November 2003
Environmental Technology
Verification Report
INTERLAB SUPPLY, LTD.
POLYTOX™
RAPID TOXICITY TESTING SYSTEM
Prepared by
Battelle
Batteiie
. . . Putting Technology To Work
Under a cooperative agreement with
U.S. Environmental Protection Agency
ETV ETV ET
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November 2003
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
InterLab Supply, Ltd.
POLYTOX™
Rapid Toxicity Testing System
by
Ryan James
Amy Dindal
Zachary Willenberg
Karen Riggs
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation by the EPA for use.
11
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
nation's air, water, and land resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, the EPA's Office of Research and Development provides data and science support that
can be used to solve environmental problems and to build the scientific knowledge base needed
to manage our ecological resources wisely, to understand how pollutants affect our health, and to
prevent or reduce environmental risks.
The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of 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. gov/etv/centers/center 1. html.
in
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. 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
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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
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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 25
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. POLYTOX™ Bacterial Cultures 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
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Table 6-le. Thallium Sulfate Percent Inhibition Results 22
Table 6-lf. 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 Responses 26
VI1
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List of Abbreviations
AMS
ASTM
ATEL
BOD
DI
DOUR
DDW
EPA
ETV
HOPE
ID
LD
mg
mL
NSDWR
%D
PE
QA
QC
QMP
SOP
ISA
Advanced Monitoring Systems
American Society for Testing and Materials
Aqua Tech Environmental Laboratories
biological oxygen demand
deionized water
dissolved oxygen uptake rate
dechlorinated drinking water from Columbus, Ohio
U.S. Environmental Protection Agency
Environmental Technology Verification
high-density polyethylene
identification
lethal dose
milligram
milliliter
National Secondary Drinking Water Regulations
percent difference
performance evaluation
quality assurance
quality control
quality management plan
standard operating procedure
technical systems audit
Vlll
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental 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 InterLab Supply, Ltd. POLYTOX™ rapid toxicity
testing system. Rapid toxicity testing systems were identified as a priority technology
verification category through the AMS Center stakeholder process.
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of environ-
mental monitoring technologies for air, water, and soil. This verification report provides results
for the verification testing of POLYTOX™. Following is a description of POLYTOX™, based
on information provided by the vendor. The information provided below was not subjected to
verification in this test.
POLYTOX™ (Figure 2-1) uses the respiration of microorganisms to indicate the toxicity of a
water or wastewater stream. When activated in water, the mixture of bacterial cultures in
POLYTOX™ begins to "breathe" like all other living organisms. They breathe in oxygen and
respire carbon dioxide. The inhibitory effect of toxicants in potable tap water (or any water-
based medium) to the bacterial cultures in POLYTOX™ is measured by evaluating the culture's
respiration rate in the presence of different concentrations of toxicants. The respiration rate is
the oxygen consumed by aerobic and facultative cultures (the dissolved oxygen update
rate—DOUR) and is expressed as milligrams (mg) of oxygen consumed per liter per minute.
The DOUR is determined by measuring the dissolved oxygen concentration at 19 and 21
minutes after adding the POLYTOX™ microbial mixture to 300 milliliters (mL) of a drinking
water sample. The DOUR of each drinking water sample is compared to a baseline DOUR
measured at the beginning of each day by
adding POLYTOX™ to a clean water
matrix and measuring the oxygen concen-
trations in a manner similar to the test
samples. For this verification test, the
vendor provided YSI 5000 and 5100
dissolved oxygen probes.
The POLYTOX™ test components
include standard 300-mL biological
oxygen demand (BOD) bottle(s) and a
dissolved oxygen probe (with stirrer) and
meter. The probe must fit snugly into the
neck of the BOD bottle, eliminating all
headspace. Also required, but not included
in the test kit, are an aeration device and
Figure 2-1. POLYTOX™ Bacterial Cultures
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one- and two-liter containers for aerating the deionized (DI) water (control). A thermometer and
a stopwatch are also provided.
