US EPA Environmental Technology Verification Report - CheckLight Ltd ToxScreen-II Test Kit


                      June 2006
    Environmental Technology
    Verification Report

    CHECKLIGHT LTD.
    ToxScREEN-ll TEST KIT
            Prepared by
             Battelle
            Balteiie
           7/ji? 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
f,EPA , Baiteiie
The Business of Innovation
U.S. Environmental Protection Agency
ETV Joint Verification Statement
TECHNOLOGY TYPE: Rapid Toxicity Testing System

APPLICATION: Detecting Toxicity in Drinking Water

TECHNOLOGY
NAME: ToxScreen-II

COMPANY: CheckLight Ltd.

ADDRESS: Kolner Strasse 6 PHONE: (972) 4 9930530
P.O. Box 72 FAX: (972) 4 9533176
Qiryat-Tiv'on 36000
Israel

WEB SITE: www.checklight.co.il
E-MAIL: info@checklight.co.il
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 CheckLight Ltd. ToxScreen-II 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, ToxScreen-II 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 ToxScreen-II 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.

ToxScreen-II 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
     CheckLight Ltd. recommends that a 50% 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 50% 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 that exceeded
     50%

 •   False negative responses—contaminants that were reported as producing less than 50% and/or were not
     significantly different from the negative control 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).

ToxScreen-II was verified by analyzing a dechlorinated drinking water sample from Columbus, Ohio (DDW),
fortified with contaminants (at concentrations ranging from lethal levels to concentrations up to one million
times less than the lethal dose) and interferences  (metals possibly present as a result of the water treatment
processes). Dechlorinated water was used because free chlorine kills the bacteria within the ToxScreen II
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 ToxScreen-II to detect
toxicity, as well as to measure the precision of ToxScreen-II results. The response of ToxScreen-II 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 sodium chloroacetate for the
Pro-Organic Buffer samples and copper chloride for the Pro-Metal Buffer samples); and negative control
samples, which consisted of the unspiked DDW.

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QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a
technical systems audit, a performance evaluation audit, and a data quality audit of 10% of the test data.

This verification statement, the full report on which it is based, and the test/QA plan for this verification test
are all available at www.epa.gov/etv/centers/centerl.html.

TECHNOLOGY DESCRIPTION

The following description of the ToxScreen-II Test Kit is based on information provided by the vendor. This
technology description was not verified in this test.

ToxScreen-II provides on-site detection of organic and inorganic toxicants, such as heavy metals; pesticides;
herbicides; chlorinated hydrocarbons; polychlorinated biphenyls; benzene, toluene, ethylbenzene, and
xylenes; and phencyclidine. ToxScreen-II can be used in both field and laboratory testing. Typical
applications include effluent toxicity testing; surface and ground water screening for changes in water quality;
and raw drinking water monitoring for early warning of dangerous spills, accidents, and
sabotage/bioterrorism.

Under proper conditions, luminous bacteria emit high and steady levels of luminescence. Chemical and
biological toxicants that affect cell respiration, electron transport systems, adenosine triphosphate generation,
and the rate of protein or lipid synthesis alter the level of luminescence. Similarly, agents that affect a cell's
integrity and membrane function have a strong effect on luminescence. Hence, toxicants of different
characteristics such as pesticides, herbicides, chlorinated hydrocarbons, and heavy metals exert a measurable
effect on a bacterial luminescence system. By comparing the luminescence level obtained in a suspected toxic
sample with that obtained in a clean water control sample after a short period of incubation, very low
concentrations of a broad range of toxicants can be detected. To detect toxicants in water samples,
ToxScreen-II uses a highly sensitive variant of Photobacterium leiognathi and two assay buffers: one for
detecting heavy metals (Pro-Metal Buffer) and the other for organic pollutants (Pro-Organic Buffer). When
used concurrently, these buffers are designed to discriminate between the presence of organic and metal
toxicants at submilligram per liter concentrations.

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VERIFICATION RESULTS
Pro-Organic Buffer
Parameter
Contaminants in
DDW
Potential
interferences in
DDW
False positive
response
False negative
response
Ease of use
Field portability
Throughput
Compound
Aldicarb
Botulinum
toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium
sulfate
VX
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Lethal
Dose (LD)
Cone.
(mg/L)
260
0.3
240
250
1,400
2,800
15
1.4
2,800
2
Cone.
(mg/L)
0.5
0.6
0.15
0.25
2.5
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
50
-87
75
100
70
83
68(a)
-6
66
-3
LD/10
-26
14
17
100
23
-10
9
-202
13
-5
LD/100
0
16
4
95
3
-32
1
4
9
2
LD/1,000
-50
-54
1
72
1
-20
10
15
-1
-6
Average Inhibition (%)
Initial
Analysis
-4
3
0
0
-1
Reanalysis(b)
-12
5
-6
3
NR
Range of
Standard
Deviations
(%)
12-18
21-58
2-5
0-2
3-29
2-10
2-5
10-68
3-6
3-9
Toxicity
Thresh.
(mg/L)

ND
ND
24
0.25
140
1,400
ND
ND
28
ND
Standard Deviation (%)
Initial
Analysis
4
3
1
4
3
Reanalysis(b)
1
15
4
3
NR
None of the disinfection by-product samples produced an inhibition significantly greater than 50%, the
inhibition level suggested by CheckLight Ltd. to conclusively determine toxicity.
Aldicarb, botulinum toxin complex B, soman, and VX produced an inhibition that either did not exceed
50% or were not significantly different from the negative control at the lethal dose concentrations. For
ricin in the Pro-Organic buffer, the inhibition of the lethal dose was significantly different from the
negative control, but not significantly different from the inhibition generated by the preservative blank.
ToxScreen-II included clearly written instructions with good illustrations. The contents of the
ToxScreen-II were well labeled, making it easy to follow the instructions. A minimum of three hours
was required to rehydrate the bacteria, which must be stored at -14°C prior to rehydration. After
rehydration, the bacteria can be used for up to seven days; however, the vendor suggested using them
within one day. Overall, the ToxScreen-II was easy to use, making it likely that a person with no
formal scientific training could conduct the tests.
ToxScreen-II was transported from a laboratory to a storage room to simulate operation in a non-
laboratory location. It was tested with cyanide at the lethal dose concentration, and the results
generated (>90% inhibition) were very similar to those obtained in the laboratory. No carrying case
was provided with ToxScreen-II (one is available for purchase from Checklight Ltd.); however, all
materials except the luminometer were transported in a small cardboard box. The box and luminometer
were easily carried by one person, and setup for analysis took less than 10 minutes.
Approximately 25 analyses were completed each hour using both buffers, and approximately 1,000
samples could be processed per kit.
ND = Significant inhibition was not detected.
NR = Not reanalyzed.
(a) Inhibition was not significantly different from the preservative blank.
(b) Potential interferences were reanalyzed due to four suspect negative inhibitions during the initial analysis with the Pro-
Metal buffer.

