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:
ToxScreen-II
COMPANY:
CheckLight, Ltd.
ADDRESS:
P.O.Box 72 PHONE: (972) 4 9930530

Qiryat Tiv'on 36000, Israel FAX: (972) 4 9533176
WEB SITE:
http://www.checklight.co.il/
E-MAIL:
info@checklight.co.il
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 ToxScreen-II.
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
between July 14 and August 22, 2003, various contaminants were added to separate drinking water samples and

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analyzed by ToxScreen-II. 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 10,000 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 ToxScreen-II to detect toxicity at various concentrations of contaminants, as well as to
measure the precision of the ToxScreen-II results. The response of ToxScreen-II 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 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 sodium chloroacetate for the pro-
organic buffer samples and copper chloride for the pro-metal buffer samples); 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 ToxScreen-II was provided by the vendor and was not subjected to verification in this
test.
Luminous bacteria are self-maintained luminescent units that, under proper conditions, emit high and steady levels
of luminescence. Chemo-physical and biological toxicants that affect cell respiration, electron transport systems,
adenosine triphosphate generation, and the rate of protein or lipid synthesis alter the level of luminescence.
Similarly, agents that affect the cell's integrity, and especially membrane function, have a strong effect on in vivo
luminescence.
ToxScreen-II uses luminous bacteria, Photobacterium leiognathi, and special assay conditions to detect toxicants
in water samples. When used in concurrent tests, two assay buffers (one for detecting organic pollutants
[pro-organic buffer] and the other for detecting heavy metals [pro-metal buffer]) are used to discriminate between
the presence of organic and metal toxicants at sub milligram-per-liter concentrations. Inhibition of greater than
50% using either buffer is considered a positive result. A positive result in the pro-organic buffer suggests that the
contaminant causing the toxicity is organic, while a positive result in the pro-metal buffer suggests that the
contaminant is a metal. First, a freeze-dried bioassay reagent is hydrated with the provided hydration buffer and,
after five minutes, transferred into the provided storage buffer. The suspended reagent is maintained at 4°C until
use (aliquots can be drawn for up to seven days). Next, pro-organic and pro-metal concentrated assay buffers are
added separately to individual aliquots of source water samples, as well as to two aliquots of the reference sample.
Then, aliquots of suspended reagent are rapidly dispensed into test cuvettes. Finally, after 90 minutes of incubation
at ambient temperature, luminescence is measured using a portable luminometer. Changes in luminescence
(compared with the reference sample) reflect water toxicity.

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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 ToxScreen-II test kit contains stoppered vials holding freeze-dried luminous bacteria, empty test tubes,
hydration buffer, storage buffer, pro-organic concentrated assay buffer, pro-metal concentrated assay buffer, and
positive control solutions of copper chloride and sodium chloroacetate. A repeat dispenser (1 to 100 microliters
[|iL]) and 10- to 1,000-|iL pipettes and tips are required, but not provided in the kit. The price of the ToxScreen-II
test kit (1,000 single tests) is $300. The luminometer for reading the results of the bioassays costs $2,895. The
luminometer can be integrated with a personal computer for data acquisition, evaluation, and storage. The
luminometer is 5.9 x 11.0 x 6.7 inches and weighs approximately one pound. An insulating styrofoam case is
available for an additional cost to conduct field tests.
VERIFICATION OF PERFORMANCE
Endpoint and Precision/Toxicity Threshold: The table below presents ToxScreen-II 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.
Pro-Organic Buffer
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
LD/10,000
Contaminants
in DDW
Aldicarb
280
69
38
58
11
NA(a)
2-15
0.28
Colchicine
240
83
20
34
55
-31
3^18
0.24
Cyanide
250
99
85
99
68
14
0-29
0.25
0.025(b)
14
-6
NA
NA
NA
18-27
NA
Dicrotophos
1,400
99
99
81
41
41
0-18
0.14
Thallium sulfate
2,400
-10
52
-12
-32
NA
10-80
ND(C)
Botulinum
toxin(d)
0.30
69
47
-15
-8
NA
14-23
0.030
Ricin(e)
15
50
10
30
-10
NA
6-28
15
Soman
0.068(f)
5
-1
-9
-17
NA
9-31
ND
VX
0.22
-8
-5
-13
-59
NA
6-30
ND
Potential
interferences
in DDW
Interference
Cone.
(mg/L)
Average Inhibitions at a
Single Concentration (%)
Standard
Deviation
(%)

Aluminum
0.36
16
41
Copper
0.65
59
7
Iron
0.069
29
23
Manganese
0.26
-23
7
Zinc
3.5
-68
79
(a)	NA = Data not collected at this concentration level for this contaminant.
^	0.025 mg/L is not a lethal dose concentration, but it is used to describe additional concentrations of cyanide that were analyzed.
(c)	ND = Not detectable.
(d)	Lethal dose solution also contained 3 mg/L phosphate and 1 mg/L sodium chloride.
(e)	Lethal dose solution also contained 3 mg/L phosphate, 26 mg/L sodium chloride, and 2 mg/L sodium azide.
®	Due to the degradation of soman in water, the stock solution confirmation analysis confirmed that the concentration of the lethal
dose was 23% of the expected concentration of 0.30 mg/L.

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Pro-Metal Buffer
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
LD/10,000
Contaminants
in DDW
Aldicarb
280
-21
5
-17
-46
NA(a)
1-7
ND(b)
Colchicine
240
-42
-72
-83
-79
4
5-12
ND
Cyanide
250
99
48
80
35
31
1-13
0.025
0.025(c)
31
3
NA
NA
NA
5-8
NA
Dicrotophos
1,400
31
-76
-50
-32
-52
9-23
1,400
Thallium
sulfate
2,400
98
69
16
6
NA
1-9
240
Botulinum
toxin(d)
0.30
-44
1
-57
-97
NA
9-22
ND
Ricin(e)
15
-89
-5
-93
-89
NA
12-74
ND
Soman
0.068™
-15
-47
-62
-55
NA
3-71
ND
VX
0.22
-38
-19
-21
-31
NA
11-31
ND
Potential
interferences
in DDW
Interference
Cone.
(mg/L)
Average Inhibitions at a
Single Concentration (%)
Standard
Deviation
(%)

Aluminum
0.36
-26
19
Copper
0.65
94
2
Iron
0.069
3
32
Manganese
0.26
22
7
Zinc
3.5
100
0
(a)	NA = Data not collected at this concentration level for this contaminant,
w	ND = Not detectable.
(c)	0.025 mg/L is not a lethal dose concentration, but it is used to describe additional concentrations of cyanide that were analyzed.
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Field Portability: A single concentration of cyanide was analyzed in the field and the laboratory. Similar
performance was observed at both locations. Inhibition in the field was 97% ± 1% and in the laboratory was 68%
± 3% for the pro-organic buffer. The pro-metal buffer inhibition in the field was 61 % ± 6% and in the laboratory
was 35% ± 7%. ToxScreen-II was transported to the field in a vinyl bag that was provided by the vendor, but was
not included as part of the test kit.
Other Performance Factors: The pictorial instruction manual was useful, and instrument operation was
straightforward. Although the operators for this test had scientific backgrounds, based on the observations of the
verification test coordinator, operators with little technical training would probably be able to successfully
analyze sample sets. The operators analyzed approximately 25 samples per hour.
Original signed by Gabor J. Kovacs 11/19/03
Gabor J. Kovacs	Date
Vice President
Environmental Sector
Battelle
Original signed by Timothy E. Oppelt	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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