The dimensions of the POLYTOX™ test kit are 8 inches x 8 inches x 4 inches. With all
necessary components, the kit size is approximately 18 inches x 18 inches x 16 inches. The
dissolved oxygen probe and meter are 9-l/2 inches x 8-V2 inches x 6 inches. When a large number
of tests are performed, data can be downloaded directly from the dissolved oxygen meter to a
laptop or desktop computer for manipulation into a usable form. The suggested price of the
POLYTOX™ culture is $147 for 20 tests. The dissolved oxygen probe and meter provided by
the vendor for use during testing cost approximately $1,600 for the complete unit.
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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
Jischeri), 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 DOUR in
the presence of the contaminants.
As part of this verification test, POLYTOX™ 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 POLYTOX™ 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) POLYTOX™ 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.
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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
POLYTOX™ 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 significantly greater than the inhibition
reported for unspiked American Society for Testing and Materials (ASTM) Type n DI water
samples (zero inhibition)
• Field portability
• Ease of use
• Throughput.
3.2 Test Design
POLYTOX™ was used to analyze the DDW sample fortified with contaminants at concentra-
tions ranging from lethal levels to concentrations several orders of magnitude times less than the
lethal dose. The lethal dose concentration was determined by calculating the concentration of
each contaminant 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 and VX the stock solution confirmation showed degradation in the water; therefore, the
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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 (endpoints) from four replicates of
each contaminant at each concentration level were evaluated to assess the ability of
POLYTOX™ to detect toxicity at various concentrations of contaminants, as well as to measure
the precision of POLYTOX™ results.
The response of POLYTOX™ 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,
POLYTOX™ 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 four concentration levels (made using the DDW) were analyzed
for each contaminant using POLYTOX™. 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 EDI water;
positive control samples, which consisted of ASTM Type n 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. Cyanide was suggested by the vendor for use as the positive
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Table 3-2. Summary of Quality Control and Contaminant Test Samples
DDW fortified
with contaminants
Field location
DDW fortified
with potential
interferences
Disinfectant
by-products
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum toxin*-1
Ricin(c)
Soman
VX
Cyanide
Aluminum
Copper
Iron
Manganese
Zinc
Chloramination by-
products
Chlorination by-products
280; 28; 2.8
240; 24; 2.4; 0.24
250; 0.25; 0.0025;
0.00025
1,400; 140; 14; 1.4
2,400; 240; 24; 2.4
0.30; 0.030; 0.0030;
0.00030
15; 1.5; 0.15; 0.015
0.18(d); 0.018;
0.0018; 0.00018
0.088(d); 0.0088;
0.00088; 0.000088
0.25
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 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4 per concentration level
4
4
4
4
4
4
4
4
W NS = Samples not fortified with any contaminant or potential interference.
^ Lethal dose solution also contained 3 mg/L phosphate and 1 mg/L sodium chloride.
(c) Lethal dose solution also contained 3 mg/L phosphate, 26 mg/L sodium chloride, and 2 mg/L sodium azide.
(d) Due to the degradation of soman and VX in water, the stock solution confirmation analysis confirmed that the
concentration of the lethal dose was 61% of the expected concentration of 0.30 mg/L for soman, and 44% of the
expected concentration of 0.20 mg/L for VX.
control sample. While performance limits were not placed on the results, inhibition of at least
50% indicated to the operator that POLYTOX™ was functioning properly. The negative control
sample was used to set a background inhibition of the DDW, the matrix in which each test
sample was prepared.
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3.3.2 Drinking Water Fortified with Contaminants
Approximately 150 liters (L) 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 n DI 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. Exceptions to this include
aldicarb, which was non-inhibitory at the 10- and 100-fold dilution levels, so no additional
dilutions were analyzed, and cyanide, which was diluted by factors of 10s and 106 from the
lethal concentration to reach non-inhibitory levels. Table 3-2 lists each concentration 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 POLYTOX™ to by-products of the chlorination process as
potential interferences, the unspiked DDW (same as the negative control) was used since the
water sample originated from a utility that uses chlorination as its disinfectant procedure. In a
similar 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 microorganisms within the POLYTOX™ 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 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
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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 tech-
nology was kept by Battelle.