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Pro-Metal Buffer
Parameter
Contaminant
s in DDW
Potential
interferences
in DDW
False
positive
response
False
negative
response
Compound
Aldicarb
Botulinum toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Lethal
Dose (LD)
Cone.
(mg/L)
260
0.3
240
250
1,400
2,800
15.0
1.4
2,800
2.0
Cone.
(mg/L)
0.5
0.6
0.15
0.25
2.5
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
-19
-185
12
89
55
98
3
-55
79
5
LD/10
-7
-121
2
64
-1
2
-2
17
53
-11
LD/100
33
-91
8
44
-10
-10
2
-66
27
-11
LD/1,000
-31
-40
-9
19
-3
-7
2
-4
4
-3
Average Inhibition (%)
Initial
Analysis
-395
-299
-399
-368
86
Reanalysis(a)
-13
30
-8
5
NR
Range of
Standard
Deviations
(%)
10-35
18-104
2-6
1-7
2-4
0-4
2-4
13-22
1-4
5-10
Toxicity
Thresh.
(mg/L)

ND
ND
ND
0.25
140
700
ND
ND
28
ND
Standard Deviation (%)
Initial
Analysis
29
26
18
15
0
Reanalysis(a)
3
4
3
4
NR
Neither the chlorination nor chloramination samples generated an inhibition greater than 50%.
However, the chloramination sample generated a result that indicated an enhancement in luminescence
(i.e., a negative inhibition), which, according to Checklight Ltd., can also indicate toxicity.
The inhibition of the chloramination by-products was -75% ± 20% with DI water as the negative
control. If a contaminant causing a 75% inhibition had been present in this water and DI water was used
as the negative control, the inhibition would have been close to 0% — a false negative response. This
underscores the need to use negative control samples that are as similar as possible to the samples being
analyzed. A second type of false negative response occurred (for aldicarb, colchicine, botulinum toxin,
ricin, soman, and VX) when the inhibition was not greater than 50% in the presence of a lethal dose of
contaminant.
ND = Significant inhibition was not detected.
NR = Not reanalyzed.
See the Pro-Organic Buffer table for descriptions for ease of use, field portability, and throughput.
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Original signed by Gregory A. Mack 6/22/06 Original signed by Andrew P. Avel 8/7/06
Gregory A. Mack Date Andrew P. Avel Date
Vice President Acting Director
Energy, Transportation, and Environment Division National Homeland Security Research Center
Battelle Office of Research and Development
U.S. Environmental Protection Agency
NOTICE: ETV verifications are based on an evaluation of technology performance under specific,
predetermined criteria and the appropriate quality assurance procedures. EPA and Battelle make no expressed or
implied warranties as to the performance of the technology and do not certify that a technology will always
operate as verified. The end user is solely responsible for complying with any and all applicable federal, state,
and local requirements. Mention of commercial product names does not imply endorsement.
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June 2006
Environmental Technology Verification
Report

ETV Advanced Monitoring Systems Center

CheckLight Ltd.
ToxScreen-ll 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 4
3.1 Test Samples 6
3.1.1 Quality Control Samples 6
3.1.2 Drinking Water Fortified with Contaminants 6
3.1.3 Drinking Water Fortified with Potential Interferences 7
3.2 Test Procedure 7
3.2.1 Test Sample Preparation and Storage 7
3.2.2 Test Sample Analysis Procedure 9
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 17
5.3 False Positive/Negative Responses 17
5.4 Other Performance Factors 18
Chapter 6 Test Results 19
6.1 Endpoints and Precision 19
6.1.1 Contaminants 19
6.1.2 Potential Interferences 34
6.1.3 Precision 37
6.2 Toxicity Threshold 37
6.3 False Positive/Negative Responses 37
6.4 Other Performance Factors 39
6.4.1 Ease of Use 39
6.4.2 Field Portability 40
6.4.3 Throughput 41
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Chapter 7 Performance Summary 42
Chapters References 45

Figures

Figure 2-1. CheckLight Ltd. ToxScreen-II Test Kit 2
Tables

Table 3-1. Contaminants and Potential Interferences 5
Table 3-2. Summary of Quality Control and Contaminant Test Samples 8
Table 3-3. Stock Solution Confirmation Results 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-la. Aldicarb Percent Inhibition Results 20
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results 21
Table 6-lc. Colchicine Percent Inhibition Results 22
Table 6-ld. Cyanide Percent Inhibition Results 23
Table 6-le. Cyanide Percent Inhibition Results—Additional Dilutions 24
Table 6-lf. Dicrotophos Percent Inhibition Results 25
Table 6-lg. Dicrotophos Percent Inhibition Results—Additional Dilutions 25
Table 6-lh. Nicotine Percent Inhibition Results 26
Table 6-li. Nicotine Percent Inhibition Results—Additional Dilutions 26
Table 6-lj. Ricin Percent Inhibition Results 27
Table 6-lk. Soman Percent Inhibition Results 28
Table 6-11. Thallium Sulfate Percent Inhibition Results 29
Table 6-lm. VX Percent Inhibition Results 30
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results 32
Table 6-3. Potential Interferences Results 35
Table 6-4. Potential Interference Results—Reanalysis 36
Table 6-5. Toxicity Thresholds 38
Table 6-6. False Negative Responses 39
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
ID identification
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 CheckLight Ltd. ToxScreen-II 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 ToxScreen-II. Following is a description of ToxScreen-II,
based on information provided by the vendor. The information provided below was not verified
in this test.

ToxScreen-II (Figure 2-1) provides on-
site detection of a wide range of organic
and inorganic toxicants, such as heavy
metals; pesticides; herbicides;
chlorinated hydrocarbons;
polychlorinated biphenyls; benzene,
toluene, ethylbenzene, and xylenes; and
phencyclidine. ToxScreen-II can be used
in both field and laboratory testing.
Typical applications include effluent
toxicity testing; surface and ground
water screening for changes in water
quality; and raw drinking water
monitoring for early warning of
dangerous spills, accidents, and
sabotage/bioterrorism.

Under proper conditions, luminous
bacteria emit high and steady levels of
luminescence. Chemical and biological
toxicants that affect cell respiration,
electron transport systems, adenosine
triphosphate generation, and the rate of protein or lipid synthesis alter the level of luminescence.
Similarly, agents that affect a cell's integrity and membrane function have a strong effect on
luminescence. Hence, toxicants of different characteristics such as pesticides, herbicides,
chlorinated hydrocarbons, and heavy metals exert a dramatic and measurable effect on a bacterial
luminescence system. By comparing the luminescence level obtained in a suspected toxic sample
Figure 2-1. CheckLight Ltd. ToxScreen-II Test
Kit
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with that obtained in a clean water control sample after a short period of incubation, very low
concentrations of a broad range of toxicants can be detected.

To detect toxicants in water samples, ToxScreen-II uses a highly sensitive variant of
Photobacterium leiognathi and two assay buffers: one for detecting heavy metals (Pro-Metal
buffer) and the other for organic pollutants (Pro-Organic buffer). When used concurrently, these
buffers are designed to discriminate between the presence of organic and metal toxicants at sub-
milligram per liter concentrations.

The ToxScreen-II luminometer is 150 millimeters (mm) wide by 280 mm deep by 170 mm high
and weighs approximately two kilograms. The test kit comes with stoppered vials holding freeze-
dried luminous bacteria, hydration buffer, storage buffer, Pro-Metal concentrated assay buffer,
Pro-Organic concentrated assay buffer, concentrated positive control solutions, and empty test
tubes. The portable luminometer costs $3,950, and a starter kit including reagents for 1,000
single tests costs $550.
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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, ToxScreen-II 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 ToxScreen-II 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) ToxScreen-II 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. Dechlorinated
water was used because free chlorine inhibits the photosynthetic process that ToxScreen II depends on to
indicate toxicity and can degrade the contaminants during storage. 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.

ToxScreen-II 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 CheckLight Ltd. recommends that a 50% inhibition is required for a
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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
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 50% inhibition threshold

• 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 that exceeded 50%

• False negative responses—contaminants that were reported as producing less than 50%
inhibition and/or were not significantly different from the negative control when the
contaminant was present at lethal concentrations or negative inhibition that could cause
falsely low inhibition results.

• Other performance factors (sample throughput, ease of use, reliability).

