June 2006
Environmental Technology
Verification Report
Lab_Bell Inc.
LuminoTox PECs Test Kit
Prepared by
Battelle
Baiteiie
The Business of Innovation
Under a cooperative agreement with
EPA U.S. Environmental Protection Agency
ETV ETVETV

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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:
COMPANY:
ADDRESS:
WEB SITE:
E-MAIL:
LuminoTox PECs
LabBell Inc.
2263, avenue du College PHONE: (819) 539-8508, ext. 107
Shawinigan, Quebec FAX: (819) 539-8880
CANADA G9N 6V8
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 LabBell Inc. LuminoTox photosynthetic enzymatic complexes (PECs) 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 PECs 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 PECs 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 PECs 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 a negative background inhibition that
caused falsely low inhibition
¦	Other performance factors (sample throughput, ease of use, reliability).
The LuminoTox PECs 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 inhibits the
photosynthetic process that the LuminoTox PECs Test Kit depends on to indicate toxicity and can degrade the
contaminants during storage. Inhibition results (endpoints) from four replicates of each contaminant at each
concentration level were evaluated to assess the ability of the LuminoTox PECs Test Kit to detect toxicity, as
well as to measure the precision of the LuminoTox PECs Test Kit results. The response of the LuminoTox
PECs 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 atwww.epa.gov/etv/centers/centerl.html.
TECHNOLOGY DESCRIPTION
The following description of the LuminoTox PECs Test Kit is based on information provided by the vendor.
This technology description was not verified in this test.
The LuminoTox PECs Test Kit is a portable biosensor that indicates the presence of toxic chemicals in water.
It uses PECs that have been stabilized through a method patented by Lab Bell Inc. The PECs are membranes
isolated from chloroplasts that are as simple to use as a chemical, but react more rapidly than a living
organism because toxic compounds do not have to penetrate the cell wall of an organism. The photosynthetic
electron chain is what is inhibited by contamination. When stimulated by light, the PECs emit fluorescence.
The LuminoTox PECs Test Kit measures the fluorescence parameters produced both in background water and
samples containing contaminants. Decreases in fluorescence parameters as a result of the presence of toxic
contamination are expressed as percent inhibition.
The LuminoTox PECs Test Kit consists of the LuminoTox analyzer, a bottle of PECs for 50 tests, reaction
buffer, a blank water control, and a positive control. Also provided are disposable syringes in which the test is
performed and fabric syringe covers to protect the reaction from light. Aluminum foil can be used as a light
protector.
The LuminoTox analyzer is 21.6 by 12.7 by 7.6 centimeters and weighs 1 kilogram. It is battery-operated and
is portable. The analyzer has a built-in RS-232 serial port outlet, which can also be used for transferring data
to a spreadsheet (which was not done during this test), and is compatible with a printer. A total of 100
measurements can be stored in the internal memory. The rechargeable battery operates for eight hours. Each
kit costs $89, and the 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
26
2
0
-2
1-3
260
Botulinum toxin
complex B
0.3
0
-5
-8
-12
2-6
ND
Colchicine
240
2
-3
-2
-6
1-5
ND
Cyanide
250
47
31
-8
-7
1-7
25
Dicrotophos
1,400
3
10
8
4
2-4
ND
Nicotine
2,800
77
80
6
9
1-6
280
Ricin
15
2
5
-7
-10
4-9
ND
Soman
1.4
-5
-6
0
-1
4-6
ND
Thallium sulfate
2,800
63
19
-3
-12
2-7
280
VX
2
-5
-8
-2
3
3-5
ND
Potential
interferences
in DDW
Interference
Cone.
(mg/L)
Average Inhibition
(%)
Standard
Deviation (%)

