June 2006
Environmental Technology
Verification Report
ABRAXIS
ABRATOX KIT
Prepared by
Battelle
Batrene
/m? Business of Innovation
Under a cooperative agreement with
U.S. Environmental Protection Agency
ET1/ET1/ET1/
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June 2006
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Abraxis
AbraTox Kit
by
Mary Schrock
Ryan James
Amy Dindal
Zachary Willenberg
Karen Riggs
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement and/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 six environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received EPA funding to plan,
coordinate, and conduct such verification tests for "Advanced Monitoring Systems for Air,
Water, and Soil" and report the results to the community at large. Information concerning this
specific environmental technology area can be found on the Internet at http://www.epa.gov/etv/
centers/center 1 .html.
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. We would also like to thank
Karen Bradham, U.S. EPA National Exposure Research Laboratory; Steve Allgeier, U.S. EPA
Office of Water; Ricardo DeLeon, Metropolitan Water District of Southern California; Yves
Mikol, New York City Department of Environmental Protection; and Stanley States, Pittsburgh
Water and Sewer Authority, for their careful review of the test/quality assurance plan and this
verification report.
IV
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Contents
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
Chapter 1 Background 1
Chapter 2 Technology Description 2
Chapters Test Design 4
3.1 Test Samples 6
3.1.1 Quality Control Samples 6
3.1.2 Drinking Water Fortified with Contaminants 6
3.1.3 Drinking Water Fortified with Potential Interferences 7
3.2 Test Procedure 7
3.2.1 Test Sample Preparation and Storage 7
3.2.2 Test Sample Analysis Procedure 7
3.2.3 Stock Solution Confirmation Analysis 9
Chapter 4 Quality Assurance/Quality Control 12
4.1 Quality Control of Stock Solution Confirmation Methods 12
4.2 Quality Control of Drinking Water Samples 12
4.3 Audits 13
4.3.1 Performance Evaluation Audit 13
4.3.2 Technical Systems Audit 13
4.3.3 Audit of Data Quality 14
4.4 QA/QC Reporting 14
4.5 Data Review 15
Chapter 5 Statistical Methods and Reported Parameters 16
5.1 Endpoints and Precision 16
5.2 Toxicity Threshold 17
5.3 False Positive/Negative Responses 17
5.4 Other Performance Factors 18
Chapter 6 Test Results 19
6.1 Endpoints and Precision 19
6.1.1 Contaminants 19
6.1.2 Potential Interferences 32
6.1.3 Precision 32
6.2 Toxicity Threshold 34
6.3 False Positive/Negative Responses 34
6.4 Other Performance Factors 35
6.4.1 Ease of Use 35
6.4.2 Field Portability 36
6.4.3 Throughput 36
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Chapter 7 Performance Summary 37
Chapters References 38
Figures
Figure 2-1. Abraxis AbraTox Kit 2
Tables
Table 3-1. Contaminants and Potential Interferences 5
Table 3-2. Summary of Quality Control and Contaminant Test Samples 8
Table 3-3. Stock Solution Confirmation Results 10
Table 3-4. Water Quality Parameters 11
Table 4-1. Summary of Performance Evaluation Audit 14
Table 4-2. Summary of Data Recording Process 15
Table 6-la. Aldicarb Percent Inhibition Results 20
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results 21
Table 6-lc. Colchicine Percent Inhibition Results 22
Table 6-ld. Cyanide Percent Inhibition Results 23
Table 6-le. Dicrotophos Percent Inhibition Results 24
Table 6-1 f Nicotine Percent Inhibition Results 24
Table 6-lg. Nicotine Percent Inhibition Results—Additional Dilutions 25
Table 6-1 h. Ricin Percent Inhibition Results 26
Table 6-li. Ricin Percent Inhibition (Compared to Preservative Blank) 26
Table 6-lj. Soman Percent Inhibition Results 28
Table 6-lk. Thallium Sulfate Percent Inhibition Results 29
Table 6-11. VX Percent Inhibition Results 29
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results 30
Table 6-3. Potential Interferences Results 33
Table 6-4. Toxicity Thresholds 34
Table 6-5. False Negative Responses 35
VI
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List of Abbreviations
AMS Advanced Monitoring Systems
ASTM American Society for Testing and Materials
ATEL Aqua Tech Environmental Laboratories
cm centimeter
DI deionized water
DDW dechlorinated drinking water from Columbus, Ohio
DPD n,n-di ethyl-p-phenylenediamine
EPA U.S. Environmental Protection Agency
ETV Environmental Technology Verification
HDPE high-density polyethylene
LD lethal dose
mM millimolar
uL microliter
mg/L milligram per liter
mL milliliter
mm millimeter
NSDWR National Secondary Drinking Water Regulations
%D percent difference
PE performance evaluation
QA quality assurance
QC quality control
QMP quality management plan
SOP standard operating procedure
TSA technical systems audit
vn
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental
technologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative
technologies by developing test plans that are responsive to the needs of stakeholders,
conducting field or laboratory tests (as appropriate), collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality
assurance (QA) protocols to ensure that data of known and adequate quality are generated and
that the results are defensible.
The EPA's National Exposure Research Laboratory and its verification organization partner,
Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center
recently evaluated the performance of the Abraxis AbraTox Kit. Rapid toxicity technologies
were identified as a priority 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
environmental monitoring technologies for air, water, and soil. This verification report provides
results for the verification testing of the kit. Following is a description of the AbraTox, based on
information provided by the vendor. The information provided below was not verified in this
test.
The AbraTox Kit (Figure 2-1) is an in vitro testing system that uses a naturally occurring and
non-pathogenic bioluminescent bacteria Vibrio fischeri (strain NRRL-B-11177) to determine the
toxicity of water-soluble samples. Vibrio fischeri, when properly grown, emits light as part of its
metabolic pathway; the emitted light is an indication of the metabolic status of the bacterium.
Differences in the amount of light produced can therefore be correlated to bacterial metabolism.
Toxic compounds interfere with the
metabolic process, resulting in a
reduction of light emission. The
reduction of light emitted is proportional
to the toxicity of the sample—the more
toxic the sample, the greater percentage
of light reduction.
The AbraTox Vibrio fischeri reagent
vials are supplied freeze dried. To
analyze the water samples, the vials are
reconstituted with 2.5 milliliter (mL) of
cold reconstitution solution and allowed
to hydrate under refrigerated conditions
for 30 minutes. Meanwhile, 800 micro-
liters (uL) of the water sample to be
Figure 2-1. Abraxis AbraTox Kit
analyzed are added to test cuvettes, followed by the addition of 100 uL of osmotic adjusting
buffer, and allowed to incubate in the refrigerated incubation chamber for at least 15 minutes.
Then, 100 uL of the diluted bacteria are added to a negative control and to each test sample and
incubated in the refrigerated incubation chamber for 15 to 60 minutes. Luminescence is then
measured using a portable luminometer. Significant changes in luminescence compared to the
negative control (or reference sample) reflect the toxicity of a sample.
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The AbraTox Kit contains six vials of freeze-dried bacteria, two vials of reconstitution solution,
one bottle of Osmotic Adjusting Buffer, and one vial of positive and negative control. Test
cuvettes, a repeater pipette (100 uL), and a 200 to 1,000 uL pipette and tips are required but not
provided.
The box containing the AbraTox Kit has dimensions of 18 by 13 by 8 centimeters (cm). The
AbraTox luminometer is 20 by 8 by 5 cm, uses 2 AA batteries, and weighs 0.3 kilograms. It can
be integrated (although it was not during this test) with a personal computer for data acquisition,
evaluation, and storage. The price of the AbraTox Kit (150 single tests) is $250, the luminometer
is $2,000, and the incubation chamber is $250.
