could be used with either chloraminated or chlorinated water since neither sample completely
inhibits the background light production and, therefore, background light remains for the
subsequent detection of contaminants. 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 on that water.
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Table 6-4. Disinfection By-Product Background Light Production
Potential
Interferences
Chlorination
by-products
Chloramination
by-products
N
60
4
Average Light
Units
1,043
2,817
Standard
Deviation
233
201
6.1.3 Precision
Across all the contaminants and potential interferences, the standard deviation (not relative
standard deviation) was measured for each set of four replicates to evaluate the Chem-IQ Tox™
Test Kit precision. Out of 78 opportunities, the standard deviation of the four replicate inhibition
measurements was less than 10% 50 times (64% of the time ), between 10% and 20% 19 times
(24% of the time), and greater than 20% 9 times (11%). Overall, 88% of the time, the standard
deviations were less than 20%. 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 6-5 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
concentrations are for colchicine at 24 mg/L and cyanide at 25 mg/L.
6.3 False Positive/Negative Responses
Because the DI water samples did not allow detectable light to be generated, the inhibition of the
chlorination and chloramination by-product samples could not be determined. This makes the
availability of a non-contaminated drinking water sample for use as a negative control mandatory
for analysis using the Chem-IQ Tox™ Test Kit. The absolute light units generated by both types
of these samples were measured. As mentioned in Section 6.1.2, adequate background light is
present to detect subsequent contamination; however, DI water may not be used as a negative
control. If samples are analyzed daily, a good practice to follow would be to archive a negative
control sample each day in case of contamination the next day.
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Table 6-5. Toxicity Thresholds
Contaminant
Aldicarb
Botulinum toxin complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Concentration (mg/L)
ND
ND
24
25
ND
28
ND
ND
2,800
ND
ND = Significant inhibition was not detected.
Table 6-6 shows the false negative responses, which are described in Section 5.3. Botulinum
toxin complex B, ricin, and VX did not exhibit a detectable inhibition at the lethal concentration.
Table 6-6. 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
no
no
yes
no
yes
yes
no
yes
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6.4 Other Performance Factors
6.4.1 Ease of Use
The Chem-IQ Tox™ Test Kit instructions were clearly written; but, because they were very
detailed, a condensed summary with only the necessary steps may be helpful. The contents of the
Chem-IQ Tox™ Test Kit were well identified with labels on the vials. Overall, the test was not
difficult to perform, but becoming efficient at analyzing several samples simultaneously required
practice. The analysis procedure required one reagent to be sonicated, while another reagent was
thawed after storage in a freezer. In one instance, the reagent vial broke during thawing.
A handheld fluorometer was provided by Aqua Survey. The fluorometer was easy to use, but
required calibration precisely one minute prior to analysis of each sample set. The electronic
readout was user-friendly, and only one number needed to be recorded. The fluorometer was
easily wiped clean and required no routine maintenance other than calibration. Other
miscellaneous items required include a micropipettor with various sized tips, a sonicator for use
during reagent preparation, and a freezer for reagent storage.
No formal scientific education would be required to use the Chem-IQ Tox™ Test Kit, but good
laboratory skills, especially pipetting, would be beneficial. Verification testing staff were able to
operate the Chem-IQ Tox™ Test Kit after a training session lasting approximately two hours.
Approximately 10 mL of liquid waste were generated per sample, along with leftover reagents.
In addition, one chamber per six samples, reagent vials, pipette tips, and cuvettes were generated
as solid waste. It was not clear whether the reagents should be considered hazardous waste.
