THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
4»EPA Batteiie
U.S. Environmental Protection Agency ^e Business of Innovation
ETV Joint Verification Statement
TECHNOLOGY TYPE: Enzymatic Test Kit
APPLICATION: Detecting Chemical Agents, Carbamate Pesticides, and
Organophosphate Pesticides in Drinking Water
TECHNOLOGY
NAME: Neuro-IQ Tox Test Kit™
COMPANY: Aqua Survey, Inc.
ADDRESS: 469 Point Breeze Road PHONE: (908) 788-8700
Flemington, NJ 08822 FAX: (908) 788-9165
WEB SITE: www.aquasurvey.com
E-MAIL: hayes@aquasurvey.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 Aqua Survey's Neuro-IQ Tox Test Kit™. 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 Neuro-IQ Tox Test Kit™ to detect
chemical agents, carbamate pesticides, and organophosphate (OP) pesticides in drinking water (DW). This
verification test assessed the performance of the Neuro-IQ Tox Test Kit™ 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 Neuro-IQ Tox Test Kit™ 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 fulvic acids spiked into
ASTM Type II DI water. 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 fulvic 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. All
DW samples were dechlorinated prior to use. QC samples included method blank (MB) samples and control
water samples, as prescribed by the Neuro-IQ Tox Test Kit™ protocol. 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 Neuro-IQ Tox
Test Kit™. 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 Neuro-IQ Tox Test Kit™. 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 Neuro-IQ Tox Test Kit™ 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 Neuro-IQ Tox Test Kit™ 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 Neuro-IQ Tox Test Kit™ is based on information provided by the vendor.
This technology description was not verified in this test.
The Neuro-IQ Tox Test Kit™ tests water supplies for the presence of contaminants in drinking water in
sufficient concentrations to cause harm to humans. The Neuro-IQ-Tox Test Kit™ is
acetocholine/cholinesterase based and detects contaminants of interest by interrupting an enzymatic reaction.
The presence or absence of contaminants at significant concentrations is predicted by adding two reagents to
water samples and measuring the drop in pH after three minutes. This test is generally performed in replicates
of up to four. If the pH of the test samples is higher (> 0.2 pH units) than the control water sample's three-
minute pH reading, this indicates the possible presence of a significant threat contaminant concentration.
The test can be conducted by a technician with basic laboratory skills. Data are recorded on a scorecard
provided with the kit.
Enough reagent is provided with the Neuro-IQ Tox Test Kit™ to assay up to 400 test water samples. The
Neuro-IQ-Tox Test Kit™ retails for $300.
VERIFICATION RESULTS
To allow for testing of all of the samples prescribed for this verification test, differences in pH were
calculated on a sample by sample basis. In addition, three samples were tested with each control water
sample since each type of sample need only to be tested in triplicate for this verification test. These changes
were recommended by the vendor.
Only qualitative (positive, negative) results were used to calculate the parameters presented in the following
tables for the Neuro-IQ Tox Test Kit™. Qualitative results were determined based on the difference between
the control water pH value and the sample pH value. If the sample pH value was > 0.2 pH units above the
control water's pH value, then the sample was concluded to be a positive result, indicating the presence of the
contaminant in the sample. If the test sample pH value was < 0.2 pH units above the control water's pH
value, then a negative result was recorded for that sample.
Accuracy was assessed by evaluating how often the Neuro-IQ Tox Test Kit™ result was positive in the
presence of a concentration above the limit of detection (LOD). Contaminant-only PT samples were used for
this analysis. LODs were not available for any of the contaminants tested in this verification test. Thus, all
analyzed contaminant-only PT samples greater than the concentration level where consistent negative results
were obtained were used for accuracy calculations. This level was defined at 1.4 mg/L for dicrotophos.
Results for VX, GB, GD, and aldicarb were not consistently negative at any level; thus, all analyzed PT
samples were included in the accuracy calculations.
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 Neuro-IQ Tox Test Kit™'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.
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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 Neuro-IQ Tox Test Kit™'s
performance such as ease of use and sample throughput were evaluated through observations made during
testing. Also addressed were qualitative observations of the verification staff from both the technical and non-
technical operators' perspective.