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Chapter 3
Test Design
The objective of this verification test of rapid toxicity technologies was to evaluate their ability
to detect certain toxins and to determine their susceptibility to interfering chemicals in a
controlled experimental matrix. Rapid toxicity technologies do not identify or determine the
concentration of specific contaminants, but serve as a screening tool to quickly determine
whether water is potentially toxic.
As part of this verification test, the AbraTox Kit was subjected to various concentrations of
contaminants such as industrial chemicals, pesticides, rodenticides, pharmaceuticals, nerve
agents, and biological toxins. Each contaminant was added to separate drinking water samples
and analyzed. In addition to determining whether the AbraTox Kit can detect the toxicity caused
by each contaminant, its response to interfering compounds such as water treatment chemicals
and by-products in clean drinking water, was evaluated. Table 3-1 shows the contaminants and
potential interferences that were evaluated during this verification test.
This verification test was conducted from August to December 2005 according to procedures
specified in the Test/QA Plan for Verification of Rapid Toxicity Technologies including
Amendments 1 and 2.(1) The AbraTox Kit was verified by analyzing a dechlorinated drinking
water sample from Columbus, Ohio (hereafter in this report referred to as DDW), fortified with
various concentrations of the contaminants and interferences shown in Table 3-1. Where
possible, the concentration of each contaminant or potential interference was confirmed
independently by Aqua Tech Environmental Laboratories (ATEL), Marion, Ohio, or by Battelle,
depending on the analyte.
The AbraTox was evaluated by
• Endpoints and precision—percent inhibition for all concentration levels of contaminants and
potential interfering compounds and precision of replicate analyses
• Toxicity threshold for each contaminant—contaminant level at which higher concentrations
generate inhibition significantly greater than the negative control and lower concentrations
do not
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Table 3-1. Contaminants and Potential Interferences
Category
Biological toxins
Botanical pesticide
Carbamate pesticide
Industrial chemical
Nerve agents
Organophosphate pesticide
Pharmaceutical
Potential interferences
Rodenticide
Contaminant
Botulinum toxin complex B, ricin
Nicotine
Aldicarb
Cyanide
Soman, VX
Dicrotophos
Colchicine
Aluminum, copper, iron, manganese, zinc, chloramination
by-products, and chlorination by-products
Thallium sulfate
• False positive responses—chlorination and chloramination by-product inhibition with respect
to unspiked American Society for Testing and Materials (ASTM) Type II deionized (DI)
water samples
• False negative responses—contaminants that were reported as producing inhibition similar to
the negative control when present at lethal concentrations or negative inhibition that could
cause falsely low inhibition
• Other performance factors (sample throughput, ease of use, reliability).
The AbraTox Kit was used to analyze the DDW samples fortified with contaminants at
concentrations ranging from lethal levels to concentrations up to 100,000 times less than the
lethal dose. The lethal dose of each contaminant was determined by calculating the concentration
at which 250 milliliters (mL) of water would probably cause the death of a 154-pound person.
These calculations were based on toxicological data available for each contaminant that are
presented in Amendment 2 of the test/QA plan.(1) Inhibition results (endpoints) from four
replicates of each contaminant at each concentration level were evaluated to assess the ability of
the AbraTox to detect toxicity at various concentrations of contaminants, as well as to measure
the precision of the AbraTox results.
The response of the AbraTox Kit to compounds used during the water treatment process
(identified as potential interferences in Table 3-1) was evaluated by analyzing separate aliquots
of DDW fortified with each potential interference at one-half of the concentration limit
recommended by the EPA's National Secondary Drinking Water Regulations (NSDWR)(2)
guidance. For analysis of by-products of the chlorination process, the unspiked DDW was
analyzed because Columbus, Ohio, uses chlorination as its disinfectant procedure. For the
analysis of by-products of the chloramination process, a separate drinking water sample was
obtained from the Metropolitan Water District of Southern California (LaVerne, California),
which uses chloramination as its disinfection process. The samples were analyzed after residual
chlorine was removed using sodium thiosulfate. Sample throughput was measured based on the
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number of samples analyzed per hour. Ease of use and reliability were determined based on
documented observations of the operators.
3.1 Test Samples
Test samples used in the verification test included drinking water and quality control (QC)
samples. Table 3-2 shows the number and type of samples analyzed. QC samples included
method blanks and positive and negative control samples. The fortified drinking water samples
were prepared from a single drinking water sample collected from the Columbus, Ohio, system.
The water was dechlorinated using sodium thiosulfate and then fortified with various
concentrations of contaminants and interferences. The DDW containing the potential
interferences was analyzed at a single concentration level, while at least four dilutions were
analyzed for each contaminant using the AbraTox Kit. Mixtures of contaminants and possible
interfering compounds were not analyzed.
3.1.1 Quality Control Samples
QC samples included method blanks, positive controls, negative controls, and preservative
blanks. The method blank samples consisted of ASTM Type IIDI water and were used to ensure
that no sources of contamination were introduced in the sample handling and analysis
procedures. A positive control sample was included in the AbraTox Kit and was used as
provided from the vendor. While performance limits were not placed on the results, significant
inhibition for the positive control sample indicated to the operator that the AbraTox Kit was
functioning properly. Two negative control samples were included. One was provided by the
vendor. The second consisted of unspiked DDW and was used to set a background inhibition of
the DDW, the matrix in which each test sample was prepared. To ensure that the preservatives in
the contaminant solutions did not have an inhibitory effect, preservative blank samples were
prepared. These preservative blanks consisted of DDW fortified with a concentration of
preservative equivalent to that in the test solutions of botulinum toxin complex B, ricin, soman,
and VX.
3.1.2 Drinking Water Fortified with Contaminants
Approximately 50 liters of Columbus, Ohio, tap water were collected in a low-density
polyethylene container. The water was dechlorinated with sodium thiosulfate. Dechlorination
was confirmed by adding an n,n-diethyl-p-phenylenediamine (DPD) tablet to a 10-mL aliquot of
the water. Lack of color development in the presence of DPD indicated that the water was
dechlorinated. All subsequent test samples were prepared from this DDW.
A stock solution of each contaminant was prepared in DDW at concentrations at or above the
lethal dose level. The stock solution was further diluted to obtain one sample containing the
lethal dose concentration for each contaminant and three additional samples with concentrations
10, 100, and 1,000 times less than the lethal dose. Additional concentrations of some contam-
inants were prepared and analyzed for two reasons: one was because of the large difference in
response between two concentration levels. For example, if only one dilution level was almost
completely inhibitory and the next dilution level was non-inhibitory, several intermediate
concentrations were analyzed to better determine the toxicity threshold of that contaminant. The
other reason was because sometimes the lowest concentration analyzed was mostly inhibitory,
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thus, not providing even an estimate of the toxicity threshold. For these contaminants, additional
tenfold dilutions were analyzed to more accurately determine the toxicity threshold. Table 3-2
lists each concentration level and the number of samples analyzed at each level.
3.1.3 Drinking Water Fortified with Potential Interferences
Individual aliquots of the DDW were fortified with one-half the concentration specified by the
EPA's NSDWR for each potential interference. Table 3-2 lists the interferences, along with the
concentrations at which they were tested. Four replicates of each of these samples were analyzed.
To test the sensitivity of the AbraTox Kit to by-products of the chlorination process as potential
interferences, the unspiked DDW (same as the negative control) was used since the water sample
originated from a utility that uses chlorination as its disinfectant procedure. In a similar manner,
by-products of the chloramination process were evaluated using a water sample from the
Metropolitan Water District of Southern California. The residual chlorine in both of these
samples was removed using sodium thiosulfate, and then the samples were analyzed in replicate
with no additional fortification of contaminants.
