June 2006
Environmental Technology
Verification Report
Lab_Bell Inc.
LuminoTox SAPS Test Kit
Prepared by
Battelle
The Ihimul':-.- *''' I HiKivation
Under a cooperative agreement with
B—glU
¦¦¦
UTT U.S. Environmental Protection Agency
ETV ETVET

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THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
ŁEPA
U.S. Environmental Protection Agency
Balteiie
The Business of Innovation
ETV Joint Verification Statement
TECHNOLOGY TYPE:	Rapid Toxicity Testing System
APPLICATION:	Detecting Toxicity in Drinking Water
TECHNOLOGY
NAME:	LuminoTox SAPS
COMPANY:
ADDRESS:
Lab_Bell Inc.
2263, avenue du College PHONE: (819) 539-8508, ext. 107
Shawinigan, Quebec FAX: (819) 539-8880
CANADA G9N 6V8
WEB SITE:
E-MAIL:
www.lab-bell.com
info@lab-bell.com
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 Lab_Bell Inc. LuminoTox stabilized aqueous photosynthetic systems (SAPS) 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, LuminoTox SAPS Test Kit was subjected to various
concentrations of contaminants such as industrial chemicals, pesticides, rodenticides, pharmaceuticals, nerve
agents, and biological toxins. Each contaminant was added to separate drinking water samples and analyzed.
In addition to determining whether LuminoTox SAPS Test Kit could detect the toxicity caused by each
contaminant, its response to interfering compounds, such as water treatment chemicals and by-products in
clean drinking water, was evaluated.
LuminoTox SAPS Test Kit was evaluated by
¦	Endpoints and precision—percent inhibition for all concentration levels of contaminants and potential
interfering compounds and precision of replicate analyses
¦	Toxicity threshold for each contaminant—contaminant level at which higher concentrations generate
inhibition significantly greater than the negative control and lower concentrations do not
¦	False positive responses—chlorination and chloramination by-product inhibition with respect to
unspiked American Society for Testing and Materials Type II deionized water samples
¦	False negative responses—contaminants that were reported as producing inhibition similar to 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).
The LuminoTox SAPS Test Kit was verified by analyzing a dechlorinated drinking water sample from
Columbus, Ohio (DDW), fortified with contaminants (at concentrations ranging from lethal levels to
concentrations up to 1,000 times less than the lethal dose) and interferences (metals possibly present as a
result of the water treatment processes). Dechlorinated water was used because free chlorine above lppm
inhibits the photosynthetic process that the LuminoTox SAPS Test Kit depends on to indicate toxicity and
can degrade the contaminants during storage. Inhibition (endpoints) from four replicates of each contaminant
at each concentration level were evaluated to assess the ability of the LuminoTox SAPS Test Kit to detect
toxicity, as well as to measure the precision of the LuminoTox SAPS Test Kit results. The response of the
LuminoTox SAPS Test Kit to possible interferents was evaluated by analyzing them at one-half of the
concentration limit recommended by the EPA's National Secondary Drinking Water Regulations guidance.
For analysis of by-products of the chlorination process, the unspiked DDW was analyzed because Columbus,
Ohio, uses chlorination as its disinfectant procedure. For the analysis of by-products of the chloramination
process, a separate drinking water sample was obtained from the Metropolitan Water District of Southern
California (LaVerne, California), which uses chloramination as its disinfection process. The samples were
analyzed after residual chlorine was removed using sodium thiosulfate. Sample throughput was measured
based on the number of samples analyzed per hour. Ease of use and reliability were determined based on
documented observations of the operators.
Quality control samples included method blank samples, which consisted of American Society for Testing
and Materials Type II deionized water; positive control samples (fortified with atrazine); and negative
control samples, which consisted of the unspiked DDW.
QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a
technical systems audit, a performance evaluation audit, and a data quality audit of 10% of the test data.

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This verification statement, the full report on which it is based, and the test/QA plan for this verification test
are all available at www.epa.gov/etv/centers/centerl.html.
TECHNOLOGY DESCRIPTION
The following description of the LuminoTox SAPS Test Kit Test Kit is based on information provided by the
vendor. This technology description was not verified in this test.
The LuminoTox SAPS Test Kit is a portable biosensor that uses SAPS activated by light absorption to
recognize toxic chemicals in water. SAPS are activated at a wavelength of 470 nanometers, and fluorescence
emission is read at wavelengths longer than 700 nanometers. SAPS are whole algae (Chlorella vulgaris) that
fluoresce when photosynthesis (the conversion of electromagnetic energy into stored chemical energy) is
activated by light absorption. Some of the absorbed energy is emitted as fluorescence, which is the signal
measured by the LuminoTox SAPS Test Kit. The photosynthetic electron chain is inhibited by a broad
spectrum of organic molecules (ureas, azides, phenols, quinones or amide derivatives, polyaromatic
hydrocarbons, polychlorinated biphenyls), redox species, cyanides, and metallic cations. The LuminoTox
SAPS Test Kit measures the fluorescence produced both in background water and samples containing
contaminants. Decreases in fluorescence as a result of adding toxic contamination are expressed as percent
inhibition.
Although other SAPS could be used in the LuminoTox analyzer, Lab_Bell uses Chlorella vulgaris, which is
concentrated by centrifugation in the middle of its exponential growth curve and stored at 4°C for a few
weeks. Prior to analysis, SAPS must be activated in room light for 90 minutes at ambient temperature. The
LuminoTox test is performed in the dark (in a covered syringe) by exposing 100 microliters of SAPS solution
to 2 milliliters of test sample for 10 minutes. In this short period of time, permeable molecules acting directly
on the photosynthetic electron chain are detected at low concentrations. Prolonged incubation allows the
detection of less permeable molecules.
The LuminoTox SAPS Test Kit consists of the LuminoTox analyzer, a bottle of SAPS for 50 tests, two vials
of organic standards (positive controls to ensure that the SAPs are fully functional), and one vial of distilled
water (for blank samples). Also provided are disposable syringes in which the test is performed and fabric
syringe covers to protect the reaction from light. The analyzer is 21.6 by 12.7 by 7.6 centimeters and weighs
1 kilogram. The analyzer is battery-operated, is equipped with a RS-232 serial port for transferring data, and
can be connected to a printer (not done during this test). A total of 100 measurements can be stored in the
internal memory. The rechargeable battery operates for eight hours. Reagents (including buffers and positive
and negative controls) for approximated 50 analyses cost $106, while the LuminoTox analyzer costs
approximately $7,500.

