November 2003
Environmental Technology
Verification Report
HlDEXOY
BloTox™
RAPID TOXICITY TESTING SYSTEM
Prepared by
Battelle
Banene
. . . Putting Technology To Work
Under a cooperative agreement with
Vy tHr\ U.S. Environmental Protection Agency
ETV ETV ET
-------
November 2003
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Hidex Oy
BioTox™
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.
11
-------
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 assess-
ment. Under a cooperative agreement, Battelle has received 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/centerl.html.
111
-------
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 17
5.5 Other Performance Factors 18
-------
6 Test Results 19
6.1 Endpoints and Precision 19
6.1.1 Contaminants 19
6.1.2 Potential Interferences 19
6.1.3 Precision 26
6.2 Toxicity Threshold 26
6.3 False Positive/Negative Responses 26
6.4 Field Portability
27
6.5 Other Performance Factors 28
7 Performance Summary 29
8 References 30
Figures
Figure 2-1. Triathler™ Luminometer with Injector 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-ld. Dicrotophos Percent Inhibition Results 21
VI
-------
Table 6-le. Thallium Sulfate Percent Inhibition Results 22
Table 6-If. Botulinum Toxin Percent Inhibition Results 22
Table 6-lg. Ricin Percent Inhibition Results 23
Table 6-lh. Soman Percent Inhibition Results 23
Table 6-li. VX Percent Inhibition Results 24
Table 6-2. Potential Interferences Results 25
Table 6-3. Toxicity Thresholds 26
Table 6-4. False Negative Results 27
vn
-------
List of Abbreviations
AMS
ASTM
ATEL
DI
DDW
EPA
ETV
HOPE
ID
LD
\iL
mL
NSDWR
%D
PE
QA
QC
QMP
TSA
Advanced Monitoring Systems
American Society for Testing and Materials
Aqua Tech Environmental Laboratories
deionized water
dechlorinated drinking water from Columbus, Ohio
U.S. Environmental Protection Agency
Environmental Technology Verification
high-density polyethylene
identification
lethal dose
microliter
milliliter
National Secondary Drinking Water Regulations
percent difference
performance evaluation
quality assurance
quality control
quality management plan
technical systems audit
Vlll
-------
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 Hidex Oy BioTox™ rapid toxicity testing system used
in conjunction with the Hidex Oy Triathler™ luminometer. Rapid toxicity testing systems were
identified as a priority technology verification category through the AMS Center stakeholder
process.
-------
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 BioTox™. Following is a description of BioTox™, based on
information provided by the vendor. The information provided below was not subjected to
verification in this test.
BioTox™ luminescent toxicity screening uses the Triathler™ luminometer, together with the
freeze-dried BioTox™ reagent, to determine the inhibitory effect of water-soluble samples,
including suspensions of solid samples. The BioTox™ reagent contains naturally luminescent
Vibrio fischeri, which produce luciferase as a part of their metabolic pathway. Luciferase
catalyzes the oxidation of a long-chain aldehyde and coenzyme, flavin mono-nucleotide.
Substances affecting any part of the metabolic pathway of the bacteria directly affect the amount
of light they emit. Toxic compounds interfere with this metabolic process, resulting in a
reduction of light emission. To determine the toxicity of a sample, changes in light output are
measured with the Triathler™ luminometer.
Sample dilutions and a control sample (2% sodium chloride) are
pipetted into test tubes (500 microliters [|J,L] each), and the
Triathler™ injector is filled with the V. fischeri reagent. The
tube containing the control sample is placed in the Triathler™
luminometer, and 500 |J,L of the reagent are measured and
injected. The measurement is taken after 5 seconds. The tube is
set aside, and the same procedure is repeated for each sample.
After a 30-minute reaction time, the tubes are shaken, and
end-point readings from the control and each sample are
measured. The inhibition of each sample dilution is calculated.
The BioTox™ kit, which provides for 144 measurements,
contains six vials of freeze-dried V. fischeri reagent, six vials of
reagent diluent (12.5 milliliters [mL] each), and one 50-mL
bottle of concentrated sample diluent.
