THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
&EPA
PROGRAM ^
ETV
Baiteiie
U.S. Environmental Protection Agency punjng Tcchnology To Work
ETV Joint Verification Statement
TECHNOLOGY TYPE: RAPID TOXICITY TESTING SYSTEM
APPLICATION: DETECTING TOXICITY IN DRINKING WATER
TECHNOLOGY NAME: Microtox®
COMPANY: Strategic Diagnostics Inc.
ADDRESS: 111 Pencader Drive PHONE: 302-456-6789
Newark, Delaware 19702 FAX: 302-456-6782
WEB SITE: http://www.sdix.com/
E-MAIL: bferguson@sdix.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
Battelle 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 the Microtox® testing system.
VERIFICATION TEST DESCRIPTION
Rapid toxicity technologies use bacteria, enzymes, or small crustaceans that produce 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 took place
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between July 14 and August 22, 2003, various contaminants were added to separate drinking water samples and
analyzed by Microtox®. Responses to interfering compounds in clean drinking water also were 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 Microtox® to detect toxicity at various concentrations of contaminants, as well as to
measure the precision of the Microtox® results. The response of Microtox® 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.
Quality control samples included method blank samples, which consisted of American Society for Testing and
Materials Type II deionized water; positive control samples fortified with zinc sulfate or phenol; 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.
TECHNOLOGY DESCRIPTION
The following description of Microtox® was provided by the vendor and was not subjected to verification in this
test.
Microtox® is an in vitro testing system that uses bioluminescent bacteria to detect toxins in air, water, soil, and
sediment. Microtox® is a metabolic inhibition test that provides both acute toxicity and genotoxic analyses.
Microtox® uses a strain of naturally occurring luminescent bacteria. Vibrio fischeri. Vibrio fischeri are non-
pathogenic, marine, luminescent bacteria that are sensitive to a wide range of toxicants. When properly grown,
luminescent bacteria produce light as a by-product of their cellular respiration. Cell respiration is fundamental to
cellular metabolism and all associated life processes. Bacterial bioluminescence is tied directly to cell respiration,
and any inhibition of cellular activity (toxicity) results in a decreased rate of respiration and a corresponding
decrease in the rate of luminescence.
The Microtox® Model 500 Analyzer was tested as a stand-alone instrument along with the Microtox® reagent. The
Vibrio fischeri are supplied in a standard freeze-dried (lyophilized) state and, to analyze water samples, are
reconstituted in a salt solution, 2.5 milliliters (mLs) of the water sample are diluted with 250 microliter (|iL) of a
Microtox® reagent, then approximately 1 mL of water sample is added to 100 |iL of the reconstituted bacteria.
Luminescence readings are taken prior to adding the drinking water and then at 5 and 15 minutes after the
addition. When analyzing unknown samples, it is recommended that inhibition data be collected at both time
intervals to determine the most appropriate data collection time since the rates can vary depending on how the
toxicant affects the bacteria. Results are displayed as absolute light units.
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To determine whether a contaminant caused detectable inhibition, the inhibition exhibited by drinking water
spiked with a contaminant was compared to the inhibition exhibited by the unspiked drinking water. Four
replicates of each spiked sample were analyzed. A result was considered positive if the inhibition of the water
sample spiked with a contaminant plus or minus the standard deviation of four replicates did not include the
inhibition of the unspiked drinking water.
The temperature-controlled Microtox® maintains the test organisms and samples at a standard temperature of 15°C
As such, the Microtox® must be operated in a laboratory setting at ambient temperatures of between 15 and 30°C.
It detects light intensity at 490 nanometers, the wavelength emitted by the bacteria. Microtox® can be used with
Microtox®Omni™ software and a personal computer to collect, analyze, track, and store test data. Microtox®
weighs 21 pounds, measures 7-1/8 inches x 15-3/8 inches x 16-1/8 inches, and runs on 120/240 volts alternating
current. Microtox® Model 500 costs $17,895, and the reagents cost $360 for approximately 200 samples.
VERIFICATION OF PERFORMANCE
Endpoint and Precision/Toxicity Threshold: The table below shows the Microtox® percent inhibition data and
range of standards deviations for the contaminants and potential interferences that were tested. The toxicity
thresholds also are shown for each contaminant tested.
Parameter
Compound
Lethal
Dose (LD)
Cone.
(mg/L)
Average Inhibitions at Concentrations
Relative to the LD Concentration
(%)
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)
LD
LD/10
LD/100
LD/1,000
Contaminants in
DDW
Aldicarb
280
81
31
4
3
2-5
28
Colchicine
240
12
2
5
3
1-3
240
Cyanide
250
100
96
46
8
0^1
0.25
Dicrotophos
1,400
80
34
6
2
2-4
140
Thallium
sulfate
2,400
32
17
6
4
1-6
240
Botulinum
toxin(a)
0.30
-4
0
-1
-2
1-5
ND(b)
Ricin(c)
15
-1
-4
0
-2
2-4
ND
Soman
0.068(d)
0
-2
0
-4
3^
ND
VX
0.22
6
-2
9
3
2-18
ND
Potential
interferences in
DDW
Interference
Cone.
(mg/L)
Average Inhibitions at a
Single Concentration
(%)
Standard
Deviation
(%)
Aluminum
0.36
1
5
Copper
0.65
61
1
Iron
0.069
-5
2
Manganese
0.26
9
3
Zinc sulfate
3.5
28
1
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water from a system disinfected by chlorination produced an inhibition that was not significantly greater than zero
allowing further contamination to be detected in that particular matrix. No inhibition (false negative responses)
was detected for lethal doses of botulinum toxin, ricin, soman, and VX.
Other Performance Factors: The Microtox® pictorial manual was useful, initial light measurements served as a
good check of bacterial health and instrument operation, sample handling was easy, and sample throughput was
15 samples per hour. Microtox® was not tested in a non-laboratory setting because it is designed to be only a
laboratory benchtop instrument. 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 follow the
instructions to analyze samples successfully.
Original signed by Gabor J. Kovacs 11/13/03
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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