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VERIFICATION RESULTS
Parameter
Compound
Lethal
Dose (LD)
Cone.
(mg/L)
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)
LD
LD/10
LD/100
LD/1,000
Contaminants in
DDW
Aldicarb
260
50
14
5
0
1-3
26
Botulinum
toxin
Complex B
0.3
-10
-6
-5
1
1-8
ND
Colchicine
240
0
4
0
3
1-5
ND
Cyanide
250
17
10
7
1
2-3
250
Dicrotophos
1,400
4
-11
-12
-10
1-2
ND
Nicotine
2,800
34
10
1
3
1-4
280
Ricin
15
0
1
-4
3
2-6
ND
Soman
1.4
-2
1
2
0
2-3
ND
Thallium
sulfate
2,800
0
1
-3
-4
2-3
ND
VX
2
5
3
-1
2
2-5
ND
Potential
interferences in
DDW
Interference
Cone.
(mg/L)
Average Inhibition
(%)
Standard
Deviation (%)
se positive. All disinfection
contamination.
Aluminum
0.5
1
4
Copper
0.6
3
1
Iron
0.15
1
2
Manganese
0.25
1
3
Zinc
2.5
-1
4
False positive
response
None of the LuminoTox SAPS Test Kit responses were considered fal
by-product test samples left enough fluorescence for inhibition due to
False negative
response
Botulinum toxin, colchicine, dicrotophos, ricin, soman, thallium sulfate, and VX exhibited non-
detectable responses at the lethal dose concentration.
Ease of use
The LuminoTox SAPS Test Kit contained detailed instructions and clear illustrations. The
contents were well identified with labels on the vials. Storage requirements were stated in the
instructions and on the reagent vials. Preparation of the test samples for analysis was
straightforward. The necessity to record four numbers as raw data was somewhat burdensome;
however, Lab_Bell has indicated this procedure is being modified. No formal scientific education
would be required to use the LuminoTox SAPS Test Kit.
Field portability
The LuminoTox SAPS Test Kit was transported from a laboratory setting to a storage room for
the field portability evaluation. The limiting factor for testing in the field would be the
approximately 90 minutes required to allow the SAPS to be exposed to light prior to testing. The
LuminoTox SAPS Test Kit was tested with one contaminant, cyanide, at the lethal dose
concentration. The results of the test were very similar to the laboratory results. Inhibition in the
laboratory was 17% ± 2%, and in the non-laboratory location, 16% ± 4%.
Throughput
Approximately 20 analyses were completed per hour, and 50 samples could be analyzed with the
supplies contained in one LuminoTox SAPS Test Kit.
ND = Significant inhibition was not detected.

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Original signed by Gregory A. Mack	6/22/06
Gregory A. Mack	Date
Vice President
Energy, Transportation, and Environment Division
Battelle
Original signed by Andrew P. Avel	8/7/06
Andrew P. Avel	Date
Acting Director
National Homeland Security Research Center
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
Lab Bell Inc.
LuminoTox SAPS 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.
ii

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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.
iii

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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. We would also like to thank
Karen Bradham, U.S. EPA National Exposure Research Laboratory; Steve Allgeier, U.S. EPA
Office of Water; Ricardo DeLeon, Metropolitan Water District of Southern California; Yves
Mikol, New York City Department of Environmental Protection; and Stanley States, Pittsburgh
Water and Sewer Authority, for their careful review of the test/quality assurance plan and/or this
verification report.
iv

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Contents
Page
Notice	ii
Foreword	iii
Acknowledgments	iv
List of Abbreviations	vii
Chapter 1 Background	1
Chapter 2 Technology Description	2
Chapter 3 Test Design	4
3.1	Test Samples	6
3.1.1	Quality Control Samples	6
3.1.2	Drinking Water Fortified with Contaminants	6
3.1.3	Drinking Water Fortified with Potential Interferences	8
3.2	Test Procedure	8
3.2.1	Test Sample Preparation and Storage	8
3.2.2	Test Sample Analysis Procedure	8
3.2.3	Stock Solution Confirmation Analysis	9
Chapter 4 Quality Assurance/Quality Control	12
4.1	Quality Control of Stock Solution Confirmation Methods	12
4.2	Quality Control of Drinking Water Samples	12
4.3	Audits	13
4.3.1	Performance Evaluation Audit	13
4.3.2	Technical Systems Audit	13
4.3.3	Audit of Data Quality	14
4.4	QA/QC Reporting	14
4.5	Data Review	14
Chapter 5 Statistical Methods and Reported Parameters	16
5.1	Endpoints and Precision	16
5.2	Toxicity Threshold	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	29
6.1.3	Precision	29
6.2	Toxicity Threshold	29
6.3	False Positive/Negative Responses	31
6.4	Other Performance Factors	32
6.4.1	Ease of Use	32
6.4.2	Field Portability	32
6.4.3	Throughput	33
v

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Chapter 7 Performance Summary	34
Chapter 8 References	35
Figures
Figure 2-1. Lab_Bell Inc. LuminoTox SAPS Test Kit	2
Tables
Table 3-1. Contaminants and Potential Interferences	5
Table 3-2. Summary of Quality Control and Contaminant Test Samples	7
Table 3-3. Stock Solution Confirmation Results	10
Table 3-4. Water Quality Parameters	11
Table 4-1. Summary of Performance Evaluation Audit	14
Table 4-2. Summary of Data Recording Process	15
Table 6-la. Aldicarb Percent Inhibition Results	20
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results	20
Table 6-lc. Colchicine Percent Inhibition Results	21
Table 6-ld. Cyanide Percent Inhibition Results	22
Table 6-le. Dicrotophos Percent Inhibition Results	23
Table 6-lf. Nicotine Percent Inhibition Results	23
Table 6-lg. Ricin Percent Inhibition Results	24
Table 6-lh. Soman Percent Inhibition Results	25
Table 6-li. Thallium Sulfate Percent Inhibition Results	26
Table 6-lj. VX Percent Inhibition Results	26
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results	27
Table 6-3. Potential Interferences Results	32
Table 6-4. Toxicity Thresholds	31
Table 6-5. False Negative Responses	32
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 -phenylenedi amine
EPA
U.S. Environmental Protection Agency
ETV
Environmental Technology Verification
HDPE
high-density polyethylene
LD
lethal dose
mM
millimolar
[jL
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
SAPS
stabilized aqueous photosynthetic systems
SOP
standard operating procedure
TSA
technical systems audit
vii

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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental
technologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative
technologies by developing test plans that are responsive to the needs of stakeholders,
conducting field or laboratory tests (as appropriate), collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality
assurance (QA) protocols to ensure that data of known and adequate quality are generated and
that the results are defensible.
The EPA's National Exposure Research Laboratory and its verification organization partner,
Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center
recently evaluated the performance of the Lab_Bell Inc. LuminoTox stabilized aqueous
photosynthetic systems (SAPS), hereafter referred to as the LuminoTox SAPS Test Kit. Rapid
toxicity technologies were identified as a priority verification category through the AMS Center
stakeholder process.
1

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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This verification report provides
results for the verification testing of the LuminoTox SAPS. Following is a description of the
LuminoTox SAPS Test Kit, based on information provided by the vendor. The information
provided below was not verified in this test.
The LuminoTox SAPS Test Kit (Figure 2-1) is a portable biosensor that uses SAPS activated by
light absorption to recognize toxic chemicals in water. SAPS are activated at a wavelength of
470 nanometers, and fluorescence emission is read at wavelengths longer than 700 nanometers.
SAPS are whole algae {Ch lore I la vulgaris) that fluoresce when photosynthesis (the conversion of
electromagnetic energy into stored chemical energy) is activated by light absorption. Some of the
absorbed energy is emitted as fluorescence, which is the signal measured by the LuminoTox
SAPS Test Kit. The photosynthetic electron chain is inhibited by a broad spectrum of organic
molecules (ureas, azides, phenols, quinones or amide derivatives, polyaromatic hydrocarbons,
polychlorinated biphenyls), redox species, cyanides, and metallic cations. The LuminoTox SAPS
Test Kit measures the fluorescence produced both in background water and samples containing
contaminants. Decreases in fluorescence parameters as a result of adding toxic contamination are
expressed as percent inhibition.
Although other SAPS could be
used in the LuminoTox analyzer,
Lab_Bell uses Chi or ell a vulgaris,
which is concentrated by
centrifugation in the middle of its
exponential growth curve and
stored at 4°C for a few weeks.
Prior to analysis, SAPS must be
activated in room light for
90 minutes at ambient
temperature. The LuminoTox test
is performed in the dark (in a
covered syringe) by exposing
100 microliters of SAPS solution
to 2 milliliters of test sample for