3.4.2 Test Sample Analysis Procedure
Each day, prior to analyzing the test samples, the oxygen probes used in conjunction
POLYTOX™ were calibrated by adjusting the probe to 100% dissolved oxygen when it was
placed in BOD bottles containing ASTM Type n DI water that had been aerated for at least
30 minutes. Then a background sample was analyzed by inserting the oxygen probes into BOD
bottles containing only DDW and recording oxygen concentrations at 19 and 21 minutes after
inserting the probes. The last step prior to sample analysis was to analyze a baseline sample to
determine the dissolved oxygen uptake rate of unspiked DDW, the matrix in which each test
sample was prepared. This was done by starting a stopwatch and adding the contents of one
POLYTOX™ container to a BOD bottle and approximately 300 mL of DDW to fill the BOD
bottle. The oxygen probes were inserted, and, according to the vendor protocol, oxygen concen-
trations were recorded every two minutes until Minute 18 and every minute until Minute 24.
Test samples were analyzed in a manner identical to the baseline except that only oxygen
concentrations at Minutes 19 and 21 were recorded. For both the baseline and test samples, only
the data points at Minutes 19 and 21 were used for the calculation of DOUR. The additional
data acquired during the analysis of the baseline sample were not used for any additional
calculations.
For each contaminant, POLYTOX™ 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 POLYTOX™, a single concentration level of cyanide, prepared in
the same way as the other DDW samples, was analyzed in replicate by POLYTOX™ 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
POLYTOX™. Both held bachelor's degrees in the sciences and spent approximately eight hours
with the vendor to become accustomed to performing tests using POLYTOX™.
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 back-
ground levels of the contaminants and potential interferences measured in the DDW sample,
which were all non-detect or negligible.
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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)
EPASW846(8141A)(5)
EPA 200.8(6)
(a)
(a)
(<0
(<0
Average Concentration
± Standard Deviation N
= 4 (mg/L)
280 ±28
NA(b)
250 ±15
1,400 ±140
2,400 ± 24
NA
NA
0.184(d)± 0.001
0.088 ±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.
^ NA = Not applicable.
(G:I 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 61% of the expected concentration of 0.30 mg/L and
for VX was 44% of the expected concentration of 0.20 mg/L.
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.
10
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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 (disinfected
Method 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)
EPA 552.2(11)
0.1NTU(a)
2.5 mg/L
364 [irnho
42 mg/L
7.65
112 mg/L
190 |ig/L
52.8 |ig/L
75.7 |ig/L
Dechlorinated
St. Petersburg, Florida,
Tap Water (disinfected by
chloramination)
0.3 NTU
2.9 mg/L
460 [irnho
97 mg/L
7.95
160 mg/L
83 |ig/L
2.4 fig/L
13.5 |ig/L
(a) NTU = nephelometric turbidity unit.
11
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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center^ 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 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 n DI water was analyzed once by
POLYTOX™ for approximately every 20 drinking water samples that were analyzed. The
method blank samples were used as baseline samples to compare the inhibition produced by the
DDW with that produced by the method blank. A baseline sample of unspiked DDW was
analyzed to obtain a DOUR of unspiked DDW to compare the results with test samples prepared
in DDW. A negative control sample (unspiked DDW) was analyzed with approximately every
four samples and compared to the DDW baseline analyzed on the same day to determine its
inhibition. Since the negative controls were compared with a baseline sample of unspiked DDW
(same as the negative control), its inhibition should be near zero. Therefore, the scatter of the
negative control results around zero show the precision of POLYTOX™ near its detection limit.
A positive control sample of 8 mg/L cyanide 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
12
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cause greater than approximately 50% inhibition, it would indicate to the operator that
POLYTOX™ was operating incorrectly. For 15 positive control samples, the average inhibition
was 85% ± 7%, indicating the proper functioning of POLYTOX™.
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:
%D = — xlOO%
A (1)
whereMis the absolute value of the difference between the measured and the prepared concen-
tration and^4 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
Contaminant
Potential
interference
Aldicarb
Cyanide
Dicrotophos
Thallium sultate
Aluminum
Copper
Iron
Manganese
Zinc
Average Measured
Concentration ±
Standard Deviation
(mg/L)
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
Actual
Concentration
(mg/L)
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.:
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
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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 Battelle
events
Sample preparation Battelle
(dates, procedures,
concentrations)
Test parameters Battelle
(contaminant
concentrations,
location, etc.)