Aluminum
0.5
0
4
Copper
0.6
70
1
Iron
0.15
7
6
Manganese
0.25
5
6
Zinc
2.5
12
5
False positive
response
Both the chlorinated and chloraminated disinfection by-product samples produced an inhibition
significantly greater than the negative control and, therefore, were considered false positive
responses. However, the disinfectant by-product samples produced an inhibition of less than 15%,
leaving enough fluorescence available for subsequent inhibition due to contamination.
False negative
response
Botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, and VX exhibited non-detectable
responses at the lethal dose concentration.
Ease of use
The LuminoTox PECs Test Kit contained detailed instructions and clear illustrations. The contents
of the LuminoTox PECs Test Kit 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. However, the PECs had to be stored in ice between every sample analysis to
keep them from coming to room temperature, which was somewhat inconvenient because the
melting ice caused the lab bench and operators' hands to be wet most of the time. The necessity to
record four numbers as raw data was somewhat burdensome; however, Lab Bell has indicated that it
is modifying this. No formal scientific education would be required to use the LuminoTox PECs
Test Kit.
Field
portability
The LuminoTox PECs Test Kit was transported from a laboratory setting to a storage room for the
field portability evaluation. The LuminoTox PECs 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 47% ± 1%, and in the non-laboratory location, 51% ± 1%.
Throughput
Approximately 20 analyses were completed per hour, and approximately 50 samples could be
analyzed with the supplies contained in one LuminoTox PECS 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 PECs 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	3
3.1	Test Samples	5
3.1.1	Quality Control Samples	5
3.1.2	Drinking Water Fortified with Contaminants	5
3.1.3	Drinking Water Fortified with Potential Interferences	7
3.2	Test Procedure	7
3.2.1	Test Sample Preparation and Storage	7
3.2.2	Test Sample Analysis Procedure	7
3.2.3	Stock Solution Confirmation Analysis	8
Chapter 4	Quality Assurance/Quality Control	11
4.1	Quality Control of Stock Solution Confirmation Methods	11
4.2	Quality Control of Drinking Water Samples	11
4.3	Audits	12
4.3.1	Performance Evaluation Audit	12
4.3.2	Technical Systems Audit	12
4.3.3	Audit of Data Quality	13
4.4	QA/QC Reporting	13
4.5	Data Review	13
Chapter 5 Statistical Methods and Reported Parameters	15
5.1	Endpoints and Precision	15
5.2	Toxicity Threshold	16
5.3	False Positive/Negative Responses	16
5.4	Other Performance Factors	17
Chapter 6 Test Results	18
6.1	Endpoints and Precision	18
6.1.1	Contaminants	18
6.1.2	Potential Interferences	27
6.1.3	Precision	29
6.2	Toxicity Threshold	29
6.3	False Positive/Negative Responses	30
6.4	Other Performance Factors	30
6.4.1	Ease of Use	30
6.4.2	Field Portability	31
6.4.3	Throughput	32
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Chapter 7 Performance Summary	33
Chapter 8 References	34
Figures
Figure 2-1. Lab_Bell Inc. LuminoTox PECs Test Kit	2
Tables
Table 3-1. Contaminants and Potential Interferences	4
Table 3-2. Summary of Quality Control and Contaminant Test Samples	6
Table 3-3. Stock Solution Confirmation Results	9
Table 3-4. Water Quality Parameters	10
Table 4-1. Summary of Performance Evaluation Audit	13
Table 4-2. Summary of Data Recording Process	14
Table 6-la. Aldicarb Percent Inhibition Results	19
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results	19
Table 6-lc. Colchicine Percent Inhibition Results	20
Table 6-Id. Cyanide Percent Inhibition Results	21
Table 6-le. Dicrotophos Percent Inhibition Results	22
Table 6-1 f. Nicotine Percent Inhibition Results	22
Table 6-1 g. Ricin Percent Inhibition Results	23
Table 6-1 h. Soman Percent Inhibition Results	24
Table 6-1 i. Thallium Sulfate Percent Inhibition Results	25
Table 6-lj. Thallium Sulfate Percent Inhibition Results—Additional Dilutions	25
Table 6-1 k. VX Percent Inhibition Results	26
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results	27
Table 6-3. Potential Interferences Results	28
Table 6-4. Toxicity Thresholds	30
Table 6-5. False Negative Responses	31
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
(_iL
microliter
mg/L
milligram per liter
mL
milliliter
mm
millimeter
NSDWR
National Secondary Drinking Water Regulations
%D
percent difference
PE
performance evaluation
PECs
photosynthetic enzymatic complexes
QA
quality assurance
QC
quality control
QMP
quality management plan
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 photosynthetic enzymatic
complexes (PECs), hereafter referred to as the LuminoTox PECs 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 PECs Test Kit. Following is a description of
the LuminoTox PECs Test Kit, based on information provided by the vendor. The information
provided below was not verified during this test.
The LuminoTox PECs Test Kit (Figure 2-1) is a portable biosensor that indicates the presence of
toxic chemicals in water. It uses PECs that have been stabilized through a method patented by
Lab_Bell Inc. The PECs are membranes isolated from chloroplasts that are as simple to use as a
chemical, but react more rapidly than a living organism because toxic compounds do not have to
penetrate the cell wall of an organism. The photosynthetic electron chain is what is inhibited by
contamination. When stimulated by light, the PECs emit fluorescence. The LuminoTox PECs
Test Kit measures the fluorescence parameters produced both in background water and samples
containing contaminants. Decreases in fluorescence parameters as a result of the presence of
toxic contamination are expressed as a percent inhibition.
The LuminoTox PECs Test Kit consists
of the LuminoTox analyzer, a bottle of
PECs for 50 tests, reaction buffer, a blank
water control, and a positive control. Also
provided are disposable syringes in which
the test is performed and fabric syringe
covers to protect the reaction from light.
Aluminum foil can be used as a light
protector.
The LuminoTox analyzer is 21.6 by 12.7
by 7.6 centimeters and weighs 1 kilo-
gram. It is battery-operated and is
portable. The analyzer has a built-in RS-232 serial port outlet, which can also be used for
transferring data to a spreadsheet (which was not done during this test) and is compatible with a
printer. A total of 100 measurements can be stored in the internal memory. The rechargeable
battery operates for eight hours. Each kit costs $89, and the analyzer costs approximately $7,500.
Figure 2-1. Lab_Bell Inc. LuminoTox PECs Test
Kit
2

