THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM ^
ETV
SEPA
U.S. Environmental Protection Agency
Batteiie
. . . Putting Technology To Work
ETV Joint Verification Statement
TECHNOLOGY TYPE:
RAPID TOXICITY TESTING SYSTEM
APPLICATION:
DETECTING TOXICITY IN DRINKING WATER
TECHNOLOGY NAME:
ToxTrak™
COMPANY:
Hach Company
ADDRESS:
P.O. Box 389
PHONE: 970-669-3050
Loveland, CO 80539
FAX: 970-669-2932
WEB SITE:
http://www.hach.com/
E-MAIL:
mhaubric @ hach.com
The U.S. Environmental Protection Agency (EPA) supports 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.
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 pre-
paring 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 seven technology areas under ETV, is operated by
Batteiie in cooperation with EPA's National Exposure Research Laboratory. The AMS Center has recently
evaluated the performance of rapid toxicity testing systems used to detect toxicity in drinking water. This
verification statement provides a summary of the test results for ToxTrak™
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VERIFICATION TEST DESCRIPTION
Rapid toxicity technologies use bacteria, enzymes, or small crustaceans that product light or use oxygen at a steady
rate in the absence of toxic contaminants. Toxic contaminants in drinking water are indicated by a change in the
color or intensity of light or by a change in the rate of oxygen use. As part of this verification test, which, for this
technology, took place between July 14 and September 12, 2003, various contaminants were added to separate
drinking water samples and analyzed by ToxTrak™. Response to interfering compounds in clean drinking water
also was evaluated. Dechlorinated drinking water samples from Columbus, Ohio, (DDW) were fortified with
contaminants at concentrations ranging from lethal levels to levels 1,000 times less than the lethal dose and
analyzed. Endpoint and precision, toxicity threshold for each contaminant, false positive/negative responses, ease
of use, and sample throughput were evaluated.
Inhibition results (endpoints) from four replicates of each contaminant at each concentration level were evaluated
to assess the ability of the ToxTrak™ to detect toxicity at various concentrations of contaminants, as well as to
measure the precision of the ToxTrak™ results. The response of ToxTrak™ to compounds used during the water
treatment process (interfering compounds) was evaluated by analyzing separate aliquots of DDW fortified with
each potential interferent at approximately 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, unspiked
DDW was analyzed because Columbus, Ohio, uses chlorination as its disinfectant procedure. For the analysis of
by-products of the chloramination process, a separate drinking water sample from St. Petersburg, Florida, which
uses chloramination as its disinfection process, was obtained. The samples were analyzed after residual chlorine
was removed using sodium thiosulfate. Sample throughput was measured based on the number of samples
analyzed per hour. Ease of use and reliability were determined based on documented observations of the operators
and the verification test coordinator.
The test/QA plan for this verification test describes only a quantitative evaluation of the percent inhibition data
generated by each technology. The ToxTrak™ manufacturer indicated during the review of this report that a
qualitative data evaluation also should be performed to describe how a typical user is more likely to interpret and
use the ToxTrak™ results. Specifically, the manufacturer suggested that the percent inhibition results for each
concentration level of each contaminant also be evaluated as a qualitative indicator of whether or not a toxic
contaminant is present. The manufacturer stated that the percent inhibition results for each contaminant do not
necessarily increase linearly with the concentration of the contaminant but, depending on the contaminant, can at
times be represented by a non-linear relationship that may exhibit parabolic functionality that increases in
response, up to a certain concentration, but then begins to decrease. Therefore, in addition to the quantitative
evaluation of the data, a qualitative evaluation was performed.
Quality control samples included method blank samples, which consisted of American Society for Testing and
Materials Type II deionized water; positive control samples, which were provided 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. EPA QA staff also
performed a technical systems audit while testing was being conducted.
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TECHNOLOGY DESCRIPTION
The following description of ToxTrak™ was provided by the vendor and was not subjected to verification in this
test.
ToxTrak™ system is a colorimetric test based on resazurin dye chemistry. Resazurin is a redox-active dye that,
when reduced, changes color from blue to pink. Resazurin is in the oxidized, blue state at the beginning of the test.
