November 2003
Environmental Technology
Verification Report
Hach Company
ToxTrak™
Rapid Toxicity Testing System
Prepared by
Battel le
Batteiie
Putting Technology To Work
Under a cooperative agreement with
SEPA U.S. Environmental Protection Agency
ETV ElV ElV

-------
November 2003
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Hach Company
ToxTrak™
Rapid Toxicity Testing System
by
Ryan James
Amy Dindal
Zachary Willenberg
Karen Riggs
Battelle
Columbus, Ohio 43201

-------
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

-------
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 seven 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. ep a. go v/et v/centers/center 1. html.
iii

-------
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. Many thanks go to Battelle's Medical
Research and Evaluation Facility for providing the facilities for and personnel capable of
working with chemical warfare agents and biotoxins. 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/QA plan and this verification
report.
iv

-------
Contents
Page
Notice	ii
Foreword 	 iii
Acknowledgments 	 iv
List of Abbreviations 	 viii
1	Background 	 1
2	Technology Description 	2
3	Test Design and Procedures 	4
3.1	Introduction	4
3.2	Test Design 	5
3.3	Test Samples 	6
3.3.1	Quality Control Samples 	6
3.3.2	Drinking Water Fortified with Contaminants	8
3.3.3	Drinking Water Fortified with Potential Interferences	8
3.4	Test Procedure	8
3.4.1	Test Sample Preparation and Storage	8
3.4.2	Test Sample Analysis Procedure 	9
3.4.3	Stock Solution Confirmation Analysis	9
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 		15
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	Field Portability		18
5.5	Other Performance Factors 		18
v

-------
6	Test Results		19
6.1	Endpoints and Precision 		19
6.1.1	Contaminants		19
6.1.2	Potential Interferences 	25
6.1.3	Precision 	27
6.2	Toxicity Threshold	27
6.3	False Positive/Negative Responses 	28
6.4	Field Portability	28
6.5	Other Performance Factors 	29
7	Performance Summary	30
8	References 	31
Figures
Figure 2-1. ToxTrak™ Rapid Toxicity Testing System 	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. Dose 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. Colchicine Percent Inhibition Results 	20
Table 6-lc. Cyanide Percent Inhibition Results 	21
Table 6-Id. Dicrotophos Percent Inhibition Results	22
vi

-------
Table 6-le.	Thallium Sulfate Percent Inhibition Results	22
Table 6-If.	Botulinum Toxin Percent Inhibition Results 	23
Table 6-lg.	Ricin Percent Inhibition Results 	23
Table 6-lh.	Soman Percent Inhibition Results 	24
Table 6-li.	VX Percent Inhibition Results	25
Table 6-2.	Potential Interference Results	26
Table 6-3.	Toxicity Thresholds	27
Table 6-4.	False Negative Responses 	28
vii

-------
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
EPA
U.S. Environmental Protection Agency
ETV
Environmental Technology Verification
HDPE
high-density polyethylene
ID
identification
LD
lethal dose
mg
milligram
mL
milliliter
NSDWR
National Secondary Drinking Water Regulations
%D
percent difference
PE
performance evaluation
QA
quality assurance
QC
quality control
QMP
quality management plan
SOP
standard operating procedure
TSA
technical systems audit
viii

-------
Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental tech-
nologies 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 tech-
nologies 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 Hach Company ToxTrak™ rapid toxicity testing
system. Rapid toxicity testing systems were identified as a priority technology verification
category through the AMS Center stakeholder process.
1

-------
Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of environ-
mental monitoring technologies for air, water, and soil. This verification report provides results
for the verification testing of ToxTrak™. Following is a description of ToxTrak™, based on
information provided by the vendor. The information provided below was not subjected to
verification in this test.
ToxTrak™ is a colorimetric test based on resazurin dye chemistry. Resazurin is a redox-active
dye that, when reduced, changes color from blue to pink. Resazurin is in the oxidized, blue state
at the beginning of the test. The bacteria oxidize the glucose added to the sample with the dye and
reduce the resazurin. The resazurin is first reduced by two electrons to resomfin, which is pink.
Resorufin can be further reduced by two electrons to dihydroresorufin, which is colorless.
Dihydroresorufin can be reoxidized by atmospheric oxygen to resorufin. To prevent interference,
readings must be taken before a significant amount of resorufin has been reduced. This inhibition
or acceleration of resazurin reduction is taken as an indication of toxicity in the test. Substances
that are toxic to bacteria can inhibit their metabolism and thus inhibit the rate of resazurin
reduction. If the reaction time is too long, the indicator is too far reduced and interference will
result.
ToxTrak™ (Figure 2-1) uses an accelerant
(gluteraldehyde) to reduce the reaction time, thus
preventing oxygen interference and allowing the
use of a lower level of inoculum to reduce the
dye. Because of the decrease in turbidity resulting
from a smaller inoculum, the absorbance of the
dye can be read on a colorimeter or spectro-
photometer without removing the bacterial cells
from the light path. This alleviates the need for
organic extraction and/or centrifugation. With the
reduced turbidity, the color change of the dye can
be distinguished visually. Also, the decreased
reaction time eliminates the interference caused
by overreduction of the dye.
ToxTrak™ works with different species of
bacteria (including both Gram positive and Gram negative species) or mixed cultures. The
ToxTrak™ kit includes 12 reusable sample cells with caps, several capsules of dried bacteria,
'yr
Figure 2-1. ToxTrak™ Rapid Toxicity
Testing System
2

