THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
ŁEPA
U.S. Environmental Protection Agency
PROGRAM jMk
ET#
Baneue
The Business of Innovation
ETV Joint Verification Statement
TECHNOLOGY TYPE: Enzymatic Test Kit
APPLICATION:
TECHNOLOGY
NAME:
COMPANY:
ADDRESS:
WEB SITE:
Detecting Chemical Warfare Agents, Carbamate
Pesticides, and Organophosphate Pesticides in Drinking
Water
OP-Stick Sensor
Protein-Biosensor
IPBS-CNRS	PHONE: +33(0)561 175 837
205 rte de Narbonne	FAX: +33(0)561 175 994
31077 Toulouse cedex, France
www.protein-biosensor.com
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 Protein-Biosensor's OP-Stick Sensor. This verification statement provides a summary of
the test results.

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VERIFICATION TEST DESCRIPTION
The objective of this verification test was to evaluate the ability of the OP-Stick Sensor to detect chemical
agents, carbamate pesticides, and organophosphate (OP) pesticides as contaminants in drinking water (DW).
This verification test assessed the performance of the OP-Stick Sensor relative to accuracy; false positive and
negative rates; precision; potential matrix and interference effects; and various operational factors including
operator observations, ease of use, and sample throughput from both a technical and non-technical operators'
perspective. The OP-Stick Sensor was evaluated using VX, sarin (GB), and soman (GD) (chemical agents);
aldicarb (carbamate pesticide); and dicrotophos (OP pesticide) in performance test (PT) and DW samples.
Quality Control (QC) samples were also included as part of the test matrix to ensure the integrity of the test.
PT samples included individual contaminants spiked into American Society for Testing and Materials
(ASTM) Type II deionized (DI) water at five different concentrations: the lethal dose concentration for each
contaminant, along with dilutions at approximately 10, 100, 1,000, and 10,000 times less than the lethal dose.
PT samples also included potential interferent samples containing a single concentration (10 times less than
the lethal dose) of the contaminant of interest in the presence of calcium (Ca) and magnesium (Mg) spiked
into ASTM Type II DI water, and humic and fiilvic acids spiked into ASTM Type II DI water. The vendor
provided a limit of detection (LOD) of <100 mg/L, which is less than one tenth of the LD50 (lethal dose for
half of the test subjects) for aldicarb. Therefore, the potential interferent samples were fortified at the LD50
level for aldicarb (260 mg/L). Each interferent mixture was prepared at two concentration levels: near the
upper limit of what would be expected in drinking water (250 milligrams per liter (mg/L) total concentration
for Ca and Mg, 5 mg/L total concentration for humic and fiilvic acids) and at a mid-low range of what would
be expected (50 mg/L total concentration for Ca and Mg, 1 mg/L total concentration for humic and fulvic
acids). Interferent PT samples were also analyzed without the addition of any contaminant. DW samples
consisted of chlorinated filtered surface water, chlorinated unfiltered surface water, chlorinated filtered
groundwater, and chloraminated filtered surface water collected from four geographically distributed
municipal water sources (OH, NY, FL, and CA, respectively). DW samples were analyzed before adding any
contaminant and after fortification with each individual contaminant at 10 times less than the lethal dose of
that contaminant, with the exception of aldicarb. As explained above, the DW samples were fortified at the
LD50 level for aldicarb (260 mg/L). All DW samples were dechlorinated prior to use. QC samples included
method blank (MB) samples and positive and negative controls, as supplied with the OP-Stick Sensor. All
samples were tested in triplicate.
The lethal dose of each contaminant was determined by calculating the concentration at which 250 milliliters
(mL) of water is likely to cause the death of a 70-kilogram (kg) person based on human oral LD50 (lethal dose
for half of the test subjects) data. Human oral LD50 data were not available for aldicarb, so rat oral LD50 data
were used instead. Lethal dose values are provided in the contaminant results tables below. Samples were
tested blindly by Battelle technical operators who were trained by the vendor in the use of the OP-Stick
Sensor. Contaminants were tested individually, and stock solutions of each contaminant were prepared
separately in ASTM Type II DI water. To minimize the loss of analytes to hydrolysis, contaminant stock
solutions prepared in DI water were made on a daily basis. In some cases, reference solutions were prepared
in ASTM Type II DI water using the stock solutions to prepare the test samples. In other cases, the actual
stock solutions were submitted for concentration confirmation by the respective reference analysis.
A subset of the samples was also tested by a non-technical operator using the OP-Stick Sensor. The non-
technical operator was someone with little to no laboratory experience who would be representative of a first
responder. For this test, the non-technical operator was a State of Ohio certified firefighter with Hazardous
Waste Operations and Emergency Response (HAZWOPER) training. The non-technical operator was trained
in the use of the OP-Stick Sensor by another Battelle staff person who was trained by the vendor. Only MB
samples and non-toxic control samples were analyzed as part of the operational factors assessment. As the
OP-Stick Sensor may be used by first-responders, its performance was evaluated under simulated first-
response conditions by having the operator dressed in a Level B protective suit, neoprene latex gloves, boots,
and a self-contained breathing apparatus (SCBA). The operator had prior experience working in personal
protective equipment (PPE). One set of MB samples was also tested without the use of PPE. Ease of use from
the perspective of the operator was documented both with and without the PPE.