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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 PECs 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 PECs
Test Kit can detect the toxicity caused by each contaminant, its response to interfering
compounds such as water treatment chemicals and by-products in clean drinking water, was
evaluated. Table 3-1 shows the contaminants and potential interferences that were evaluated
during this verification test.
This verification test was conducted from August to December 2005 according to procedures
specified in the Test/QA Plan for Verification of Rapid Toxicity Technologies including
Amendments 1 and 2.^ The LuminoTox PECs 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 PECs 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
3

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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 percent inhibition
¦	Other performance factors (sample throughput, ease of use, reliability).
The LuminoTox PECs 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."' Inhibition (endpoints)
from four replicates of each contaminant at each concentration level were evaluated to assess the
ability of the LuminoTox PECs Test Kit to detect toxicity at various concentrations of
contaminants, as well as to measure the precision of the LuminoTox PECs Test Kit results.
The response of the LuminoTox PECs 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
4

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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
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 the LuminoTox PECs 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 II DI 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 PECs 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 PECs 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. Additional concentrations of thallium sulfate
were prepared and analyzed because of the large difference in response between two concen-
tration levels. One dilution level was almost completely inhibitory and the next dilution level
was non-inhibitory, so several intermediate concentrations were analyzed to better determine the
toxicity threshold of that contaminant. Table 3-2 lists each concentration level and the number of
samples analyzed at each level.
5

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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
15
Positive control
Used as provided in kit, 0.2 mg/L
atrazine
15
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
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; 2,100; 1,400; 700; 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
60
NA = not applicable, samples not fortified with any preservative, contaminant, or potential interference.
6

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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 PECs 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
inhibits the photosynthetic process that the LuminoTox PECs 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 II DI water. The
positive control sample, 0.2 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 add the reaction buffer to the bottle of dried PECs, swirl,
and then wait for 15 minutes before shaking well to finalize the dissolution of the PECs. Next,
2 mL of each control and test sample were each taken up into 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 j_iL of the PECs solution to each control and water sample, each syringe was mixed by
7

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inverting five times. The solutions were allowed to react for 10 minutes, and the content of the
syringe was added to a cuvette residing within the LuminoTox analyzer. The cover was closed
for one minute, and the sample readings were taken from the LuminoTox 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 analyzer. 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 analyzer.
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.
8

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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®
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(5)
2.63 ±0.05
0.27
NA = Not applicable.
No standard method available. QA audits and balance calibration assured accurately prepared solutions.
® Target concentration was highest concentration for each contaminant or interference on Table 3-2.
9