-------
lauryl tryptose broth for culturing the bacteria, 50 ToxTrak™ Reagent Powder Pillows,
15 milliliters (mLs) of ToxTrak™ accelerator solution, 20 sterile transfer pipettes, a test tube rack,
forceps, five germicidal cloths, a lab marker, illustrated instructions, and a carrying case.
The percent inhibition results are only a relative measurement. They do not represent a true
quantitative measurement of toxic concentration. The percent inhibition does not necessarily
increase in direct proportion to the concentration of contaminants. To determine the toxicity
threshold of a toxin, it is possible to make tenfold dilutions of the sample and determine the
percent inhibition for the dilutions until the sample is diluted sufficiently so that no inhibition is
observed. Due to the many variables involved in the test, the limits of detection are on the order of
10% inhibition. Percent inhibition results more than 10% or more negative than -10% should be
considered toxic. The percent inhibition results of several samples should be evaluated before
determining whether or not a sample is toxic. Consistently detectable results indicate a high
likelihood of toxicity.
For this verification test, the vendor provided a Hach DR/4000V spectrophotometer for the
laboratory-based colorimeter measurements and a Hach DR/890 handheld colorimeter for the
non-laboratory measurements. Any colorimeter that can analyze samples at a wavelength at or
near 603 nanometers could be used in conjunction with the ToxTrak™ reagents. The ToxTrak™
kit costs $280, and reagent sets cost $100. The reagent set can be used with the test kit, a
spectrophotometer, or a colorimeter. The spectrophotometer used in this verification test cost
$3,950.
3

-------
Chapter 3
Test Design and Procedures
3.1 Introduction
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. Rapid toxicity technologies use bacteria (e.g., Vibrio fischeri), enzymes (e.g.,
luciferase), or small crustaceans (e.g., Daphnia magna) that either directly, or in combination
with reagents, produce a background level of light or use dissolved oxygen at a steady rate in the
absence of toxic contaminants. Toxic contaminants in water are indicated by a change in the color
or intensity of light produced or by a decrease in the dissolved oxygen uptake rate in the presence
of the contaminants.
As part of this verification test, ToxTrak™ 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 ToxTrak™ can detect the toxicity caused by each
contaminant, its response to interfering compounds in clean drinking water, such as water
treatment chemicals and by-products, was evaluated. Table 3-1 shows the contaminants and
potential interferences that were evaluated during this verification test.
This verification test was conducted according to procedures specified in the Test/QA Plan for
Verification of Rapid Toxicity Technologies.(1) ToxTrak™ was verified by analyzing a
dechlorinated drinking water (DDW) sample from Columbus, Ohio, fortified with various
concentrations of the contaminants and interferences shown in Table 3-1. Hereafter in this report,
DDW will refer to dechlorinated drinking water from Columbus, Ohio. 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.
4

-------
Table 3-1. Contaminants and Potential Interferences
Category
Contaminant
Carbamate pesticide
aldicarb
Pharmaceutical
colchicine
Industrial chemical
cyanide
Organophosphate pesticide
dicrotophos
Rodenticide
thallium sulfate
Biological toxins
botulinum toxin, ricin
Nerve agents
soman, VX
Potential interferences
aluminum, copper, iron, manganese, zinc,
chloramination by-products, and chlorination
by-products
ToxTrak™ was evaluated by
•	Endpoint and precision—quantitative evaluation of the percent inhibition and precision for all
concentration levels of contaminants and potential interfering compounds; also a qualitative
evaluation of the presence or absence of each contaminant and potential interference at each
concentration level
•	Toxicity threshold for each contaminant
•	False negative responses—contaminants that were reported as producing inhibition results
similar to the negative control when the contaminant was present at lethal concentrations
•	False positive responses—occurrence of inhibition significantly greater than the inhibition
reported for unspiked American Society for Testing and Materials (ASTM) Type II deionized
(DI) water samples (zero inhibition)
•	Field portability
•	Ease of use
•	Throughput.
3.2 Test Design
ToxTrak™ was used to analyze the DDW sample fortified with contaminants at concentrations
ranging from lethal levels to concentrations 1,000 times less than the lethal dose. The lethal dose
of each contaminant was determined by calculating the concentration at which 250 mL of water
would probably cause the death of a 154-pound person. These calculations were based on
toxicological data available for each contaminant. For soman, the stock solution confirmation
5

-------
showed degradation in the water; therefore, the concentrations analyzed were less than
anticipated. Whether the concentration is still a lethal dose, as is the case for all contaminants,
depends on the characteristics of the individual person and the amount of contaminant ingested.
Inhibition results from four replicates of each contaminant at each concentration level were
evaluated quantitatively to assess the ability of ToxTrak™ to detect toxicity at various
concentrations of contaminants, as well as to measure the precision of ToxTrak™ results.
Additionally, a qualitative evaluation of the data was performed to determine if the ToxTrak was
able to indicate the presence of each contaminant at various concentration levels.
The response of ToxTrak™ 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 approximately 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 from St.
Petersburg, Florida, which uses chloramination as its disinfection process, was obtained. 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 and the
verification test coordinator. In addition to comprehensive testing in Battelle laboratories,
ToxTrak™ was operated in the basement of a Columbus, Ohio, home to test its ability to be
transported and operated in a non-laboratory setting.
3.3 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. Using this DDW (Columbus, Ohio, dechlorinated drinking
water), individual solutions containing each contaminant and potential interference were prepared
and analyzed. The DDW containing the potential interferences was analyzed at a single concen-
tration level, while four concentration levels (made using the DDW) were analyzed for each
contaminant using ToxTrak™. Mixtures of contaminants and interfering compounds were not
analyzed. One concentration level of cyanide was analyzed in the field setting.
3.3.1 Quality Control Samples
QC samples included method blank samples, which consisted of ASTM Type IIDI water; positive
control samples, which consisted of ASTM Type II DI water or DDW (depending on vendor
preference) fortified with a contaminant and concentration selected by the vendor; and negative
6

-------
Table 3-2. Summary of Quality Control and Contaminant Test Samples


Concentration

Type of Sample
Sample Characteristics
Levels (mg/L)
No. of Sample Analyses

Method blank
NS(a)
21

Positive control
5,000
24
Quality control
(formaldehyde)



Negative control (unspiked
NS
38

DDW)



Aldicarb
280; 28; 2.8; 0.28
4 per concentration level

Colchicine
240; 24; 2.4; 0.24
4 per concentration level

Cyanide
250; 25; 2.5; 0.25
4 per concentration level

Dicrotophos
1,400; 140; 14; 1.4
4 per concentration level

Thallium sulfate
2,400; 240; 24; 2.4
4 per concentration level
DDW fortified
with contaminants
Botulinum toxin03'
0.30; 0.030; 0.0030;
0.00030
4 per concentration level

