THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
vvEPA
U.S. Environmental Protection Agency
Batreiie
Trtc Business o/ Innovation
ETV Joint Verification Statement
TECHNOLOGY TYPE: Rapid Toxicity Testing System
APPLICATION: Detecting Toxicity in Drinking Water
TECHNOLOGY
NAME:
COMPANY:
ADDRESS:
WEB SITE:
E-MAIL:
AbraTox Kit
Abraxis
54 Steamwhistle Drive
Warminster, PA 18974
www.abraxiskits.com
info@abraxiskits.com
PHONE: (215) 357-3911
FAX: (215) 357-5232
The U.S. Environmental Protection Agency (EPA) has established the Environmental Technology Verification
(ETV) Program to facilitate the deployment of innovative or improved environmental technologies through
performance verification and dissemination of information. The goal of the ETV Program is to further
environmental protection by accelerating the acceptance and use of improved and cost-effective technologies.
ETV seeks to achieve this goal by providing high-quality, peer-reviewed data on technology performance to
those involved in the design, distribution, financing, permitting, purchase, and use of environmental
technologies. Information and ETV documents are available at www.epa.gov/etv.
ETV works in partnership with recognized standards and testing organizations, with stakeholder groups
(consisting of buyers, vendor organizations, and permitters), and with individual technology developers. The
program evaluates the performance of innovative technologies by developing test plans that are responsive to
the needs of stakeholders, conducting field or laboratory tests (as appropriate), collecting and analyzing data,
and preparing peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality
assurance (QA) protocols to ensure that data of known and adequate quality are generated and that the results
are defensible.
The Advanced Monitoring Systems (AMS) Center, one of six technology areas under ETV, is operated by
Battelle in cooperation with EPA's National Exposure Research Laboratory. The AMS Center evaluated the
performance of the Abraxis AbraTox Kit. This verification statement provides a summary of the test results.
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VERIFICATION TEST DESCRIPTION
Rapid toxicity technologies use various biological organisms and chemical reactions to indicate the presence
of toxic contaminants. The toxic contaminants are indicated by a change or appearance of color or a change in
intensity. As part of this verification test, 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 could detect the toxicity caused by each contaminant, its response to
interfering compounds, such as water treatment chemicals and by-products in clean drinking water, was
evaluated.
The AbraTox Kit was evaluated by
• Endpoints and precision—percent inhibition for all concentration levels of contaminants and potential
interfering compounds and precision of replicate analyses
• Toxicity threshold for each contaminant—contaminant level at which higher concentrations generate
inhibition significantly greater than the negative control and lower concentrations do not
• False positive responses—chlorination and chloramination by-product inhibition with respect to
unspiked American Society for Testing and Materials Type II deionized water samples
• False negative responses—contaminants that were reported as producing inhibition similar to the
negative control when present at lethal concentrations (the concentration at which 250 milliliters of
water would probably cause the death of a 154-pound person) or negative background inhibition that
caused falsely low inhibition
• Other performance factors (sample throughput, ease of use, reliability).
The AbraTox Kit was verified by analyzing a dechlorinated drinking water sample from Columbus, Ohio
(DDW), fortified with contaminants (at concentrations ranging from lethal levels to concentrations up to
100,000 times less than the lethal dose) and interferences (metals possibly present as a result of the water
treatment processes). Dechlorinated water was used because free chlorine kills the bacteria within the
AbraTox reagent and can degrade the contaminants during storage. Inhibition results (endpoints) from four
replicates of each contaminant at each concentration level were evaluated to assess the ability of the AbraTox
Kit to detect toxicity, as well as to measure the precision of the AbraTox Kit results. The response of the
AbraTox Kit to possible interferents was evaluated by analyzing them at one-half of the concentration limit
recommended by the EPA's National Secondary Drinking Water Regulations guidance. For analysis of by-
products of the chlorination process, the unspiked DDW was analyzed because Columbus, Ohio, uses
chlorination as its disinfectant procedure. For the analysis of by-products of the chloramination process, a
separate drinking water sample was obtained from the Metropolitan Water District of Southern California
(LaVerne, California), which uses chloramination as its disinfection process. The samples were analyzed after
residual chlorine was removed using sodium thiosulfate. Sample throughput was measured based on the
number of samples analyzed per hour. Ease of use and reliability were determined based on documented
observations of the operators.
Quality control samples included method blank samples, which consisted of American Society for Testing
and Materials Type II deionized water; positive control samples (supplied by the vendor); and negative
control samples, which consisted of the unspiked DDW.
QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a
technical systems audit, a performance evaluation audit, and a data quality audit of 10% of the test data.
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This verification statement, the full report on which it is based, and the test/QA plan for this verification test
are all available atwww.epa.gov/etv/centers/centerl.html.
TECHNOLOGY DESCRIPTION
The following description of the AbraTox Kit is based on information provided by the vendor. This
technology description was not verified in this test.
The AbraTox Kit 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 microliters (uL) of the water sample to be 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 the test sample.
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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VERIFICATION RESULTS
Parameter
Contaminants in
DDW
Potential
interferences in
DDW
False positive
response
False negative
response
Ease of use
Field portability
Throughput
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
U(a)
31(a)
14
-32
LD/10 LD/100
21
-11
-39
71
10
-3
g(a)
-24
20
-15
Average Inhibition
-4
32
7
1
15
-3
-40
2
43
14
-2
-100
-10
5
2
LD/1,000
12
-2
-51
25
26
4
?(a)
-1
6
-7
Standard Deviation
10
11
3
6
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
1.4°°
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.
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.
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%.
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.
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Original signed by Gregory A. Mack
Gregory A. Mack Date
Vice President
Energy, Transportation, and Environment Division
Battelle
6/22/06 Original signed by Andrew P.Avel
Andrew P. Avel
Acting Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
8/7/06
Date
NOTICE: ETV verifications are based on an evaluation of technology performance under specific,
predetermined criteria and the appropriate quality assurance procedures. EPA and Battelle make no expressed or
implied warranties as to the performance of the technology and do not certify that a technology will always
operate as verified. The end user is solely responsible for complying with any and all applicable federal, state,
and local requirements. Mention of commercial product names does not imply endorsement.
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