Figure 2-1. LabJRell Inc. LuminoTox SAPS Test Kit
2

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10 minutes. In this short period of time, permeable molecules acting directly on the
photosynthetic electron chain are detected at low concentrations. Prolonged incubation allows
the detection of less permeable molecules.
The LuminoTox SAPS Test Kit consists of the LuminoTox analyzer, a bottle of SAPS for
50 tests, two vials of organic standards (positive controls to ensure that the SAPS are fully
functional), and one vial of distilled water (for blank samples). Also provided are disposable
syringes in which the test is performed and fabric syringe covers to protect the reaction from
light. The analyzer is 21.6 by 12.7 by 7.6 centimeters and weighs 1 kilogram. The analyzer is
battery-operated, is equipped with a RS-232 serial port for transferring data, and can be
connected to a printer (not done during this test). A total of 100 measurements can be stored in
the internal memory. The rechargeable battery operates for eight hours. Reagents (including
buffers and positive and negative controls) for 50 analyses cost $106, while the LuminoTox
analyzer costs approximately $7,500.
3

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Chapter 3
Test Design
The objective of this verification test of rapid toxicity technologies was to evaluate their ability
to detect certain toxins and to determine their susceptibility to interfering chemicals in a
controlled experimental matrix. Rapid toxicity technologies do not identify or determine the
concentration of specific contaminants, but serve as a screening tool to quickly determine
whether water is potentially toxic.
As part of this verification test, the LuminoTox SAPS Test Kit was subjected to various
concentrations of contaminants such as industrial chemicals, pesticides, rodenticides,
pharmaceuticals, nerve agents, and biological toxins. Each contaminant was added to separate
drinking water samples and analyzed. In addition to determining whether the LuminoTox SAPS
Test Kit could detect the toxicity caused by each contaminant, its response to interfering
compounds such as water treatment chemicals and by-products in clean drinking water was
evaluated. Table 3-1 shows the contaminants and potential interferences that were evaluated
during this verification test.
This verification test was conducted from August to December 2005 according to procedures
specified in the Test/QA Plan for Verification of Rapid Toxicity Technologies including
Amendments 1 and 2.(1) The LuminoTox SAPS Test Kit was verified by analyzing a
dechlorinated drinking water sample from Columbus, Ohio (hereafter in this report referred to as
DDW), fortified with various concentrations of the contaminants and interferences shown in
Table 3-1. Where possible, the concentration of each contaminant or potential interference was
confirmed independently by Aqua Tech Environmental Laboratories (ATEL), Marion, Ohio, or
by Battelle, depending on the analyte.
The LuminoTox SAPS Test Kit was evaluated by
¦	Endpoints and precision—percent inhibition for all concentration levels of contaminants and
potential interfering compounds and precision of replicate analyses
¦	Toxicity threshold for each contaminant— contaminant level at which higher concentrations
generate inhibition significantly greater than the negative control and lower concentrations
do not
4

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Table 3-1. Contaminants and Potential Interferences
Category
Contaminant
Biological toxins
Botulinum toxin complex B, ricin
Botanical pesticide
Nicotine
Carbamate pesticide
Aldicarb
Industrial chemical
Cyanide
Nerve agents
Soman, VX
Organophosphate pesticide
Dicrotophos
Pharmaceutical
Colchicine
Potential interferences
Aluminum, copper, iron, manganese, zinc, chloramination
by-products, and chlorination by-products
Rodenticide
Thallium sulfate
¦	False positive responses—chlorination and chloramination by-product inhibition with respect
to unspiked American Society for Testing and Materials (ASTM) Type II deionized (DI)
water samples
¦	False negative responses—contaminants that were reported as producing inhibition similar to
the negative control when present at lethal concentrations or negative inhibition that could
cause falsely low inhibition
¦	Other performance factors (sample throughput, ease of use, reliability).
The LuminoTox SAPS Test Kit was used to analyze the DDW samples fortified with
contaminants at concentrations ranging from lethal levels to concentrations up to 1,000 times less
than the lethal dose. The lethal dose of each contaminant was determined by calculating the
concentration at which 250 milliliters (mL) of water would probably cause the death of a
154-pound person. These calculations were based on toxicological data available for each
contaminant that are presented in Amendment 2 of the test/QA plan.(1) Inhibition results
(endpoints) from four replicates of each contaminant at each concentration level were evaluated
to assess the ability of the LuminoTox SAPS Test Kit to detect toxicity at various concentrations
of contaminants, as well as to measure the precision of the LuminoTox SAPS Test Kit results.
The response of the LuminoTox SAPS Test Kit to compounds used during the water treatment
process (identified as potential interferences in Table 3-1) was evaluated by analyzing separate
aliquots of DDW fortified with each potential interference at one-half of the concentration limit
recommended by the EPA's National Secondary Drinking Water Regulations (NSDWR)(2)
guidance. For analysis of by-products of the chlorination process, the unspiked DDW was
analyzed because Columbus, Ohio, uses chlorination as its disinfectant procedure. For the
analysis of by-products of the chloramination process, a separate drinking water sample was
obtained from the Metropolitan Water District of Southern California (LaVerne, California),
which uses chloramination as its disinfection process. The samples were analyzed after residual
chlorine was removed using sodium thiosulfate. Sample throughput was measured based on the
5

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number of samples analyzed per hour. Ease of use and reliability were determined based on
documented observations of the operators.
3.1 Test Samples
Test samples used in the verification test included drinking water and quality control (QC)
samples. Table 3-2 shows the number and type of samples analyzed. QC samples included
method blanks and positive and negative control samples. The fortified drinking water samples
were prepared from a single drinking water sample collected from the Columbus, Ohio, system.
The water was dechlorinated using sodium thiosulfate and then fortified with various concen-
trations of contaminants and interferences. The DDW containing the potential interferences was
analyzed at a single concentration level, while at least four dilutions were analyzed for each
contaminant using the LuminoTox SAPS Test Kit. Mixtures of contaminants and possible
interfering compounds were not analyzed.
3.1.1	Quality Control Samples
QC samples included method blanks, positive controls, negative controls, and preservative
blanks. The method blank samples consisted of ASTM Type IIDI water and were used to ensure
that no sources of contamination were introduced in the sample handling and analysis
procedures. A positive control sample was included in the LuminoTox SAPS Test Kit and was
used as provided from the vendor. While performance limits were not placed on the results,
inhibition significantly greater than the negative control for the positive control sample indicated
to the operator that the LuminoTox SAPS Test Kit was functioning properly. The negative
control consisted of unspiked DDW and was used to set a background inhibition of the DDW,
the matrix in which each test sample was prepared. To ensure that the preservatives in the
contaminant solutions did not have an inhibitory effect, preservative blank samples were
prepared. These preservative blanks consisted of DDW fortified with a concentration of
preservative equivalent to that in the test solutions of botulinum toxin complex B, ricin, soman,
and VX.
3.1.2	Drinking Water Fortified with Contaminants
Approximately 50 liters of Columbus, Ohio, tap water were collected in a low-density
polyethylene container. The water was dechlorinated with sodium thiosulfate. Dechlorination
was confirmed by adding an n,n-diethyl-p-phenylenediamine (DPD) tablet to a 10-mL aliquot of
the water. Lack of color development in the presence of DPD indicated that the water was
dechlorinated. All subsequent test samples were prepared from this DDW.
A stock solution of each contaminant was prepared in DDW at concentrations at or above the
lethal dose level. The stock solution was further diluted to obtain one sample containing the
lethal dose concentration for each contaminant and three additional samples with concentrations
10, 100, and 1,000 times less than the lethal dose. Table 3-2 lists each concentration level and the
number of samples analyzed at each level.
6