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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.
Testing was conducted from November 2005 through February 2006. This verification statement, the full
report on which it is based, and the test/QA plan for this verification test are all available at
www.epa.gov/etv/centers/centerl.html.
TECHNOLOGY DESCRIPTION
The following description of the OP-Stick Sensor is based on information provided by the vendor. This
technology description was not verified in this test.
The OP-Stick Sensor is an enzymatic colorimetric assay designed for detecting organophosphate (including
thiophosphate) and carbamate (OP/C) pesticide residues in water, soil, and food. This technology had not been
used to test for chemical warfare agents (CWA) prior to this verification test. This assay is a field diagnostic test
that measures acetylcholinesterase (AChE) activity and is based on an enzyme engineered for increased
sensitivity to OP and C pesticides.
When not in the presence of inhibiting pesticides, AChE hydrolyzes acetylthiocholine to thiocholine, which reacts
with a colorimetric substrate on a test stick to produce a brown color. In the presence of OP/Cs (which are
oxidized during the test to an "oxon" form), AChE is irreversibly inhibited and color formation is reduced or
absent depending on the pesticide concentration. The intensity of the brown color is inversely proportional to
OP/C concentration.
Detection limits for the various OP/Cs differ depending on their ability to inhibit the enzyme. Combinations of
various OP/Cs will have an additive effect on the inhibition assay. The test allows screening without any
laboratory analysis of the sample. Positive tests would need confirmation by further analysis for qualitative and
quantitative assay.
One OP-Stick Sensor kit is composed of three tubes each labeled with a colored sticker and one test stick. Tube 1
(labeled yellow) contains an oxidizing agent for phosphorothioate activation in an "oxon" form. Tube 2 (labeled
blue) contains a neutralizing agent to avoid denaturation of AChE by the reagent from Tube 1. Tube 3 (labeled
red) contains the chromogen reagent. The OP-Stick Sensor kit is 10 by 5 by 2 centimeters. The price of the kit is
approximately $20.
VERIFICATION RESULTS
Accuracy was assessed by evaluating how often the OP-Stick Sensor result was positive in the presence of a
concentration above the limit of detection (LOD). Contaminant-only PT samples were used for this analysis.
For aldicarb, the vendor-provided LOD was >100 mg/L. LODs were not available for dicrotophos, VX, GB,
and GD. For these compounds, all analyzed contaminant-only PT samples greater than the concentration
level where consistent negative results were obtained were used for calculations. Results for VX, GB, and
GD were not consistently negative at any concentration level; thus, all analyzed PT samples were included in
the accuracy calculations. For dicrotophos, consistent negative results were observed at 1.4 mg/L; therefore
only contaminant-only PT samples with concentrations above this level were used to calculate accuracy.
A false positive response was defined as a response indicating the presence of a contaminant when the PT
interferent or DW sample was not spiked with contaminant. A false negative response was defined as a
response indicating the absence of a contaminant when the sample was spiked with a contaminant at a
concentration greater than the OP-Stick Sensor's LOD or consistent negative response level, as defined
above. Spiked PT (contaminant and interferent) samples and spiked DW samples were included in the
analysis. The precision of three replicates of each sample set was assessed by calculating the overall number
of consistent responses for all the sample sets. Operational aspects of the OP-Stick Sensor's performance such
as ease of use and sample throughput were evaluated through observations made during testing. Also