Reagent injection and data acquisition can be performed by a
computer connected to the Triathler™ luminometer. The
dimensions of the Triathler™ luminometer are 10 inches by
10 inches by 6 inches, and it weighs approximately 10 pounds.
Figure 2-1. Triathler™
Luminometer with Injector
-------
It can only be operated on 110-volt alternating current electricity. The BioTox™ kit (which is
sufficient for 150 to 250 measurements) costs $128, the Triathler™ injector costs $1,950, and the
luminometer with liquid scintillation counter costs $6,950.
-------
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 magnd) 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, BioTox™ was subjected to various concentrations of contamin-
ants 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 BioTox™ 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.(l) BioTox™ 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 interference was confirmed independently by Aqua Tech
Environmental Laboratories (ATEL), Marion, Ohio, or by Battelle, depending on the analyte.
-------
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
BioTox™ was evaluated by
• Endpoint and precision—percent inhibition for all concentration levels of contaminants and
potential interfering compounds and precision of replicate analyses
• 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 results 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
BioTox™ 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
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,
5
-------
depends on the characteristics of the individual person and the amount of contaminant ingested.
Inhibition results (endpoints) from four replicates of each contaminant at each concentration
level were evaluated to assess the ability of BioTox™ to detect toxicity at various concentrations
of contaminants, as well as to measure the precision of BioTox™ results.
The response of BioTox™ 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,
BioTox™ 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 concen-
trations 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
concentration level, while at least four dilutions (made using the DDW) were analyzed for each
contaminant using BioTox™. 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 n DI 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 control samples, which consisted of the unspiked DDW. The method blank samples
were used to help ensure that no sources of contamination were introduced in the sample
handling and analysis procedures. Zinc sulfate was suggested by the vendor for use as the
-------
Table 3-2. Summary of Quality Control and Contaminant Test Samples
Type of Sample
Quality control
Sample Characteristics
Method blank
Positive control (zinc sulfate)
Negative control (unspiked
DDW)
Concentration
Levels (mg/L)
NS(a)
25
NS
No. of Sample Analyses
17
23
39
Aldicarb
Colchicine
Cyanide
Dicrotophos
nrinjf ..f , Thallium sulfate
DDW fortified
with contaminants Botulinum toxin*'
280; 28; 2.8; 0.28
240; 24; 2.4; 0.24
250; 25; 2.5; 0.25
1,400; 140; 14; 1.4
2,400; 240; 24; 2.4
0.30; 0.030; 0.0030;
0.00030
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
Field location
DDW fortified
with potential
interferences
Disinfectant
by-products
Ricin(c)
Soman
VX
Cyanide
Aluminum
Copper
Iron
Manganese
Zinc
Chloramination by-products
Chlorination by-products
15; 1.5; 0.15; 0.015
0.068(d)
0.22; 0.022; 0.0022;
0.00022
2.5
0.36
0.65
0.069
0.26
3.5
NS
NS
4 per concentration level
4 per concentration level
4 per concentration level
4
4
4
4
4
4
4
4
(a) NS = Samples not fortified with any contaminant or potential interference.
-------
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 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 BioTox™ 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 BioTox™ 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
of 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 n
DI water. The positive control samples were made using ASTM Type n DI water 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.
-------
3.4.2 Test Sample Analysis Procedure
The BioTox™ reagents were used in conjunction with the Hidex Triathler™ luminometer. The
Triathler™ luminometer was equipped with an injector that was used to add the BioTox™
reagents to the water sample being analyzed. To analyze the test samples, the Vibrio fischeri were
reconstituted in a 2% salt solution and placed in a beaker. The intake tubing from the injector
was placed in the solution of bacteria. The test samples were prepared by adding 450 |J,L of
sample (made in DDW) and 50 |J,L of 20% sodium chloride to a sample cuvette. The cuvette was
then placed into the Triathler™ luminometer, and 500 |J,L of bacteria were injected into the
cuvette. After approximately 5 seconds, the luminescence was recorded. The cuvette was inserted
into the Triathler™ luminometer 30 minutes later for the final luminescence measurement.