ToxScreen-II was used to analyze the DDW samples fortified with contaminants at
concentrations ranging from lethal levels to concentrations up to one million times less than the
lethal dose. The lethal dose of each contaminant was determined by calculating the concentration
at which 250 milliliters (mL) of water would probably cause the death of a 154-pound person.
These calculations were based on toxicological data available for each contaminant that are
presented in Amendment 2 of the test/QA plan.(1) Inhibition results (endpoints) from four
replicates of each contaminant at each concentration level were evaluated to assess the ability of
ToxScreen-II to detect toxicity at various concentrations of contaminants, as well as to measure
the precision of ToxScreen-II results.

The response of ToxScreen-II 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

5
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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.
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
concentrations 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 ToxScreen-II. Solutions were analyzed using both the Pro-
Organic and Pro-Metal buffers. 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. Positive control samples consisted of ASTM Type II DI water fortified with a
vendor-specified contaminant at a vendor-specified concentration level. Sodium chloroacetate
(Pro-Organic) and copper chloride (Pro-Metal) were used as positive control samples throughout
the verification test with their respective buffer solutions. While performance limits were not
placed on the results, an inhibition of approximately 50% for these positive control samples
indicated to the operator that ToxScreen-II was functioning properly. The negative control
samples consisted of unspiked DDW and were 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. Additional concentrations of some contam-
inants were prepared and analyzed for two reasons: one was because of the large difference in
response between two concentration levels. For example, if only one dilution level was almost
completely inhibitory and the next dilution level was non-inhibitory, several intermediate
concentrations were analyzed to better determine the toxicity threshold of that contaminant. The
other reason was because sometimes the lowest concentration analyzed was mostly inhibitory,
thus, not providing even an estimate of the toxicity threshold. For these contaminants, additional
tenfold dilutions were analyzed to more accurately determine the toxicity threshold. Table 3-2
lists each concentration level and the number of samples analyzed at each level.

3.1.3 Drinking Water Fortified with Potential Interferences

Individual aliquots of the DDW were fortified with one-half the concentration specified by the
EPA's NSDWR for each potential interference. Table 3-2 lists the interferences, along with the
concentrations at which they were tested. Four replicates of each of these samples were analyzed.
To test the sensitivity of ToxScreen-II 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 ToxScreen-H 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 were labeled only with sample identification numbers so that the
operators did not know their content.
7
-------
Table 3-2. Summary of Quality Control and Contaminant Test Samples
Type of Sample

Quality control

DDW fortified with
contaminants

DDW fortified with

Disinfectant
by-products
Sample Characteristics
Method blank
(ASTM Type II water)
Positive control:
Pro-Organic
Positive control:
Pro-Metal
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
1 : 100 dilution of sodium
chloroacetate stock provided
in kit
1 : 100 dilution of copper
chloride stock 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)
0.30; 0.030; 0.0030; 0.00030
mg/L
240; 24; 2.4; 0.24 mg/L
250; 25; 2.5; 0.25; 0.025;
0.0025; 0.00025 mg/L
1,400; 1,000; 500; 140; 14;
1.4; mg/L
2,800; 2,100; 1,400; 700; 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
16
16 (Pro-Organic test only)
15 (Pro-Metal test only)
56
4
4 with VX, 4 with soman
4 each at concentration: lethal
dose (LD) (cone, at left), LD/10,
LD/100, andLD/1,000
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
56
NA = not applicable, samples not fortified with any preservative, contaminant, or potential interference.
-------
3.2.2 Test Sample Analysis Procedure

To analyze the test samples, the luminescent marine bacteria Photobacterium leiognathi (strain
SB) were reconstituted with hydration buffer, incubated at ambient temperature for
approximately 5 minutes, then transferred into storage buffer and mixed well. The rehydrated
bacteria were stored at 4°C until use. The bacteria were prepared the afternoon before their use
for all tests, with the exception of the field portability test. For the field portability test, two sets
of bacteria were prepared. One set was prepared approximately 3 hours before use and the
second set approximately 24 hours before use to assess the performance of the minimum
incubation time (3 hours) against the more standard time (24 hours) used during this testing
program. Once the bacteria were properly rehydrated and incubated, 800 microliters ((^L) of the
test sample were added to a sample cuvette along with 200 ^iL of either the Pro-Metal or Pro-
Organic buffer, and this combination was mixed. Then, 10 (^L of rehydrated bacteria were added
to each water/buffer solution, mixed well, and incubated at ambient temperature for 60 minutes.
After 60 minutes, luminescence was measured. The luminescence of the test sample was
compared with that of the negative control to determine percent inhibition.

For each contaminant, a minimum of the lethal dose concentration and three additional
concentration levels were analyzed four times using ToxScreen-II. Only one concentration of
each potential interference was analyzed four times. The luminescence was recorded, and the
percent inhibition was calculated for each sample. Two operators performed all the analyses
using ToxScreen-II. 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
ToxScreen-II.

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)
(a)
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
-------
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
-------
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 ToxScreen-
II for approximately every 20 drinking water samples that were analyzed. Because an 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 also was analyzed once for
approximately every 20 drinking water samples. While performance limits were not placed on
the results of the positive control sample, the vendor informed Battelle that, if the positive
control samples did not cause inhibition that was significantly greater than DI water, it would
indicate to the operator that ToxScreen-H was not functioning properly. For 16 sodium
chloroacetate (Pro-Organic) positive control samples and 15 copper chloride (Pro-Metal) positive
12
-------
control samples, inhibition results of 50% ± 14% and 75% ± 18%, respectively, were measured
after a 60-minute incubation. These inhibition values indicated the proper functioning of
ToxScreen-II. 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 concen-
tration of the PE sample, and the nominal concentration of that sample was calculated using the
following equation:

%D=— xlOO% C1)
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(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.
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 luminometer provided with the ToxScreen-II reported the absolute light units for each
sample analyzed. Each DDW 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-=—— xlOO% (2)
y Li negative control j

Where L is the absolute light units produced for each test sample and L negative control is the average
negative control of the four negative control samples analyzed in the same sample set as the
subject test sample. The negative control sample was always DDW, except when the inhibition
of the disinfectant by-products was being determined, in that case, ASTM Type IIDI water
served as the control sample.

The standard deviation (SD) of the results for the replicate samples was calculated, as follows,
and used as a measure of technology precision at each concentration. The standard deviation
around the average negative control results represented the variability of the inhibition caused by
the negative control water. Similarly, the standard deviation of the rest of the contaminant
concentrations represented the precision of the inhibition caused by the background water
combined with the contaminant.
SD =
(3)
16
-------
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 an
average inhibition significantly greater than the negative control. Also, each concentration level
higher than the toxicity threshold had to be significantly greater than the negative control, and
the inhibition produced by each lower concentration analyzed had to be significantly less than
that produced by the toxicity threshold concentration. Since the inhibition of the test samples was
calculated with respect to the inhibition of each negative control sample, the percent inhibition of
the negative control was always zero. A significant difference in the average inhibition at two
concentration levels required that the average inhibition at each concentration level, plus or
minus its respective standard deviation, did not overlap.

CheckLight Ltd. suggests that a 50% 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 50% 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 greater than 50% when determined with respect to DI water. Depending on the degree
of background inhibition in a sample, toxicity from subsequent contamination of that sample
may not be detectable or could be exaggerated as a result of the baseline inhibition. Drinking
water samples collected from water systems using chlorination and chloramination as the
disinfecting process were analyzed in this manner.

A response was considered false negative if, when a lethal concentration of some contaminant
was analyzed, the average inhibition did not exceed 50%, was not significantly different from the
negative control, or was not significantly different from the other concentration levels analyzed
(for lethal dose inhibition less than 100%). The inhibition of the lethal dose sample was required
to be significantly greater than the other concentration levels because it more thoroughly
incorporated the uncertainty of all the measurements made by the ToxScreen-n 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. In addition, background water samples that increased the light production of the
ToxScreen-II organisms (i.e., negative inhibition) were considered false negative because such
samples could cancel out the effect of a contaminant that inhibits light production, making it
seem that the contaminant had no toxic effect.