VX Summary Table
Parameter
Qualitative Results
Contaminant-
Only PT
Samples
Interfered PT
Samples
DW Samples
Accuracy
False Positives
False Negatives
Precision
Matrix
DI Water
Humic and Fulvic
Acids
Ca and Mg
DW
VX
Concentration
2.1 mg/L (a)
0.21 mg/L
0.021 mg/L
0.0021 mg/L
0.00021 mg/L
0.21 mg/L
0.21 mg/L
0.21 mg/L
Number
Detected/Number
of Samples
3/3
3/3
3/3
3/3
3/3
6/6
5/6
12/12
100% (15 out of 15) of the contaminant-only PT samples were
positive.
Thirteen false positive responses were obtained. Seven positive
responses were found across unspiked 1 mg/L and 5 mg/L humic
and fulvic acids as well as unspiked 50 mg/L Ca and Mg samples.
All six replicates for unspiked OH and FL DW yielded positive
results.
One false negative result was obtained for spiked PT and DW
samples. One replicate of the spiked 250 mg/L Ca and Mg samples
returned a negative result.
90% (19 out of 21) of the sample sets showed consistent results
among the individual replicates within that set.
(a)
Lethal dose.
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GB Summary Table
Parameter
Qualitative Results
Contaminant-
Only PT
Samples
Interfered PT
Samples
DW Samples
Accuracy
False Positives
False Negatives
Precision
Matrix
DI Water
Humic and Fulvic
Acids
Ca and Mg
DW
GB
Concentration
20 mg/L (a)
2.0 mg/L
0.20 mg/L
0.020 mg/L
0.0020 mg/L
2.0 mg/L
2.0 mg/L
2.0 mg/L
Number
Detected/Number
of Samples
2/3
3/3
3/3
3/3
3/3
6/6
6/6
12/12
93% (14 out of 15) of the contaminant-only PT samples were
positive.
Thirteen false positive responses were obtained. Seven positive
responses were found across unspiked 1 mg/L and 5 mg/L humic
and fulvic acids as well as unspiked 50 mg/L Ca and Mg samples.
All six replicates for unspiked OH and FL DW yielded positive
results.
One false negative result was obtained for spiked PT and DW
samples. One replicate of the spiked DI water samples at the lethal
dose returned a negative result.
90% (19 out of 21) of the sample sets showed consistent results
among the individual replicates within that set.
Lethal dose.
GD Summary Table
Parameter
Qualitative Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
False Positives
False Negatives
Precision
Matrix
DI Water
Humic and Fulvic
Acids
Ca and Mg
DW
GD
Concentration
1.4 mg/L (a)
0.14 mg/L
0.0 14 mg/L
0.0014 mg/L
0.000 14 mg/L
0.14 mg/L
0.14 mg/L
0.14 mg/L
Number
Detected/Number
of Samples
3/3
3/3
o /o
3/3
3/3
1/3
6/6
6/6
12/12
87% (13 out of 15) of the contaminant-only PT samples were
positive.
Thirteen false positive responses were obtained. Seven positive
responses were found across unspiked 1 mg/L and 5 mg/L humic
and fulvic acids as well as unspiked 50 mg/L Ca and Mg samples.
All six replicates for unspiked OH and FL DW yielded positive
results.
Two false negative results were obtained for spiked PT and DW
samples. Two replicates of the spiked DI water samples at 10,000x
less than the lethal dose (0.00014 mg/L) returned a negative result.
90% (19 out of 21) of the sample sets showed consistent results
among the individual replicates within that set.
(a) Lethal dose.
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Aldicarb Summary Table
Parameter
Qualitative Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
False Positives
False Negatives
Precision
Matrix
DI Water
Humic and Fulvic
Acids
Ca and Mg
DW
Aldicarb
Concentration
260 mg/L (a)
26 mg/L
2.6 mg/L
0.26 mg/L
0.026 mg/L
26 mg/L
26 mg/L
26 mg/L
Number
Detected/Number
of Samples
3/3
3/3
3/3
0/3
1/3
6/6
3/6
12/12
67% (10 out of 15) of the contaminant-only PT samples were
positive.