Ricin(c)
15; 1.5; 0.15; 0.015
4 per concentration level

Soman
0.15(d); 0.015;
0.0015; 0.00015
4 per concentration level

VX
0.22; 0.022; 0.0022;
0.00022
4 per concentration level
Field location
Cyanide
250
4

Aluminum
0.36
4
DDW fortified
Copper
0.65
4
with potential
interferences
Iron
0.069
4

Manganese
0.26
4

Zinc
3.5
4
Disinfectant
by-products
Chloramination by-
products
Chlorination by-products
NS
NS
4
4
(a)	NS = Samples not fortified with any contaminant or potential interference.
(b)	Lethal dose solution also contained 3 mg/L phosphate and 1 mg/L sodium chloride.
-------
3.3.2	Drinking Water Fortified with Contaminants
Approximately 150 liters of Columbus, Ohio, tap water were collected in a high-density
polyethylene (HDPE) container. The sample was dechlorinated with 0.5 milliliter (mL) of 0.4 M
sodium thiosulfate for every liter of water. All subsequent test samples were prepared from this
DDW and stored in glass containers to avoid chlorine leaching from HDPE containers.
A stock solution of each contaminant was prepared in ASTM Type IIDI water at concentrations
above the lethal dose level. The stock solution was diluted in DDW 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 concentra-
tion level and the number of samples analyzed at each level.
3.3.3	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 ToxTrak™ to by-products of the chlorination process as potential inter-
ferences, 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 test
involving the by-products of the chloramination process, an additional water sample was obtained
from St. Petersburg, Florida, a city that uses chloramination as its disinfectant procedure. 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.
3.4 Test Procedure
3.4.1 Test Sample Preparation and Storage
A drinking water sample was collected as described in Section 3.3.2 and, because free chlorine
kills the bacteria within the ToxTrak™ reagent and can degrade the contaminants during storage,
was immediately dechlorinated with sodium thiosulfate. Prior to preparing each stock solution,
dechlorination of the water sample was qualitatively confirmed by adding an n,n-diethyl-p-
phenylenediamine tablet to a 25-mL aliquot of the DDW. Once dechlorination was confirmed, all
the contaminant samples, potential interference samples, and negative control QC samples were
made from this DDW, while the method blank sample was prepared from ASTM Type II DI water.
The positive control samples were made using the DDW in Class A volumetric glassware. All QC
samples were prepared prior to the start of the testing and stored at room temperature for a
maximum of 60 days. The aliquots of DDW containing the contaminants were prepared within
seven days of testing and stored in the dark at room temperature without chemical preservation.
Aliquots to be analyzed by each technology were placed in uniquely labeled sample containers.
The sample containers were assigned an identification (ID) number. A master log of the samples
and sample ID numbers for each technology was kept by Battelle.
8
-------
3.4.2	Test Sample Analysis Procedure
In preparation for the analysis of test samples, a capsule containing dried bacteria was crushed at
the bottom of a container filled with lauryl tryptose broth, and the solution was incubated at 35°C
overnight. To analyze a test or control sample, 5 mL of sample, one ToxTrak™ Reagent Powder
Pillow, two drops of accelerator solution, and 0.5 mL of the bacteria solution that had been
incubated overnight were added to a sample cell. The cell was capped and mixed by shaking, and
was then placed in the spectrometer for absorbance measurement. An initial measurement was
made on all cells within the sample set. The spectrometer reported absorbances for each measure-
ment. The absorbance of the control sample, unspiked DDW, was periodically measured. When its
absorbance had decreased by 0.400 to 0.700 absorbance units, the absorbances of the rest of the
test samples were measured a second time. The reaction time was 90 to 120 minutes.
For each contaminant, ToxTrak™ analyzed the lethal dose concentration and three additional
concentration levels four times. Only one concentration of potential interference was analyzed. To
test the field portability of ToxTrak™, a single concentration level of cyanide, prepared in the same
way as the other DDW samples, was analyzed in replicate by ToxTrak™ in the basement of a
Columbus, Ohio, home. Sample analysis procedures were performed in the same way as during
testing in the laboratory. Two operators performed all the analyses using ToxTrak™. Both held
bachelor's degrees in the sciences and spent approximately one hour with the vendor to become
accustomed to performing tests using ToxTrak™ and the accompanying spectrometers.
3.4.3	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—
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 concentrations 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 reporting 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 the
concentration of trihalomethanes, haloacetic acids, and total organic halides; turbidity; dissolved
organic carbon content; pH; alkalinity; specific conductivity; and hardness. 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 in St. Petersburg, Florida, representing a water system using chloramination as the
disinfecting process.
9
-------
Table 3-3. Dose Confirmation Results

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



Aldicarb
EPA 531.1(3)
280 ± 28
<0.0007
Colchicine
(a)
NA03'
NA
Cyanide
EPA 335.1(4)
250 ± 15
0.008
Dicrotophos
EPA SW846 (8141A)®
1,400 ± 140
<0.002
Thallium sulfate
EPA 200.8(6)
2,400 ± 24
<0.001
Botulinum toxin
(a)
NA
NA
Ricin
(a)
NA
NA
Soman
(<0
0.15(d)± 0.001
<0.05
VX
(<0
0.22 ± 0.02
<0.05
Potential Interference