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addressed were qualitative observations of the verification staff from both the technical and non-technical
operators' perspective.
VX Summary Table




Parameter
Matrix
VX Concentration
Number
Detected/Number of
Samples
Qualitative Results
Contaminant-Only
PT Samples
DI Water
2.1 mg/L (a)
0.21 mg/L
0.021 mg/L
0.0021 mg/L
0.00021 mg/L
3/3
0/3
0/3
0/3
2/3

Interferent PT
Humic and Fulvic Acids
0.21 mg/L
3/6

Samples
Ca and Mg
0.21 mg/L
5/6

DW Samples
DW
0.21 mg/L
10/12
Accuracy
33% (5 out of 15) of the contaminant-only PT samples gave positive results
during testing at 0.00021 to 2.1 mg/L VX. Six inconclusive results were
observed in the nine replicates of the contaminant-only PT samples at and
below the concentration level of 0.021 mg/L VX.
False Positive Rate
No false positive results (0 out of 24) were observed during the testing with
VX.
False Negative Rate
Seven false negative results out of 39 samples were observed during testing
with VX: one replicate of the 0.021 mg/L VX in DI water PT sample, and
three replicates each of the 0.21 mg/L VX in DI water PT sample and the
0.21 mg/L VX in 1 mg/L humic and fulvic acid solution interferent sample.
Precision
62% (13 out of 21) of the sample sets showed consistent results among the
individual replicates within each set during testing with VX.
(a) Lethal dose




GB Summary Table




Parameter
Matrix
GB Concentration
Number
Detected/Number of
Samples
Qualitative Results
Contaminant-Only
PT Samples
DI Water
20 mg/L (a)
2.0 mg/L
0.2 mg/L
0.02 mg/L
0.002 mg/L
3/3
3/3
3/3
0/3
0/3

Interferent PT
Humic and Fulvic Acids
2.0 mg/L
6/6

Samples
Ca and Mg
2.0 mg/L
4/6

DW Samples
DW
2.0 mg/L
10/12
Accuracy
60% (9 out of 15) of the contaminant-only PT samples gave positive results
during testing with GB. Four inconclusive results were observed at the 0.02
and 0.002 mg/L GB concentration levels, with two negative results
observed at the 0.002 mg/L GB concentration level.
False Positive Rate
No false positive results (0 out of 24) were observed during testing with
GB.
False Negative Rate
Two false negative results out of 39 samples were observed during testing
with GB. These samples were at the lowest concentration of the
contaminant-only PT samples, fortified at 0.002 mg/L.
Precision
71% (15 out of 21) of the sample sets showed consistent results among the
individual replicates with each set during testing with GB.
w Lethal dose

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GD Summary Table
Parameter
Matrix
GD Concentration
Number
Detected/Number of
Samples
Qualitative Results
Contaminant-Only
PT Samples
DI Water
1.4 mg/L (a)
0.14 mg/L
0.014 mg/L
0.0014 mg/L
0.00014 mg/L
1/3
3/3
0/3
0/3
0/3
Interferent PT
Samples
Humic and Fulvic Acids
0.14 mg/L
4/6
Ca and Mg
0.14 mg/L
6/6
DW Samples
DW
0.14 mg/L
8/12
Accuracy
27% (4 out of 15) of the contaminant-only PT samples gave positive results
during testing at concentrations of 0.00014 to 1.4 mg/L GD. Seven
inconclusive results were observed at the concentration level of 0.014 mg/L
GD and below. Two negative results were observed at the lowest
concentration level tested, 0.00014 mg/L GD.
False Positive Rate
No false positive results (0 out of 24) were observed during testing with
GD.
False Negative Rate
Two false negative results (2 out of 39) were observed during testing with
GD. These results were observed at the lowest concentration level tested,
0.00014 mg/L GD.
Precision
57% (12 out of 21) of the sample sets showed consistent results among the
individual replicates within that set during testing with GD.
w Lethal dose
Aldicarb Summary Table
Parameter
Matrix
Aldicarb
Concentration
Number
Detected/Number of
Samples
Qualitative Results
Contaminant-Only
PT Samples
DI Water
260 mg/L (a)
26 mg/L
2.6 mg/L
0.26 mg/L
0.026 mg/L
3/3
0/3
0/3
0/3
0/3
Interferent PT
Samples
Humic and Fulvic Acids
260 mg/L
6/6
Ca and Mg
260 mg/L
6/6
DW Samples
DW
260 mg/L
12/12
Accuracy
100%) (3 out of 3) of the contaminant-only PT samples at 260 mg/L gave
positive results during testing with aldicarb. The vendor provided an LOD
of >100 mg/L, therefore none of the other concentration levels were
included in the calculation of accuracy.
False Positive Rate
One false positive result (out of 24 results) was observed during testing
with aldicarb. This positive result was observed in a 250 mg/L Ca and Mg
solution into which no aldicarb was spiked. The other two results for this
sample set were two negative results.
False Negative Rate
No false negative results (0 out of 27) were observed during testing with
aldicarb.
Precision
95%o (20 out of 21) of the sample sets showed consistent results among the
individual replicates with each set during testing with aldicarb. The one set
which did not have consistent results was the unfortified 250 mg/L Ca and
Mg solution.
w Lethal dose