17
-------
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.
18
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Chapter 6
Test Results
6.1 Endpoints and Precision

Tables 6-la-m present the percent inhibition data for 10 contaminants; and Table 6-2 gives the
percent inhibition data 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 results 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 bacteria and were considered non-toxic.

6.1.1 Contaminants

The contaminants that were analyzed by ToxScreen-II during this verification test produced
results that differed depending on whether the Pro-Organic or Pro-Metal buffer was used
(Tables 6-la-m). The inhibition in both buffers was determined for each contaminant at the
concentration levels indicated in the tables. Since the buffers were developed to enhance the
sensitivity of specific classes of compounds (metal or organic pollutants), the results were
expected to show this difference.

In the Pro-Organic buffer, all the contaminants except aldicarb, botulinum toxin complex B,
soman, and VX exhibited an inhibition that was significantly larger than the negative control.
Aldicarb generated a positive average inhibition at only the lethal dose concentration. However,
the uncertainties around all of the aldicarb measurements, including the negative control, were
rather large; therefore, not even the lethal dose sample was significantly different from the
negative control. Thallium sulfate produced a detectable inhibition at the top three concentration
levels, colchicine and dicrotophos generated a detectable inhibition for the lethal dose and the
first tenfold dilution concentration level, while nicotine generated a detectable inhibition at the
lethal dose concentration. Additional dilutions for cyanide, dicrotophos, and nicotine were
performed to more closely determine the toxicity threshold (Tables 6-le, g, and i). ToxScreen-II
was especially sensitive to cyanide. The first four concentrations that were analyzed (250, 25,
2.5, 0.25 mg/L) produced an inhibition that was significantly larger than the negative control;
19
-------
Table 6-la. Aldicarb Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.26
2.6
26
260
(Lethal Dose)
Pro-Organic Buffer
Inhibition
(%)
54
-6
7
-55
-62
-55
-33
-50
-10
-9
3
14
-48
-29
-22
-5
34
42
60
65
Average
(%)
0
-50
0
-26
50
Standard
Deviation
(%)
45
12
12
18
15
Pro-Metal Buffer
Inhibition
(%)
31
-13
-48
30
-28
-55
-42
1
53
44
-19
55
-8
7
-24
-5
-22
-30
-10
-11
Average
(%)
0
-31
33
-7
-19
Standard
Deviation
(%)
38
24
35
13
10
20
-------
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
0.3
(Lethal Dose)
Lethal Dose
Preservative
Blank
Pro-Organic Buffer
Inhibition
(%)
-42
-21
-3
65
-77
-92
-79
32
-16
31
23
25
45
5
20
-15
-29
-104
-133
83
52
-51
7
-31
-21
-93
-120
-73
55
-43
12
-24
Average
(%)
0
-54
16
14
-87
-6
-77
0
Standard
Deviation
(%)
46
58
21
25
44
46
42
43
Pro-Metal Buffer
Inhibition
(%)
7
12
53
-73
-70
-9
-33
-51
-102
-10
-96
-154
-104
-107
-138
-136
-217
-274
-215
-35
-256
-129
-220
-243
-2
-20
-1
57
-14
27
-3
10
Average
(%)
0
-40
-91
-121
-185
-212
9
0
Standard
Deviation
(%)
52
26
60
18
104
57
33
18
Shading indicates inhibition calculated with respect to the preservative blank.
21
-------
Table 6-lc. Colchicine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.24
2.4
24
240
(Lethal Dose)
Pro-Organic Buffer
Inhibition
(%)
-2
3
-2
1
4
0
2
-2
1
(a)
7
3
16
11
23
19
74
78
73
73
Average
(%)
0
1
4
17
75
Standard
Deviation
(%)
3
3
3
5
2
Pro-Metal Buffer
Inhibition
(%)
3
-1
-1
-1
-17
-6
-6
-6
1
11
9
10
-1
3
-1
7
13
14
9
12
Average
(%)
0
-9
8
2
12
Standard
Deviation
(%)
2
6
4
4
2
Deleted -98% because it was an outlier.
22
-------
Table 6-ld. Cyanide Percent Inhibition Results
Concentration
(mg/L)
Negative Control
0.25
2.5
25
250
(Lethal Dose)
Negative Control
(3 -hour incubation
250
(3 -hour incubation)
Negative Control
(24-hour incubation)
250
(24-hour incubation)
Pro-Organic Buffer
Inhibition
(%)
-3
0
-3
6
72
69
71
74
95
95
95
96
100
100
100
100
100
100
100
100
-9
-3
12
1
98
99
100
99
10
-7
-11
8
98
98
97
98
Average
(%)
0
72
95
100
100
0
99
0
98
Standard
Deviation
(%)
4
2
0
0
0
9
1
11
0
Pro-Metal Buffer
Inhibition
(%)
-l
-3
11
-7
17
18
19
20
46
43
42
44
75
62
59
62
89
88
88
89
-22
-1
4
19
90
91
92
91
0
3
3
-6
95
97
98
95
Average
(%)
0
19
44
64
89
0
91
0
96
Standard
Deviation
(%)
8
2
2
7
1
17
1
4
1
Shading indicates results from field portability testing.
23
-------
Table 6-le. Cyanide Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
0.00025
0.0025
0.025
0.25
Pro-Organic Buffer
Inhibition
(%)
-4
12
-14
6
-15
-9
-10
-11
-16
-12
-9
-14
-10
-17
-15
-15
32
29
31
34
Average
(%)
0
-11
-13
-14
31
Standard
Deviation
(%)
11
3
3
3
2
24
-------
Table 6-If. Dicrotophos Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
1.4
14
140
1,400
(Lethal Dose)
Pro-Organic Buffer
Inhibition
(%)
-l
l
-2
2
4
6
0
-5
2
1
2
8
12
27
26
29
84
25
84
84
Average
(%)
0
1
3
23
70
Standard
Deviation
(%)
2
5
3
7
29
Pro-Metal Buffer
Inhibition
(%)
l
0
-2
2
-6
-3
2
-5
-11
-8
-8
-15
-1
2
-3
-1
57
54
54
54
Average
(%)
0
-3
-10
-1
55
Standard
Deviation
(%)
2
4
3
2
2
Table 6-lg. Dicrotophos Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
140
500
1,000
1,400
(Lethal Dose)
Pro-Organic Buffer
Inhibition
(%)
2
-1
2
-3
4
-1
3
1
7
7
9
9
15
20
20
23
27
22
29
25
Average
(%)
0
2
8
19
26
Standard
Deviation
(%)
2
2
1
4
3
Pro-Metal Buffer
Inhibition
(%)
l
-2
2
-1
8
8
10
8
13
15
14
14
21
20
25
20
21
18
18
21
Average
(%)
0
8
14
21
19
Standard
Deviation
(%)
2
1
1
2
2
25
-------
Table 6-lh. Nicotine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Pro-Organic Buffer
Inhibition
(%)
19
-9
-2
-8
-17
-16
-22
-27
-41
-20
-36
-31
-9
-18
-16
3
82
84
84
81
Average
(%)
0
-20
-32
-10
83
Standard
Deviation
(%)
13
5
9
10
2
Pro-Metal Buffer
Inhibition
(%)
-2
-4
6
0
-5
-5
-10
-8
-16
-7
-10
-9
3
-1
1
3
98
98
99
98
Average
(%)
0
-7
-10
2
98
Standard
Deviation
(%)
4
2
4
2
0
Table 6-li. Nicotine Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
700
1,400
2,100
2,800
(Lethal Dose)
Pro-Organic Buffer
Inhibition
(%)
2
25
-6
-20
14
21
32
20
79
77
79
80
55
58
61
55
64
67
60
66
Average
(%)
0
22
79
57
64
Standard
Deviation
(%)
19
7
2
3
3
Pro-Metal Buffer
Inhibition
(%)
-6
3
0
3
20
14
12
16
75
75
71
68
85
86
84
84
96
96
96
96
Average
(%)
0
16
72
85
96
Standard
Deviation
(%)
4
3
3
1
0
26
-------
Table 6-lj. Ricin Percent Inhibition Results
Concentration
(mg/L)
Negative Control
0.015
(Lethal Dose/
1,000)
Lethal Dose/1,000
Preservative Blank
0.15
(Lethal Dose/ 100)
Lethal Dose/100
Preservative Blank
1.5
(Lethal Dose/10)
Lethal Dose/10
Preservative Blank
15
(Lethal Dose)
Lethal Dose
Preservative Blank
Pro-Organic Buffer
Inhibition
(%)
-l
-6
-1
9
12
16
4
7
12
12
9
22
-2
3
4
0
-1
3
6
5
7
11
9
7
22
13
14
26
67
66
68
73
67
63
66
69
Average
(%)
0
10
14
1
3
9
18
68
66
Standard
Deviation
(%)
6
5
6
3
3
2
6
3
2
Pro-Metal Buffer
Inhibition
(%)
0
-4
-2
6
0
2
2
4
1
3
-1
1
3
4
-1
0
-2
0
-3
-1
3
-5
-6
1
3
5
3
-1
7
-1
3
1
3
9
-4
-5
Average
(%)
0
2
1
2
-2
-2
2
3
1
Standard
Deviation
(%)
4
2
1
2
1
4
2
3
6
27
-------
Table 6-Ik. Soman Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.0014
0.014
0.14
1.4
(Lethal Dose)
Lethal Dose
Preservative
Blank
Pro-Organic Buffer
Inhibition
(%)
3
-18
34
-20
-8
25
43
0
33
-30
-18
33
-304
-160
-175
-170
-2
-5
-21
2
13
-27
4
-64
Average
(%)
0
15
4
-202
-6
-18
Standard
Deviation
(%)
25
23
33
68
10
35
Pro-Metal Buffer
Inhibition
(%)
-25
27
2
-4
-14
14
-13
-3
-80
-62
-44
-78
-14
24
40
17
-40
-55
-80
-47
41
31
46
-10
Average
(%)
0
-4
-66
17
-55
27
Standard
Deviation
(%)
21
13
17
22
17
26
28
-------
Table 6-11. Thallium Sulfate Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Pro-Organic Buffer
Inhibition
(%)
0
0
-2
2
3
-4
0
-3
2
11
8
16
21
9
12
11
68
68
66
63
Average
(%)
0
-1
9
13
66
Standard
Deviation
(%)
2
3
6
5
3
Pro-Metal Buffer
Inhibition
(%)
2
0
0
-1
7
0
7
2
32
27
23
25
53
51
55
54
79
81
78
79
Average
(%)
0
4
27
53
79
Standard
Deviation
(%)
1
4
4
2
1
29
-------
Table 6-lm. VX Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.002
0.02
0.2
2.0
(Lethal Dose)
Lethal Dose
Preservative
Blank
Pro-Organic Buffer
Inhibition
(%)
l
-2
3
-2
-6
-4
-4
-10
1
-3
10
2
-10
-8
-4
1
3
-8
-13
6
-12
-12
-9
-5
Average
(%)
0
-6
2
-5
-3
-9
Standard
Deviation
(%)
2
3
5
5
9
3
Pro-Metal Buffer
Inhibition
(%)
l
5
-8
2
-6
3
-1
-9
-24
-11
-11
1
-18
-5
-4
-18
8
5
9
-3
-3
-20
-21
2
Average
(%)
0
-3
-11
-11
5
-10
Standard
Deviation
(%)
5
5
10
7
6
12
therefore, additional dilutions had to be performed to reach a concentration that did not produce
detectable inhibition. These additional dilutions confirmed that the lowest concentration that
generated detectable inhibition was 0.25 mg/L. Interestingly, upon reanalysis, the 0.25 mg/L
sample produced less than half of the inhibition that it did upon the initial analysis. There wasn't
a clear reason for this.

