September 2006
Environmental Technology
Verification Report
AQUA SURVEY, INC.
NEURO-IQ Tox TEST KIT™
Prepared by
Battelle
Batreiie
ine Business of Innovation
Under a cooperative agreement with
trr\
U.S. Environmental Protection Agency
ET1/ET1/ET1/
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September 2006
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Aqua Survey, Inc.
Neuro-IQ Tox Test Kit™
by
Stephanie Buehler
Raj Mangaraj
Amy Dindal
Zachary Willenberg
Karen Riggs
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation by the EPA for use.
11
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
nation's air, water, and land resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, the EPA's Office of Research and Development provides data and science support that
can be used to solve environmental problems and to build the scientific knowledge base needed
to manage our ecological resources wisely, to understand how pollutants affect our health, and to
prevent or reduce environmental risks.
The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of six environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received EPA funding to plan,
coordinate, and conduct such verification tests for "Advanced Monitoring Systems for Air,
Water, and Soil" and report the results to the community at large. Information concerning this
specific environmental technology area can be found on the Internet at http://www.epa.gov/
etv/centers/centerl .html.
in
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. Many thanks go to Battelle's
Hazardous Materials Research Center for providing the facilities for and personnel capable of
working with chemical warfare agents. We sincerely appreciate the contribution of drinking
water samples from the Metropolitan Water District of Southern California (Paul Rochelle and
Melinda Stalvey), the New York Department of Environmental Protection (Virginia Murray),
and Orange County Utilities, Orlando, Florida (Theresa Slifko and Liza Robles). We would also
like to thank Armah de la Cruz (U.S. EPA, National Exposure Research Laboratory), Ricardo
DeLeon (Metropolitan Water District of Southern California), Yves Mikol (New York City
Department of Environmental Protection), and Helen Schurz Rogers (Centers for Disease
Control and Prevention National Center for Environmental Health) for their careful review of the
test/QA plan and this verification report.
IV
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Contents
Page
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
Chapter 1 Background 1
Chapter 2 Technology Description 2
Chapters Test Design 3
3.1 Introduction 3
3.2 Test Samples 3
3.2.1 PT Samples 4
3.2.2 DW Samples 5
3.2.3 QC Samples 6
3.2.4 Operational Factors 6
3.3 Verification Schedule 7
3.4 Test Procedure 7
3.4.1 Test Sample Preparation and Storage 7
3.4.2 Test Sample Analysis Procedure 7
3.4.3 Drinking Water Characterization 8
Chapter 4 Quality Assurance/Quality Control 10
4.1 Sample Chain-of Custody Procedures 10
4.2 QC Samples 10
4.3 Equipment/Calibration 12
4.4 Characterization of Stock Solutions 12
4.5 Audits 13
4.5.1 Performance Evaluation Audit 13
4.5.2 Technical Systems Audit 13
4.5.3 Audit of Data Quality 14
4.6 QA/QC Reporting 14
4.7 Data Review 14
Chapters Statistical Methods and Reported Parameters 16
5.1 Accuracy 16
5.2 False Positive/False Negative Rates 16
5.3 Precision 17
5.4 Potential Matrix and Interferent Effects 17
5.5 Operational Factors 17
Chapter 6 Test Results 18
6.1 Accuracy 18
6.2 False Positive/False Negative Rates 20
6.3 Precision 26
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6.4 Potential Matrix and Interferent Effects 26
6.4.1 Interferent PT Samples 26
6.4.2 DW Samples 27
6.5 Operational Factors 27
6.5.1 Technical Operators 27
6.5.2 Non-Technical Operator 28
Chapter 7 Performance Summary 30
Chapter 8 References 37
Figures
Figure 2-1. Neuro-IQ Tox Test Kit™ 2
Figure 6-1. Side View of PPE Worn by Non-Technical Operator 29
Figure 6-2. Testing of the Neuro-IQ Tox Test Kit™ with the Non-Technical Operator Wearing
PPE 29
Tables
Table 3-1. Lethal Dose of Target Contaminants 4
Table 3-2. Performance Test Samples 5
Table 3-3. Drinking Water Samples 6
Table 3-4. ATEL Water Quality Characterization of Drinking Water Samples 9
Table 4-1. Reference Methods for Target Contaminants and Interferents 11
Table 4-2. Performance Evaluation Samples and Percent Difference 13
Table 4-3. Summary of Data Recording Process 15
Table 6-1. Contaminant-Only PT Sample Results 19
Table 6-2a. VX False Positive/Negative Results 21
Table 6-2b. GB False Positive/Negative Results 22
Table 6-2c. GD False Positive/Negative Results 23
Table 6-2d. Aldicarb False Positive/Negative Results 24
Table 6-2e. Dicrotophos False Positive/Negative Results 25
Table 7-1. VX Summary Table 31
Table 7-2. GB Summary Table 32
Table 7-3. GD Summary Table 33
Table 7-4. Aldicarb Summary Table 34
Table 7-5. Dicrotophos Summary Table 35
vi
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List of Abbreviations
AMS
ASTM
ATEL
Ca
DI
DPD
DW
BCD
EPA
ETV
GB
GC
GD
HAZWOPER
HOPE
HMRC
ICP
kg
L
LC
LD50
LOD
LRB
MB
Mg
mg/L
mL
MS
Hg/L
uMHO
NaOH
NDR
Advanced Monitoring Systems
American Society for Testing and Materials
Aqua Tech Environmental Laboratories, Inc.