Laboratory
record books
Laboratory
record books
Start/end of test, and
at each change of a
test parameter
When each sample
was prepared
Laboratory
record books
When set or
changed
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
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
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Chapter 5
Statistical Methods and Reported Parameters
The statistical methods presented in this chapter were used to verify the performance parameters
listed in Section 3.1.
5.1 Endpoints and Precision
For both baseline and sample analysis using POLYTOX™, dissolved oxygen concentrations
were measured at 19 and 21 minutes after the water was added to POLYTOX™. DOURs were
calculated from these results using the following equation
DOUR = ;9mi"2;mi" (2)
2mm
where C19min and C21min are the dissolved oxygen concentrations measured at 19 and 21 minutes.
Percent inhibitions (%T) were calculated by comparing the baseline DOUR to the sample DOUR
as follows:
HI = 1 -samp 10Q% (3)
^ DOURbaselme )
During this testing, DOURbackgroundwas always zero. For all of the test samples, DOURhaseline is the
baseline DOUR measurement using unspiked DDW analyzed at the start of each testing day.
The only exceptions to this were the disinfectant by-product samples, which were compared to
an ASTM Type n DI water baseline.
The standard deviation (S) of the results for the replicate samples was calculated, as follows, and
used as a measure of technology precision at each concentration.
[7 ~\
—, ±(lk -I}'
n—]k=1\ * / J
\1/2
—, lk - (4)
*
16
-------
where n is the number of replicate samples, Ik is the percent inhibition measured for the A*h
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
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 different from the negative control. The average inhibition of the
50 negative control samples analyzed using POLYTOX™ was 3% ± 15%; therefore, for any
result to be significantly different from that of the negative control, the inhibition would have to
be greater than 18%.
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
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 n DI water. Drinking water samples collected
from water systems using chlorination and chloramination as the disinfecting process were
analyzed in this manner. Since the inhibition of ASTM Type n DI water is defined as zero, for
the same reason as described in Section 5.2, samples significantly different than zero would
have to have an inhibition greater than 18%.
A response was considered false negative when POLYTOX™ was subjected to a lethal concen-
tration of some contaminant in the DDW and did not indicate inhibition significantly greater
than the negative control and the other concentration levels analyzed. Requiring the inhibition of
the lethal dose sample to be significantly greater than the negative control (3% ± 15%) and the
other concentration levels more thoroughly incorporated the uncertainty of all the measurements
made by POLYTOX™ in determining a false negative response.
5.4 Field Portability
The results obtained from the measurements made on DDW samples in the laboratory and field
setting were compiled independently and compared to assess the performance of the
POLYTOX™ 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 POLYTOX™ in a non-laboratory setting were made by the verification test
17
-------
coordinator and operators. Factors such as 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 6-1 a-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. Samples that produced
negative percent inhibition values indicated that the sample caused an increase in the respiration
of the bacteria within POLYTOX™ relative to the negative control and was considered not toxic
to the bacteria.
6.1.1 Contaminants
The contaminants that were analyzed by POLYTOX™ during this verification test resulted in
percent inhibition data that varied considerably among the contaminants. The percent
inhibitions for thallium sulfate and ricin were significantly different from the negative control (3
± 15%) for only the highest concentration level (lethal dose). POLYTOX™ was especially
sensitive to cyanide at concentrations near the lethal dose. Cyanide concentrations as low as
0.25 mg/L (one thousand times less concentrated than the lethal dose) produced an average
percent inhibition of 61%. No inhibition significantly greater than the negative control was
produced by aldicarb, colchicine, dicrotophos, botulinum toxin, soman, and VX. The lethal
concentration of ricin was significantly different from the negative control, but not significantly
different from the lower concentrations analyzed. However, one low result (13%) of the four
replicates heavily influenced the average inhibition. If that outlier was removed, the lethal
concentration was significantly different from results from the lower concentration levels. The
three lowest concentrations of ricin produced average inhibitions ranging from 16 to 26%, but
the uncertainty (7 to 12%) suggests that these results are not significantly different from the
negative control.
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 POLYTOX™. All five potential interferences exhibited percent inhibitions that
were not significantly different from the negative control samples.