The bacteria oxidize the glucose added to the sample with the dye and reduce the resazurin. The resazurin is first
reduced by two electrons to resorufin, which is pink. Resorufin can be further reduced by two electrons to
dihydroresorufin, which is colorless. Dihydroresorufin can be reoxidized by atmospheric oxygen to resorufin. To
prevent interference, readings must be taken before a significant amount of resorufin has been reduced. This
inhibition or acceleration of resazurin reduction is taken as an indication of toxicity in the test. Substances that are
toxic to bacteria can inhibit their metabolism and thus inhibit the rate of resazurin reduction. If the reaction time is
too long, the indicator is too far reduced and interference will result. Percent inhibition results of several replicate
results that are greater than 10% inhibition or more negative than -10% are indications of toxicity, according to the
vendor's protocol. The presence/absence data trend among the four replicates was evaluated to determine if
ToxTrak™ consistently indicated the presence (or absence) of the contaminants at the measured concentrations.
Three out of four positive responses were required to indicate the presence of a contaminant at that concentration
level. If two results were positive and two negative, the overall result was not considered a positive or a negative
result.
ToxTrak™ works with different species of bacteria (including both Gram positive and Gram negative species) or
mixed cultures. The ToxTrak™ kit includes 12 reusable sample cells with caps, several capsules of dried bacteria,
lauryl tryptose broth for culturing the bacteria, 50 ToxTrak™ Reagent Powder Pillows, 15 milliliters (mLs) of
ToxTrak™ accelerator solution, 20 sterile transfer pipettes, a test tube rack, forceps, five germicidal cloths, a lab
marker, illustrated instructions, and a carrying case. For this verification test, the vendor provided a Hach
DR/4000V spectrophotometer for the laboratory-based colorimeter measurements and a Hach DR890 handheld
colorimeter for the field measurements. Any colorimeter that can analyze samples at a wavelength at or near
603 nanometers could be used in conjunction with the ToxTrak™ reagents. The ToxTrak™ kit costs $280, and
reagent sets cost $100. The reagent kit can be used with the test kit, a spectrophotometer, or a colorimeter The
DR/4000V spectrophotometer used in this verification test cost $3,950.
VERIFICATION OF PERFORMANCE
Endpoint and Precision/Toxicity Threshold: The table below presents ToxTrak™ percent inhibition data and
the range of standard deviations for the contaminants and potential interferences that were tested. The toxicity
thresholds also are shown for each contaminant tested.
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Parameter
Compound
Lethal
Dose
(LD)
Cone.
(mg/L)
Average Percent Inhibitions at
Concentrations Relative to the LD
Concentration (Qualitative Result:
"+" = present= absent)
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)
LD
LD/10
LD/100
LD/1,000
Quan.
Qual.
Contaminants
in DDW
Aldicarb
280
-16 (+)
-7 (-)
12 (+)
-11 (NC)W
3-24
ND(b)
280
Colchicine
240
14 (+)
8 (NC)
-3 (-)
8 (NC)
3-24
ND
240
Cyanide
250
72 (+)
11 (+)
-6 (-)
-10(-)
7-17
250
25
Dicrotophos
1,400
-60 (+)
-53 (+)
-37 (+)
-12 (NC)
14-82
ND
14
Thallium sulfate
2,400
-104 (+)
-37 (+)
-21 (+)
-38 (+)
22-62
ND
2.4
Botulinum toxin(c)
0.30
10 (NC)
5 (-)
6 (-)
18 (+)
6-16
ND
ND
Ricin(d)
15
-32 (+)
-38 (+)
-33 (+)
-45 (+)
11-27
ND
0.015
Soman
0.15
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Other Performance Factors: The pictorial manual was useful, sample handling was easy, and sample throughput
was approximately 25 samples per hour. Although the operators had scientific backgrounds, based on the
observations of the verification test coordinator, operators with little technical training would probably be able to
analyze sample using only the instruction manual as a guide.
Original signed by Gabor J. Kovacs 11/19/03
Gabor J. Kovacs Date
Vice President
Environmental Sector
Battelle
Original signed by Timothy E. Qppelt 12/1/03
Timothy E. Oppelt Date
Director
National Homeland Security Research Center
U.S. Environmental Protection Agency
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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