Absolute light units were reported by the Triathler™ luminometer. For each contaminant,
BioTox™ 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 BioTox™, a single concentration level of cyanide, prepared in the same way as the other
DDW samples, was analyzed in replicate by BioTox™ 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 BioTox™. Both held bachelor's
degrees in the sciences and took part in a conference call with the vendor to become accustomed
to operating BioTox™.
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 interferent 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 back-
ground 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.
-------
Table 3-3. Dose Confirmation Results
Contaminant
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum toxin
Ricin
Soman
VX
Method
EPA531.1(3)
(a)
EPA335.1(4)
EPA SW846 (8141A)(5)
EPA 200.8(6)
(a)
(a)
(c)
(c)
Average Concentration
± Standard Deviation
N = 4 (mg/L)
280 ± 28
NAW
250 ± 15
1,400± 140
2,400 ± 24
NA
NA
0.068(d) ± 0.001
0.22 ± 0.001
Background in
DDW Sample
(mg/L)
<0.0007
NA
0.008
<0.002
<0.001
NA
NA
<0.05
<0.05
Potential Interference
Aluminum
Copper
Iron
Manganese
Zinc
EPA 200.8
EPA 200.8
EPA 200.8
EPA 200.8
EPA 200.8
0.36 ±0.01
0.65 ± 0.01
0.069 ± 0.008
0.26 ± 0.01
3.5 ± 0.35
<0.10
0.011
<0.04
<0.01
0.30
(a) No standard method available. QA audits and balance calibration assured accurately prepared solutions.
(b) NA = Not applicable.
-------
Table 3-4. Water Quality Parameters
Parameter
Turbidity
Organic carbon
Specific conductivity
Alkalinity
pH
Hardness
Total organic halides
Total trihalomethanes
Total haloacetic acids
Dechlorinated Columbus,
Ohio, Tap Water
Method (disinfected by chlorination)
EPA180.1(7)
SM5310(8)
SM2510(8)
SM 2320(8)
EPA150.1(9)
EPA 130.2(9)
SM 5320B(8)
EPA 524.2(10)
EPA552.2(11)
0.1NTU(a)
2.5 mg/L
364 |j,mho
42 mg/L
7.65
112 mg/L
190|ig/L
52.8 \Lg/L
75.7 \Lg/L
Dechlorinated
St. Petersburg, Florida,
Tap Water (disinfected by
chloramination)
1.3 NTU
1.7 mg/L
502 |j,mho
90 mg/L
7.80
177 mg/L
110 \Lg/L
20.1 |ig/L
7.6 u.g/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 standard operating procedure. 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 performed. QA audits and balance calibrations assured that solutions for
these compounds were accurately prepared.
4.2 Quality Control of Drinking Water Samples
A method blank sample consisting of ASTM Type IIDI water was analyzed once by BioTox™
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 is 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 zero, it would indicate to the operator that BioTox™ was
operating incorrectly. For 23 positive control samples, the average inhibition was 58% ± 39%,
indicating the proper functioning of BioTox™.
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 n DI 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 concentra-
tion 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 700%
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
(mg/L) (mg/L)
Contaminant
Potential
interference
Aldicarb
Cyanide
Dicrotophos
Thallium sulfate
Aluminum
Copper
Iron
Manganese
Zinc
0.00448 ± 0.000320
0.207 ± 0.026
0.00728 ± 0.000699
0.090 ± 0.004
0.512 ±0.013
0.106 ±0.002
0.399 ± 0.004
0.079 ± 0.003
0.106 ±0.016
0.00500
0.200
0.00748
0.100
0.500
0.100
0.400
0.100
0.100
Percent
Difference
11
4
3
10
2
6
0.30
21
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
Sample
preparation (dates,
procedures,
concentrations)
Test parameters
(contaminant
concentrations,
location, etc.)