19
-------
Table 6-1 a. Aldicarb Percent Inhibition Results'
(a)
Concentration
(mg/L)
2.8
28
280
(Lethal Dose)
Inhibition
(%)
-9
-32
-16
6
0
-3
-13
27
26
19
16
Average Standard Deviation
-16 12
-3 8
22 5
Only three concentration levels of aldicarb were analyzed.
Table 6-1 b. Colchicine Percent Inhibition Results
Concentration
(mg/L)
0.24
2.4
24
240
(Lethal Dose)
Inhibition
(%)
-18
-4
-18
22
-9
-21
6
1
-24
-13
-7
-7
Average Standard Deviation
-13 6
-9 23
-6 12
-13 8
20
-------
Table 6-1 c. Cyanide Percent Inhibition Results
Concentration
(mg/L)
0.00025
0.0025
0.25
250
(Lethal Dose)
0.25
(Field Location)
Inhibition Average
11
15
7
10
9
3
7
57
62
62
64
84
84 86
87 86
87
61
67
61
67
Table 6-ld. Dicrotophos Percent Inhibition
Concentration
(mg/L)
1.4
14
140
1,400
(Lethal Dose)
Inhibition Average
11
30
13
10
8
16
19
30
-38
15 8
23 8
33
-8
~o "5
Standard Deviation
3
6
3
2
3
Results
Standard Deviation
10
9
31
4
-10
21
-------
Table 6-1 e. Thallium Sulfate
Concentration
(mg/L)
2.4
24
240
2,400
(Lethal Dose)
Inhibition
-13
0
25
0
-3
-6
6
9
3
22
19
25
16
25
34
34
41
Table 6-lf. Botulinum Toxin
Concentration
(mg/L)
0.00030
0.0030
0.030
0.30
(Lethal Dose)
Inhibition
16
0
0
18
8
20
16
14
12
20
-5
-5
-4
24
-9
10
Percent Inhibition Results
Average Standard Deviation
2 14
3 7
20 4
34 6
Percent Inhibition Results
Average Standard Deviation
9 10
14 5
6 12
3 15
22
-------
Table 6-1 g. Ricin Percent Inhibition Results
Concentration Inhibition Average Standard Deviation
(mg/L)
16
0.015 \l
5
0.15
1.5
15
(Lethal Dose)
Table 6-lh. Soman
Concentration
(mg/L)
0.00018
0.0018
0.018
0.18(a)
(Lethal Dose)
30
20 25
18
37
36
14
18
48
56 AA
44
13
59
Percent Inhibition Results
Inhibition Average
-32
-11
14 "6
5
-5
M
14
-16
37
10 6
-5
5
5
-5 1
0
7
12
21
Standard Deviation
20
11
23
5
(a) Due to the degradation of soman in water, the stock solution confirmation analysis
confirmed that the concentration of the lethal dose was 61% of the expected
concentration of 0.30 mg/L.
23
-------
Table 6-1 i. VX Percent Inhibition Results
Concentration
(mg/L)
0.000088
0.00088
0.0088
0.088(a)
(Lethal Dose)
Inhibition Average Standard Deviation
(%) (%) (%)
-4
9
-10 °
5
17
4
-5 °
-15
26
39
-5 19
15
17
22 4
-10
-15
9
14
19
19
Due to the degradation of VX in water, the stock solution confirmation analysis
confirmed that the concentration of the lethal dose was 44% of the expected
concentration of 0.20 mg/L.
All of the contaminant and potential interference samples were prepared in the DDW and
compared with the baseline sample of unspiked DDW. Therefore, any background inhibition in
the DDW should be accounted for in the percent inhibition calculation. To investigate whether
POLYTOX™ is sensitive to by-products of the disinfecting processes, dechlorinated drinking
water samples from water systems that use chlorination and chloramination were analyzed and
compared with ASTM Type n DI water as the baseline sample. This determination is crucial
because the ability of POLYTOX™ to detect toxicity is dependent on its baseline DOUR in a
clean drinking water matrix. If clean drinking water produces 100% inhibition of DOUR, the
detection of subsequently added contaminants would not be possible. On average, the
chlorinated sample exhibited inhibitions of 10 ± 15%, while the chloraminated sample exhibited
inhibitions of 27% ± 4%. This suggests that by-products of either disinfection process that may
be present in drinking water could interfere with POLYTOX™ results if they are compared with
baseline measurements in ASTM Type n DI water. If a matrix similar to the drinking water
sample being analyzed is used as the baseline sample, there would probably be no interference.