In the Pro-Metal buffer, colchicine, cyanide, dicrotophos, nicotine, and thallium sulfate exhibited
an inhibition that was significantly greater than the negative control. Colchicine produced an
inhibition that was significantly greater than the negative control at the 240- and 2.4-mg/L
concentration levels. However, samples at 24 and 0.24 mg/L were not significantly greater than
the negative control. All four cyanide concentrations generated an inhibition significantly
different from the negative control. Inhibition produced by dicrotophos were significantly
different from the negative control only at the lethal dose level, so additional dilutions were
performed to elucidate the toxicity threshold of dicrotophos (Table 6-lg); and, during the
additional analyses, all four concentrations that were analyzed (between 1,400 mg/L and
140 mg/L) generated an inhibition significantly larger than the negative control. During the
analysis of the additional dilutions, the lethal dose sample produced an inhibition of 19% ± 2%
compared to an inhibition of 55% ± 2% during the first analysis. There was no clear reason for
this discrepancy. Similarly, upon initial analysis of nicotine, only the lethal dose generated an
inhibition that was significantly greater than zero. Upon analysis of several concentrations
30
-------
between the lethal dose and the first tenfold dilution (Table 6-li), it was shown that nicotine
concentrations between the lethal dose and 700 mg/L generated an inhibition significantly
greater than zero. As in the Pro-Organic buffer, thallium sulfate produced a detectable inhibition
at the top three concentration levels analyzed.

It is important to note that the botulinum toxin complex B, ricin, soman, and VX stock solutions
used to prepare the test samples for this verification test 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 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
ToxScreen-II 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 with the contaminants that were not stored in preservatives.
This technique, however, could indicate that ToxScreen-II 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.

During the initial analysis of the preservative blanks (Table 6-2), the only sample that generated
an inhibition significantly different from the unfortified negative controls was the sample repre-
senting the ricin preservative, with an inhibition of 61% ± 3% in the Pro-Organic buffer.

Therefore, for the ricin test samples, all of the preservative blanks were diluted with the same
concentration of preservatives as the test samples containing ricin. For the other contaminant test
samples, only the samples containing preservatives equivalent to those of the lethal dose were
analyzed with the contaminant samples.

Using the Pro-Organic buffer, the inhibition of botulinum toxin complex B was not significantly
different from the DDW negative control or the preservative blank. Note that the lethal dose
concentration sample did generate an average inhibition that was considerably more negative
than the lethal dose preservative blank. According to Checklight Ltd., it is possible that this
increase in luminescence could indicate possible toxicity. However, during this evaluation, the
large uncertainty surrounding these average results make it difficult to be confident that the
31
-------
average inhibition is different from the controls. Checklight Ltd. also noted that it is possible that
botulinum toxin complex B inhibition is linear over a smaller concentration range than was
evaluated during this verification test. While that is true, the higher concentrations would be
expected to generate some level of toxicity if the lower concentrations did. In the case of
botulinum toxin complex B, the large relative variability at each concentration level made any
distinctions from the negative control difficult to determine. In the Pro-Metal buffer, the negative
average inhibition of the botulinum toxin complex B samples increased as the sample
concentration increased. Unexpectedly, the preservative blank also generated a very large
negative inhibition (-212% ± 57%), while previously it had generated an inhibition of -10% ±
2%. The uncertainties surrounding this inhibition data were rather large. As a result there was no
clear trend of distinctly increasing or decreasing inhibition with concentration, and the inhibition
was all negative. However, because the preservative blank analyzed with this set of test samples
contained the same preservative concentration as the lethal dose sample, the inhibition of the
0.3-mg/L sample was calculated with respect to the preservative blank. When performing the
calculation in this

Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results
Preservative
Blank
Negative
Control
Ricin
Soman/VX(a)
Botulinum
Toxin
Complex B
Pro-Organic Buffer
Inhibition
(%)
-5
0
0
5
59
58
62
66
0
-6
-4
3
-7
-6
5
8
Average
(%)
0
61
-2
0
Standard
Deviation
(%)
4
3
4
8
Pro-Metal Buffer
Inhibition
(%)
-4
-1
-1
6
14
0
-1
-3
2
-5
-4
0
-11
-8
-9
-12
Average
(%)
0
3
-2
-10
Standard
Deviation
(%)
4
8
3
2
(a)
Soman and VX use the same preservative

way, the inhibition of the preservative blank was 0% ±18%, and the inhibition of the lethal dose
of botulinum toxin complex B was 9% ± 33%, indicating the lack of a toxic effect from
botulinum toxin complex B. The large negative inhibition when calculated with respect to the
DDW negative control did indicate that, during this set of analyses, the preservative in the
botulinum toxin complex B samples was causing enhanced luminescence in both the preservative
blank sample and the botulinum toxin complex B samples. Further, even though no additional
preservative blank samples were analyzed, results from the less concentrated botulinum toxin
complex B samples generated progressively fewer negative results as the concentration
decreased. Direct comparison of the lethal dose botulinum toxin complex B sample and the
preservative blank showed that the botulinum toxin complex B exhibited no toxicity, strongly
32
-------
suggesting that the decreasing enhanced luminescence shown by the increased dilutions of the
botulinum toxin complex B really indicates the increased dilution of the preservative.

As explained previously, the original analysis of the ricin preservative blank test samples
resulted in the preservative blank producing a 61% ± 3% inhibition with respect to the DDW
negative control shown in Table 6-2 in the Pro-Organic buffer. Therefore, equivalent dilutions of
the preservative blank samples were analyzed with the ricin test samples for both the Pro-
Organic and the Pro-Metal buffers. With respect to the negative control without preservative, the
15 mg/L sample (68% ± 3%) and the 1.5 mg/L sample (9% ± 2%) generated detectable
inhibition. The preservative blank samples corresponding to these contaminant concentrations
generated very similar inhibition data. The 15-mg/L preservative blank generated an inhibition of
66% ± 2%, and the 1.5 mg/L preservative blank generated an inhibition of 18% ± 6%; showing
that the ricin preservatives, rather than the ricin, probably contribute to a toxic effect on the
ToxScreen-II organisms. For ricin in the Pro-Metal buffer, neither the ricin test samples nor the
preservative blanks showed an inhibition significantly different from the negative control,
indicating that neither ricin nor the ricin preservatives inhibit the ToxScreen-II organisms in the
Pro-Metal buffer.

For soman in the Pro-Organic buffer, only the samples at the 0.14-mg/L concentration generated
an inhibition significantly different from the negative control. The average inhibition at that
concentration was -202% ± 68%. At the other concentrations, as well as for the preservative
blank, inhibition was not detectable. As for botulinum toxin complex B, Checklight Ltd.
indicated that this enhancement of luminescence could indicate a toxic effect at this
concentration; but, again, during this evaluation, the fact that none of the other three
concentration levels generated a change from the negative control in either direction didn't seem
to support this. Checklight Ltd. also stated that it is possible that the indication of soman's
toxicity is linear over a smaller range than was analyzed during this test. While true, if that is the
case, it seems unlikely that a higher concentration would not exhibit any inhibition at all. The
large negative inhibition for the 0.14 mg/L concentration level was not easily explained;
however, the variability in the ToxScreen-II results seemed somewhat higher during the soman
analyses than it had been throughout the rest of the verification test. In the Pro-Metal buffer, the
0.014- and the 1.4-mg/L samples both generated an inhibition significantly different from the
negative control; however, both were negative. Also, there was no clear trend of positive (or
negative) inhibition with concentration; that is, some of the concentrations exhibited an average
inhibition that was negative and some that was positive, making an evaluation of the toxic effect
of soman difficult to determine. The inhibition for the preservative blank was not significantly
different from the negative control, so there did not seem to be a toxic effect from the
preservative.

For VX in the Pro-Organic buffer, the average inhibition at each concentration was within 10%
of that of the negative control, and none of the concentrations (including the preservative blank)
generated a positive inhibition that was significantly different from the negative control. Samples
at two concentrations generated an inhibition that was negative and significantly different from
the negative control; but again, no matter what the concentration, all of the average inhibition
results were within 10% of the negative control, which indicates a minimal toxic effect. In the
Pro-Metal buffer, none of the samples (including the preservative blank) exhibited an inhibition
that was significantly different from the negative control, indicating a lack of toxic effect for all
samples.

33
-------
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
ToxScreen-II to detect toxicity is dependent on the bacteria's background light production in
whatever drinking water matrix is being used. If the background drinking water sample produces
100% inhibition of light, inhibition caused by contaminants could not be detected. However,
even if a drinking water sample generated some degree of inhibition, it can still be used as the
background sample provided there is adequate background light available to indicate the
presence of subsequent contamination.

Table 6-3 presents the results from the samples that were analyzed to test the effect of potential
interferences on ToxScreen-II. In the Pro-Organic buffer, none of the potential interferences
exhibited an inhibition significantly different from the negative control. In the Pro-Metal buffer,
four out of the five metal solutions exhibited a large negative inhibition, while the zinc solution
exhibited an 86% inhibition. There was no obvious explanation for the negative inhibition
because the positive control analyzed with that sample set (a copper solution) exhibited positive
(62%) inhibition as was expected, and the background luminescence from the negative control
was rather typical. A negative inhibition, which indicates an increase in light production by the
ToxScreen-II bacteria, caused by these solutions does not necessarily mean that these
compounds will interfere with the analysis. A direct interference would cause all of the
background luminescence to be inhibited. In this case, a DDW sample with similar
concentrations of these metals would likely be amenable to the ToxScreen-II because there
would actually be an increase in background light that could potentially be inhibited by
contaminants. However, for zinc, the background luminescence was 86% depleted, leaving not
much available luminescence for inhibition due to contamination. Because of the large, negative
inhibition, these four possible interferences were reanalyzed using freshly prepared samples
(Table 6-4). During this second analysis, only the copper test sample generated an inhibition that
was significantly larger than the negative control. In the Pro-Metal buffer, iron and aluminum
produced a slightly negative inhibition, while the average inhibition of manganese was not
different from the negative control. In the Pro-Organic buffer, these possible interferences
generated an inhibition that was either just slightly negative or not significantly different from
the negative control, very similar to the results during the initial analyses of these samples. It is
not clear why the results for the Pro-Metal buffer were so different during the initial analysis.
34
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Table 6-3. Potential Interferences Results
Potential
Interferences
Negative
control
(Metals)
Aluminum
Copper
Iron
Manganese
Zinc
Negative
control
(By-products)
Chlorination
by-products
Chloramination
by-products

Concen-
tration
(mg/L)
NA
0.5
0.6
0.15
0.25
2.5
NA
NA
NA
Pro-Organic Buffer
Inhibition
(%)
l
-2
-1
1
-5
-6
-7
2
7
1
4
0
1
0
1
-1
6
-2
-2
-4
-2
-4
-4
3
-2
4
1
-2
(a)
-2
-5
0
-6
Average
(%)
0
-4
3
0
0
-1
0
18
-3
Standard
Deviation
(%)
1
4
3
1
4
3
3
26
3
Pro-Metal Buffer
Inhibition
(%)
4
9
-11
-3
-353
-400
-405
-420
-277
-288
-296
-336
-401
-373
-406
-414
-358
-381
-351
-381
86
86
86
86
-1
4
-6
3
(a)
-89
-94
-63
-54
Average
(%)
0
-395
-299
-399
-368
86
0
13
-75
Standard
Deviation
(%)
9
29
26
18
15
0
5
34
20
NA = Not applicable.
(a) Average inhibition across all DDW negative control samples (N=56).
35
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Table 6-4. Potential Interference Results—Reanalysis
Potential
Interferences
Negative
control
Aluminum
Copper
Iron
Manganese