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Table 3-2. Summary of Quality Control and Contaminant Test Samples
Type of Sample
Sample
Characteristics
Concentration Levels
No. of Sample Analyses
Quality control
Method blank
(ASTM Type II water)
NA
14
Positive control
Used as provided in kit, 0.01 mg/L
atrazine
14
Negative control
(unspiked DDW)
NA
60
Preservative blank:
botulinum toxin
complex B
0.015 millimolar (mM) sodium citrate
4
Preservative blank:
VX and soman
0.21% isopropyl alcohol
4 with VX, 4 with soman
Preservative blank:
ricin
0.00024% NaN3, 0.45 mM NaCl,
0.03 mM phosphate
4

Thallium sulfate
2,800; 280; 28; 2.8 mg/L
4 per concentration level
DDW fortified with
contaminants
Aldicarb
260; 26; 2.6; 0.26 milligrams/liter
(mg/L)
4 per concentration level
Botulinum toxin
complex B
0.3; 0.03; 0.003; 0.0003 mg/L
4 per concentration level
Colchicine
240; 24; 2.4; 0.24 mg/L
4 per concentration level
Cyanide
250; 25; 2.5; 0.25 mg/L
4 per concentration level
Dicrotophos
1,400; 140; 14; 1.4; mg/L
4 per concentration level
Nicotine
2,800; 280; 28; 2.8 mg/L
4 per concentration level
Ricin
15; 1.5; 0.15; 0.015 mg/L
4 per concentration level
Soman
1.4; 0.14; 0.014; 0.0014 mg/L
4 per concentration level
Thallium sulfate
2,800; 280; 28; 2.8 mg/L
4 per concentration level
VX
2.0; 0.2; 0.02; 0.002 mg/L
4 per concentration level
DDW fortified with
potential interferences
Aluminum
0.5 mg/L
4
Copper
0.6 mg/L
4
Iron
0.15 mg/L
4
Manganese
0.25 mg/L
4
Zinc
2.5 mg/L
4
Disinfectant
by-products
Chloramination by-
products
NA
4
Chlorination by-
products
NA
56
NA = not applicable, samples not fortified with any preservative, contaminant, or potential interference.
7

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3.1.3 Drinking Water Fortified with Potential Interferences
Individual aliquots of the DDW were fortified with one-half the concentration specified by the
EPA's NSDWR for each potential interference. Table 3-2 lists the interferences, along with the
concentrations at which they were tested. Four replicates of each of these samples were analyzed.
To test the sensitivity of the LuminoTox SAPS Test Kit to by-products of the chlorination
process as potential interferences, the unspiked DDW (same as the negative control) was used
since the water sample originated from a utility that uses chlorination as its disinfectant
procedure. In a similar manner, 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
over 1 ppm inhibits the photosynthetic process that the LuminoTox SAPS Test Kit depends on to
indicate toxicity 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 sample, 0.01 mg/L atrazine, was provided by the vendor. All QC
samples were prepared prior to the start of the testing and stored at room temperature. The
stability of each contaminant for which analytical methods are available was confirmed by
analyzing it three times over a two-week period. Throughout this time, each contaminant
maintained its original concentration to within approximately 25%. Therefore, the aliquots of
DDW containing the contaminants were prepared within two weeks of testing and were stored at
room temperature without chemical preservation. The contaminants without analytical methods
were analyzed within 48 hours of their preparation. To maintain the integrity of the test, test
samples provided to the operators were labeled only with sample identification numbers so that
the operators did not know their content.
3.2.2	Test Sample Analysis Procedure
The first step of sample analysis was to allow the LuminoTox SAPS to sit in the light at room
temperature for 90 minutes. Next, 2 mL of each control and water sample (both at room
temperature) were taken up into individual 3-mL syringes that were then covered with an opaque
cloth provided by the vendor or with aluminum foil. A sample set typically included one method
blank, one positive control sample, four replicates of the negative control, and four replicates
each of four or five concentrations of contaminant. After adding 100 |iL of the SAPS solution to
each control and water sample, each syringe was mixed by inverting five times. The solutions
8

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were allowed to react for 10 minutes, and the content of the syringe was added to a cuvette
residing within the LuminoTox SAPS Test Kit analyzer. The cover was closed for one minute,
and the sample reading (reported in light units that were converted to percent inhibition) was
taken from the LuminoTox SAPS Test Kit analyzer. The analyzer generated four readings, two
absolute fluorescence readings and an efficiency and an inhibition reading. If the proper control
sample (one very similar to the test sample) was entered into the analyzer, the percent inhibition
could be obtained directly. Two operators performed all the analyses using the LuminoTox
SAPS Test Kit. One operator performed testing with contaminants that did not require special
chemical and biological agent training and one performed testing with those that did. Both held
bachelor's degrees in the sciences and were trained by the vendor to operate the LuminoTox
SAPS Test Kit.
3.2.3 Stock Solution Confirmation Analysis
The concentrations of the contaminant and interfering compound stock solutions were verified
with standard analytical methods, with the exception of colchicine, ricin, and botulinum toxin
complex B—contaminants without standard analytical methods. Aliquots to be analyzed by
standard methods were preserved as prescribed by the method. In addition, the same standard
methods were used to measure the concentration of each contaminant/potential interference in
the unspiked DDW so that background concentrations of contaminants or potential interferences
were accounted for within the displayed concentration of each contaminant/potential interference
sample. Table 3-3 lists the standard methods used to measure each analyte; the results from the
stock solution confirmation analyses (obtained by analyzing the lethal dose concentration for the
contaminants and the single concentration that was analyzed for the potential interferences); and
the background levels of the contaminants and potential interferences measured in the DDW
sample, which were all non-detect or negligible.
Standard methods were also used to characterize several water quality parameters such as
alkalinity; dissolved organic carbon content; specific conductivity; hardness; pH; concentration
of haloacetic acids, total organic carbon, total organic halides, and trihalomethanes; and
turbidity. Table 3-4 lists these measured water quality parameters for both the water sample
collected in Columbus, Ohio, representing a water system using chlorination as the disinfecting
process, and the water sample collected at the Metropolitan Water District of Southern
California, representing a water system using chloramination for disinfection.
9

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Table 3-3. Stock Solution Confirmation Results