24
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Table 6-2. Potential Interferences Results
Concentration Inhibition Average Standard Deviation
Compound (mg/L) (%) (%) '°/-
Aluminum 0.36
Copper 0.65
Iron 0.069
3
o
-17 "8
-17
3
0
10
0
3
* 7
5
20
11
4
9
Manganese 0.26
Zinc
15
35 10 11
13
8
3
Chlorination NA(a) ^ 1Q 15
by-products
31
Chloramination .... 21 ._ .
U J 4. NA 00 27 4
by-products 28
28
(a) Sample not fortified with any contaminant or potential interferent.
^ Chlorination by-product data averaged over the negative control results with respect to the inhibition of
ASTM Type IIDI water (N = 11).
6.1.3 Precision
Across all the contaminants, the standard deviation was measured and reported for each set of
four replicates to evaluate POLYTOX™ precision. For the test samples, the standard deviations
ranged from 2% to 31%. Out of 43 sample sets, 22 had standard deviations less than 10%, 17
had standard deviations of between 10% and 20%, and four were greater than 20%. However,
when the average inhibition of the sample was greater than 20% (with the exception of ricin),
the standard deviations were always below 10%. These precision results were consistent with
those of the negative and positive control samples, where the average inhibition for the negative
control samples was 3 ± 15% and 85% ± 7% for the positive control sample. When the
inhibition was high, the uncertainty was low; and, near the detectable limit, the uncertainty was
high.
25
-------
6.2 Toxicity Threshold
Table 6-3 gives the toxicity thresholds (i.e., lowest concentration of contaminant with percent
inhibition significantly greater than the negative control) for each contaminant. The lowest
toxicity threshold concentration was for cyanide at 0.25 mg/L, indicating that POLYTOX™ was
most sensitive to cyanide. For aldicarb, colchicine, dichrotophos, botulinum toxin, soman, and
VX, inhibition significantly greater than the negative control was not detected, regardless of the
concentration level, indicating that the technology was not highly responsive to these
contaminants.
Table 6-3. Toxicity Thresholds
Contaminant Concentration (mg/L)
Aldicarb ND(a)
Colchicine ND
Cyanide 0.25
Dicrotophos ND
Thallium sulfate 2,400
Botulinum toxin ND
Ricin 15
Soman ND
VX ND
-------
A false negative response is when a lethal dose of contaminant is present in the drinking water
sample and the inhibition is not significantly different from the negative control. Table 6-4 gives
these results. The inhibition induced by lethal doses of cyanide, thallium sulfate, and ricin was
detectable by POLYTOX™, while aldicarb, colchicine, dicrotophos, botulinum toxin, soman,
and VX did not indicate inhibition significantly greater than the negative control, indicating
false negative results.
Table 6-4. False Negative Responses
Lethal Dose
Concentration False Negative
Contaminant (mg/L) Response
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum toxin
Ricin
Soman
VX
280
240
250
1,400
2,400
0.30
15
0.18(a)
0.088(a)
yes
yes
no
yes
no
yes
no
yes
yes
Due to the degradation of soman and VX in water, the stock solution
confirmation analysis confirmed that the concentration of the lethal dose
of soman was 61% of the expected concentration of 0.30 mg/L and of VX
was 44% of the expected concentration of 0.20 mg/L.
6.4 Field Portability
POLYTOX™ was used to prepare and analyze a single concentration of cyanide in replicate at a
field location to examine its use in a non-laboratory setting. POLYTOX™, the oxygen probe and
meter, and necessary accessories were transported to the field location in a medium-sized
cardboard box because a single carrying case was not provided by the vendor. At the field
location, the oxygen probe was operated, using batteries as the power source, on a small table in
the basement of a house. A flat, sturdy surface is necessary to operate POLYTOX™ because of
the requirement for using BOD bottles and oxygen probes. Also, because some overflow of the
water sample occurs when the oxygen probe is inserted, a secondary container was used to
contain the planned spill. Since 300 mL of sample are required for each sample, a sizable waste
container was necessary for field work. Table 6-1 c shows the results for the cyanide samples
analyzed at the field location, along with the results of the cyanide samples analyzed in the
laboratory. The concentration of the solution analyzed in the field was 0.25 mg/L. The inhibition
produced in the field was 64% ± 3%, and the inhibition produced in the laboratory at the same
concentration was 61% ± 3%. These inhibitions are not significantly different from one another.