Aluminum
EPA 200.8
0.36 ±0.01
<0.10
Copper
EPA 200.8
0.65 ±0.01
0.011
Iron
EPA 200.8
0.069 ± 0.008
<0.04
Manganese
EPA 200.8
0.26 ±0.01
<0.01
Zinc
EPA 200.8
3.5 ±0.35
0.30
(a)	No standard method available. QA audits and balance calibration assured accurately prepared solutions.
(b)	NA = Not applicable.
(c)	Purity analyses performed on chemical and biological agent materials using Battelle standard operating procedures.
(d)	The result of the dose confirmation analysis for soman was 51% of the expected concentration of 0.30 mg/L.
10
-------
Table 3-4. Water Quality Parameters
Dechlorinated
Dechlorinated Columbus, St. Petersburg, Florida,
Ohio, Tap Water (disinfected Tap Water (disinfected by
Parameter
Method
by chlorination)
chloramination)
Turbidity
EPA 180.1(7)
0.1 NTU(a)
0.3 NTU
Organic carbon
SM 5310(8)
2.5 mg/L
2.9 mg/L
Specific conductivity
SM 2510(S)
364 |imho
460 |imho
Alkalinity
SM 2320(S)
42 mg/L
97 mg/L
pH
EPA 150.1(9)
7.65
7.95
Hardness
EPA 130.2(9)
112 mg/L
160 mg/L
Total organic halides
SM 5320B(8)
190 |ig/L
83 |ig/L
Total trihalomethanes
EPA 524.2(10)
52.8 |ig/L
2.4 |ig/L
Total haloacetic acids
EPA 552.2(11)
75.7 |ig/L
13.5 |ig/L
(a) NTU = nephelometric turbidity unit.
11
-------
Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center(12) and the test/QA plan for this verification test.(1)
4.1 Quality Control of Stock Solution Confirmation Methods
The stock solutions for aldicarb, cyanide, dicrotophos, and thallium sulfate were analyzed using a
standard reference method at ATEL. 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 and soman, the confirmation analyses were performed
at Battelle using a Battelle SOP. Calibration standard recoveries of VX and soman were always
between 69% and 130%, and most of the time were between 90% and 100%. Standard analytical
methods for colchicine, ricin, and botulinum toxin were not available and, therefore, were not per-
formed. 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 ToxTrak™
for approximately every 20 drinking water samples that were analyzed. These samples set a
baseline response for a clean water matrix. A negative control sample (unspiked DDW) was
analyzed with approximately every four samples. The inhibitions of the test samples were
calculated with respect to the negative control samples analyzed within the same analysis set.
Therefore, any inhibition significantly greater than zero was due to the contaminants and not the
DDW matrix. A positive control sample also was analyzed once for approximately every 20
drinking water samples. While performance limits were not placed on the results of the positive
control sample, the vendor informed Battelle that, if the positive control samples did not cause
inhibition significantly greater than 50%, it would indicate to the operator that ToxTrak™ was
operating incorrectly. In 27 positive control samples analyzed, the average inhibition was 80%
with a standard deviation of 27%, indicating the proper functioning of ToxTrak™.
12
-------
4.3 Audits
4.3.1 Performance Evaluation Audit
The concentration of the standards used to prepare the contaminant and potential interferences
was confirmed by analyzing solutions of each analyte prepared in ASTM Type IIDI water from
two separate commercial vendors using the confirmation methods. The standards from one source
were used to prepare the stock solutions during the verification test, while the standards from a
second source were used exclusively to confirm the accuracy of the measured concentration of the
first source. The percent difference (%D) between the measured concentration of the performance
evaluation (PE) sample and the prepared concentration of that sample was calculated using the
following equation:
M
%D = — x 100%
A	(1)
where M is the absolute value of the difference between the measured and the prepared concen-
tration and A is the prepared concentration. The %D between the measured concentration of the
PE standard and the prepared 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.
Given the lack of confirmation methodology for some of the contaminants in this verification test,
PE audits were not performed for all of the contaminants. 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. To assure the purity of the other standards,
documentation, such as certificates of analysis, was obtained for colchicine, botulinum toxin, and
ricin. In the case 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 technical systems audit (TSA) to ensure that the
verification test was performed in accordance with the test/QA plan(1) and the AMS Center
QMP.(12) As part of the audit, the Battelle Quality Manager reviewed the contaminant standard
and stock solution confirmation methods, compared actual test procedures with those specified in
the test/QA plan, and reviewed data acquisition and handling procedures. Observations and
findings from this audit were documented and submitted to the Battelle verification test
coordinator for response. No findings were documented that required any significant action. The
records concerning the TSA are permanently stored with the Battelle Quality Manager.
13
-------
Table 4-1. Summary of Performance Evaluation Audit


Average Measured




Concentration ±




Standard Deviation
Actual Concentration
Percent


(mg/L)
(mg/L)
Difference

Aldicarb
0.00448 ± 0.000320
0.00500
11

Cyanide
0.207 ± 0.026
0.200
4
Contaminant
Dicrotophos
0.00728 ± 0.000699
0.00748
3