Method
Average Concentration ± Standard
Deviation N = 4 (mg/L)(b)
Background in
DDW (mg/L)
Contaminant



Aldicarb
Battelle method
260 ±7
<0.005
Botulinum toxin
complex B
(a)
NA
NA
Colchicine
(a)
NA
NA
Cyanide
EPA 335.3(3)
249 ±4
296 ± 26 (field portability)
0.006
Dicrotophos
Battelle method
1,168 ± 18
<3.0
Nicotine
Battelle method
2,837 ± 27
<0.01
Ricin
(a)
NA
NA
Soman
Battelle method
1.3 ±0.1 (10/18/05)
1.16 ±0.06 (10/21/05)
<0.025
Thallium sulfate
EPA 200.8(4)
2,469 ±31
<0.001
VX
Battelle method
1.89 ±0.08 (10/17/05)
1.77 ±0.03 (10/20/05)
<0.0005
Potential
Interference



Aluminum
EPA 200.7(5)
0.50 ± 0.02
<0.2
Copper
EPA 200.7(5)
0.60 ± 0.03
<0.02
Iron
EPA 200.7(5)
0.155 ±0.006
<0.04
Manganese
EPA 200.7(5)
0.281 ±0.008
<0.01
Zinc
EPA 200.7(S)
2.63 ± 0.05
0.27
NA = Not applicable.
(a)	No standard method available. QA audits and balance calibration assured accurately prepared solutions.
(b)	Target concentration was highest concentration for each contaminant or interference on Table 3-2.
10

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Table 3-4. Water Quality Parameters
Parameter
Method
Dechlorinated Columbus,
Ohio, Tap Water
(disinfected by
chlorination)
Dechlorinated Southern
California Tap Water
(disinfected by
chloramination)
Alkalinity (mg/L)
SM 2320 B(6)
40
71
Specific conductivity
((j,mho)
SM 2510 B(6)
572
807
Hardness (mg/L)
EPA 130.2(7)
118
192
pH
EPA 150.1(7)
7.6
8.0
Total haloacetic acids
((xg/L)
EPA 552.2(8)
32.8
17.4
Dissolved organic
carbon (mg/L)
SM 5310 B(6)
2.1
2.9
Total organic carbon
(mg/L)
SM 5310 B(6)
2.1
2.5
Total organic halides
(Mg/L)
SM 5320B(6)
220
170
Total trihalomethanes
(Mg/L)
EPA 524.2(9)
74.9
39.2
Turbidity (NTU)
SM 2130(10)
0.1
0.1
NTU = nephelometric turbidity unit.
11

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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center*11} and the test/QA plan for this verification test.(1)
4.1	Quality Control of Stock Solution Confirmation Methods
The stock solutions for the contaminants cyanide and thallium sulfate and for the potential
interferences aluminum, magnesium, zinc, iron, and copper were analyzed at ATEL using
standard reference methods. As part of ATEL's standard operating procedures (SOPs), various
QC samples were analyzed with each sample set. These included matrix spike, laboratory control
spike, and method blank samples. According to the standard methods used for the analyses,
recoveries of the QC spike samples analyzed with samples from this verification test were within
acceptable limits of 75% to 125%, and the method blank samples were below the detectable
levels for each analyte. For VX, soman, aldicarb, nicotine, and dicrotophos, the confirmation
analyses were performed at Battelle using a Battelle SOP or method. Calibration standard
recoveries of VX and soman were always between 62% and 141%, and most of the time were
between 90% and 120%. Dicrotophos standard recoveries ranged from 89% to 122%. Aldicarb
standard recoveries ranged from 95% to 120%. Nicotine standard recoveries ranged from 96% to
99%. Standard analytical methods for colchicine, ricin, and botulinum toxin complex B were not
available and, therefore, not performed. QA audits and balance calibrations assured that solutions
for these compounds were accurately prepared.
4.2	Quality Control of Drinking Water Samples
A method blank sample consisting of ASTM Type IIDI water was analyzed once by the
LuminoTox SAPS Test Kit for approximately every 20 drinking water samples that were
analyzed. Because inhibition has to be calculated with respect to a control sample, none were
calculated for the method blank samples. The method blanks were used as the control for
calculating the background inhibition of the DDW for the disinfection by-product evaluation. A
positive control sample of 0.01 mg/L atrazine 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, an inhibition significantly greater than zero indicated to the operator that
the LuminoTox SAPS Test Kit was functioning properly. For 14 positive control samples, an
inhibition of 28% ±12% was measured. This is further discussed in Section 6.1.1. A negative
12

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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=—x 100%	(!)
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 inter-
ference was commercially available and when methods were available to perform the confirma-
tion; 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

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Table 4-1. Summary of Performance Evaluation Audit


Measured
Concentration
(mg/L)
Nominal
Concentration
(mg/L)
%D
Contaminant
Aldicarb
0.057
0.050
14
Cyanide
1,025
1,000
3
Dicrotophos
1.10
1.00
10
Nicotine
0.120
0.100
20
Thallium
1,010
1,000
1
Potential
interference
Aluminum
960
1,000
4
Copper
1,000
1,000
0
Iron
960
1,000
4
Manganese
922
1,000
8
Zinc
1,100
1,000
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." " 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

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Table 4-2. Summary of Data Recording Process
Data to be
Recorded
Responsible
Party
Where
Recorded
How Often
Recorded
Disposition of Data '1
Dates, times of test
events
Battelle
Laboratory
record books
Start/end of test,
and at each change
of a test parameter
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
Sample
preparation (dates,
procedures,
concentrations)
Battelle
Laboratory
record books
When each sample
was prepared
Used to confirm the
concentration and
integrity of the samples
analyzed; procedures
entered into laboratory
record books
Test parameters
(contaminant
concentrations,
location, etc.)
Battelle
Laboratory
record books
When set or
changed
Used to organize/check
test results, manually
incorporated in data
spreadsheets as
necessary
Stock solution
confirmation
analysis, sample
analysis, chain of
custody, and
results
Battelle or
contracted
laboratory
Laboratory
record books,
data sheets, or
data acquisition
system, as
appropriate
Throughout sample
handling and
analysis process
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 LuminoTox SAPS Test Kit reported two values of absolute
fluorescence units for each sample analyzed. One of the values (Fi) represented a lower intensity
fluorescence measurement and the other value (F2) represented a higher intensity fluorescence
measurement. These two measurements were used to calculate a percent inhibition with respect
to the negative control. This was done using the following equations, which were provided by
the vendor:
.	F 2 sample — Fi sample	(0\
efficiency = —=		^
F 2 negative control
% inhibition ¦
f 77	^	(3)
x 100%
^	'sample
E negative control J
where efficiency (E) is a measure of the fluorescence produced by the SAPS with respect to the
average high-intensity fluorescence measurement values produced by the replicate negative
control samples f2 negative control ) an^ the percent inhibition is the relative decrease in
fluorescence production with respect to the average efficiency for four negative control samples
(E negative control)- As shown in the above equations, efficiency is calculated directly from the raw
data, while the percent inhibition is calculated from the efficiencies. The response of the negative
control samples is accounted for in the calculation of percent inhibition of each sample.
Therefore, the percent inhibition of the four negative control samples within each sample set
always averaged zero percent. The negative control sample was always DDW, except when the
inhibition of the disinfection 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 the LuminoTox SAPS Test Kit's precision at each concentration. The
standard deviation around the average negative control results represented the variability of the
16