3.2 Test Procedure
The procedures for preparing, storing, and analyzing test samples and confirming stock solutions
are provided below.
3.2.1 Test Sample Preparation and Storage
A drinking water sample was collected as described in Section 3.1.2 and, because free chlorine
kills the bacteria within the AbraTox reagent and can degrade the contaminants during storage,
was immediately dechlorinated with sodium thiosulfate. Dechlorination of the water sample was
qualitatively confirmed by adding a DPD tablet to a 10-mL aliquot of the DDW. All the
contaminant samples, potential interference samples, preservative blanks, and negative control
QC samples were made from this water sample, while the method blank sample was prepared
from ASTM Type IIDI water. The positive and negative control samples included in the
AbraTox Kit were used as provided. All QC samples were prepared prior to the start of testing
and stored at room temperature. The stability of each contaminant for which analytical methods
are available was confirmed by analyzing it three times over a two-week period. Throughout this
time, each contaminant maintained its original concentration to within approximately 25%.
Therefore, the aliquots of DDW containing the contaminants were prepared within two weeks of
testing and were stored at room temperature without chemical preservation. The contaminants
without analytical methods were analyzed within 48 hours of their preparation. To maintain the
integrity of the test, test samples provided to the operators were labeled only with sample
identification numbers so that the operators did not know their content.
3.2.2 Test Sample A nalysis Procedure
To analyze the test samples, 800 uL of the test sample were added to a sample cuvette. Then,
100 jiL of osmotic adjusting buffer were pipetted into all cuvettes and mixed well. The cuvettes
were incubated at 15°C for 30 minutes. As soon as the sample cuvettes were set aside for
incubation, the bioluminescent bacteria Vibriofischeri (strain NRRL-B-11177) were
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Table 3-2. Summary of Quality Control and Contaminant Test Samples
Type of Sample
Quality control
DDW fortified with
contaminants
DDW fortified with
potential interferences
Disinfectant
by-products
Sample
Characteristics
Method blank
(ASTM Type II water)
Positive control
(distilled water)
Negative control
(25% methanol)
Negative control
(unspiked DDW)
Preservative blank:
botulinum toxin
complex B
Preservative blank:
VX and soman
Preservative blank:
ricin
Aldicarb
Botulinum toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Aluminum
Copper
Iron
Manganese
Zinc
Chloramination by-
products
Chlorination by-
products
Concentration Levels
NA
Used as provided by the vendor
Used as provided by the vendor
NA
0.015 millimolar (mM) sodium citrate
0.21% isopropyl alcohol
0.00024% NaN3, 0.00045 molar
NaCl, O.OSmM phosphate
260; 26; 2.6; 0.26 milligrams/liter
(mg/L)
0.3; 0.03; 0.003; 0.0003 mg/L
240; 24; 2.4; 0.24; 0.024;
0.0024 mg/L
250; 25; 2.5; 0.25 mg/L
1,400; 140; 14; 1.4; mg/L
2,800; 2,100; 1,400; 700; 280; 28;
2.8 mg/L
15; 1.5; 0.15; 0.015 mg/L
1.4; 0.14; 0.014; 0.0014 mg/L
2,800; 280; 28; 2.8 mg/L
2.0; 0.2; 0.02; 0.002 mg/L
0.5 mg/L
0.6 mg/L
0.15 mg/L
0.25 mg/L
2.5 mg/L
NA
NA
No. of Sample Analyses
15
15
15
60
4
4 with VX, 4 with soman
4
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 per concentration level
4
4
4
4
4
4
60
NA = not applicable, samples not fortified with any preservative, contaminant, or potential interference.
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reconstituted with reconstitution solution and equilibrated at 4°C for 30 minutes. After the
30-minute incubation period, 100 jiL of the reconstituted bacteria were added to the sample
cuvettes. After the bacteria were added, the samples were incubated for another 30 min at 4°C,
and the luminescence was measured and recorded. The luminescence was compared with that of
the negative control to determine percent inhibition. The bacteria were prepared the day of use
for all tests.
For each contaminant, a minimum of the lethal dose concentration and three additional
concentration levels were analyzed four times using the AbraTox Kit. Only one concentration of
each potential interference was analyzed four times. The luminescence was recorded, and the
percent inhibition was calculated for each sample. Two operators performed all the analyses
using the AbraTox Kit. One operator performed testing with contaminants that did not require
special chemical and biological agent training and one performed testing with those that did.
Both held bachelor's degrees in the sciences and were trained by the vendor to operate the
AbraTox Kit.
3.2.3 Stock Solution Confirmation Analysis
The concentrations of the contaminant and interfering compound stock solutions were verified
with standard analytical methods, with the exception of colchicine, ricin, and botulinum toxin
complex B—contaminants without standard analytical methods. Aliquots to be analyzed by
standard methods were preserved as prescribed by the method. In addition, the same standard
methods were used to measure the concentration of each contaminant/potential interference in
the unspiked DDW so that background concentrations of contaminants or potential interferences
were accounted for within the displayed concentration of each contaminant/potential interference
sample. Table 3-3 lists the standard methods used to measure each analyte; the results from the
stock solution confirmation analyses (obtained by analyzing the lethal dose concentration for the
contaminants and the single concentration that was analyzed for the potential interferences); and
the background levels of the contaminants and potential interferences measured in the DDW
sample, which were all non-detect or negligible.
Standard methods were also used to characterize several water quality parameters such as
alkalinity; dissolved organic carbon content; specific conductivity; hardness; pH; concentration
of haloacetic acids, total organic carbon, total organic halides, and trihalomethanes; and
turbidity. Table 3-4 lists these measured water quality parameters for both the water sample
collected in Columbus, Ohio, representing a water system using chlorination as the disinfecting
process, and the water sample collected at the Metropolitan Water District of Southern
California, representing a water system using chloramination for disinfection.
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Table 3-3. Stock Solution Confirmation Results
Contaminant
Aldicarb
Botulinum toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Potential
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Method
Battelle
method
(a)
(a)
EPA335.3(3)
Battelle
method
Battelle
method
(a)
Battelle
method
EPA 200.8(4)
Battelle
method
EPA 200.7(5)
EPA 200.7(5)
EPA 200.7(5)
EPA 200.7(5)
EPA 200.7(5)
Average Concentration ± Standard
Deviation N = 4 (mg/L)00
260 ±7
NA
NA
249 ±4
296 ± 26 (field portability)
1,168±18
2,837 ±27
NA
1.3 ±0.1 (10/18/05)
1.16 ±0.06 (10/21/05
2,469 ±31
1.89 ±0.08 (10/17/05)
1.77 ±0.03 (10/20/05)
0.50 ±0.02
0.60 ±0.03
0.155 ±0.006
0.281 ±0.008
2.63 ±0.05
Background in
DDW (mg/L)
<0.005
NA
NA
0.006
<3.0
<0.01
NA
<0.025
<0.001
<0.0005
<0.2
<0.02
<0.04
<0.01
0.27
NA = Not applicable.
(a) No standard method available. QA audits and balance calibration assured accurately prepared solutions.
(b) Target concentration was highest concentration for each contaminant or interference on Table 3-2.