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Dicrotophos Summary Table
Parameter
Matrix
Dicrotophos
Concentration
Number
Detected/Number of



Samples



1400 mg/L (a)
3/3

Contaminant-Only
PT Samples
DI Water
140 mg/L
14 mg/L
3/3
3/3
Qualitative Results


1.4 mg/L
0.14 mg/L
0/3
0/3

Interferent PT
Humic and Fulvic Acids
140 mg/L
6/6

Samples
Ca and Mg
140 mg/L
6/6

DW Samples
DW
140 mg/L
12/12
Accuracy
100% (9 out of 9) of the contaminant-only PT samples gave positive results
during testing with dicrotophos. Consistent negative results were observed
at and below the concentration level of 1.4 mg/L dicrotophos, therefore
only concentrations above this level were used to calculate accuracy.
False Positive Rate
No false positive results (0 out of 24) were observed during testing with
dicrotophos.
False Negative Rate
No false negative results (0 out of 30) were observed during testing with
dicrotophos.
Precision
100% (21 out of 21) of the sample sets showed consistent results among the
individual replicates within each set during the testing of dicrotophos.
w Lethal dose
Operational Factors:
Technical Operators
The Protein Biosensor OP-Stick Sensor was operated by one Battelle technician throughout testing with the
pesticides and by a different Battelle technician throughout testing with chemical warfare agents. The
technicians were trained by the vendor in the operation of the test kit. Both technicians had extensive
laboratory experience. The operators commonly observed that the tape on the bottom of the sticks is
extremely difficult to remove. Since the test samples may be potentially hazardous, it may not be acceptable
to remove the tape by hand.
Some variability within the production lots of kits was observed. The first lot of OP-Stick kits showed spots
that were various shades of yellow, grey, or green, not only black or white as the instructions indicated they
should be. This made it very difficult to discern the result for a particular sample, leading to inconclusive
results. The second lot of OP-Stick kits that were used toward the end of testing was much more reactive.
The reference spot on these tubes showed a deep black color, and the indicator spot was either a deep black or
plain white. These results were less subjective and much easier to read. Sample throughput varied with the
operator as multiple samples can be analyzed simultaneously. Physical accommodations (i.e., hood space or
table space) and operator preference for sample size may affect sample throughput.
Non-Technical Operator
Unspiked DI water samples were tested on the Protein Biosensor OP-Stick by a non-technical operator both
with and without PPE. During testing with the PPE on, the samples were analyzed while the operator wore
full PPE, consisting of a Level B suit, neoprene latex gloves, boots and SCBA. The SCBA was worn
throughout the entire testing procedure by the non-technical operator (only during the tests in which PPE
was to be donned) to represent the physical burden borne by a similarly outfitted first responder. However,
the operator ran the air from the SCBA only part of the time during testing to conserve the tank. Including set
up and operation, the time required for a test was approximately 1.5 to 2 hours; an operator equipped with a
SCBA would have to obtain a new tank of air for the duration of the test. A gloved operator would also have
trouble removing the tape on the OP-Stick. The operator had to use tweezers to remove the tape. The length
of time for the test and the need to manipulate the OP-Stick make its use difficult for users wearing PPE, such
as first responders.

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Original signed by Gregory Mack	9/18/06
Gregory A. Mack	Date
Vice President
Energy, Transportation, and Environment Division
Battelle
Original signed by Andrew Avel	10/2/06
Andrew P. Avel	Date
Acting Director
National Homeland Security Research Center
Office of Research and Development
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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