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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
1
n ~ 1 *=i
{>* - '):
11/2
(4)
where n is the number of replicate samples, h is the percent inhibition measured for the kth
sample, and I is the average percent inhibition of the replicate samples. Because the average
inhibition was frequently near zero for this data set, relative standard deviations often would
have greatly exceeded 100%, making the results difficult to interpret. Therefore, the precision
results were left in the form of standard deviations of the percent inhibition so the reader could
easily view the uncertainty around the average percent inhibition for results that were both near
zero and significantly larger than zero.
5.2 Toxicity Threshold
The toxicity threshold was defined as the lowest concentration of contaminant to exhibit a
percent inhibition significantly greater than the negative control. Also, each concentration level
higher than the toxicity threshold had to be significantly greater than the negative control, and
the inhibition produced by each lower concentration analyzed had to be significantly less than
that produced by the toxicity threshold concentration. Since the inhibition of the test samples was
calculated with respect to the inhibition of each negative control sample, the percent inhibition of
the negative control was always zero. A significant difference in the inhibition at two
concentration levels required that the average inhibition at each concentration level, plus or
minus its respective standard deviation, did not overlap.
5.3 False Positive/Negative Responses
A response was considered false positive if an unspiked drinking water sample produced an
inhibition significantly greater than zero when determined with respect to DI water. Depending
on the degree of inhibition in the sample, toxicity from subsequent contamination of that sample
may not be detectable or could be exaggerated as a result of the baseline inhibition. Drinking
water samples collected from water systems using chlorination and chloramination as the
disinfecting process were analyzed in this manner. An inhibition was considered significantly
different from zero if the average inhibition, plus or minus the standard deviation, did not
overlap with the zero inhibition plus or minus the standard deviation.
A response was considered false negative when the LuminoTox SAPS Test Kit, subjected to a
lethal concentration of some contaminant in the DDW, did not indicate inhibition significantly
greater than the negative control (zero inhibition) and the other concentration levels analyzed
(for lethal dose inhibition less than 100%). The inhibition of the lethal dose sample was required
to be significantly greater than other concentration levels because it more thoroughly
incorporated the uncertainty of all the measurements made by the LuminoTox SAPs Test Kit in
determining a false negative result. A difference was considered significant if the average
inhibition plus or minus the standard deviation did not encompass the value or range of values
17

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that were being compared. In addition, background water samples that increased the light
production of the LuminoTox SAPS Test Kit 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.
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-j 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 SAPS and were considered non-toxic.
6.1.1 Contaminants
The contaminants that generated an inhibition significantly greater than the negative control
included aldicarb, cyanide, and nicotine. Aldicarb and cyanide generated an inhibition
significantly greater than the negative control at the highest three concentrations. In both, some
of the three concentrations exhibited an inhibition that, while significantly different from the
negative control, was not significantly different from the concentration level(s) below it.
Consequently, the toxicity threshold, which must be significantly different from the
concentration levels above and below it, was 26 mg/L for aldicarb and 250 mg/L for cyanide.
Nicotine generated a detectable inhibition at the two highest concentrations analyzed
(2,800 mg/L and 280 mg/L). Colchicine, dicrotophos, and thallium sulfate had no detectable
inhibition.
It is important to note that the botulinum toxin complex B, ricin, soman, and VX stock solutions
used to prepare the test samples were stored in various preservatives that included sodium azide,
sodium chloride, and sodium phosphate for ricin; sodium citrate only for botulinum toxin
complex B; and isopropyl alcohol for soman and VX. During the previous ETV test of this
technology category, the preservatives were not accounted for in the negative control; therefore,
the results from each test should be interpreted accordingly. The results for this test are more
thorough because they show the sensitivity (or lack thereof) to both the preservative and the
contaminant. In the in the earlier verification test, toxicity could have been the result of either.
19

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Table 6-la. Aldicarb Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)

0


Negative Control
0
0
2
3

-2


Positive Control
8w



1


0.26
4
0
3
0

-3



5


2.6
6
5
2
7

2



13


26
15
14
1
14

13



49


260
51
50
1
(Lethal Dose)
51

48


TT)	
Positive control percent inhibition less than suggested by Lab_Bell.
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
0
0
6
-5
8
-3
Positive Control
41

0.0003
4
1
4
3
1
-5
0.003
0
-5
4
-8
-5
-9
0.03
-8
-6
1
-5
-6
-6
0.3
(Lethal Dose)
-6
-10
8
-21
-11
-1
Lethal Dose
Preservative
Blank
-4
-6
3
-10
-6
-3
20

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Table 6-lc. Colchicine Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)

2


Negative Control
-3
0
2
0

1


Positive Control
9
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Table 6-ld. Cyanide Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
Positive Control
0.25
-2
-1
38
-4
0
2.5
10
25
ll
12
10
250
(Lethal Dose)
20
14
19
17
17
Field Portability
Negative Control
Field Portability
Positive Control
Field Portability
250
-l
40
12
15
21
16
16
22

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Table 6-le. Dicrotophos Percent Inhibition Results
Concentration
Inhibition
Average
Standard
(mg/L)
(%)
(%)
Deviation (%)

6


Negative Control
0
0
4
0

-5


Positive Control
32W



-10


1.4
-10
-10
1
-9

-11



-12


14
-10
-12
1
-13

-12



-11


140
-12
-11
1
-11

-10



6


1,400
1
4
2
(Lethal Dose)
6

3


TT)	
Positive control percent inhibition less than suggested by Lab_Bell.
Table 6-If. Nicotine Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)

-3


Negative Control
-2
0
3
1

3


Positive Control
28(a)



5


2.8
-3
3
4
4

7



2


28
-2
1
3
-1

6



11


280
7
10
2
10

11



35


2,800
34
34
1
(Lethal Dose)
34

34


TTv	
Positive control percent inhibition less than suggested by Lab_Bell.
23

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Table 6-lg. Ricin Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)

-8


Negative Control
9
0
9
6

-8


Positive Control
22
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Table 6-lh. Soman Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)

0


Negative Control
l
0
1
0

-i


Positive Control
33
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Table 6-li. Thallium Sulfate Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)

-2


Negative Control
0
0
3
4

-1


Positive Control
27
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Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results
Preservative
Blank
Inhibition
(%)
Average
(%)
Standard
Deviation
(%)
Negative
Control
-l
0
3
-2
(b)
3
Positive Control
8a

