THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
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:
RAPIDTOXKIT
Strategic Diagnostics Inc.
Ill Pencader Drive
Newark, DE 19702
www.sdix.com
jambrosius@sdix.com
PHONE: (302) 456-6789
FAX: (302) 456-6782
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 Strategic Diagnostics Inc. RAPIDTOXKIT. 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 RAPIDTOXKIT 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 RAPIDTOXKIT 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 RAPIDTOXKIT 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. Note that
Strategic Diagnostics Inc. recommends that a 30% inhibition is required for a conclusive indication of
toxicity. During this test, a thorough evaluation of the toxicity threshold was performed. Therefore, the
toxicity threshold was determined with respect to the negative control rather than the 30% inhibition
threshold
• False positive responses—chlorination and chloramination by-product inhibition exceeding 30% 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 results less than 30%
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 RAPIDTOXKIT 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 one
million 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 larval crustacean within the
RAPIDTOXKIT 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
RAPIDTOXKIT to detect toxicity, as well as to measure the precision of the RAPIDTOXKIT results. The
response of the RAPIDTOXKIT 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 (vendor-specified); and negative control
samples, which consisted of the unspiked DDW.
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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.
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 RAPIDTOXKIT is based on information provided by the vendor. This
technology description was not verified in this test.
The RAPIDTOXKIT uses larvae of the anostracan crustacean T. platyurus to detect freshwater (including
drinking water) contamination. The RAPIDTOXKIT bioassays are performed in disposable test tubes using
T. platyurus hatched from cysts. Cyst hatching must begin 30 to 45 hours prior to performing the test. The
T. platyurus are exposed to samples for 15 minutes to one hour, after which a suspension of red microspheres
is added. The organisms ingest the microspheres, resulting in a deep red color in their digestive tracts.
Stressed (intoxicated) organisms either fail to take up particles altogether or ingest at a much lower rate. The
presence or the absence of colored microspheres in the digestive tract of the larval crustaceans is observed
under a stereomicroscope, and data are recorded on a sheet supplied with the RAPIDTOXKIT. The total
number of T. platyurus in the control (standard freshwater) well(s), and the number of T. platyurus that have
taken up the red particles are counted, and the fraction of larval crustaceans affected by the contaminant is
defined as the percent inhibition. As a guideline, 30 percent inhibition of particle uptake is considered a
threshold for the presence of potentially toxic compounds in the water.
Each test kit includes three 1-milliliter test tubes containing cysts of T. platyurus, one bottle of standard
freshwater, three hatching vessels, six sub-sampling tubes, 48 test tubes, six test tube holders, one vial with
red microspheres, one vial with fixative, six observation plates, six transparent covers for observation plates, a
blue plastic sheet and grid designed to be placed under plates to aid in observing and scoring test organisms,
standard operating procedure booklet, bench protocol, six sheets for scoring test results and calculating mean
inhibition of particle uptake, and a specification sheet containing batch numbers and shelf lives of kit
components. Materials required but not provided as part of the kit include a 25°C incubator with 4,000-lux
constant illumination, a dissection microscope with minimum 10X magnification, and an overhead light
source for the microscope. The complete RAPIDTOXKIT, adequate for 7 to 15 water samples each,
depending on the sample size, measures 30 centimeters by 25 centimeters by 10 centimeters and costs $196.
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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
100
4
56
100
100
100
27
100
100
99
LD/10
100
-51
13
100
100
100
14
99
100
10
Average
Inhibition (%)
29
100
20
-11
24
LD/100
100
-40
26
100
100
100
-2
100
79
4
LD/1,000
53
-32
28
51
-4
100
6
-2
29
22
Standard
Deviation (%)
6
0
4
11
9
Range of
Standard
Deviations
(%)
0-10
6-23
9-13
0-17
0-6
0
1-6
0-6
0-19
1-6
Toxicity
Thresh.
(mg/L)
0.26
ND
240
0.25
14
0.28
15
0.007
28
1.5
No results from the RAPIDTOXKIT were considered false positive because inhibition in the
chlorinated and chloraminated drinking water samples was always less than 30%.
Only botulinum toxin complex B exhibited inhibition less
dose concentration.
than 30% when analyzed at a lethal
The RAPIDTOXKIT contained clearly written instructions and illustrations. The contents of the
RAPIDTOXKIT were well identified. The only problem, other than the difficulty opening some
containers, was a slight difficulty getting the cysts out of the tubes with the recommended 1 mL
of water. Manually counting the number of red organisms under the microscope was tedious
when the results from many samples were determined one after the other over a few hours.
Overall, the RAPIDTOXKIT was easy to use, making it likely that a person with no formal
scientific training could conduct the tests.
The RAPIDTOXKIT was not evaluated for field portability.
Not including the 30 to 45-hour cyst-hatching period, approximately 25 analyses (including
method blanks and positive and negative controls) were completed in three hours. A maximum of
45 samples could be processed per kit.
ND = Significant inhibition was not detected.
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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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