Concen-
tration
(mg/L)
NA
0.5
0.6
0.15
0.25
Pro-Organic Buffer
Inhibition
(%)
4
12
-14
6
-12
-13
-13
-10
17
17
1
-16
-11
-5
-6
-3
1
1
3
6
Average
(%)
0
-12
5
-6
3
Standard
Deviation
(%)
11
1
15
4
3
Pro-Metal Buffer
Inhibition
(%)
2
-1
2
0
-13
-17
-10
-11
30
25
31
33
-12
-6
-9
-6
10
8
2
2
Average
(%)
0
-13
30
-8
5
Standard
Deviation
(%)
2
3
4
3
4
NA = Not applicable.

To investigate whether the ToxScreen-II is sensitive to by-products of disinfecting processes,
DDW samples from water systems that use chlorination and chloramination were analyzed and
compared with ASTM Type n DI water as the control sample (these results are presented in
Table 6-3). In the absence of a background water sample, it seems likely that DI water may be
used as a "clean water" control; therefore, it would be helpful to know what the results would be
if this is done. In the Pro-Organic buffer, the sample from the water supply disinfected with
chlorination exhibited an inhibition of 18% ± 26% (N=56), while the sample from the water
supply disinfected by chloramination exhibited an inhibition of -3% ± 3% on four replicates. The
difference in the number of replicates is because the dechlorinated water was used as the
negative control with each sample set; therefore, much more data were collected on that water.
This suggests that samples that have been disinfected using either process are not likely to
interfere with ToxScreen-II results because the inhibition caused by the "clean" drinking water
matrix left most of the light to potentially be inhibited by contamination. For the Pro-Metal
buffer, the inhibition of the sample from the water supply disinfected by chlorination was 13% ±
34%, and the inhibition of the sample from the water supply disinfected by chloramination was -
75% ± 20%. In the former case, interference is unlikely because of the weak inhibition caused by
the background; while, in the latter case, the inhibition could be underestimated unless the
negative control sample is very similar to the background water sample. For example, if a
contaminant that exhibited approximately 75% inhibition was placed in water from a
36
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chloraminated system, and ASTM Type n DI water was used as the reference sample, the
percent inhibition would be approximately zero. However, if the chloraminated water was used
as the negative control, an appropriate inhibition would be determined. Overall, as long as a
similar negative control sample is used for the Pro-Metal buffer, water disinfected using either
process is not likely to interfere with the ToxScreen-II results.

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 ToxScreen-II
precision. Out of 170 opportunities, the standard deviation of the four replicate measurements
was less than 10% 122 times (72%), between 10% and 20% 24 times (14%), and greater than
20% 24 times (14%). Overall the standard deviation was less than 10% more than twice as often
as not. 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
concentration was for cyanide at 0.25 mg/L when both the Pro-Metal Pro-Organic buffers were
used. Note that a concentration level was not determined to be detectable unless all the
concentrations below it generated a significantly smaller inhibition.
6.3 False Positive/Negative Responses

The chlorination and chloramination by-product samples at times generated results that were
significantly different from the negative control; but, on average, the inhibition of both types of
water was less than 50%, therefore not exceeding the inhibition level suggested by CheckLight
Ltd. as a minimum for determining toxicity without being considered a false positive result.
Since the background inhibition is not complete, it can be accounted for by using negative
control samples that are very similar to the water being analyzed. If samples are analyzed daily, a
good practice would be to archive a negative control sample each day in case of contamination
the next day.
37
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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)
Pro-Organic
ND
ND
24
0.25
140
1,400
ND
ND
28
ND
Pro-Metal
ND
ND
ND
0.25
140
700
ND
ND
28
ND
ND = Significant inhibition was not detected.

The inhibition of the sample from the water system disinfected by chloramination was -75% ±
20% when using the Pro-Metal buffer. According to Checklight Ltd., a negative inhibition can
also indicate toxicity in a sample. If this is the case, this should be considered a false positive
result. Ironically, it seems that this phenomenon also introduces the possibility of a false negative
response if the reference sample is not similar to the water sample. If ASTM Type IIDI water
was used as the reference sample, and a contaminant in a chloraminated water sample caused a
75% inhibition, the inhibition would be approximately zero—a false result. In this case, using a
reference sample similar to the water sample would solve the problem, but the possibility of false
negative results must be considered if ASTM Type II water is used as the reference. A second
type of false negative result could occur when a lethal dose of contaminant is present in the water
sample and the inhibition is not at least 50%—the lower limit for a positive response according
to Checklight Ltd—and significantly different from the negative control. Table 6-6 gives these
results. The lethal dose concentration of aldicarb, botulinum toxin complex B, colchicine, ricin,
soman, and VX produced an inhibition that either did not exceed 50% or that was not
significantly different from the negative control in at least one of the two buffers used by
ToxScreen-II. For ricin in the Pro-Organic buffer, the inhibition of the lethal dose was
significantly different from the negative control, but not significantly different from the
inhibition generated by the preservative blank
38
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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)
280
0.30
240
250
1,400
2,800
15
1.4
2,800
2.0
False Negative Response(a)
Pro-Organic Buffer
yes
yes
no
no
no
no
(b)
yes
no
yes
Pro-Metal Buffer
yes
yes
yes
no
no
no
yes
yes
no
yes
(a) Defined as the lethal dose sample having <50% inhibition or not exhibiting an inhibition significantly different
from the control.
(b) When compared with the negative control, the ricin was detectable at the lethal dose, however, when that result
was compared with the preservative blank, the results was falsely negative.
6.4 Other Performance Factors
6.4.1 Ease of Use

The ToxScreen-II contained instructions with clearly written information and illustrations.
Contents of ToxScreen-II were well identified with labels on the vials with the exception of the
bacteria. The bacteria vial was labeled on the outer box, but not on the vial itself. Storage
requirements were marked on all outer packages. Storage conditions were also marked on the
buffer solution vials, but were not on the bacteria or positive control stock vials. Overall, the
packaging was easy to open, with the exception of a wax seal on the bacteria vial. All procedures
could be carried out at room temperature and were not sensitive to light. ToxScreen-II requires
that all samples be analyzed twice, once with the Pro-Organic buffer and once with the Pro-
Metal buffer; however, these two tests could be run in parallel.

Prior to rehydration, the bacteria need to be stored at -14° C while all of the other reagents
required storage at 2 to 4°C. The procedure required a three-hour wait between bacteria
rehydration and testing; however, the vendor recommended that, for optimal performance, the
bacteria should be rehydrated the day before use. After preparation, the hydrated bacteria can be
used for up to seven days. The freeze-dried bacteria have a shelf-life of one year, while the shelf
life of the buffer reagents is eight months when refrigerated.
39
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All equipment was supplied with ToxScreen-II except for pipettes with tips and the ASTM Type
II water used to prepare reagents. The luminometer was easy to use and required no calibration
before use. The digital display was easy to read, and only one number needed to be recorded. On
occasion, consecutive readings of the same sample resulted in a wide range of relative light units.
The luminometer was easily wiped clean and did not require any routine maintenance.

