THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
f,EPA , Baiteiie
The Business of Innovation
U.S. Environmental Protection Agency
ETV Joint Verification Statement
TECHNOLOGY TYPE: Rapid Toxicity Testing System
APPLICATION: Detecting Toxicity in Drinking Water
TECHNOLOGY
NAME: ToxScreen-II
COMPANY: CheckLight Ltd.
ADDRESS: Kolner Strasse 6 PHONE: (972) 4 9930530
P.O. Box 72 FAX: (972) 4 9533176
Qiryat-Tiv'on 36000
Israel
WEB SITE: www.checklight.co.il
E-MAIL: info@checklight.co.il
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 CheckLight Ltd. ToxScreen-II Test Kit. This verification statement provides a summary
of the test results.
-------
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, ToxScreen-II 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 ToxScreen-II 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.
ToxScreen-II 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
CheckLight Ltd. recommends that a 50% 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 50% inhibition threshold
• False positive responses—chlorination and chloramination by-product inhibition with respect to
unspiked American Society for Testing and Materials Type II deionized water samples that exceeded
50%
• False negative responses—contaminants that were reported as producing less than 50% and/or were not
significantly different from 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).
ToxScreen-II 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 bacteria within the ToxScreen II
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 ToxScreen-II to detect
toxicity, as well as to measure the precision of ToxScreen-II results. The response of ToxScreen-II 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 (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.
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 ToxScreen-II Test Kit is based on information provided by the vendor. This
technology description was not verified in this test.
ToxScreen-II provides on-site detection of organic and inorganic toxicants, such as heavy metals; pesticides;
herbicides; chlorinated hydrocarbons; polychlorinated biphenyls; benzene, toluene, ethylbenzene, and
xylenes; and phencyclidine. ToxScreen-II can be used in both field and laboratory testing. Typical
applications include effluent toxicity testing; surface and ground water screening for changes in water quality;
and raw drinking water monitoring for early warning of dangerous spills, accidents, and
sabotage/bioterrorism.
Under proper conditions, luminous bacteria emit high and steady levels of luminescence. Chemical 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 a cell's
integrity and membrane function have a strong effect on luminescence. Hence, toxicants of different
characteristics such as pesticides, herbicides, chlorinated hydrocarbons, and heavy metals exert a measurable
effect on a bacterial luminescence system. By comparing the luminescence level obtained in a suspected toxic
sample with that obtained in a clean water control sample after a short period of incubation, very low
concentrations of a broad range of toxicants can be detected. To detect toxicants in water samples,
ToxScreen-II uses a highly sensitive variant of Photobacterium leiognathi and two assay buffers: one for
detecting heavy metals (Pro-Metal Buffer) and the other for organic pollutants (Pro-Organic Buffer). When
used concurrently, these buffers are designed to discriminate between the presence of organic and metal
toxicants at submilligram per liter concentrations.
The ToxScreen-II luminometer is 150 millimeters (mm) wide by 280 mm deep by 170 mm high and weighs
approximately two kilograms. The test kit comes with stoppered vials holding freeze-dried luminous bacteria,
hydration buffer, storage buffer, Pro-Metal concentrated assay buffer, Pro-Organic concentrated assay buffer,
concentrated positive control solutions, and empty test tubes. The portable luminometer costs $3,950, and a
starter kit including reagents for 1,000 single tests costs $550.
-------
VERIFICATION RESULTS
Pro-Organic Buffer
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
surf ate
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
50
-87
75
100
70
83
68(a)
-6
66
-3
LD/10
-26
14
17
100
23
-10
9
-202
13
-5
LD/100
0
16
4
95
3
-32
1
4
9
2
LD/1,000
-50
-54
1
72
1
-20
10
15
-1
-6
Average Inhibition (%)
Initial
Analysis
-4
3
0
0
-1
Reanalysis(b)
-12
5
-6
3
NR
Range of
Standard
Deviations
(%)
12-18
21-58
2-5
0-2
3-29
2-10
2-5
10-68
3-6
3-9
Toxicity
Thresh.