10
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Table 3-4. Water Quality Parameters
Parameter
Alkalinity (mg/L)
Specific conductivity
(umho)
Hardness (mg/L)
pH
Total haloacetic acids
(Mg/L)
Dissolved organic
carbon (mg/L)
Total organic carbon
(mg/L)
Total organic halides
(Mg/L)
Total trihalomethanes
(Mg/L)
Turbidity (NTU)
Method
SM 2320 B(6)
SM2510B(6)
EPA 130.2(7)
EPA150.1(7)
EPA 552.2(8)
SM5310B(6)
SM5310B(6)
SM 5320B(6)
EPA 524.2(9)
SM2130(10)
Dechlorinated Columbus,
Ohio, Tap Water
(disinfected by
chlorination)
40
572
118
7.6
32.8
2.1
2.1
220
74.9
0.1
Dechlorinated Southern
California Tap Water
(disinfected by
chloramination)
71
807
192
8.0
17.4
2.9
2.5
170
39.2
0.1
NTU = nephelometric turbidity unit.
11
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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center(11) and the test/QA plan for this verification test.(1)
4.1 Quality Control of Stock Solution Confirmation Methods
The stock solutions for the contaminants cyanide and thallium sulfate and for the potential
interferences aluminum, magnesium, zinc, iron, and copper were analyzed at ATEL using a
standard reference method. As part of ATEL's standard operating procedures (SOPs), various
QC samples were analyzed with each sample set. These included matrix spike, laboratory control
spike, and method blank samples. According to the standard methods used for the analyses,
recoveries of the QC spike samples analyzed with samples from this verification test were within
acceptable limits of 75% to 125%, and the method blank samples were below the detectable
levels for each analyte. For VX, soman, aldicarb, nicotine, and dicrotophos, the confirmation
analyses were performed at Battelle using a Battelle SOP or method. Calibration standard
recoveries of VX and soman were always between 62% and 141%, and most of the time were
between 90% and 120%. Dicrotophos standard recoveries ranged from 89% to 122%. Aldicarb
standard recoveries ranged from 95% to!20%. Nicotine standard recoveries ranged from 96% to
99%. Standard analytical methods for colchicine, ricin, and botulinum toxin complex B were not
available and, therefore, not performed. QA audits and balance calibrations assured that solutions
for these compounds were accurately prepared.
4.2 Quality Control of Drinking Water Samples
A method blank sample consisting of ASTM Type IIDI water was analyzed once by the
AbraTox Kit for approximately every 20 drinking water samples that were analyzed. Because
inhibition has to be calculated with respect to a control sample, none were calculated for the
method blank samples. The method blanks were used as the control for calculating the inhibition
of the DDW for the disinfecting by-product evaluation. A positive control sample was provided
by the vendor and was analyzed once for approximately every 20 drinking water samples. While
performance limits were not placed on the results of the positive control sample, the vendor
informed Battelle that, if the positive control samples did not cause significant inhibition, it
would indicate to the operator that the AbraTox Kit was not functioning properly. For 15 positive
control samples, an average inhibition of 98% ± 2% was measured. These inhibition values
12
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indicated the proper functioning of the AbraTox Kit. A negative control sample (unspiked DDW)
was analyzed with approximately every four samples. The percent inhibition calculation for each
sample incorporated the average inhibition of the negative control samples analyzed with that
particular sample set; therefore, by definition, the average inhibition of four negative control
samples was 0%. A negative control supplied by the vendor was analyzed with approximately
every 20 samples. This negative control provided luminescence readings similar to the method
blank and DDW negative control.
4.3 Audits
A performance evaluation (PE) audit, a technical systems audit (TSA), and an audit of data
quality were performed for this verification test.
4.3.1 Performance Evaluation Audit
The accuracy of the reference method used to confirm the concentration of the stock solutions of
the contaminants and potential interferences was confirmed by analyzing solutions of each
analyte from two separate commercial vendors. The standards from one source were used to
prepare the stock solutions during the verification test, while the standards from a second source
were analyzed as the PE sample. The percent difference (%D) between the measured concen-
tration of the PE sample, and the nominal concentration of that sample was calculated using the
following equation:
%£>=—xlOO% (!)
A
where Mis the absolute value of the difference between the measured and the nominal concen-
tration, and^4 is the nominal concentration. The %D between the measured concentration of the
PE standard and the nominal concentration had to be less than 25% for the measurements to be
considered acceptable. Table 4-1 shows the results of the PE audit for each compound. All %D
values were less than 25.
PE audits were performed when more than one source of the contaminant or potential
interference was commercially available and when methods were available to perform the
confirmation; therefore, PE audits were not performed for all of the contaminants. To assure the
purity of the other standards, documentation, such as certificates of analysis, was obtained for
colchicine, botulinum toxin complex B, and ricin. In the cases of VX and soman, which were
obtained from the U.S. Army, the reputation of the source, combined with the confirmation
analysis data, provided assurance of the concentration analyzed.
4.3.2 Technical Systems A udit
The Battelle Quality Manager conducted a TSA to ensure that the verification test was performed
in accordance with the test/QA plan*-1-* and the AMS Center QMP.(11) As part of the audit, the
Battelle Quality Manager reviewed the contaminant standard and stock solution confirmation
methods, compared actual test procedures with those specified in the test/QA plan, and reviewed
data acquisition and handling procedures. Observations and findings from this audit were
13
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Table 4-1. Summary of Performance Evaluation Audit
Contaminant
Potential
interference
Aldicarb
Cyanide
Dicrotophos
Nicotine
Thallium
Aluminum
Copper
Iron
Manganese
Zinc
Measured
Concentration
(mg/L)
0.057
1,025
1.10
0.120
1,010
960
1,000
960
922
1,100
Nominal
Concentration
(mg/L)
0.050
1,000
1.00
0.100
1,000
1,000
1,000
1,000
1,000
1,000
%D
14
3
10
20
1
4
0
4
8
10
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.
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/1 ^ 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 they were used to calculate,
evaluate, or report verification results. Table 4-2 summarizes the types of data recorded. The
review was performed by a technical staff member involved in the verification test, but not the
staff member who originally generated the record. The person performing the review added
his/her signature or initials and the date to a hard copy of the record being reviewed.
Table 4-2. Summary of Data Recording Process
Data to be
Recorded
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
Responsible
Party
Battelle
Battelle
Battelle
Battelle or
contracted
laboratory
Where
Recorded
Laboratory
record books
Laboratory
record books
Laboratory
record books
Laboratory
record books,
data sheets, or
data acquisition
system, as
appropriate
How Often
Recorded
Start/end of test,
and at each change
of a test parameter
When each sample
was prepared
When set or
changed
Throughout sample
handling and
analysis process
Disposition of Data(a)
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
All activities subsequent to data recording were carried out by Battelle.
15
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Chapter 5
Statistical Methods and Reported Parameters
The statistical methods presented in this chapter were used to verify the performance parameters
listed in Section 3.
5.1 Endpoints and Precision
The luminometer provided with the AbraTox Kit reported the absolute light units for each
sample analyzed. Each test sample was compared with a negative control sample that, for this
verification test, was unspiked DDW. This comparison was made by accounting for the
inhibition of the negative control in the calculation of the percent inhibition. Therefore, the
percent inhibition of the four negative control samples within each sample set always averaged
zero. The percent inhibition for each sample was calculated using the following equation:
% inhibition = 1 - =—^ x 100% (2)
I L negative control J
Where Zsampie is the absolute light units generated by each test sample, and Lnegative control is the
average number of light units produced across the four negative control samples analyzed in the
same sample set as the subject test sample. For this test, the negative control sample was always
DDW, except when the inhibition of the disinfectant by-products was being determined, in that
case, ASTM Type IIDI water served as the control sample.