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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
(fjjnho)
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
(l-Lg/L)
EPA 552.2®
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.
10

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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center^ and the test/QA plan for this verification test.®
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 II DI water was analyzed once by the
LuminoTox PECs 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 inhibition of the DDW for the disinfecting by-product evaluation. A positive
control sample of 0.2 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, the vendor informed Battelle that, if the positive control samples did not cause
significant inhibition, it would indicate to the operator that the LuminoTox PECs Test Kit was
not functioning properly. For 15 positive control samples, an inhibition of 69% ± 6% was
11

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measured. These inhibition values seemed to indicate the proper functioning of the LuminoTox
PECs Test Kit. A negative control sample (unspiked DDW) was analyzed with approximately
every four samples. The percent inhibition calculation for each sample incorporated the average
inhibition of the negative control samples analyzed with that particular sample set; therefore, by
definition, the average inhibition of four negative control samples was 0%.
4.3 Audits
A performance evaluation (PE) audit, a technical systems audit (TSA), and an audit of data
quality were performed for this verification test.
4.3.1	Performance Evaluation Audit
The accuracy of the reference method used to confirm the concentrations of the stock solutions
of the contaminants and potential interferences was confirmed by analyzing solutions of each
analyte from two separate commercial vendors. The standards from one source were used to
prepare the stock solutions during the verification test, while the standards from a second source
were analyzed as the PE sample. The percent difference (%D) between the measured
concentration of the PE sample, and the nominal concentration of that sample was calculated
using the following equation:
o/oD=—x 100%	(!)
A
where Mis 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^ and the AMS Center QMP/11^ As part of the audit, the
Battelle Quality Manager reviewed the contaminant standard and stock solution confirmation
methods, compared actual test procedures with those specified in the test/QA plan, and reviewed
data acquisition and handling procedures. Observations and findings from this audit were
documented and submitted to the Battelle Verification Test Coordinator for response. No
findings were documented that required any significant action. The records concerning the TSA
are permanently stored with the Battelle Quality Manager.
12

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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/11^ Once the assessment report was prepared, the Battelle
Verification Test Coordinator ensured that a response was provided for each adverse finding or
potential problem and implemented any necessary follow-up corrective action. The Battelle
Quality Manager ensured that follow-up corrective action was taken. The results of the TSA
were sent to the EPA.
4.5	Data Review
Records generated in the verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-2 summarizes the types of data recorded. The
review was performed by a technical staff member involved in the verification test, but not the
staff member who originally generated the record. The person performing the review added
his/her signature or initials and the date to a hard copy of the record being reviewed.
13

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Table 4-2. Summary of Data Recording Process
Data to be
Recorded
Responsible
Party
Where
Recorded
How Often
Recorded
Disposition of Data'8'
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
,;ii All activities subsequent to data recording were carried out by Battelle.
14

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Chapter 5
Statistical Methods and Reported Parameters
The statistical methods presented in this chapter were used to verify the performance parameters
listed in Section 3.
5.1 Endpoints and Precision
The fluorescence analyzer provided with the LuminoTox PECs Test Kit reported two values of
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 - F
rr- •	2 sample	1 sample
efficiency = 		
F.
2 negative control
% inhibition ¦
1-=-
E
sample
\
E
xl00%
(2)
(3)
negative control j
where efficiency (Ł) is a measure of the fluorescence produced by the PECs with respect to the
average high-intensity fluorescence measurement values produced by the replicate negative
control samples (F2 negative control) and 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 efficiency. 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 II DI
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 PECs Test Kit precision at each concentration. The
15

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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.
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 PECs 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 the other concentration levels because it more thoroughly
1/2
(4)
where n is the number of replicate samples, h is the percent inhibition measured for the &th
16

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incorporated the uncertainty of all the measurements made by the LuminoTox PECs 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
that were being compared. In addition, background water samples that increased the light
production of the LuminoTox PECs 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.
17