Thallium
0.090 ± 0.004
0.100
10

sulfate




Aluminum
0.512 ±0.013
0.500
2

Copper
0.106 ±0.002
0.100
6
Potential
interference
Iron
0.399 ± 0.004
0.400
0.30

Manganese
0.079 ± 0.003
0.100
21

Zinc
0.106 ±0.016
0.100
6
The EPA Quality Manager also conducted a TSA to ensure that the verification test was
performed in accordance with the test/QA plan(1) and the AMS Center QMP.(12) As part of the
audit, the EPA Quality Manager compared actual test procedures with those specified in the
test/QA plan and reviewed data acquisition and sample preparation records and procedures. No
significant findings were observed during the EPA TSA. The records concerning the TSA are
permanently stored with the EPA Quality Manager.
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.(12) 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.
14
-------
4.5 Data Review
Records generated in the verification test were reviewed before these records 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 initials and the date to a hard copy of the record being reviewed.
Table 4-2. Summary of Data Recording Process
Data to be
Recorded
Responsible
Party
Where
Recorded
How Often
Recorded
Disposition of Data(a)
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
(a) All activities subsequent to data recording were carried out by Battelle.
15
-------
Chapter 5
Statistical Methods and Reported Parameters
The statistical methods presented in this chapter were used to verify the performance parameters
listed in Section 3.1.
5.1 Endpoints and Precision
Initial and final absorbance readings were recorded from the spectrometer for each sample
analyzed. Each DDW sample containing contaminants was compared with a negative control
sample that, for this verification test, was unspiked DDW. This comparison was made by
accounting for the background inhibition of the DDW when calculating the percent inhibition.
Each test sample was compared to a negative control sample analyzed in the same set as the test
sample. The percent inhibition was calculated using the following equation:
% inhibition
j ^44sample
AA
V	control J
xlOO	(2)
Apinai Ajnitial
where A represents absorbance measurements made using the spectrometers.
The test/QA plan for this verification test(1) describes only a quantitative evaluation of the percent
inhibition data generated by each technology. The ToxTrak™ manufacturer indicated during the
review of this report that a qualitative data evaluation should also be performed to describe how a
typical user is more likely to interpret and use the ToxTrak™ results. Specifically, the manu-
facturer suggested that the percent inhibition results for each concentration level of each
contaminant also be evaluated as a qualitative indicator of whether or not a toxic contaminant is
present. The manufacturer stated that the percent inhibition results for each contaminant do not
necessarily increase linearly with the concentration of the contaminant but, depending on the
contaminant, can at times be represented by a non-linear relationship that may exhibit parabolic
functionality that increases in response, up to a certain concentration, but then begins to decrease.
Per the manufacturer's instructions, percent inhibition results greater than 10% or less than -10%
are considered an indication of the presence of the contaminant. Results between and including
-10% and 10% indicate the absence of toxic contaminants. The presence/absence data trend
among the four replicates was evaluated to determine if ToxTrak™ consistently indicated the
16
-------
presence (or absence) of the contaminants at the measured concentrations. Three out of four
positive responses were required to indicate the presence of a contaminant at that concentration
level. If two results were positive and two negative, the overall result was not considered a
positive or a negative result.
The standard deviation (S) of the percent inhibition results for the replicate samples was
calculated, as follows, and used as a measure of technology precision at each concentration:
-11/2
/ n / —\2
5= ——
' k=lK
n ,	-x2
X /, -/
Ir — 1^	'
(3)
where n is the number of replicate samples, Ik is the percent inhibition measured for the kth
sample, and I is the average percent inhibition of the replicate samples. Because the average
inhibitions were 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 so the reader could easily view the uncertainty
around the average for results that were both near zero and significantly larger than zero. A
quantitative evaluation of precision was not appropriate for the qualitative results. However,
reproducibility was shown, to a limited extent, by the requirement that three out of four replicates
at a single concentration level must exhibit positive results for that concentration level to be
identified as detectable.
5.2	Toxicity Threshold
The toxicity threshold was defined as the lowest concentration of contaminant to exhibit a
percent inhibition significantly different from the negative control. For the quantitative results, the
toxicity threshold was calculated as the concentration at which the results were significantly less
than -10% or significantly greater than 10%, and the inhibition produced by each lower
concentration level was significantly less than that produced by the toxicity threshold
concentration. For the qualitative results, the toxicity threshold was defined as the lowest
concentration level that exhibited percent inhibitions either more negative than -10% or more than
10% in at least three out of four replicates. For both the quantitative and qualitative results, the
concentration levels higher than the toxicity threshold were required to meet their respective
detectability requirements.
5.3	False Positive/Negative Responses
A response was considered false positive if an unspiked drinking water sample produced an
inhibition significantly different from zero, as described above, when determined with respect to
ASTM Type IIDI water. Depending on the degree of inhibition in the sample, toxicity due to
subsequent contamination of that sample may not be detectable or could be exaggerated as a
result of the baseline inhibition. To test for this possibility, the percent inhibition of the unspiked
drinking water was determined with respect to ASTM Type II DI water. Drinking water samples
collected from water systems using chlorination and chloramination as the disinfecting process
were analyzed in this manner. A qualitative result was considered false positive if the water
17
-------
sample exhibited percent inhibitions results that were either less than -10% or more than 10% in
at least 3 out of 4 replicates when analyzed with respect to ASTM Type IIDI water.
A response was considered false negative when ToxTrak™ was subjected to a lethal concentration
of some contaminant in the DDW and did not indicate significant inhibition as defined in
Section 5.2 and significantly different from the other concentration levels analyzed. Requiring the
inhibition of the lethal dose sample to be significantly greater than the negative control and the
other concentration levels more thoroughly incorporated the uncertainty of all the measurements
made by ToxTrak™ in determining a false negative response. A qualitative response was consid-
ered false negative if the lethal dose concentration level did not exhibit percent inhibitions that
were either less than -10% or more than 10% in at least 3 out of 4 replicates.
5.4 Field Portability
The results obtained from the measurements made on DDW samples in the laboratory and in the
field were compiled independently and compared to assess the performance of the ToxTrak™
under different analysis conditions. Means and standard deviations of the endpoints generated in
both locations were used to make the comparison. Also, qualitative observations of ToxTrak™ in a
non-laboratory setting were made by the verification test coordinator and operators. Factors such
as the ease of transport and set-up, demand for electrical power, and space requirement were
documented.
5.5 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 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
-------
Chapter 6
Test Results
6.1 Endpoints and Precision
Tables 6-la-i present the percent inhibition data for nine contaminants, and Table 6-2 presents
data for five potential interferences and the drinking water samples disinfected by both
chlorination and chloramination. 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 calculated according to the test/QA
plan. Samples that produced negative percent inhibition values indicated an increase in
metabolism by the bacteria relative to the negative control. According to the vendor literature, any
sample that causes inhibition significantly greater than 10% or more negative than -10% should
be considered toxic. A qualitative interpretation of each replicate sample, although not planned,
also is included, with a "+" sign, indicating the presence of a contaminant at that concentration
level (i.e., percent inhibition results that are greater than 10% or less than -10%), or a sign
indicating the absence of a contaminant at that concentration level (i.e., percent inhibition results
that are between -10% and 10%). The presence/absence data trend among the four replicates was
evaluated to determine if ToxTrak™ consistently indicated the presence (or absence) of the
contaminants at the measured concentrations. Three out of four positive responses were required
to indicate the presence of a contaminant at that concentration level. If two results were positive
and two negative, the overall result was not considered a positive or a negative result.
6.1.1 Contaminants
The contaminants that were analyzed by ToxTrak™ during this verification test produced results
with a high degree of variability, making it difficult to quantitatively interpret the data. The only
contaminant that met the requirements for quantitative detection as defined in Section 5.2 was
cyanide. Cyanide was consistently detected at only the 250-mg/L level. The percent inhibitions of
the two highest concentrations of thallium sulfate were significantly more negative than -10%, but
these concentrations levels were not distinguishable from another. The rest of the contaminants
were mostly not significantly more negative than -10% or significantly more positive than 10%.
A qualitative evaluation of the percent inhibition results revealed that the presence of a toxic
contaminant was indicated for aldicarb at 2.8 and 280 mg/L, colchicine at 240 mg/L, cyanide at
25 and 250 mg/L, dicrotophos at 14, 140, and 1,400 mg/L, thallium sulfate and ricin at all four
19
-------
Table 6-la. Aldicarb Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/ Overall
Absence Result
7
0.28 j5 -11 22
-41
NC(a)
+
+
-3
-13
2.8 12 24
39
+
+
+
+
-3
28 .32 -7 17
8
+
-11
28° -16
(Lethal Dose) -17
-19
+
+
+
+
+
(a) NC = Not consistently positive or negative.
Table 6-lb. Colchicine Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/ Overall
Absence Result
10
11
0.24 8 3
4
+
-8
-4
2.4 2 "3 4
-5
-
-15
5
24 8 24
41
+
NC(a)
+
11
240 9
14 5
(Lethal Dose) 16
21
+
+
+
+
(a) NC = Not consistently positive or negative.
20
-------
Table 6-lc. Cyanide Percent Inhibition Results