The standard deviation (SD) of the results for the replicate samples was calculated, as follows,
and used as a measure of technology precision at each concentration. The standard deviation
around the average negative control results represented the variability of the inhibition caused by
the negative control water. Similarly, the standard deviation of the rest of the contaminant
concentrations represented the precision of the inhibition caused by the background water
combined with the contaminant.
n —
(3)
16
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where n is the number of replicate samples, h is the percent inhibition measured for the Jt
sample, and / is the average percent inhibition of the replicate samples. Because the average
inhibition was frequently near zero for this data set, relative standard deviations often would
have greatly exceeded 100%, making the results difficult to interpret. Therefore, the precision
results were left in the form of standard deviations of the percent inhibition so the reader could
easily view the uncertainty around the average percent inhibition for results that were both near
zero and significantly larger than zero.
5.2 Toxicity Threshold
The toxicity threshold was defined as the lowest concentration of contaminant to exhibit a
percent inhibition significantly greater than the negative control. Also, each concentration level
higher than the toxicity threshold had to be significantly greater than the negative control, and
the inhibition produced by each lower concentration analyzed had to be significantly less than
that produced by the toxicity threshold concentration. Since the inhibition of the test samples was
calculated with respect to the inhibition of each negative control sample, the percent inhibition of
the negative control was always zero. A significant difference in the inhibition at two
concentration levels required that the average inhibition at each concentration level, plus or
minus its respective standard deviation, did not overlap.
5.3 False Positive/Negative Responses
A response was considered false positive if an unspiked drinking water sample produced an
inhibition significantly greater than zero when determined with respect to DI water. Depending
on the degree of inhibition in the sample, toxicity from subsequent contamination of that sample
may not be detectable or could be exaggerated as a result of the baseline inhibition. Drinking
water samples collected from water systems using chlorination and chloramination as the
disinfecting process were analyzed in this manner. An inhibition was considered significantly
different from zero if the average inhibition, plus or minus the standard deviation, did not
overlap with the zero inhibition plus or minus the standard deviation.
A response was considered false negative when the AbraTox Kit, subjected to a lethal
concentration of some contaminant in the DDW, did not indicate inhibition significantly greater
than the negative control (zero inhibition) and the other concentration levels analyzed (for lethal
dose inhibition less than 100%). The inhibition of the lethal dose sample was required to be
significantly greater than the other concentration levels because it more thoroughly incorporated
the uncertainty of all the measurements made by the AbraTox Kit in determining false negative
results. A difference was considered significant if the average inhibition plus or minus the
standard deviation did not encompass the value or range of values that were being compared. In
addition, background water samples that increased the light production of the AbraTox Kit
organisms (i.e., negative inhibition) were considered false negative because such samples could
cancel out the effect of a contaminant that inhibits light production, making it seem that the
contaminant had no toxic effect.
17
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5.4 Other Performance Factors
Ease of use (including clarity of the instruction manual, user-friendliness of software, and overall
convenience) was qualitatively assessed throughout the verification test through documented
observations of the operators and Verification Test Coordinator. Sample throughput was
evaluated quantitatively based on the number of samples that could be analyzed per hour.
18
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Chapter 6
Test Results
6.1 Endpoints and Precision
Tables 6-la-l present the percent inhibition data for 10 contaminants; and Table 6-2 gives the
percent inhibition data for preservatives with a concentration similar to what would be contained
in a lethal dose of botulinum toxin complex B, ricin, soman, and VX. Given in each table are the
concentrations analyzed, the percent inhibition results for each replicate at each concentration,
and the average and standard deviation of the inhibition of the four replicates at each
concentration. Contaminant test samples that produced negative percent inhibition values
indicated an increase in light production by the bacteria and were considered non-toxic.
6.1.1 Contaminants
All the contaminants except botulinum toxin complex B, ricin, and VX exhibited some inhibition
that was significantly different from the negative control. Aldicarb and thallium sulfate generated
detectable inhibition at the two highest concentration levels analyzed, while colchicine generated
inhibition only at the lethal dose concentration. Dicrotophos generated detectable inhibition at
the lethal dose concentration and at the thousandfold dilution level, but, considering the rather
low inhibition at the two intermediate concentrations, the significance of the inhibition at the
lower concentration is questionable. Upon initial analysis, nicotine generated detectable
inhibition at concentrations only at the lethal dose. Additional dilutions were done to better
determine the toxicity threshold of nicotine. During the additional dilutions, the degree of
inhibition changed in the lethal dose sample from 43% ± 2% initially to 90% ± 5% and inhibition
was determined to be detectable down to 700 mg/L. The AbraTox Kit was especially sensitive to
cyanide. Inhibition at the highest three concentration levels (250, 25, and 2.5 mg/L) was
significantly different from the negative control. The 0.25-mg/L concentration level generated an
inhibition of 25% ± 20%, which, while a positive inhibition, was not significantly different from
the negative control because of the uncertainty around the average inhibition of the negative
control.
19
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Table 6-la. Aldicarb Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.26
2.6
26
260
(Lethal Dose)
Inhibition
(%)
8
-1
-3
-5
0
15
17
16
12
1
-7
-17
33
23
24
4
65
62
60
63
Average
(%)
0
12
-3
21
63
Standard
Deviation
(%)
6
8
12
12
2
20
-------
Table 6-lb. Botulinum Toxin Complex B Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.0003
0.003
0.03
0.3
(Lethal Dose)
Lethal Dose
Preservative
Blank
0.3
Lethal Dose
Lethal Dose
Preservative
Blank
Inhibition
(%)
-40
13
9
18
-28
8
-3
16
-23
-24
-37
-77
14
5
-3
-61
13
-3
-11
-42
-66
-27
-16
-13
34
21
15
-9
-27
3
11
13
Average
(%)
0
-2
-40
-11
-10
-30
15
0
Standard
Deviation
(%)
27
19
25
34
23
24
18
19
Shading indicates that inhibition results were calculated with
respect to the preservative blank.
21
-------
Table 6-1 c. Colchicine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.0024
0.024
0.24
2.4
24
240
(Lethal Dose)
Inhibition
(%)
9
2
-8
-3
-14
-17
-23
-27
-25
-29
-23
-24
-45
-45
-53
-63
11
3
-5
-1
-35
-38
-49
-33
16
14
17
19
Average
(%)
0
-20
-25
-51
2
-39
17
Standard
Deviation
(%)
7
6
2
9
7
7
2
22
-------
Table 6-1 d. Cyanide Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.25
2.5
25
250
(Lethal Dose)
Field
Portability
Negative
Control
Field
Portability
250
Inhibition
(%)
-31
0
13
18
38
36
29
-4
48
48
47
30
73
74
72
66
79
80
79
89
-1
4
4
-7
73
78
77
75
Average
(%)
0
25
43
71
82
0
76
Standard
Deviation
(%)
22
20
9
3
5
5
2
23
-------
Table 6-le. Dicrotophos Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
1.4
14
140
1,400
(Lethal Dose)
Inhibition
(%)
2
3
-1
-4
34
29
27
13
-4
21
20
20
18
16
13
-7
47
47
45
31
Average
(%)
0
26
14
10
42
Standard
Deviation
(%)
3
9
12
12
8
Table 6-lf. Nicotine Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Inhibition
(%)
4
5
-1
-8
6
-1
8
3
-4
-5
-1
4
-3
-13
-2
5
43
42
41
46
Average
(%)
0
4
-2
-3
43
Standard
Deviation
(%)
6
4
4
7
2
24
-------
Table 6-1 g. Nicotine Percent Inhibition Results—Additional Dilutions
Concentration
(mg/L)
Negative
Control
280
700
1,400
2,100
2,800
Inhibition
(%)
-5
11
2
-9
9
11
5
14
68
27
27
25
59
64
59
59
95
100
98
91
89
89
86
98
Average
(%)
0
10
37
60
96
90
Standard
Deviation
(%)
9
4
21
2
4
5
25
-------
Table 6-lh. Ricin Percent Inhibition (Compared to Negative Control)
Concentration
(mg/L)
Negative
Control
0.015
Lethal
Dose/1,000
Preservative
Blank
0.15
Lethal
Dose/100
Preservative
Blank
1.5
Lethal Dose/ 10
Preservative
Blank
15
Lethal Dose
Preservative
Blank
Inhibition
(%)
-40
13
9
18
-68
-50
-30
-18
-108
-55
-30
-16
-14
-24
-7
-24
-18
-10
-17
-20
-1
-15
-6
-11
-35
-10
-15
-8
-58
-42
-12
-4
-51
-42
-45
-43
Average
(%)
0
-41
-52
-17
-16
-8
-17
-29
-45
Standard
Deviation
(%)
27
22
41
8
4
6
12
25
4
26
-------
Table 6-li. Ricin Percent Inhibition Results (Compared to Preservative Blank)
Concentration
(mg/L)
0.015
Lethal
Dose/1,000
Preservative
Blank
0.15
Lethal Dose/ 100
Preservative
Blank
1.5
Lethal Dose/ 10
Preservative
Blank
15
(Lethal Dose)
Lethal Dose
Preservative
Blank
Inhibition
(%)
-10
1
15
23
-37
-2
15
24
2
-7
8
-7
-1
5
-1
-3
14
2
9
5
-15
6
2
7
-9
2
23
29
-4
2
0
2
Average
(%)
7
0
-1
0
8
0
11
0
Standard
Deviation
(%)
15
27
7
3
5
11
17
3
Each concentration level is shown directly above the preserva-
tive blank containing an equivalent amount of preservatives. The
inhibition of each pair is calculated with respect to each
preservative blank.