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Chapter 6
Test Results
6.1 Endpoints and Precision
Tables 6-la-k 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 of 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 LuminoTox PECs Test Kit and were considered non-toxic.
6.1.1 Contaminants
Aldicarb, cyanide, nicotine, and thallium sulfate generated an inhibition significantly larger than
the negative control. Cyanide and nicotine generated detectable inhibition at the two highest
concentration levels analyzed; and aldicarb and thallium sulfate (upon the initial analysis),
produced detectable inhibition only at the lethal dose concentration level. Because of the rather
large inhibition (63%) for thallium sulfate at only the lethal dose concentration, additional
dilutions were performed to more closely determine the toxicity threshold. Those results are
shown in Table 6-lj. During these additional dilutions, toxicity due to thallium sulfate was
detectable down to 280 mg/L. Interestingly, inhibition at that concentration was not detectable
during the initial analyses. Colchicine and dicrotophos did not generate a 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.
Table 3-2 details the concentrations of preservatives in the lethal dose samples of each
18

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Table 6-la. Aldicarb Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
-2
0
2
-1
2
0
0.26
-1
-2
2
-2
0
-4
2.6
1
0
1
0
-2
0
26
0
2
3
2
7
1
260
(Lethal Dose)
25
26
3
26
23
30
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
-3
0
2
0
3
-1
0.0003
-11
-12
2
-14
-10
-11
0.003
-11
-8
4
-9
-10
-1
0.03
-4
-5
3
-8
-5
-2
0.3
(Lethal Dose)
-3
0
6
3
7
-7
Lethal Dose
Preservative
Blank
-8
-10
3
-9
-13
-12
19

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Table 6-1 c. Colchicine Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
-5
0
4
-1
1
5
0.24
-8
-6
2
-5
-4
-7
2.4
-3
-2
1
-2
-2
-2
24
-1
-3
5
-9
-4
1
240
(Lethal Dose)
3
2
3
-3
5
1
20

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Table 6-1 d. Cyanide Percent Inhibition Results
Concentration
Inhibition
Average
Standard
(mg/L)
(%)
(%)
Deviation (%)

6


Negative Control
3
0
5
-3

-5



-8


0.25
-13
-7
5
-6

-1



-7


2.5
-17
o
7
-10


-1



37


25
27
31
6
37

25



47


250
49
47
1
(Lethal Dose)
47
46



3


Field Portability
1
0
3
Negative Control
0
-4



50


Field Portability
52
51
1
250
50

53


21

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Table 6-le. Dicrotophos Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
16
0
11
-9
-6
-2
1.4
2
4
4
-1
4
10
14
10
8
2
5
8
9
140
9
10
2
12
11
9
1,400
(Lethal Dose)
5
3
2
0
5
3
Table 6-lf. Nicotine Percent Inhibition Results



Standard
Concentration
Inhibition
Average
Deviation
(mg/L)
(%)
(%)
(%)

-4


Negative
-1
0
6
Control
-3

8



1


2.8
15
9
6
12

9



3


28
0
6
5
8

12



81


280
77
80
2
79

82



77


2,800
76
77
1
(Lethal Dose)
76
77


22

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Table 6-lg. Ricin Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
-2
0
2
-1
2
1
0.015
-15
-10
5
-12
-3
-8
0.15
-3
-7
4
-9
-4
-11
1.5
1
5
4
4
7
9
15
(Lethal Dose)
4
2
9
4
-10
11
Lethal Dose
Preservative
Blank
-16
-10
6
-14
-6
-5
23

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Table 6-lh. Soman Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
2
0
4
-5
4
-1
0.0014
-7
-1
5
-2
3
2
0.014
6
0
6
-7
2
-3
0.14
-7
-6
4
-9
-8
0
1.4
(Lethal Dose)
-11
-5
6
-10
1
-2
Lethal Dose
Preservative
Blank
-6
-3
6
6
-6
-6
24

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

-3


Negative
-7
0
7
Control
-1

10



-7


2.8
-20
-12
6
-14

-8



-12


28
3
-3
7
-6

2



8


280
-2
1
5
-2

-1



60


2,800
65
63
2
(Lethal Dose)
63
62


Table 6-lj. Thallium Sulfate Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
-1
0
1
-1
1
1
280
16
19
4
23
18
15
700
27
31
5
31
35
37
1,400
49
49
1
50
49
49
2,100
64
65
1
66
64
64
2,800
73
73
1
72
73
74
25