Quantitative
Qualitative
Concentration
Inhibition
Average
Standard Deviation
Presence/ Overall
(mg/L)
(%)
(%)
(%)
Absence Result

-33


+
0.25
-10
7
-5
-10
17
-

-16


+
2.5
-4
3
-6
-6
7
-

17


+
25
0
14
12
11
7
+
+
+

86


+
250
65
72
10
+
+
+
+
(Lethal Dose)
65
71

76


+
250(a)
82
83
11
+
+
+
+
(Field Location)
100
76
(a) Measurements made by Hach DR890 colorimeter.
21
-------
Table 6-Id. Dicrotophos Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/ Overall
Absence Result
-5
-33
1.4 n -12 14
0
NC(a)
+
-66
-50
14 _29 -37 28
-2
+
+
+
+
-22
-155
140 22 -53 69
-12
+
+
+
+
+
-51
1,400 475 60 X?
(Lethal Dose) -32
17
+
+
+
+
+
(a) NC = Not consistently positive or negative.
Table 6-le. Thallium Sulfate Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/A Overall
bsence Result
-94
2.4 J -38 44
-52
+
+
+
+
-17
^ /T
24 " -21 22
-40
+
+
+
+
-21
240 -37 22
-55
+
+
+
+
+
-26
2 400 -145
/t iVr, ^ -104 62
(Lethal Dose) -83
-163
+
+
+
+
+
22
-------
Table 6-If. Botulinum Toxin Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/ Overall
Absence Result
17
-3
0.00030 25 18 15
31
+
+
+
+
3
24
0.0030 , 6 14
6
-10
+
5
12
5 6
0.030
-3
+
5
0.30 -9
(Lethal Dose) 17
29
NC(a)
+
+
(a) NC = Not consistently positive or negative.
Table 6-lg. Ricin Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/ Overall
Absence Result
-32
-35
0.015 3? -45 20
-75
+
+
+
+
+
-18
-13
0.15 ?3 -33 27
-28
+
+
+
+
+
-22
-33
1.5 _44 -38 13
-51
+
+
+
+
+
-35
^ -32 11
(Lethal Dose) -17
-34
+
+
+
+
+
23
-------
Table 6-lh. Soman Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/ Overall
Absence Result
-5 -10 6
-5
0.00015
-16
NC(a)
+
+
-21 -21 3
-18
0.0015 _23
-24
+
+
+
+
+
-29 -24 6
90
0.015 ;24
-16
+
+
+
+
+
-1
0 1500 -20
A
(Lethal Dose) -13
9
NC
+
(a)	NC = Not consistently positive or negative.
(b)	Due to the degradation of soman in water, the stock solution confirmation analysis confirmed that the
concentration of the lethal dose was 51% of the expected concentration of 0.30 mg/L.
24
-------
Table 6-li. VX Percent Inhibition Results
Quantitative
Qualitative
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
Presence/ Overall
Absence Result
7
1
0.00022 22 9 9
5
+
-17
10
0.0022 . -6 12
-6
-13
+
NC(a)
+
-3
2
0.022 -5 12
-22
+
-16
°*22 _24 16 8
(Lethal Dose) -5
-19
+
+
+
+
(a) NC = Not consistently positive or negative.
concentration levels, botulinum toxin at only the lowest concentration level (0.00030 mg/L),
soman at 0.0015 and 0.015 mg/L, and VX at 0.22 mg/L. These qualitative results indicating the
presence of a toxic contaminant did not consistently correlate with increasing concentration,
which is a performance trend that is noted in the manufacturer's literature.
6.1.2 Potential Interferences
Table 6-2 presents the results from the samples that were analyzed to test the effect of potential
interferences on ToxTrak™. Quantitatively, aluminum, copper, zinc, and manganese exhibited
percent inhibitions not significantly greater than 10% or significantly less than -10%, indicating
little or no response to these compounds, while iron exhibited an inhibition of -36% ± 23%,
indicating a slightly elevated response. Qualitative evaluation of these results revealed the
presence of a toxic contaminant in response to only iron.
All of the contaminant and potential interference samples were prepared in the DDW and
compared with unspiked DDW. Therefore, any background inhibition in the DDW was corrected
by subtracting the inhibition caused by the negative control sample. To investigate whether
ToxTrak™ is sensitive to by-products of disinfecting processes, dechlorinated drinking water
samples from water systems that use chlorination and chloramination were analyzed and com-
pared with ASTM Type IIDI water as the control sample. This determination is crucial because
the ability of ToxTrak™ to detect toxicity is dependent on the light production of the reagents in a
25
-------
Table 6-2. Potential Interference Results
Concentration
Compound (mg/L)
Quantitative
Qualitative
Average Standard Deviation
Inhibition (%) (%) (%)
Presence/ Overall
Absence Result
Aluminum 0.36
-17
"22 -3 10
5
+
Copper 0.65
-23
I -6 14
-11
+
NC(a)
+
Iron 0.069
-37
-38 "36 23
-63
+
+
+
+
Manganese 0.26
14
24
5 11 10
2
+
+ NC
Zinc 3.5
2
_179 -17 19
-43
+ NC
+
Chlorination . T»rw
i , . NAW
by-products
(c)	45 14
(d)	-7 13
NA
Chloramination ^
by-products
-14
-12 11
-23
+
+
+
+
(a)	NC = Not consistently positive or negative.
(b)	NA = Not applicable.
(c)	Chlorination by-product data from July.
(d)	Chlorination by-product data from September.
clean drinking water matrix. Approximately half of the replicates of the water samples from the
water system using chlorination were analyzed in July (with non-chem/bio agent contaminants)
and half in September (with chem/bio agent contaminants). In July, this sample exhibited an
average inhibition of 45% ± 14% and, in September, an average inhibition of -7% ± 13%. The
reason for the difference in inhibition is not clear, given that the only difference in the analyses
was the amount of time that had passed. The significantly positive inhibition measured in July
indicates that the DDW could interfere with the ToxTrak™ results. For the water sample that uses
chloramination as the disinfection process, the inhibition with respect to ASTM Type IIDI water
was -11% ± 11%, indicating no toxicity. No interference is likely when performing analyses in