No formal scientific education would be required for operation. However, good laboratory skills,
especially pipetting, would be beneficial. Verification testing staff were able to operate
ToxScreen-II after a brief training session. Approximately 2 mL of liquid waste were generated
per sample, along with leftover bacteria and positive control dilutions. In addition, two tubes per
sample, bacteria and positive control vials, and pipette tips were generated as solid waste.
Information on whether the bacteria, buffers, or positive controls should be considered hazardous
waste was available on material safety data sheets available from Checklight Ltd.

6.4.2 Field Portability

ToxScreen-II was transported from a laboratory to a storage room to simulate a situation in
which the ToxScreen-II would be operated in a non-laboratory location. The storage room
contained several tables and light and power sources, but no other laboratory facilities. During
this evaluation of field portability, ToxScreen-II was tested with cyanide at the lethal dose
concentration. The inhibition results from this portion of the test are given in Table 6-Id. Two
sets of the lethal dose of cyanide were analyzed. The first set was analyzed after the bacteria
were incubated for 3 hours, which is the minimum suggested by the vendor, and after a 24-hour
incubation, which is what was suggested by the vendor for this verification test. For both
incubation times and for both buffers, the inhibition was greater than 90%, nearly complete
inhibition. This is similar to the results obtained during the laboratory portion of the test.
ToxScreen-II produced an inhibition of 100% and 89% for the Pro-Organic and Pro-Metal buffer
respectively.

No carrying case was provided with ToxScreen-II (there is one available for purchase from
Checklight Ltd.); however, all materials except the luminometer were transported in a small
cardboard box. The box and luminometer were easily carried by one person. ToxScreen-II was
easily set up in less than 10 minutes. The luminometer operated on battery power. While the neat
bacteria must be kept in a freezer, if the bacteria were reconstituted prior to leaving for the field,
they could be stored at refrigerator temperatures until use. ToxScreen-II instructions indicate that
the reconstituted bacteria are good for seven days if refrigerated; therefore, they could be
reconstituted ahead of time for easier transport to the field. A refrigerator or cooler would be
needed to transport the reconstituted bacteria and could also be used to transport the assay
buffers; however, the buffers could be kept at ambient temperatures for short field tests (i.e., less
than 10 hours). The following items not provided in ToxScreen-II were needed for field use: a
cooler to transport and store reagents, high-purity water to prepare positive control dilutions, a
timer or watch, and a waste container. Unless reconstituted bacteria are constantly kept available
(in a cooler), ToxScreen II would require a minimum lead time of three hours because of the
time required for bacteria rehydration. Overall ToxScreen-II was easy to transport to the field
and, with the reagents prepared ahead of time, was deployed in a matter of minutes. Analysis of
samples was performed as in the laboratory and results were within 60 minutes.
40
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6.4.3 Throughput

Approximately 25 analyses were completed using both the Pro-Organic and Pro-Metal buffers in
one hour. The 25 analyses included method blanks and positive controls, as well as test samples.
Approximately 1,000 samples could be processed per kit.
41
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Chapter 7
Performance Summary
Pro-Organic Buffer Performance Verification Results
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
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
50
-87
75
100
70
83
68(a)
-6
66
-3
LD/10
-26
14
17
100
23
-10
9
-202
13
-5
LD/100
0
16
4
95
3
-32
1
4
9
2
LD/1,000
-50
-54
1
72
1
-20
10
15
-1
-6
Average Inhibition (%)
Initial
Analysis Reanalysis(b)
-4 -12
3 5
0 -6
0 3
-1 NR
Range of
Standard
Deviations
(%)
12-18
21-58
2-5
0-2
3-29
2-10
2-5
10-68
3-6
3-9
Toxicity
Thresh.
(mg/L)
ND
ND
24
0.25
140
1,400
ND
ND
28
ND
Standard Deviation (%)
Initial
Analysis
4
3
1
4
3
Reanalysis(b)
1
15
4
3
NR
None of the potential interferences or disinfection by-product samples produced an inhibition
significantly greater than 50%, the inhibition level suggested by CheckLight Ltd. to conclusively
determine toxicity.
False negative
response
Aldicarb, botulinum toxin complex B, soman, and VX produced an inhibition that either did not
exceed 50% or were not significantly different from the negative control at the lethal dose
concentrations. For ricin in the Pro-Organic buffer, the inhibition of the lethal dose was
significantly different from the negative control, but not significantly different from the inhibition
generated by the preservative blank.
Ease of use
ToxScreen-II included clearly written instructions with good illustrations. The contents of the
ToxScreen-II were well labeled, making it easy to follow the instructions. A minimum of three
hours was required to rehydrate the bacteria; however, for optimal performance the vendor
suggests preparing the bacteria the day before use. The bacteria must be stored at -14°C prior to
rehydration. After rehydration, the bacteria can be used for up to seven days. Overall, the
ToxScreen-II was easy to use, making it likely that a person with no formal scientific training
could conduct the tests.
42
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Field portability
Throughput
ToxScreen-II was transported from a laboratory to a storage room to simulate operation in a non-
laboratory location. It was tested with cyanide at the lethal dose concentration, and the results
generated (>90% inhibition) were very similar to those obtained in the laboratory. No carrying
case was provided with ToxScreen-II (one is available for purchase from Checkligiht Ltd.);
however, all materials except the luminometer were transported in a small cardboard box. The
box and luminometer were easily carried by one person, and setup for analysis took less than 10
minutes.
Approximately 25 analyses were completed each hour using both buffers, and approximately
1,000 samples could be processed per kit.
ND = Significant inhibition was not detected.
NR = Not reanalyzed.
(a) Inhibition was not significantly different from the preservative blank.
(b) Potential interferences were reanalyzed due to four suspect negative inhibitions during the initial analysis with
the Pro-Metal buffer.
43
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Pro-Metal Buffer Performance Verification Results
Parameter
Contaminants in
DDW
Potential
interferences 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
15.0
1.4
2,800
2.0
Cone.
(mg/L)
0.5
0.6
0.15
0.25
2.5
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
-19
-185
12
89
55
98
3
-55
79
5
LD/10
-7
-121
2
64
-1
2
-2
17
53
-11
LD/100
33
-91
8
44
-10
-10
2
-66
27
-11
LD/1,000
-31
-40
-9
19
-3
-7
2
-4
4
-3
Average Inhibition (%)
Initial
Analysis Reanalysis(a)
-395 -13
-299 30
-399 -8
-368 5
86 NR
Range of
Standard
Deviations
(%)
10-35
18-104
2-6
1-7
2-4
0-4
2-4
13-22
1-4
5-10
Toxicity
Thresh.
(mg/L)
ND
ND
ND
0.25
140
700
ND
ND
28
ND
Standard Deviation (%)
Initial
Analysis
29
26
18
15
0
Reanalysis(a)
3
4
3
4
NR
False positive
response
Neither the chlorination nor chloramination samples generated an inhibition greater than 50%.
However, the chloramination sample generated a result that indicated an enhancement in
luminescence (i.e., a negative inhibition), which, according to Checklight Ltd., can also indicate
toxicity.
False negative
response
The inhibition of the chloramination by-products was -75% ± 20% with DI water as the negative
control. If a contaminant causing a 75% inhibition had been present in this water and DI water was
used as the negative control, the inhibition would have been close to 0%—a false negative
response. This underscores the need to use negative control samples that are as similar as possible
to the samples being analyzed. A second type of false negative response occurred (for aldicarb,
colchicine, botulinum toxin complex B, ricin, soman, and VX) when the inhibition was not greater
than 50% in the presence of a lethal dose of contaminant.
ND = Significant inhibition was not detected.
NR = Not reanalyzed.
(a) Potential interferences were reanalyzed due to four suspect negative inhibitions during the initial analysis with
the Pro-Metal buffer.
See the Pro-Organic Buffer table for descriptions for ease of use, field portability, and throughput.
44
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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 EPAJ600/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.
45
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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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