Ricin
-14
-5
9
-7
-8
-8
Soman/VX00
-3
-4
4
-8
-6
1
Botulinum
Toxin Complex
B
-12
-5
9
-6
7
-9
ta) Positive control percent inhibition less than suggested by Lab_Bell.
(b) Removed -45% because result was an obvious outlier.
Table 3-2 details the concentrations of preservatives in the lethal dose samples of each
contaminant. These data could be evaluated in two ways to determine the sensitivity of the
LuminoTox SAPS to contaminants stored in preservatives. The first approach would be to
determine the inhibition of the test samples containing preservatives with respect to the back-
ground negative control, as was the case for the contaminants that were not stored in
preservatives. This technique, however, could indicate that the LuminoTox SAPS Test Kit was
sensitive to the contaminant when, in fact, it was sensitive to one of the preservatives. Since
these contaminants are only available (either commercially or from the government) in aqueous
formulations with the preservatives, this may be appropriate. The second approach would be to
fortify negative control samples with the same concentrations of preservative contained in all the
samples so that the inhibition resulting from the preservatives could be subtracted from the
inhibition caused by the contaminant. This approach would greatly increase the number of
samples required for analysis. Therefore, for this test, aspects of both approaches were
incorporated without substantially increasing the number of samples. Negative control samples
fortified with a concentration of each preservative equivalent to the concentration in the lethal
dose test samples (preservative blanks) were analyzed prior to and with every set of test samples.
For those sets of test samples for which it was especially difficult to determine whether
inhibitory effects were from the contaminant or the preservative, the preservative blank was
diluted identically to all the contaminant samples and analyzed so a background subtraction
could take place if necessary.
During the initial analysis of the preservative blanks (Table 6-2), none of the preservative blank
samples generated an inhibition significantly greater than the DDW negative control. Because
the preservatives apparently do not have toxic effects at the lethal dose concentration, no
additional preservative blanks were analyzed to determine whether there were toxic effects from
each individual concentration level. Each concentration level was evaluated and compared with
27

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the negative control to determine any toxic effects. The lethal dose preservative blank was
determined with each contaminant sample set and is shown with each contaminant inhibition
regardless of the result of the initial preservative blank analysis. Neither the contaminant test
samples nor the preservative blank differed significantly from the negative control for botulinum
toxin complex B, ricin, soman, or VX. Therefore, none of these contaminants caused detectable
inhibition.
A positive control sample was analyzed with every set of analyses and, overall, the positive
control inhibition was somewhat inconsistent throughout the test of the LuminoTox SAPS Test
Kit. Prior to the verification test, Battelle had not been informed of a defined performance
criterion for the positive control; so, if an inhibition greater than the negative control was
generated, the testing staff considered the LuminoTox SAPS Test Kit to be operating properly.
The average inhibition across all of the positive control samples was 28% ± 12%, with each
positive control exhibiting more inhibition than its associated negative control. After the
completion of the test, Lab_Bell expressed concern that the positive control samples did not
always generate a percent inhibition as high as expected. Battelle was then provided a detailed
protocol that stated that the 0.01-mg/L solution of atrazine was expected to have an inhibition of
approximately 43% ± 5%. Had this information been available at the time of testing, the sample
sets not meeting the required positive control inhibition would have been reanalyzed once to try
to bring the control into the acceptable range as defined by Lab_Bell. However, because this was
not available to Battelle until after testing, the data are being reported as collected, and the tables
containing data from sample sets with positive control data less than 38% are noted as such in a
footnote. Three of the positive controls had an inhibition of 40% and above, four were between
30% and 40%, and seven were less than 30%. Three sample sets contained positive controls that
generated an inhibition less than 10% (aldicarb—8%, colchicine—9%, and the preservative
blanks—8%). Despite the low positive control inhibition, the three highest concentration levels
of aldicarb generated detectable inhibition; therefore, in that case, a positive control inhibition of
8% seemed to indicate the adequate functioning of the LuminoTox SAPS. Additionally, nicotine,
with a positive control response of 28% had detectable inhibition at the top two concentrations.
The low positive control inhibition may indicate a lower sensitivity of the LuminoTox SAPS
Test Kit than when a positive control inhibition of greater than 40% is obtained, but at least for
aldicarb and nicotine, the sensitivity seemed to be adequate.
The preservative blank inhibition also seems to suggest that the positive control inhibition was
adequate for confirming the function of the technology. The lethal dose preservative blank was
analyzed once prior to and once with the analysis of the contaminant samples. During the first
analysis prior to contaminant testing, the positive control inhibition was 8%. During subsequent
contaminant testing, every applicable positive control inhibition was higher (botulinum toxin
complex B—41%, ricin—22%, soman—33%, and VX—22%) and all of the lethal dose
preservative blanks generated an inhibition that was either the same as or extremely similar to
what was determined during the first analysis, therefore confirming the inhibition results from
the initial analysis that may have otherwise been in question because of the low positive control
inhibition. Additionally, the repeatability of results across all of the contaminants was very
good. Eighty-six percent of the time the standard deviation was less than 5% inhibition. This
suggests that even an inhibition of 8 or 9% is likely a significant inhibition with respect to the
negative control. All positive control results are reported along with their respective contaminant
set in Tables 6-la through 6-lj.
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6.1.2	Potential Interferences
All of the potential interference samples were prepared in DDW and compared with the negative
control to determine the level of inhibition. This determination is crucial because the ability of
the LuminoTox SAPS Test Kit to detect toxicity is dependent on the background fluorescence
production in whatever drinking water matrix is being used. If the background drinking water
sample completely inhibits background fluorescence, inhibition caused by contaminants could
not be detected. Table 6-3 presents the results from the samples that were analyzed to test the
effect of potential interferences on the LuminoTox SAPS Test Kit. Of the five metal solutions
that were evaluated as possible interferences, none exhibited an inhibition that was significantly
different from the DDW negative control. Therefore, it seems that there is little risk of
interference for these metals because enough fluorescence is produced for inhibition as a result
of contamination.
To investigate whether the LuminoTox SAPS Test Kit 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 IIDI water as the control sample. In the absence of a
background water sample, it seems likely that DI water may be used as a "clean water" control;
therefore, it would be helpful to know what the results would be if this is done. The sample from
the water supply disinfected by chlorination (N=56) exhibited an average inhibition of -8% ±
23%, while the sample from the water supply disinfected by chloramination exhibited an
inhibition of 0% ± 5% 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. These inhibition data suggest that samples disinfected by
either process are not likely to interfere with the LuminoTox SAPS Test Kit results.
6.1.3	Precision
Across all the contaminants and potential interferences, the standard deviation (not relative
standard deviation) was measured and reported for each set of four replicates to evaluate the
LuminoTox SAPS Test Kit precision. Out of 78 opportunities, the standard deviation of the four
replicate inhibition measurements was less than 5% inhibition 67 times (86% of the time),
between 5% and 10% inhibition 10 times (13% of the time), and greater than 10% inhibition just
1 time (1%). As described in Section 3.2.2, the analysis procedure required that each replicate
undergo the entire analysis process; therefore, the measurement of precision represents the
precision of the analysis method performed on a single water sample on a given day. The
precision does not reflect the repeatability of the method across more than one day or more than
one preparation of reagents or more than one operator.
6.2 Toxicity Threshold
Table 6-4 gives the toxicity thresholds, as defined in Section 5.2, for each contaminant. Note the
difference between detectability with respect to the negative control and the toxicity threshold
with respect to the other concentration levels analyzed. A contaminant concentration level can
have an inhibition significantly different from the negative control (thus detectable), but if its
inhibition is not
29

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Table 6-3. Potential Interferences Results
Potential
Interferences
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard Deviation
(%)
Negative control
(Metals)
Positive Control
(Metals)
Aluminum
NA
NA
0.5
-3
0
1
3
45
-3
2
0
5
Copper
0.6
Iron
0.15
2
2
-1
-1
Manganese
0.25
3
4
-3
-2
Zinc
2.5
-4
-1
5
-2
Negative control
(By-products)
Positive control
(By-products)
Chlorination
by-products
NA
NA
NA
1
0
-3
2
34'
«
(b)
23
Chloramination
by-products
NA
-4
0
-3
7
NA = Not applicable.
/a\
Positive control percent inhibition less than suggested by Lab_Bell.
(b) Average inhibition across all DDW negative control samples (N=56).
30