Stock solution
confirmation
analysis, sample
analysis, chain of
custody, and
results
Battelle
Battelle
Laboratory
record books
Laboratory
record books
Start/end of test,
and at each change
of a test parameter
When each sample
was prepared
Battelle
Battelle or
contracted
laboratory
Laboratory
record books
Laboratory
record books,
data sheets, or
data acquisition
system, as
appropriate
When set or
changed
Throughout sample
handling and
analysis process
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
Used to confirm the
concentration and
integrity of the samples
analyzed, procedures
entered into laboratory
record books
Used to organize/check
test results, manually
incorporated in data
spreadsheets as
necessary
Transferred to
spreadsheets/agreed
upon report
(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
The Triathler™ reports the absolute light units for each sample analyzed. Light measurements
were recorded at 5 seconds after bacteria injection and then again 30 minutes later. The percent
inhibition (%/) of each sample was calculated by comparing the inhibition of light in the test
sample to that in a control sample using the following equations:
k = (2)
T
%I=\1 -- F— 1x100% (3)
k-T,
J
where k is the correction factor for the control sample, CF and Cl are the final and initial light
measurements of the control sample, respectively; and TF and 7) are the final and initial light
measurements of the test samples. For all but the chlorinated and chloraminated by-product
samples, which used ASTM Type IIDI water as the control sample, DDW was used as the
control sample.
The standard deviation (S) of the results for the replicate samples were calculated, as follows, and
used as a measure of technology precision at each concentration.
S =
1 ,
n — lk=i
(4)
where n is the number of replicate samples, Ik is the percent inhibition measured for the kth
sample, and / 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
16
-------
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.
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 level had to be significantly less than that
produced by the toxicity threshold concentration, based on the standard deviation around the
average inhibitions. Since the inhibition of the negative control sample was subtracted from the
inhibition of each sample, the percent inhibition of the negative control was always zero. 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.
5.3 False Positive/Negative Responses
A response would be considered false positive if an unspiked drinking water sample produced an
inhibition significantly greater than zero when determined with respect to ASTM Type n DI
water. Depending on the degree of inhibition in the sample, toxicity due to subsequent con-
tamination 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. An inhibition was considered significantly different from zero if the
average inhibition, plus or minus the standard deviation, did not include zero.
A response was considered false negative when BioTox™ was subjected to a lethal concentration
of some contaminant in the DDW and did not indicate inhibition significantly greater than the
negative control (zero inhibition) and 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 BioTox™ in determining a false negative response. A difference was considered signifi-
cant if the average inhibition plus or minus the standard deviation did not encompass the value or
range of values that were being compared.
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 BioTox™
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 BioTox™ in
a non-laboratory setting were made by the verification test coordinator and operators. Factors
17
-------
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 chlorina-
tion 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. Samples that produced negative
percent inhibition values indicated an increase in light production by the bacteria relative to the
negative control for any of the concentrations.
6.1.1 Contaminants
Two contaminants that were analyzed by BioTox™ during this verification test produced
inhibitions significantly different from the negative control for each sample set. The inhibition
samples containing cyanide increased in proportion to the concentration in the sample, with the
two highest concentrations of cyanide causing inhibition significantly greater than the negative
control and the other concentration levels. For thallium sulfide, only the highest concentration
sample exhibited inhibition distinguishable from the negative control and the other concentration
levels. The other seven contaminants did not cause inhibition significantly greater than the
negative control.
6.1.2 Potential Interferences
Iron exhibited percent inhibitions near zero (Table 6-2), indicating little or no response to these
compounds; while aluminum and manganese exhibited slightly higher average inhibitions with
relatively large uncertainties. Zinc exhibited a considerably higher inhibition of 48%, and copper
exhibited almost complete inhibition. Copper and zinc may cause inhibitions that interfere with
the BioTox™ results.