27
-------
Table 6-lj. Soman Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.0014
0.014
0.14
1.4
(Lethal Dose)
Lethal Dose
Preservative
Blank
1.4
(Lethal Dose)
Lethal Dose
Preservative
Blank
Inhibition
(%)
-9
-1
5
4
-12
-4
-4
18
-11
-9
-6
-15
-22
-31
-15
-29
8
10
-8
4
-50
-43
-45
-21
34
36
23
31
-8
-4
-2
14
Average
(%)
0
-1
-10
-24
4
-40
31
0
Standard
Deviation
(%)
6
13
4
7
8
13
6
9
Shaded inhibition calculated with respect to the preservative blank.
28
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Table 6-1 k Thallium Sulfate Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
2.8
28
280
2,800
(Lethal Dose)
Inhibition
(%)
6
1
1
-8
6
6
5
7
-1
2
10
9
23
18
17
21
23
6
14
11
Average
(%)
0
6
5
20
14
Standard
Deviation
(%)
6
1
5
3
7
Table 6-11. VX Percent Inhibition Results
Concentration
(mg/L)
Negative
Control
0.002
0.02
0.2
2
(Lethal Dose)
Lethal Dose
Preservative
Blank
Inhibition
(%)
8
-4
5
-9
-5
-13
-12
1
-6
-8
8
15
-38
-24
-4
8
-30
-30
-41
-27
-17
-7
-1
-11
Average
(%)
0
-7
2
-15
-32
-9
Standard
Deviation
(%)
8
6
11
21
6
7
29
-------
Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results
Preservative
Blank
Negative
Control
Ricin
Soman/VX(a)
Botulinum
Toxin
Complex B
Inhibition
(%)
-29
-7
23
13
29
17
19
21
(b)
-18
-3
10
-9
-4
-25
-33
Average
(%)
0
21
-4
-18
Standard
Deviation
(%)
23
5
14
14
^> Soman and VX use the same preservative.
(b) Removed -98% as an outlier.
It is important to note that the botulinum toxin complex B, ricin, soman, and VX stock solutions
used to prepare the test samples were stored in various preservatives that included sodium azide,
sodium chloride, and sodium phosphate for ricin; sodium citrate only for botulinum toxin
complex B, and isopropyl alcohol for soman and VX. During the previous ETV test of this
technology category, the preservatives were not accounted for in the negative control; therefore,
the results from each test should be interpreted accordingly. The results for this test are more
thorough because they show the sensitivity (or lack thereof) to both the preservative and the
contaminant. In the in the earlier verification test, toxicity could have been the result of either.
Table 3-2 details the concentrations of preservatives in the lethal dose samples of each
contaminant. These data could be evaluated in two ways to determine the sensitivity of the
AbraTox Kit to contaminants stored in preservatives. The first approach would be to determine
the inhibition of the test samples containing preservatives with respect to the background
negative control, as was the case for the contaminants that were not stored in preservatives. This
technique, however, could indicate that AbraTox Kit was sensitive to the contaminant when, in
fact, it was sensitive to one of the preservatives. Since these contaminants are only available
(either commercially or from the government) in aqueous formulations with the preservatives,
this may be appropriate. The second approach would be to fortify negative control samples with
the same concentrations of preservative contained in all the samples so that the inhibition
resulting from the preservatives could be subtracted from the inhibition caused by the
contaminant. This approach would greatly increase the number of samples required for analysis.
Therefore, for this test, aspects of both approaches were incorporated without substantially
increasing the number of samples. Negative control samples fortified with a concentration of
each preservative equivalent to the concentration in the lethal dose test samples (preservative
blanks) were analyzed prior to analyzing any test samples. For those sets of test samples for
which it was especially difficult to determine whether inhibitory effects were from the
contaminant or the preservative, the preservative blank was diluted identically to all the
30
-------
contaminant samples and analyzed with them so a background subtraction could take place if
necessary.
During the initial analysis of the preservative blanks (Table 6-2), the only sample that generated
inhibition significantly different from the unfortified negative controls was the sample
representing the ricin preservative, with an inhibition of 21% ± 5%. Subsequently, for the ricin
samples, all of the preservative blanks were diluted with the same concentration of preservatives
as the test samples containing ricin. For the other contaminant tests, only the preservative blank
containing preservatives equivalent to those in the lethal dose sample were analyzed with the
contaminant samples.
The inhibition of the botulinum toxin complex B test samples was not significantly different
from the negative control. In addition, the lethal dose preservative blank was not significantly
different from the negative control. However, the average inhibition of the preservative blank
was somewhat more negative (-30% ± 24%) than when it was analyzed prior to analysis of the
contaminant samples (-18 ± 14%) (Table 6-2). Because of this, the inhibition of the lethal dose
contaminant solution was also calculated with respect to the preservative blank. Calculated in
this way, the inhibition of the lethal dose of the contaminant solution was 15% ± 18% , which
was not significantly different compared to the preservative blank (0% ± 19%) when calculated
with respect to itself. Because the highest concentration of botulinum toxin complex B analyzed
was not significantly different from the preservative blank, it is unlikely that the lower
concentrations would be affected by testing against dilutions of the preservative blank.
Therefore, no additional dilutions of the preservative blanks were analyzed.
As mentioned above, the ricin preservative blank generated a detectable inhibition prior to the
contaminant analysis and equivalent dilutions of the preservative blank samples were analyzed
with the ricin samples. The inhibition of each ricin sample was calculated with respect to the
preservative blank of the appropriate concentration. As Table 6-li shows, the ricin sample
inhibition was not significantly different from the corresponding preservative blank. When
analyzed with the contaminant samples, the inhibition of the lethal dose level preservative blank
was -45% ± 4%, compared with 21% ± 5% during its initial analysis (see Table 6-2). There is no
explanation for this result. Nonetheless, because of the rather large uncertainty in the measure-
ments, even when the inhibition of the contaminant samples was calculated with respect to the
negative control (Table 6-lh) rather than with respect to their preservative blanks, the inhibition
of the ricin samples was not detectable.