The agreement of the field results with those in the laboratory indicate that POLYTOX™
27
-------
functioned similarly at both locations. POLYTOX™ can be kept at room temperature prior to
use, making it convenient for long-term field deployment.
6.5 Other Performance Factors
The step-by-step pictorial instruction manual for POLYTOX™, combined with a half-day
training session with the vendor, enabled operators to become quickly adept at analyzing
samples. POLYTOX™ was very straightforward to operate. Baseline and background measure-
ments took approximately one hour to complete. After those were complete, the ETV operators
analyzed three samples per hour. Each sample required approximately 300 mL of water; there-
fore, 8 L of waste were generated each day when analyzing large sample sets. The membranes on
the oxygen probes were changed every 40 to 50 samples. Although the operators had scientific
backgrounds, based on observations of the verification test coordinator, an operator with little
technical training would probably be able to successfully analyze drinking water samples using
POLYTOX™.
28
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Chapter 7
Performance Summary
Parameter
Contaminants in
DDW
Potential
interferences in
DDW
Compound
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum
toxm(d)
Ricm(e)
Soman
VX
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Lethal
Dose (LD)
Cone.
(mg/L)
280
240
250(c)
1,400
2,400
0.30
15
0.18®
0.088(f)
Cone.
(mg/L)
0.36
0.65
0.069
0.26
3.5
Average Inhibitions at
Concentrations Relative to the LD
Concentration (%)
LD
22
-13
86
-5
34
3
44
1
4
LD/10
o
-J
-6
61
8
20
6
26
6
19
LD/100
-16
-9
3
18
o
J
14
25
5
0
LD/1,000
NA®
-13
11
16
2
9
16
-6
0
Average Inhibitions at a
Single Concentration (%)
-8
5
7
6
11
Range of
Standard
Deviations (%)
5-12
6-23
2-6
4-31
4-14
5-15
7-21
5-23
9-19
Standard
Deviation (%)
11
4
9
5
3
Toxicity
Thresh.
(mg/L)(a)
ND
ND
0.25
ND
2,400
ND
15
ND
ND
I
False positive
response
Average inhibitions for chloraminated and chlorinated drinking water were 27% ± 4% and 10% ±
15%, respectively. The chloraminated water result was false positive because the inhibition was
significantly greater than the negative control. However, both types of water had positive average
inhibition, so a water matrix similar to the negative control should be used to compare background
inhibition due to disinfectant by-products and inhibition due to toxic contaminants.
False negative
response
At the lethal concentration level, aldicarb, colchicine, dicrotophos, botulinum toxin, soman, and VX
inhibitions were not significantly different from the negative control or inhibition was generated by
lower concentrations of the same contaminant, indicating false negatives.
Field portability
Performance in the field was similar to performance in laboratory. A flat, sturdy surface is needed
for BOD bottles and oxygen probes. Not including reference and background samples, 300 mL of
waste were generated for every sample. A carrying case was not provided. Overflow upon inserting
oxygen probe required a secondary container.
Other
performance
factors
The pictorial manual was useful, and sample throughput was three samples per hour. Each sample
required 300 mL of water; 8 L/day of waste were generated per oxygen probe. Oxygen probe
membranes changed once per 40 to 50 samples. Although the operators had scientific backgrounds,
operators with little technical training would probably be able to successfully analyze samples.
See Tables 6-la-I in the report for the precision around each individual inhibition result.
ND = Not detectable.
LD/10, LD/100, LD/1,000 concentrations for cyanide are 0.25, 0.0025, and 0.00025 mg/L respectively.
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 and VX in water, the stock solution confirmation analysis confirmed that the
concentration of the lethal dose of soman was 61 % of the expected concentration of 0.30 mg/L and of VX was
44% of the expected concentration of 0.20 mg/L.
29
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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. EPAMethod 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/600/R-95/131.
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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 IIIEPA/600/R-95/131.
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.
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