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
accounted for within the calculation of the inhibition of each sample. To investigate whether
BioTox™ is sensitive to by-products of disinfecting processes, dechlorinated drinking water
samples from water systems that use chlorination and chloramination were analyzed and
19
-------
Table 6-la. Aldicarb Percent Inhibition Results
Concentration
(mg/L)
0.28
2.8
28
Inhibition Average Standard Deviation
(%) (%)
-14
-10 -10
-10
-7
-17
-1
10
-13
7
8 °
3
(%)
3
12
9
-3
280 4
(Lethal Dose) 11
0
Table 6-lb. Colchicine Percent Inhibition Results
Concentration
(mg/L)
0.24
2.4
24
240
(Lethal Dose)
Inhibition Average Standard Deviation
-10
-11
-27
-18 '
-68
39
-10
-9 10
21
-20
-18
-23
1
-25
t -8
27
24
11
22
21
20
-------
Table 6-lc. Cyanide Percent Inhibition Results
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
9
-19
0.25 -1 12
-1
22
2.5 „ 10 9
25
250
70
65
70 61
38
97
98
96 %
94
16
2
50
2.5 „
(Field 57
Location)
59
Table 6-ld. Dicrotophos Percent Inhibition Results
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
1.4
14
140
15
-3
12 6
0
-5
-5
15 2
2
5
-1
5
7
8
9
10
4
-2
1,400 -6
(Lethal Dose) 5
11
21
-------
Table 6-le. Thallium Sulfate
Concentration
(mg/L)
2.4
24
240
2,400
(Lethal Dose)
Inhibition
3
_2
-8
-10
16
1
10
15
15
-3
26
34
49
41
38
37
Table 6- If. Botulinum Toxin
Concentration
(mg/L)
0.0003
0.003
0.03
0.30
(Lethal Dose)
Inhibition
8
0
8
-8
14
12
13
1
1
5
-5
6
3
1
14
3
Percent Inhibition Results
Average Standard Deviation
-4 6
11 7
18 16
41 6
Percent Inhibition Results
Average Standard Deviation
2 8
10 6
2 5
5 6
22
-------
Table 6-lg. Ricin Percent Inhibition Results
Concentration
(mg/L)
0.015
0.15
1.5
15
(Lethal Dose)
Inhibition
(%)
-8
0
-8
14
3
7
5
-6
3
-5
2
12
-6
5
-13
Average Standard Deviation
(%) (%)
0 10
2 6
3
-5 7
Table 6-lh. Soman Percent Inhibition Results
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
0.00068
0.0068
0.068
0.068(a)
(Lethal Dose)
4
0
7 3
0
1
2
3 l
-1
0
0
-6
7
7
9
3
2
3
3
(a) Due to the degradation of soman in water, the stock solution confirmation
analysis confirmed that the concentration of the lethal dose was 23% of the
expected concentration of 0.30 mg/L.
23
-------
Table 6-li. VX Percent Inhibition Results
Concentration Inhibition Average Standard Deviation
(mg/L) (%) (%) (%)
0.00022
0.0022
0.022
0.22
(Lethal Dose)
5
4
2
0
0
3
4
5
8
6
-4
15
3
2
-1
10
,4,
7
2
2
9
3
compared with ASTM Type IIDI water as the control sample. This determination is crucial
because the ability of BioTox™ to detect toxicity is dependent on the light production of the
reagents in a clean drinking water matrix. If clean drinking water produces 100% inhibition of
light, the detection of subsequently added contaminants would not be possible. On average, the
chlorinated sample exhibited a negative and highly variable percent inhibition. When ASTM
Type n DI water was used as the sample, there was an approximately 10% light loss over the
30-minute reaction time. When DDW was used as the sample, the BioTox™ reagent produced
more light after the 30-minute reaction time than at the 5-second measurement, which, averaged
across all 39 negative control samples, resulted in a calculated inhibition of -49% ± 33%.