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Table 6-lk. VX Percent Inhibition Results
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
-2
0
2
0
1
1
0.002
8
3
5
-5
5
3
0.02
-2
-2
5
-9
4
-1
0.2
-11
-8
4
-12
-5
-4
2
(Lethal Dose)
0
-5
3
-5
-7
-7
Lethal Dose
Preservative
Blank
5
-1
4
-2
-4
-2
contaminant. These data could be evaluated in two ways to determine the sensitivity of the
LuminoTox PECS Test Kit to contaminants stored in preservatives. The first approach would be
to determine the inhibition of the test samples containing preservatives with respect to the
background negative control, as was the case for the contaminants not stored in preservatives.
This technique, however, could indicate that the LuminoTox PECs 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 with them so a background subtraction could take place if
necessary.
26

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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 did not have toxic effects at the lethal dose concentration, no
additional dilutions of preservative blanks were required to determine whether there were toxic
effects from each individual concentration level. Each contaminant concentration level was
evaluated and compared with the negative control to determine any toxic effects. The inhibition
of the lethal dose preservative blank was determined with each contaminant sample set and is
shown with the results from each of the contaminants regardless of the result of the initial
preservative blank analysis.
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results
Preservative
Blank
Inhibition
(%)
Average
(%)
Standard
Deviation (%)
Negative Control
-3
0
7
6
5
-8
Ricin
22
12
12
21
6
-3
Soman/VX
-2
-5
3
-8
(a)
-4
Botulinum Toxin
Complex B
9
0
7
3
-6
-6
{R> Removed because it was an obvious outlier.
For all four of these contaminants, neither the contaminant sample nor the lethal dose
preservative blank generated an inhibition that was significantly greater than the negative
control. This indicated that none of these contaminants produced a toxic effect on the
LuminoTox PECs Test Kit.
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 PECs Test Kit to detect toxicity is dependent on the background light production
in whatever drinking water matrix is being used. If the background drinking water sample
completely inhibits the background light, inhibition caused by contaminants could not be
detected. Table 6-3 presents the results from the samples analyzed to test the effect of potential
interferences on the LuminoTox PECs Test Kit. Of the five metal solutions that were evaluated
as possible interferences, two, zinc (12% ± 5%) and copper (70% ± 1%), exhibited inhibition that
was significantly different from the DDW negative control (0% ± 2%). Because zinc exhibited a
very small inhibition, therefore leaving plenty of light for inhibition due to contamination, it
27

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Table 6-3. Potential Interferences Results
Potential
Interferences
Concen-
tration
(mg/L)
Inhibition
(%)
Average
(%)
Standard
Deviation
(%)


0


Negative control
NA
-2
0
2
(Metals)
0

2




-1


Aluminum
0.5
2
0
4
-4


5




69


Copper
0.6
69
70
1
71


70




1


Iron
0.15
6
7
6
15


6




6


Manganese
0.25
14
5
6
2


-1




20


Zinc
2.5
10
12
5
8


11




3


Negative control

2
0
3
(By-products)

-2

-3


Chlorination
by-products
NA
(a)
14
11


5


Chloramination
NA
7
7
2
by-products
7

10


NA = Not applicable.
= Average inhibition across all negative control samples (N=60).
28