6.4.2 Field Portability
The Chem-IQ Tox™ Test Kit 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. No carrying case was provided with the Chem-IQ
Tox™ Test Kit; however, all materials were transported by one person in a 38-centimeter by
38-centimeter by 38-centimeter cardboard box. The Chem-IQ Tox™ Test Kit was set up easily
in less than 10 minutes, with the exception of letting one reagent thaw, which took
approximately 20 minutes after removal from a freezer. A source of electricity was required for
the sonicator; however, the fluorometer runs on batteries. Longer-term field deployment would
require a freezer for storing reagents. The following items not provided in the Chem-IQ Tox™
Test Kit were needed for field use: a cooler to transport and store reagents, pipettes and tips, the
sonicator and a power source, the fluorometer, and a waste container. Overall the Chem-IQ
Tox™ Test Kit was easy to transport to the field and was deployed in a matter of minutes. The
limiting factor to testing in the field would be the approximately 20 minutes required to thaw one
of the reagents. Results were obtained within 10 minutes of starting the test.
The Chem-IQ Tox™ Test Kit was tested with one contaminant, cyanide, at the lethal dose
concentration. Interestingly, the results obtained for the lethal dose of cyanide at the non-
laboratory location (Table 6-Id) were very different from those obtained initially in the
laboratory. The inhibition measured initially was 63% ± 2%, and in the non-laboratory location it
was -37% ±11%. The positive control samples analyzed in both locations generated significant
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inhibition as would be expected. There was no indication that the Chem-IQ Tox™ Test Kit was
not functioning properly because the location of the analysis didn't seem to be related to these
unexplainable results and more expected results had been obtained during the cyanide laboratory
testing. No reanalysis was performed.
6.4.3 Throughput
Approximately 30 analyses were completed in one hour. The 30 analyses included method
blanks, positive controls, and test samples. Approximately 130 samples could be processed per
pair of Reagent One and Reagent Two vials.
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Chapter 7
Performance Summary
Parameter
Contaminants in
DDW
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
-16
-62
104
63
-55
50
-44
16
66
-44
LD/10
13
1
63
47
-30
84
-13
-8
16
-36
Average
Inhibition (%)
4
46
-26
11
34
LD/100
-33
4
17
-21
-38
71
10
19
-5
-14
LD/1,000
7
5
42
-18
-13
-3
-3
7
-25
-13
Standard Deviation
(%)
9
3
20
9
2
Range of
Standard
Deviations
(%)
9-32
3-8
2-15
2-24
8-41
1-12
4-12
1-6
3-13
8-26
Toxicity
Thresh.
(mg/L)
ND
ND
24
25
ND
28
ND
ND
2,800
ND
False positive
response
Because DI water did not generate any measurable background light, the disinfection by-product samples
could not be compared with the inhibition due to DI water. Therefore only the absolute light units
produced by the chlorinated and chloraminated samples could be measured. Both of these samples left
adequate light for subsequent inhibition due to contamination and are thus not considered to have
generated false positive results.
False negative
response
False negative responses (inhibition less than 20%) were generated for aldicarb, botulinum toxin complex
B, dicrotophos, ricin, soman, and VX when they were analyzed at the lethal dose concentration.
Ease of use
The Chem-IQ Tox™ Test Kit instructions were clearly written; but a condensed summary with only the
necessary steps may be helpful. The contents of the Chem-IQ Tox™ Test Kit were well identified. The
test was not difficult to perform, but analyzing several samples simultaneously required practice. No
formal scientific education would be required to use the Test Kit.
Field portability
The Chem-IQ Tox™ Test Kit was transported from a laboratory to a storage room to simulate a non-
laboratory location. All materials were easily transported by one person in a small cardboard box. The
Test Kit was set up in less than 10 minutes, except that Reagent Two took approximately 20 minutes to
thaw. A source of electricity was required for the sonicator, while the fluorometer ran on batteries. A
cooler to transport and store reagents, pipettes and tips, the sonicator and a power source, the fluorometer,
and a waste container were needed for field use. Results were obtained within 10 minutes of starting the
test.
Throughput
Approximately 30 analyses were completed in one hour. The 30 analyses included method blanks,
positive controls, as well as test samples. Approximately 130 samples could be processed per pair of
Reagent One and Reagent Two vials.
ND = Significant inhibition was not detected.
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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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