For this ETV verification test, the increased background light production when analyzing DDW
samples was not critical because "clean" negative control samples were analyzed with each
sample set so the test samples containing the contaminants could be directly compared with a
background sample that is guaranteed to be non-inhibitory. However, in a real-world scenario, a
"clean" background matrix identical to the sample matrix may not be available, and ASTM
Type n DI water as the control sample would be the likely replacement. This substitution would
not be appropriate to obtain accurate percent inhibition data. For example, when ASTM Type II
DI water was used as the control during this test, some "clean" DDW exhibited inhibitions of
approximately -49%. Therefore, if a contaminant was added to that matrix to cause a 49%
inhibition of the bacteria, the calculated inhibition of that sample would be zero percent,
24
-------
Table 6-2. Potential Interferences Results
Concentration Inhibition Average Standard Deviation
Interference (mg/L) (%) (%) (%)
4
Aluminum 0.36 ^ 16 12
26
19
95
Copper 0.65 ^ % 4
99
Iron
0
0.069 ~l 0
3
2
Manganese 0.26 „„ 10
10
33
Zinc 3.5 5552 48 10
52
Chlorination NA(a) (b) _49 33
By-products
14
Chloramination ... 13 10 „
„ , t NA 10 13 2
By-products 13
10
(a) NA = Not applicable.
-------
6.1.3 Precision
Across all the contaminants and potential interferences, the standard deviation was measured
and reported for each set of four replicates to evaluate the BioTox™ precision. Out of 42
opportunities, the standard deviation of the four replicate measurements was under 10% for
30 concentration levels, between 10% and 20% for nine concentration levels, and over 20% for
three concentration levels. Overall, the range of standard deviations was between 2 and 27%.
6.2 Toxicity Threshold
Table 6-3 gives the toxicity thresholds as defined in Section 5.2 for each contaminant. The lowest
toxicity threshold concentration was for thallium sulfate at 24 mg/L, indicating that BioTox™
was most sensitive to thallium sulfate. The only other contaminant that BioTox™ was able to
significantly distinguish from the negative control was cyanide at the two highest concentration
levels. For aldicarb, dicrotophos, colchicine, botulinum toxin, ricin, soman, and VX, no
inhibition greater than the negative control was detected, regardless of the concentration level,
indicating that the technology was not responsive to these contaminants.
Table 6-3. Toxicity Thresholds
Contaminant
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum toxin
Ricin
Soman
VX
(a) ND = Significant inhibition
Concentration (mg/L)
ND(a)
ND
25
ND
24
ND
ND
ND
ND
was not detected.
6.3 False Positive/Negative Responses
The drinking water sample disinfected by chloramination produced, on average, inhibition of
13% ± 2%. In the absence of a negative control sample of a very similar matrix, there is the
possibility of a slightly exaggerated inhibition due to the baseline inhibition of the drinking water
sample. As described in Section 6.1.2, BioTox™ produced false negative responses for unspiked
drinking water disinfected by chlorination when it was calculated with respect to ASTM Type n
DI water. The example described in that section provides an instance where contamination of the
DDW caused significant inhibition of the bacteria's light production; however, if ASTM Type n
26
-------
DI water was used as the control sample, the result would be reported as negligible inhibition
because of the negative background inhibition produced by the DDW. Therefore, when only
ASTM Type n DI water is used as the control for water samples in a different matrix, the risk for
a false negative response is great. Another type of false negative response is when a lethal dose
of contaminant is present in the water sample and the inhibition is not significantly different
either from the negative control or the other lower concentration levels. Only one of the two
criteria had to be met to be considered a false negative response. Table 6-4 gives these results.
The inhibition induced by lethal doses of cyanide and thallium sulfate was detectable by
BioTox™, while aldicarb, colchicine, dicrotophos, botulinum toxin, ricin, soman, and VX did
not indicate inhibition greater than the negative control, indicating false negative responses.
Table 6-4. False Negative Results
Lethal Dose
Concentration False Negative
Contaminant (mg/L) Result
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum toxin
Ricin
Soman
VX
280
240
250
1,400
2,400
0.30
15
0.068(a)
0.22
yes
yes
no
yes
no
yes
yes
yes
yes
-------
same concentration was 10% ± 9%. While these inhibitions are not the same, the field measure-
ments were made on freshly prepared solutions with a newly reconstituted batch of bacteria. The
precision of the results suggests that the BioTox™ reagent and Triathler™ luminometer
functioned similarly at the non-laboratory and laboratory locations.