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Table 6-4. Toxicity Thresholds
Contaminant
Concentration (mg/L)
Aldicarb
26
Botulinum toxin complex B
ND
Colchicine
ND
Cyanide
250
Dicrotophos
ND
Nicotine
280
Ricin
ND
Soman
ND
Thallium sulfate
ND
VX
ND
ND = Significant inhibition was not detected.
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
aldicarb at 26 mg/L.
6.3 False Positive/Negative Responses
None of the LuminoTox SAPS Test Kit results would be considered false positive because
neither the chlorination nor chloramination by-product samples were inhibitory and, therefore,
fluorescence production was adequate to allow inhibition to occur if a contaminant was present
that produced a detectable toxic effect. Since the background inhibition is not complete, it can be
accounted for by using negative control samples that are very similar to the water being
analyzed. If samples are analyzed daily, a good practice would be to archive a negative control
sample each day in case of contamination the next day.
Table 6-5 shows the LuminoTox SAPS Test Kit false negative responses, which are described in
Section 5.3. Botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, thallium sulfate,
and VX did not exhibit a detectable inhibition at the lethal concentration.
31

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Table 6-5. False Negative Responses
Contaminant
Lethal Dose
Concentration (Mg/L)
False Negative
Aldicarb
260
no
Botulinum toxin
complex B
0.30
yes
Colchicine
240
yes
Cyanide
250
no
Dicrotophos
1,400
yes
Nicotine
2,800
no
Ricin
15
yes
Soman
1.4
yes
Thallium sulfate
2,800
yes
VX
2.0
yes
6.4 Other Performance Factors
6.4.1	Ease of Use
The LuminoTox SAPS Test Kit contained detailed instructions and clear illustrations. The
contents of the LuminoTox SAPS Test Kit were well identified with labels on the vials. Storage
requirements were stated in the instructions and on the reagent vials. Overall, the test was easy to
perform; but additional practice helped the operators become accustomed to the timing involved
with running a large number of test samples.
Preparation of the test samples for analysis was straightforward. The analyzer, including a piece
of foil covering the cuvette opening, was easy to use, but the necessity to record four numbers as
raw data was somewhat burdensome. Lab Bell has indicated that this is undergoing modification.
After testing, the fluorometer was easily wiped clean and required no routine maintenance other
than selecting the SAPS mode prior to the start of sample analysis.
No formal scientific education would be required to use the LuminoTox SAPS Test Kit.
However, good laboratory skills, especially pipetting technique, would be beneficial.
Verification testing staff were able to operate the LuminoTox SAPS Test Kit after a 4-hour
training session with the vendor. With every sample, approximately 2 mL of liquid waste were
generated, along with leftover SAPS and a 3-mL disposable syringe.
6.4.2	Field Portability
The LuminoTox SAPS Test Kit was transported from a laboratory setting to a storage room for
the field portability evaluation. The storage room contained several tables and light and power
32

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sources, but no other laboratory facilities. No carrying case was provided with the LuminoTox
SAPS Test Kit; however, all materials were transported by one person in a small cardboard box.
The LuminoTox SAPS Test Kit was set up easily in less than 10 minutes, and a source of
electricity was not required since the fluorometer ran on batteries. Minimum space requirements
in the field would be a mostly flat surface of approximately 45 by 60 centimeters. The only items
needed for field use not provided in the LuminoTox SAPS Test Kit was a timer and a waste
reservoir. Overall, the LuminoTox SAPS Test Kit was easy to transport to the field and was
deployed in a matter of minutes. The limiting factor for testing in the field would be the
approximately 90 minutes required to expose the SAPS to light prior to testing. After the light
exposure, results were obtained within 10 minutes of starting the test. The LuminoTox SAPS
Test Kit was tested with one contaminant, cyanide, at the lethal dose concentration. The results
of the test (see Table 6-ld) were very similar to the laboratory results. Inhibition in the
laboratory was 17% ± 2%, and in the non-laboratory location, 16% ± 4%, suggesting that
location did not impact the performance of the LuminoTox SAPS Test Kit.
6.4.3 Throughput
Once the SAPS were prepared, approximately 20 analyses were completed per hour. The
20 analyses included method blanks and positive and negative controls, as well as test samples.
Approximately 50 samples could be analyzed with the supplies contained in one LuminoTox
SAPS Test Kit.
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Chapter 7
Performance Summary
Parameter
Compound
Lethal
Dose (LD)
Cone.
(mg/L)
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
LD/10
LD/100
LD/1,000
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)
Aldicarb
260
50
14
0
1-3
26
Botulinum toxin
complex B
0.3
-10
-6
-5
1-8
ND
Colchicine
240
1-5
ND
Cyanide
250
17
10
2-3
250
Contaminants in
DDW
Dicrotophos
1,400
-11
-12
-10
1-2
ND
Nicotine
2,800
34
10
1-4
280
Ricin
15
2-6
ND
Soman
1.4
2-3
ND
Thallium
sulfate
2,800
-3
-4
2-3
ND
VX
3
-1
Interference
Cone.
(mg/L)
Average Inhibition
(%)
Potential
interferences in
DDW
Aluminum
0.5
1
Copper
0.6
Iron
0.15
Manganese
0.25
Zinc
2.5
-1
Standard
Deviation (%)
False positive
response
None of the LuminoTox SAPS Test Kit responses were considered false positive. All disinfection by-
product test samples left enough fluorescence for inhibition due to contamination.
False negative
response
Botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, thallium sulfate, and VX exhibited
non-detectable responses at the lethal dose concentration.
Ease of use
The LuminoTox SAPS Test Kit contained detailed instructions and clear illustrations. The contents
were well identified with labels on the vials. Storage requirements were stated in the instructions and
on the reagent vials. Preparation of the test samples for analysis was straightforward. The necessity to
record four numbers as raw data was somewhat burdensome; however, this feature is being modified
according to Lab_Bell. No formal scientific education would be required to use the LuminoTox SAPS
Test Kit.
Field portability
The LuminoTox SAPS Test Kit was transported from a laboratory setting to a storage room for the
field portability evaluation. The limiting factor for testing in the field would be the approximately
90 minutes required to allow the SAPS to be exposed to light prior to testing. The LuminoTox SAPS
Test Kit was tested with one contaminant, cyanide, at the lethal dose concentration. The results of the
test were very similar to the laboratory results. Inhibition in the laboratory was 17% ± 2%, and in the
non-laboratory location, 16% ±4%.
Throughput
Approximately 20 analyses were completed per hour, and 50 samples could be analyzed with the
supplies contained in one LuminoTox SAPS Test Kit.
ND = Significant inhibition was not detected.
34

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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 I iPA/600/R-95/131.
9.	U.S. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
Spectrometry," Methods for the Determination of Organic Compounds in Drinking Water—
Supplement III, EPA/600/R-95/131.
10.	American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater, 20th edition, 1998, Washington, DC.
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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