Three false positive responses were obtained. Positive responses
were found for all replicates of the unspiked 5 mg/L humic and
fulvic acids samples.
Eight false negative results were obtained for spiked PT and DW
samples. Five samples of the spiked DI water samples returned a
negative result. All three replicates of the spiked 250 mg/L Ca and
Mg samples yielded negative results.
95% (20 out of 21) of the sample sets showed consistent results
among the individual replicates within that set.
(a) Lethal dose.
Dicrotophos Summary Table
Parameter
Qualitative
Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
False Positives
False Negatives
Precision
Matrix
DI Water
Humic and Fulvic
Acids
Ca and Mg
DW
Dicrotophos
Concentration
1400 mg/L (a)
140 mg/L
14 mg/L
1.4 mg/L
0.14 mg/L
140 mg/L
140 mg/L
140 mg/L
Number
Detected/Number of
Samples
3/3
0/3
1/3
0/3 (b)
0/3 (b)
4/6
6/6
12/12
44% (4 out of 9) of the contaminant-only PT samples above the level
of consistent negative responses were positive.
Three false positive responses were obtained.
were found for all replicates of the unspiked 5
fulvic acids samples.
Positive responses
mg/L humic and
Seven false negative results were obtained for spiked PT and D W
samples. Five samples of the spiked DI water samples returned a
negative result. Two replicates of the spiked 1 mg/L fulvic and
humic acid samples yielded negative results.
90% (19 out of 21) of the sample sets showed
among the individual replicates within that set
consistent results
(b)
Lethal dose.
Not used in accuracy calculations because samples are at or below level of consistent negative response.
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Operational Factors:
Technical Operators
The Neuro-IQ Tox Test Kit™ was operated by one Battelle technician throughout testing with the pesticides
and by a different Battelle technician throughout testing with chemical agents. Both technicians had
extensive laboratory experience. Multiple problems were encountered with the test kit operation, including a
faulty pH probe and unstable pH readings after adding Reagent B and when trying to reach a pH of 8.30. Two
reagents are used to test a water sample with the Neuro-IQ Tox Test Kit™. Reagent A is frozen and must
come to room temperature before it can be used. Reagent B has to be reconstituted with DI water before use.
Individual vials of each reagent were provided with the kit to make daily testing easier. Between the two
operators, it took an average of 64 ± 18 minutes to complete testing on a set of three samples using the Neuro-
IQ Tox Test Kit™. The operators were able to analyze between three and six sets of samples a day.
Non-Technical Operator
Unspiked DI water samples were tested on the Neuro-IQ Tox Test Kit™ by a non-technical operator both
with and without PPE. Adjusting the pH to 8.30 was not easy for the operator to accomplish and many times
that pH was exceeded. Reagent A was hard to handle with the gloves on, and the magnetic stir plate was
difficult to adjust while in full PPE. The pipettes needed for the test were cumbersome, confusing, and
difficult to use for a non-technical operator. The 50-mL beakers used for each sample were small, and the
level of the liquid in them was shallow, making it difficult, particularly while in PPE, to correctly place the
pH probe and magnetic stirrer. Testing of three MB samples while in PPE took 52 minutes, while testing of
three MB samples without PPE took 40 minutes. The test kit would be very difficult for the operator to set up
and use if no table-top surface was available in the field. A control water sample, or a water sample that is the
same matrix as the test sample but not contaminated, is needed for the Neuro-IQ Tox Test Kit™ protocol.
Obtaining such a sample could be problematic in the field. Overall, the Neuro-IQ Tox Test Kit™ would be
hard for a first-responder with no experience with the kit and no laboratory skills to use if the operator is
donned in the level of PPE used in this verification test.
Original signed by Gregory Mack
Gregory A. Mack Date
Vice President
Energy, Transportation, and Environment Division
Battelle
9/18/06 Original signed by Andrew Avel
Andrew P. Avel
Acting Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
10/2/06
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.
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