this drinking water matrix. Qualitative evaluation of these results revealed that the by-products of
chlorination (July results) and chloramination are likely to interfere with the results from
26
-------
ToxTrak™. The elevated response to these background samples indicates a potential for false
results, according to both the qualitative and quantitative data evaluations.
6.1.3 Precision
Quantitatively, the standard deviations of the replicate samples were rather large. Out of 43
opportunities, in only 12 instances were the standard deviations of four replicates smaller than
10%. Standard deviations were as high as 82% and were often greater than 15%. Precision was
not an appropriate parameter for qualitative evaluation of the data, but consistency of the reported
results was interpreted as an overall positive or negative trend in Table 6-1 a-i above.
6.2 Toxicity Threshold
In Table 6-3, the quantitative toxicity threshold as defined in Section 5.2 is presented for each
contaminant. The only contaminant meeting the quantitative definition was cyanide at a
concentration level of 250 mg/L. For aldicarb, botulinum toxin, colchicine, dichrotophos, ricin,
soman, thallium sulfate, and VX, requirements for detection were not met, regardless of the
concentration level, indicating that the technology was not highly responsive to these
contaminants.
Table 6-3. Toxicity Thresholds
Toxicity Threshold Concentration (mg/L)
Contaminant
Quantitative Evaluation
Qualitative Evaluation
Aldicarb
ND(a)
280
Colchicine
ND
240
Cyanide
250
25
Dicrotophos
ND
14
Thallium sulfate
ND
2.4
Botulinum toxin
ND
ND
Ricin
ND
0.015
Soman
ND
ND
VX
ND
0.22
«
ND = Significant inhibition was not detected.
Table 6-3 also gives the qualitative toxicity thresholds as defined in Section 5.2 for each
contaminant. ToxTrak™ indicated the presence of a toxic contaminant for botulinum toxin at
0.00030 mg/L and soman at 0.0015 mg/L, but these results were not considered to be the toxicity
threshold because ToxTrak™ did not determine the higher concentration levels of those
27
-------
contaminants to be toxic. ToxTrak™ detected the toxicity due to ricin at a concentration of 0.015
mg/L, indicating that ToxTrak™ was most sensitive to that contaminant.
6.3 False Positive/Negative Responses
In July, false positive responses were observed for unspiked drinking water from the system that
uses chlorination as its disinfectant process. In September, that same sample was largely non-
inhibitory. Therefore, there seems to be a risk of false positive responses for such samples, but it
is not clear why these results were not consistent. The water sample treated by chloramination and
then subsequently dechlorinated caused no detectable inhibition. These results were consistent for
both quantitative and qualitative data evaluation.
A false negative response was when a lethal dose of contaminant was present in the water sample,
and the inhibition was not significantly larger than 10% or more negative than -10% and was also
significantly greater than the other lower concentration levels. Table 6-4 presents the quantitative
interpretation of the false negative responses. The inhibition induced by the lethal dose of cyanide
was detectable by ToxTrak™, while the other contaminants did not indicate significant inhibition,
indicating false negative responses. Table 6-4 also presents the qualitative false negative
responses. When evaluating the data qualitatively, only botulinum toxin and soman were
considered not to be toxic contaminants at the lethal dose concentration level, and thus were
considered false negative responses.
Table 6-4. False Negative Responses
Contaminant
Lethal Dose
Concentration
(mg/L)
False Negative Response
Quantitative Evaluation Qualitative Evaluation
Aldicarb
280
yes
no
Colchicine
240
yes
no
Cyanide
250
no
no
Dicrotophos
1,400
yes
no
Thallium sulfate
2,400
yes
no
Botulinum toxin
0.30
yes
yes
Ricin
15
yes
no
Soman
0.15(a)
yes
yes
VX
0.22
yes
no
(a) Due to the degradation of soman in water, the stock solution confirmation analysis confirmed that the
concentration of the lethal dose was 51% of the expected concentration of 0.30 mg/L.
28
-------
6.4 Field Portability
The Hach DR890 colorimeter was used for the inhibition measurements at the field location. A
single concentration of cyanide was prepared and analyzed in replicate to examine the ability of
ToxTrak™ to be used in a non-laboratory setting. ToxTrak™ and necessary accessories were trans-
ported to the field in a medium-sized cardboard box because the carrying case was not provided
by the vendor. At the field location, ToxTrak™ was operated with batteries on a small table in the
basement of a house. Table 6-lc shows the results of the cyanide samples analyzed at the field
location, along with the results of the cyanide samples analyzed in the laboratory. The concen-
tration of the solution analyzed in the field was 250 mg/L. From a qualitative perspective, all of
the cyanide results at 250 mg/L produced both in the laboratory and in the field were positive
responses. The inhibition produced in the field was 83% ±11%, and the inhibition produced in
the laboratory at the same concentration was 72% ± 10%. The overlapping results indicate that
ToxTrak™ functioned similarly at the laboratory and non-laboratory locations. The ToxTrak™
reagent must be incubated overnight at 35°C prior to use. This could be problematic for field