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should be considered only a slight interference. Therefore, water samples containing similar
concentration of zinc could be used as a representative negative control sample because enough
background light for inhibition by contaminants remains even though in it is somewhat inhibited
by zinc. Copper, on the other hand, should be considered a possible interference because the
majority of background light would be inhibited by the matrix if a similar copper concentration
was present, leaving less residual light to be inhibited by contamination.
To investigate whether the LuminoTox PECs 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 II DI water as the control sample. In the absence of a
background water sample, it seems likely that DI water may be used as a "clean water" control;
therefore, it would be helpful to know what the results would be if this is done. The sample from
the water supply disinfected with chlorination (N=60) exhibited an average inhibition of 14% ±
11%, while the sample from the water supply disinfected by chloramination exhibited an
inhibition of 7% ± 2% on four replicates. The difference in 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 particular water. These inhibition data suggest that samples
disinfected by either process are not likely to interfere with the LuminoTox PECs Test Kit results
because the inhibition caused by the "clean" drinking water matrices left most of the background
fluorescence to potentially be inhibited by contamination. Even for copper, which produced a
greater inhibition, the inhibition was not complete; and it can be accounted for by using negative
control samples that are very similar to the water being analyzed. For both scenarios, 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.
6.1.3 Precision
Across all the contaminants and potential interferences, the standard deviation (not relative
standard deviation) was measured and reported for each set of four replicates to evaluate the
precision of the LuminoTox PECs Test Kit. Out of 80 opportunities, the standard deviation of the
four replicate inhibition measurements was less than 5% inhibition 51 times (63% of the time ),
between 5 and 10% inhibition 26 times (33% of the time), and greater than 10% inhibition 3
times (4%). As described in Section 3.2.2, the analysis procedure required that each replicate
undergo the entire analysis process; therefore, the measurement of precision represents the
precision of the analysis method performed on a single water sample on a given day. The
precision does not reflect the repeatability of the method across more than one day or more than
one preparation of reagents or more than one operator.
6.2 Toxicity Threshold
Table 6-4 gives the toxicity thresholds, as defined in Section 5.2, for each contaminant. Note the
difference between detectability with respect to the negative control and the toxicity threshold
with respect to the other concentration levels analyzed. A contaminant concentration level can
have an inhibition significantly different from the negative control (thus detectable), but if its
inhibition is not significantly different from the concentration levels below it, it would not be
considered the toxicity threshold because in the context of this test, its inhibition would not be
distinguishable from that of the lower concentrations. The lowest toxicity threshold
concentration was for cyanide at 25 mg/L.
29

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Table 6-4. Toxicity Thresholds
Contaminant
Concentration (mg/L)
Aldicarb
260
Botulinum toxin complex B
ND
Colchicine
ND
Cyanide
25
Dicrotophos
ND
Nicotine
280
Ricin
ND
Soman
ND
Thallium sulfate
280
VX
ND
ND = Significant inhibition was not detected.
6.3	False Positive/Negative Responses
The chlorination and chloramination by-product samples generated an inhibition that was
considered false positive because it was significantly greater than the negative control.
However, the inhibition was very slight, leaving enough fluorescence available for inhibition if
contamination was present.
Table 6-5 shows the LuminoTox PECs Test Kit false negative responses, which are described in
Section 5.3. Botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, and VX did not
exhibit a detectable inhibition at the lethal concentration.
6.4	Other Performance Factors
6.4.1 Ease of Use
The LuminoTox PECs Test Kit contained detailed instructions and clear illustrations. The
contents of the LuminoTox PECs Test Kit 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. However, the PECs had to be
stored on ice between every sample analysis to keep them from coming to room temperature.
Therefore, the operators had to open and close the cooler containing the ice every time they
needed to add PECs to a water sample or control, which was somewhat inconvenient because the
melting ice caused the lab bench and operators' hands to be wet most of the time. The analyzer,
30

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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
no
VX
2.0
yes
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 analyzer was easily wiped clean and required no
routine maintenance other than selecting the PECs mode prior to the start of sample analysis.
No formal scientific education would be required to use the LuminoTox PECs Test Kit.
However, good laboratory skills, especially pipetting technique, would be beneficial.
Verification testing staff were able to operate the LuminoTox PECs Test Kit after a 4-hour
training session with the vendor. Approximately 2 mL of liquid waste were generated per
sample, along with leftover PECs and a 3-mL disposable syringe.
6.4.2 Field Portability
The LuminoTox PECs Test Kit was transported from a laboratory setting to a storage room for
the field portability evaluation. The storage room contained several tables and light and power
sources, but no other laboratory facilities. No carrying case was provided with the LuminoTox
PECs Test Kit; however, all materials were transported by one person in a small cardboard box.
The LuminoTox PECs Test Kit was set up easily in less than 10 minutes, and a source of
electricity was not required since the analyzer was powered by 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 that were not provided in the LuminoTox PECs Test Kit
were a cooler to transport and store the PECs and a timer. These two items can be purchased
through Lab_Bell or are commercially available. In addition, no waste reservoir was provided.
Overall, the LuminoTox PECs Test Kit was easy to transport to the non-laboratory location and
was deployed in a matter of minutes. The limiting factor for testing in the field would be the
approximately 30 minutes required to allow the PECs to dissolve. After dissolution, results were
obtained within 10 minutes of starting the test. The LuminoTox PECs Test Kit was tested with
one contaminant, cyanide, at the lethal dose concentration. The results of the test (see
31