The BioTox™ reagent must be kept at approximately -20°C prior to reconstitution and, after
reconstitution, the reagent needs to be stabilized for at least two hours. The reconstituted reagent
must be used the same day. These factors could be problematic in a long-term field deployment.
6.5 Other Performance Factors
There was no formal manual with instructions for getting the injector to function properly in
conjunction with the Triathler™ luminometer. The instructions for the BioTox™ reagent and
sample preparation were clear, but initially it was not clear how to collect the data properly in the
absence of an electronic data acquisition system. Two conference calls with the vendor and
considerable effort by the verification test coordinator were necessary to determine the proper
operational procedure for the BioTox™/Triathler™. Once operational, with help from the
vendor, the button on the Triathler™ luminometer that triggers injection of the bacteria
spontaneously changed to a different button for no apparent reason. A significant amount of time
was required to figure out which button was the correct one to use. Once the correct procedure
was determined, the BioTox™/Triathler™ was easy to use and worked correctly and con-
sistently. Although the operators had scientific backgrounds, based upon observations of the
verification test coordinator, an operator with little technical training would probably be able to
analyze multiple sample sets after adequate direction on how to perform tests correctly had been
provided. Such direction may come through contact with the vendor or an improved instruction
manual. The operators were able to analyze approximately 50 samples per hour.
28
-------
Chapter 7
Performance Summary
Parameter
Contaminants
inDDW
Potential
interferences
inDDW
False positive
response
Compound
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum
toxin(c)
Ricin(d)
Soman
VX
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Lethal
Dose (LD)
Cone.
(mg/L)
280
240
250
1,400
2,400
0.30
15
0.068(e)
0.22
Cone.
(mg/L)
0.36
0.65
0.069
0.26
3.5
Average Inhibitions at
Concentrations Relative to the LD
Concentration (%)
LD
3
-8
96
2
41
5
-5
7
8
LD/10
0
-15
61
5
18
2
3
-1
3
LD/100
-1
10
10
2
11
10
2
1
5
LD/1,000
-10
-27
-1
6
-4
2
0
3
2
Average Inhibitions at a
Single Concentration (%)
16
96
0
10
48
Range of
Standard
Deviations
(%)
3-12
11-27
2-16
4-10
6-16
5-8
6-10
2-3
2-9
Standard
Deviation (%)
12
4
2
9
10
Slightly exaggerated inhibitions may result if chloraminated water, which produced 13% :
inhibitions, is analyzed with respect to ASTM Type II DI water.
Toxicity
Thresh.
(mg/L)(a)
ND(b)
ND
25
ND
24
ND
ND
ND
ND
II
b2%
False negative
response
Inhibition greater than the negative control was not detected for lethal doses of aldicarb, colchicine,
dicrotophos, botulinum toxin, ricin, soman(c), and VX. There was a -49% ± 33% inhibition for water
from the system treated by chlorination, resulting in a risk of false negative results when using
ASTM Type II DI water as the control sample.
Field
portability
The inhibition of 2.5 mg/L cyanide in the field was 57% ± 4%, and in the laboratory it was 10% ±
9%. Practically, the operation did not seem much different. However, the Triathler™ is not
equipped for use with batteries, so electricity was required. A field-portable case with batteries may
be purchased. A flat, sturdy surface is needed to operate BioTox™ because a beaker of bacteria
must be connected to the injector.
Other
performance
factors
Although determining how to operate the BioTox™/Triathler™ was difficult without an instruction
manual and required significant intervention from the vendor, it was easy to use once the correct
procedure was determined. Although the operators had scientific backgrounds, upon observation of
the test procedures, it seems likely that an operator with little technical training would probably be
able to analyze samples successfully once provided with adequate guidance in the form of contact
with the vendor or an improved instruction manual. Sample throughput was 50 samples per hour.
See Tables 6-la-i in the report for the precision around each individual inhibition result.
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 23% of the expected concentration of 0.30 mg/L.
29
-------
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 III, 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 III, EPA/6QQ/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 EPAJ600/R-95/131.
30
-------
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.
31
------- |