For soman, the preservative blank analyzed prior to the contaminant samples yielded an
inhibition of-4% ± 14% (Table 6-2)—not significantly different from that of the negative
control. Thus, dilutions of the preservative blank were not analyzed with the contaminants and
the contaminant inhibition was calculated only with respect to the negative control. None of the
soman samples exhibited inhibition significantly different from the negative control. However,
the lethal dose preservative blank that was analyzed with the contaminant samples exhibited a
negative inhibition that was significantly different from both the negative control and the
preservative blank analyzed prior to the contaminant samples. When the inhibition of the lethal
dose solution of soman was calculated with respect to the preservative blank, the inhibition was
31% ± 6%—a slight inhibition. Because of the rather modest inhibition at the lethal dose
concentration and the inconsistent results from the preservative blanks, no other dilutions of the
preservative blank were analyzed.
31
-------
For VX, with the exception of the lethal dose (-32% ± 6%), the average inhibition at each
concentration level was not significantly different from the negative control, and none of
concentrations (including the preservative blank) generated a positive inhibition that was
significantly different from the negative control.
6.1.2 Potential Interferences
All of the potential interference samples were prepared in DDW and compared with the negative
control to determine the level of inhibition. This determination is crucial because the ability of
the AbraTox Kit to detect toxicity is dependent on the background light production in whatever
drinking water matrix is being used. If the background drinking water sample completely inhibits
background light, inhibition caused by contaminants could not be detected. Table 6-3 presents
the results from the samples that were analyzed to test the effect of potential interferences on the
AbraTox Kit. Of the five metal solutions that were evaluated as possible interferences with the
AbraTox Kit, three of them, zinc (15% ± 10%), iron (7% ± 3%), and copper (32% ± 11%)
exhibited inhibition that was significantly different from the negative control (0% ± 3%). Zinc
and iron inhibition was only slightly detectable, and the copper inhibition was an average of only
32%, leaving more than half of the available background light for inhibition by contaminants.
Therefore, water samples containing similar concentrations of metals could be analyzed for
contaminants as long as a negative control sample with similar levels of metals was used to
generate a representative background inhibition. Enough background light for inhibition by
contaminants remains even though there is some inhibition caused by the metals.
To investigate whether the AbraTox Kit is sensitive to by-products of disinfecting processes,
DDW samples from water systems that use chlorination and chloramination were analyzed and
compared with ASTM Type n DI water as the control sample. In the absence of a background
water sample, it seems likely that DI water may be used as a "clean water" control; therefore, it
would be helpful to know what the results would be if this is done. The sample from the water
supply disinfected by chlorination (N=60) exhibited an average inhibition of 5% ± 16%, while
the sample from the water supply disinfected by chloramination exhibited an inhibition of 4% ±
7% on four replicates. The difference in the number of replicates is because the dechlorinated
water was used as the negative control with each sample set; therefore, much more data were
collected on that water. These inhibition data suggest that samples disinfected by either process
are not likely to interfere with the AbraTox Kit results because the inhibition caused by the
"clean" drinking water matrices left most of the light to potentially be inhibited by
contamination.
6.1.3 Precision
Across all the contaminants and potential interferences, the standard deviation (not relative
standard deviation) was measured and reported for each set of four replicates to evaluate the
AbraTox Kit precision. Out of 80 opportunities, the standard deviation of the four replicate
measurements was less than 10% 54 times (68%), between 10% and 20% 16 times (20%), and
greater than 20% 10 times (12%). There was no consistent trend concerning when the results
were repeatable. As described in Section 3.2.2, the analysis procedure required that each
replicate undergo the entire analysis process; therefore, the measurement of precision represents
the precision of the analysis method performed on a single water sample on a given day. The
precision does not reflect the repeatability of the method across more than one day or more than
one preparation of reagents or more than one operator.
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Table 6-3. Potential Interferences Results
Potential
Interferences
Negative control
(Metals)
Aluminum
Copper
Iron
Manganese
Zinc
Negative control
(By-products)
Chlorination
by-products
Chloramination
by-products
Concen-
tration
(mg/L)
NA
0.5
0.6
0.15
0.25
2.5
NA
NA
NA
Inhibition
(%)
-3
0
0
4
-18
0
-2
4
42
39
29
17
3
9
9
6
2
7
3
-7
4
23
24
11
0
2
-4
1
(a)
9
11
-4
1
Average
(%)
0
-4
32
7
1
15
0
5
4
Standard
Deviation
(%)
3
10
11
3
6
10
3%
16
7
NA = Not applicable.
(a) Average inhibition across all DDW negative control samples (N=60).
33
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6.2 Toxicity Threshold
Table 6-4 gives the toxicity thresholds, as defined in Section 5.2, for each contaminant. Note the
difference between detectability with respect to the negative control and the toxicity threshold
with respect to the other concentration levels analyzed. A contaminant concentration level can
have an inhibition significantly different from the negative control (thus detectable), but if its
inhibition is not significantly different from the concentration levels below it, it would not be
considered the toxicity threshold because, in the context of this test, the inhibition must be
different from both the negative control and all lower concentrations. Specific examples include
aldicarb (detectable at 26 mg/L, toxicity threshold of 260 mg/L) and cyanide (detectable at all
four concentration levels, but a toxicity threshold of 25 mg/L). The lowest toxicity threshold
concentration was for soman at 1.4 mg/L. Soman was not detectable when compared with the
negative control, but the lethal dose of soman was detectable when the effect of the preservative
was accounted for by comparing with the preservative blank containing an equivalent
concentration of preservatives. Apparently, the preservative in the soman stock solution has a
toxic effect on the AbraTox Kit organisms. When the effect of the preservative was subtracted
from the effect of the contaminant, a significant effect of the soman remained.
Table 6-4. Toxicity Thresholds
Contaminant
Aldicarb
Botulinum toxin complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Concentration (mg/L)
260
ND
240
25
1,400
700
ND
L4oo
280
ND
ND = Significant inhibition was not detected.
(a) Soman was only detectable if calculated with respect to the
preservative blank.
6.3 False Positive/Negative Responses
None of the AbraTox Kit results would be considered false positive because neither the
chlorination nor chloramination inhibition was, on average, significantly different from the
negative control, and, therefore, light production was adequate to allow inhibition to occur if a
contaminant was present that produced a detectable toxic effect. Since the background inhibition
is not complete, it can be accounted for by using negative control samples that are very similar to
34
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the water being analyzed. If samples are analyzed daily, a good practice would be to archive a
negative control sample each day in case of contamination the next day.
Table 6-5 shows the false negative responses, which are described in Section 5.3. Botulinum
toxin complex B, ricin, and VX did not exhibit a detectable inhibition at the lethal concentration.
Table 6-5. False Negative Responses
Contaminant
Aldicarb
Botulinum toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Lethal Dose
Concentration (Mg/L)
260
0.30
240
250
1,400
2,800
15
1.4
2,800
2.0
False Negative
no
yes
no
no
no
no
yes
no(a)
No
yes
Soman was not a false negative when compared to the preservative blank.
6.4 Other Performance Factors
6.4.1 Ease of Use
The AbraTox Kit contained clearly written instructions and illustrations, and the contents were
clearly labeled. Storage requirements were marked on the vial labels. Overall, the packaging was
easy to open. Pull-back tabs on some of the bottles occasionally had to be pried open with a
spatula. The most difficult aspect of using the AbraTox Kit was keeping the incubator at 15°C
because there was no temperature control on the incubator. The incubator was refrigerated before
use, but warmed up quickly during the 30-minute incubation time. Therefore, the incubator was
placed in the refrigerator door during the incubation time. On occasion, test readings of zero
were obtained among a series of more "normal" bioluminescence readings. The vendor
suggested that the Vibrio fischeri were not getting into the solution. The technicians were certain
that the bacteria were getting into the cuvette appropriately; but, subsequently, even more care
was taken during that step in the analysis.