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Table 6-1 d) were very similar to the laboratory results. Inhibition in the laboratory was 47% ±
1%, and in the non-laboratory location, 51% ± 1%, suggesting that location did not significantly
impact the performance of the LuminoTox PECs Test Kit.
6.4.3 Throughput
Once the PECs 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
PECS Test Kit.
32

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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
(%)
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)
LD
LD/10
LD/100
LD/1,000
Contaminants
in DDW
Aldicarb
260
26
2
0
-2
13
260
Botulinum toxin
complex B
0.3
0
-5
8
-12
2-6
ND
Colchicine
240
2
3
-2
-6
1-5
ND
Cyanide
250
47
31
8
-7
1-7
25
Dicrotophos
1,400
3
10
8
4
2-4
ND
Nicotine
2,800
77
80
6
9
1-6
280
Ricin
15
2
5
-7
-10
4-9
ND
Soman
1.4
-5
-6
0
1
4-6
ND
Thallium sulfate
2,800
63
19
3
-12
2-7
280
VX
2
-5
8
-2
3
3-5
ND
Potential
interferences
in DDW
Interference
Cone.
(mg/L)
Average Inhibition
(%)
Standard
Deviation (%)

Aluminum
0.5
0
4
Copper
0.6
70
1
Iron
0.15
7
6
Manganese
0.25
5
6
Zinc
2.5
12
5
Both the chlorinated and chloraminated disinfection by-product samples produced an inhibition
significantly greater than the negative control and, therefore, were considered false positive
responses. However, the disinfectant by-product samples produced an inhibition of less than 15%,
leaving enough fluorescence available for subsequent inhibition due to contamination.
False negative Botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, and VX exhibited non-detectable
response responses at the lethal dose concentration.
Ease of Use The LuminoTox PECs Test Kit contained detailed instructions and clear illustrations. The contents
of the LuminoTox PECs Test Kit 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. However, the PECs had to be stored in ice between every sample analysis to
keep them from coming to room temperature, which was somewhat inconvenient because the
melting ice caused the lab bench and operators' hands to be wet most of the time. The necessity to
record four numbers as raw data was somewhat burdensome. Lab Bell has indicated that it is
modifying this. No formal scientific education would be required to use the LuminoTox PECs Test
Kit.	
The LuminoTox PECs Test Kit was transported from a laboratory setting to a storage room for the
field portability evaluation. The LuminoTox PECs 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 47% + 1%, and in the non-laboratory location, 51% + 1%.
Throughput Approximately 20 analyses were completed per hour, and approximately 50 samples could be
analyzed with the supplies contained in one LuminoTox PECS Test Kit.
ND = Significant inhibition was not detected.
False positive
response
Field
Portability
33

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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-9 2/001, July 1992.
3.	U.S. EPA Method 335.3, "Cyanide, Total—Colorimetric, Automated UV," inMethodsfor
the Chemical Analysis of Water and Wastes, EPA/600/4-79/020, March 1983.
4.	U.S. EPA Method 200.8, "Determination of Trace Elements in Waters and Wastes by
Inductively-Coupled Plasma Mass Spectrometry," in Methods for the Determination of
Metals in Environmental Samples, Supplement I, EPA/600/R-94/111, 1994.
5.	U.S. EPA Method 200.7, "Trace Elements in Water, Solids, and Biosolids by Inductively
Coupled Plasma—Atomic Emission Spectrometry," EPA-821-R-01-010, January 2001.
6.	American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, 1997. Washington, DC.
7.	U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020.
8.	U.S. EPA Method 552.2, "Haloacetic Acids and Dalapon by Liquid-Liquid Extraction,
Derivatization and GC with Electron Capture Detector," Methods for the Determination of
Organic Compounds in Drinking Water—Supplement III EPA/600/R-95/131.
9.	U.S. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
Spectrometry," Methods for the Determination of Organic Compounds in Drinking Water—
Supplement III, EPA/600/R-95/131.
10.	American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater, 20th edition, 1998, Washington, DC.
34

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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.
35

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