deployment. According to the manufacturer, bacteria lypholyzed in the tubes and ready for
immediate deployment currently are available, but were not on the market when the verification
test was conducted.
6.5 Other Performance Factors
The step-by-step pictorial instruction manual for ToxTrak™ was easy to understand, which
enabled operators to become quickly adept at analyzing multiple sample sets. Although the
operators had scientific backgrounds, based on observations of the verification test coordinator,
operators with little technical training would probably be able to analyze samples using only the
instruction manual for guidance. ToxTrak™ was very straightforward to operate. The operators
analyzed approximately 25 samples per hour.
29
-------
Chapter 7
Performance Summary
Parameter
Compound
Lethal
Dose
(LD)
Cone.
(mg/L)
Average Percent Inhibitions at
Concentrations Relative to the LD
Concentration (Qualitative Result:
"+" = Present= absent)
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)'"1
LD
LD/10
LD/100
LD/1,000
Quan.
Qual.
Contaminants
in DDW
Aldicarb
280
-16 (+)
-7 (-)
12 (+)
-11 (NC)(b)
3-24
ND(C)
280
Colchicine
240
14 (+)
8 (NC)
-3 (-)
8 (-)
3-24
ND
240
Cyanide
250
72 (+)
11 (+)
-6 (-)
-10(-)
7-17
250
25
Dicrotophos
1,400
-60 (+)
-53 (+)
-37 (+)
-12 (NC)
14-82
ND
14
Thallium sulfate
2,400
-104 (+)
-37 (+)
-21 (+)
-38 (+)
22-62
ND
2.4
Botulinum toxin(d)
0.30
10 (NC)
5 (-)
6 (-)
18 (+)
6-16
ND
ND
Ricin(e)
15
-32 (+)
-38 (+)
-33 (+)
-45 (+)
11-27
ND
0.015
Soman
0.15®
-6 (NC)
-24 (+)
-21 (+)
-10 (NC)
3-13
ND
ND
VX
0.22
-16 (+)
-5 (-)
-6 (NC)
9 (-)
8-12
ND
0.22
Potential
interferences
in DDW
Interference
Cone.
(mg/L)
Average Inhibitions at a
Single Concentration (%)
Standard
Deviation
(%)
¦
Aluminum
0.36
-3 (-)
10
Copper
0.65
-6 (NC)
14
Iron
0.069
-36 (+)
23
Manganese
0.26
11 (NC)
10
Zinc
3.5
-17 (NC)
19
False positive
response
45% ± 14% inhibition in dechlorinated water from system disinfected by chlorination for samples
analyzed in July. Samples analyzed in September were non-inhibitory. The water sample from a water
system disinfected by chloramination was non-inhibitory (-11% ± 11%). Qualitative results were
consistent with quantitative results (i.e., both interpretation methods indicated false positive responses
with these matrices).
False negative
responses
According to the quantitative data interpretation, inhibition greater than the negative control was not
detected for lethal doses of any contaminant except cyanide (i.e., all contaminants except for cyanide
produced false negative results). According to the qualitative data interpretation, botulinum toxin and
soman exhibited false negative results.	
Field
portability
ToxTrak™ performance in the field was similar to its performance in the laboratory both
quantitatively and qualitatively. The carrying case was not provided by the vendor. Hach DR890
handheld colorimeter was used for field measurements. Overnight incubation of bacteria may be
inconvenient for field deployment.
Other
performance
factors
Pictorial manual was useful, sample handling was easy, and sample throughput was approximately
25 samples per hour. Although the operators had scientific backgrounds, operators with little technical
training would probably be able to analyze sample using only instruction manual as guide.	
«
(b)
(c)
Ml
(e)
ffi
See Tables 6-la-i in the report for the precision around each individual inhibition result.
NC = Not consistently positive or negative.
ND = Not detectable.
Lethal dose solution also contained 3 mg/L phosphate and 1 mg/L sodium chloride.
Lethal dose solution also contained 3 mg/L phosphate, 26 mg/L sodium chloride, and 2 mg/L sodium azide.
Due to the degradation of soman in water, the stock solution confirmation analysis confirmed that the concentration of the lethal
dose was 51% of the expected concentration of 0.30 mg/L.
30
-------
Chapter 8
References
1.	Test/QA Plan for Verification of Rapid Toxicity Technologies, Battelle, Columbus, Ohio,
June 2003.
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 531.1, "Measurement of n-Methylcarbamoyloximes and
n-Methylcarbamates in Water by Direct Aqueous Injection HPLC with Post Column
Derivatization," in Methods for the Determination of Organic Compounds in Drinking
Water—Supplement 111, EPA/600/R-95/131, 1995.
4.	U.S. EPA Method 335.1, "Cyanides, Amenable to Chlorination," in Methods for the
Chemical Analysis of Water and Wastes, EPA/600/4-79/020, March 1983.
5.	SW846 Method 8141 A, "Organophosphorous Compounds by Gas Chromatography:
Capillary Column Technique," Revision 1, September 1994.
6.	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
Organic Compounds in Drinking Water, Supplement I, EPA/600/R-94/111, 1994.
7.	U.S. EPA Method 180.1, "Turbidity (Nephelometric)," Methods for the Determination of
Inorganic Substances in Environmental Samples, EPA/600/R-93/100, 1993.
8.	American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, 1997. Washington, DC.
9.	U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020.
10.	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 111, EPA/600/R-95/131.
11.	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.
31
-------
12. Quality Management Plan (QMP)for the ETV Advanced Monitoring Systems Center,
Version 4.0, U.S. EPA Environmental Technology Verification Program, Battelle, Columbus,
Ohio, December 2002.
32
-------