(mg/L)
ND
ND
24
0.25
140
1,400
ND
ND
28
ND
Standard Deviation (%)
Initial
Analysis
4
3
1
4
o
J
Reanalysis(b)
1
15
4
3
NR
None of the disinfection by-product samples produced an inhibition significantly greater than 50%, the
inhibition level suggested by CheckLight Ltd. to conclusively determine toxicity.
Aldicarb, botulinum toxin complex B, soman, and VX produced an inhibition that either did not exceed
50% or were not significantly different from the negative control at the lethal dose concentrations. For
ricin in the Pro-Organic buffer, the inhibition of the lethal dose was significantly different from the
negative control, but not significantly different from the inhibition generated by the preservative blank.
ToxScreen-II included clearly written instructions with good illustrations. The contents of the
ToxScreen-II were well labeled, making it easy to follow the instructions. A minimum of three hours
was required to rehydrate the bacteria, which must be stored at -14°C prior to rehydration. After
rehydration, the bacteria can be used for up to seven days; however, the vendor suggested using them
within one day. Overall, the ToxScreen-II was easy to use, making it likely that a person with no
formal scientific training could conduct the tests.
ToxScreen-II was transported from a laboratory to a storage room to simulate operation in a non-
laboratory location. It was tested with cyanide at the lethal dose concentration, and the results
generated (>90% inhibition) were very similar to those obtained in the laboratory. No carrying case
was provided with ToxScreen-II (one is available for purchase from Checklight Ltd.); however, all
materials except the luminometer were transported in a small cardboard box. The box and luminometer
were easily carried by one person, and setup for analysis took less than 10 minutes.
Approximately 25 analyses were completed each hour using both buffers, and approximately 1,000
samples could be processed per kit.
ND = Significant inhibition was not detected.
NR = Not reanalyzed.
(a) Inhibition was not significantly different from the preservative blank.
(b) Potential interferences were reanalyzed due to four suspect negative inhibitions during the initial analysis with the Pro-
Metal buffer.
-------
Pro-Metal Buffer
Parameter
Contaminant
sinDDW
Potential
interferences
inDDW
False
positive
response
False
negative
response
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
15.0
1.4
2,800
2.0
Cone.
(mg/L)
0.5
0.6
0.15
0.25
2.5
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
-19
-185
12
89
55
98
o
5
-55
79
5
LD/10
-7
-121
2
64
-1
2
-2
17
53
-11
LD/100
33
-91
8
44
-10
-10
2
-66
27
-11
LD/1,000
-31
-40
-9
19
-3
-7
2
-4
4
-3
Average Inhibition (%)
Initial
Analysis
-395
-299
Reanalysis(a)
-13
30
-399 -8
-368
86
5
NR
Range of
Standard
Deviations
(%)
10-35
18-104
2-6
1-7
2-4
0-4
2-4
13-22
1-4
5-10
Toxicity
Thresh.
(mg/L)
ND
ND
ND
0.25
140
700
ND
ND
28
ND
Standard Deviation (%)
Initial
Analysis
29
26
18
15
0
Reanalysis(a)
3
4
3
4
NR
Neither the chlorination nor chloramination samples generated an inhibition greater than 50%.
However, the chloramination sample generated a result that indicated an enhancement in luminescence
(i.e., a negative inhibition), which, according to Checklight Ltd., can also indicate toxicity.
The inhibition of the chloramination by-products was -75% ± 20% with DI water as the negative
control. If a contaminant causing a 75% inhibition had been present in this water and DI water was used
as the negative control, the inhibition would have been close to 0% — a false negative response. This
underscores the need to use negative control samples that are as similar as possible to the samples being
analyzed. A second type of false negative response occurred (for aldicarb, colchicine, botulinum toxin,
ricin, soman, and VX) when the inhibition was not greater than 50% in the presence of a lethal dose of
contaminant.
ND = Significant inhibition was not detected.
NR = Not reanalyzed.
See the Pro-Organic Buffer table for descriptions for ease of use, field portability, and throughput.
-------
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.
------- |