After adding osmotic adjusting solution to the freeze-dried bacteria, the bacteria stock had to be
refrigerated for 30 minutes. Therefore, the AbraTox Kit could be used with only 30 minutes
advance notice. The Vibrio fischeri needed to be stored at -20°C prior to re-hydration. All other
reagents required refrigerator storage at 4°C. Expiration dates were listed on vial labels. The
Vibrio fischeri were consumed the day of use, other reagents were used until the vial was empty.
35
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All necessary supplies were provided with the AbraTox Kit except for pipettes with tips and the
ASTM Type II water used to prepare reagents. The luminometer was easy to use and required no
special preparation before use. The electronic readout was user-friendly, with only one number
needing to be recorded. The luminometer was easily wiped clean and did not require any routine
maintenance after three months of use.
No formal scientific training would be required to use the AbraTox Kit, but good laboratory
skills would be beneficial. Verification testing staff were able to operate the AbraTox Kit after a
brief training session. Contact information for technical assistance was included in the
instructions. One cuvette per sample, reagent vials, and pipette tips were generated as solid
waste. No guidance was provided as to whether the waste generated was hazardous or not.
6.4.2 Field Portability
The AbraTox Kit was transported from a laboratory to a storage room to simulate a situation in
which it would be operated in a non-laboratory location. The storage room contained several
tables and light and power sources, but no other laboratory facilities. The luminometer was
transported in a small box, and a small cooler was used to transport the reagents. One person
could easily carry the basic equipment provided by the vendor (luminometer, small cooler,
reagents). The AbraTox Kit was easy to set up and was operational as soon as all equipment was
laid out and the luminometer was turned on. No source of electricity was required for this short-
term field deployment since the luminometer operated on batteries and the reagents were kept in
a cooler. A long-term field deployment would need controlled temperature storage for reagents.
Minimum space requirements in the field would be a flat surface of approximately 1.5 feet by
2 feet to keep the cuvette tray level and to keep the sample solution level during luminometer
readings. A cooler was required to transport and store the reagents. Maintaining a controlled
15°C incubation temperature was a challenge. The following items not provided in the AbraTox
Kit were needed for field use: a cooler to transport and store reagents, ice packs, high-purity
water to prepare solutions, a timer, pipettes and tips, and a waste container. Overall the AbraTox
Kit was easy to transport to the field and was deployed in a matter of minutes. Results were
obtained within 30 minutes of starting the test and were very similar to those obtained in the
laboratory. The AbraTox Kit was tested with cyanide at the lethal dose concentration. In the
laboratory, the inhibition was 82% ± 5%; while at the non-laboratory location, the inhibition was
76% ± 2%, suggesting that the performance of the AbraTox Kit was not dependent on where the
analysis was performed.
6.4.3 Throughput
Approximately 25 sample analyses plus method blanks and controls were completed in one hour.
Approximately 25 samples could be processed per vial of Vibrio fischeri.
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Chapter 7
Performance Summary
Parameter
Contaminant
sinDDW
Potential
interferences
inDDW
False
positive
response
False
negative
response
Compound
Aldicarb
Botulinum toxin
complex B
Colchicine
Cyanide
Dicrotophos
Nicotine
Ricin
Soman
Thallium sulfate
VX
Interference
Aluminum
Copper
Iron
Manganese
Zinc
Lethal
Dose (LD)
Cone.
(mg/L)
260
0.3
240
250
1,400
2,800
15
1.4
2,800
2
Cone.
(mg/L)
0.5
0.6
0.15
0.25
2.5
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
63
-10
17
82
42
43
ll(a)
31(a)
14
-32
LD/10
21
-11
-39
71
10
-3
8 (a)
-24
20
-15
LD/100
o
-6
-40
2
43
14
-2
_l(a)
-10
5
2
LD/1,000
12
-2
-51
25
26
4
7(3)
-1
6
-7
Average Inhibition Standard Deviation
(%) (%)
-4 10
32 11
7 3
1 6
15 10
Range of
Standard
Deviations
(%)
2-12
19-34
2-9
3-20
8-12
2-7
5-17
4-13
1-7
6-21
Toxicity
Thresh.
(mg/L)
260
ND
240
25
1,400
700
ND
L4(a)
280
ND
No false positive results were obtained because the inhibition of the chlorination and chloramination
by-product water samples was not significantly different from that of the negative control samples.
The AbraTox Kit generated false negative responses at the lethal dose concentration for botulinum
toxin complex B, ricin, and VX.
Ease of use
The AbraTox Kit contained clearly written instructions and illustrations, and the contents were clearly
labeled. Storage requirements were marked on the vial labels. The packaging was easy to open except
for the pull-back tabs on some of the bottles. The most difficult aspect of using the AbraTox Kit was
keeping the incubator at 15°C because there was no temperature control on the incubator. Because
bacteria stock had to be refrigerated for 30 minutes, at least 30 minutes of advance notice is necessary
before using the AbraTox Kit. No formal scientific training would be required to use the AbraTox Kit.
Field
portability
The AbraTox Kit was transported from a laboratory to a storage room to simulate a situation in which it
would be operated in a non-laboratory location. The luminometer was transported in a small box, and a
small cooler was used to transport the reagents. Overall the AbraTox Kit was easy to transport to the
field and was deployed in a matter of minutes. The AbraTox Kit was tested with cyanide at the lethal
dose concentration. Results were obtained within 30 minutes of starting the test and were very similar
to those obtained in the laboratory. In the laboratory, the inhibition for the lethal dose concentration of
cyanide was 82% ± 5%; while at the non-laboratory location, the inhibition was 76% ± 2%.
Throughput
Approximately 25 sample analyses plus method blanks and controls were completed in one hour.
Approximately 25 samples could be processed per vial of Vibrio fischeri.
ND = Significant inhibition was not detected.
(a) Inhibition calculated with respect to the preservative blank.
37
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Chapter 8
References
1. Test/QA Plan for Verification of Rapid Toxicity Technologies, Battelle, Columbus, Ohio,
June 2003; Amendment 1: June 9, 2005; Amendment 2: August 19, 2005.
2. United States Environmental Protection Agency, National Secondary Drinking Water
Regulations: Guidance for Nuisance Chemicals, EPA/810/K-92/001, July 1992.
3. U. S. EPA Method 335.3, "Cyanide, Total—Colorimetric, Automated UV," in Methods for
the Chemical Analysis of Water and Wastes, EPA/600/4-79/020, March 1983.
4. U.S. EPA Method 200.8, "Determination of Trace Elements in Waters and Wastes by
Inductively-Coupled Plasma Mass Spectrometry," in Methods for the Determination of
Metals in Environmental Samples, Supplement I, EPA/600/R-94/111, 1994.
5. U.S. EPA Method 200.7, "Trace Elements in Water, Solids, and Biosolids by Inductively
Coupled Plasma—Atomic Emission Spectrometry," EPA-821-R-01-010, January 2001.
6. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, 1997. Washington, DC.
7. U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020.
8. U.S. EPA Method 552.2, "Haloacetic Acids and Dalapon by Liquid-Liquid Extraction,
Derivatization and GC with Electron Capture Detector," Methods for the Determination of
Organic Compounds in Drinking Water—Supplement 111EPA/600/R-95/131.
9. U.S. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
Spectrometry," Methods for the Determination of Organic Compounds in Drinking Water—
Supplement III, EPA/600/R-95/131.
10. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater, 20th edition, 1998, Washington, DC.
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11. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, December 2004.
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