calcium
deionized
diethyl-p-phenylene diamine
drinking water
electron capture detection
U.S. Environmental Protection Agency
Environmental Technology Verification
sarin
gas chromatography
soman
Hazardous Waste Operations and Emergency Response
high density polyethylene
Hazardous Materials Research Facility
inductively coupled plasma
kilogram
liter
liquid chromatography
lethal dose for half of test subjects
limit of detection
laboratory record book
method blank
magnesium
milligram per liter
milliliter
mass spectrometry
microgram per liter
micromho
sodium hydroxide
negative differential resistance
vn
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ng nanogram
NTU nephelometric turbidity unit
OP organophosphate
PE performance evaluation
PPE personal protective equipment
PT performance test
QA quality assurance
QC quality control
QMP quality management plan
SCBA self-contained breathing apparatus
SM standard method
SOP standard operating procedure
TSA technical systems audit
Vlll
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative 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 testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
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 EPA's National Exposure Research Laboratory and its verification organization partner,
Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center
recently evaluated the performance of the Aqua Survey, Inc., Neuro-IQ Tox Test Kit™ in
detecting chemical agents, carbamate pesticides, and organophosphate (OP) pesticides in
drinking water. Enzymatic test kits were identified as a priority technology category for
verification through the AMS Center stakeholder process.
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This verification report provides
results for testing the Neuro-IQ Tox Test Kit™. Following is a description of the Neuro-IQ Tox
Test Kit™, based on information provided by the vendor. The information provided below 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.
Figure 2-1. Neuro-IQ Tox Test Kit™
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Chapter 3
Test Design
3.1 Introduction
Enzymatic test kits, generally designed to be handheld and portable, detect the presence of
chemical agents, carbamate pesticides, and/or OP pesticides by relying on the reaction of the
cholinesterase enzyme. Under normal conditions, the enzyme reacts as expected with other
chemicals present in the test kit. The activity of the enzyme is inhibited, however, by chemical
agents, carbamate pesticides, and OP pesticides. The effects of this inhibition will then generally
lead to a color change, indicating the presence or absence of these compounds.
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 OP pesticides in drinking water. 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
• Operational factors (operator observations, ease of use, and sample throughput).
3.2 Test Samples
This test evaluated the ability of the Neuro-IQ Tox Test Kit™ to detect VX, sarin (GB), and
soman (GD) (chemical agents); aldicarb (carbamate pesticide); and dicrotophos (OP pesticide) in
performance test (PT) and drinking water (DW) samples. Quality Control (QC) samples were
also included as part of the test matrix to ensure the integrity of the test. Contaminants were
tested individually, and stock solutions of each contaminant were prepared separately in
American Society for Testing and Materials (ASTM) Type II deionized (DI) water. Samples
were prepared in the appropriate matrix using these stock solutions and analyzed on the same
day. To minimize the loss of analytes to hydrolysis, contaminant stock solutions prepared in DI
water were made on a daily basis. Chemical agent stock solutions were prepared twice daily,
once in the morning and once in the afternoon. Aliquots of each stock solution were diluted to
the appropriate concentration using volumetric glassware and volumetric or calibrated pipettes.
In some cases, reference solutions were prepared in ASTM Type II DI water using the stock
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solutions to prepare the test samples. In other cases, the actual stock solutions were submitted for
concentration confirmation by the respective reference analysis (Table 4-1). Aqua Tech
Environmental Laboratories, Inc. (ATEL) of Marion, OH performed the physiochemical
characterization for each type of DW sample along with reference analyses of the interferent
solutions. All other reference analyses were performed at Battelle.
3.2.1 PT Samples
PT samples were prepared separately in ASTM Type IIDI water for each contaminant. The first
type of PT samples consisted of ASTM Type II DI water spiked with the contaminant at five
different concentrations: the lethal dose concentration given in Table 3-1 for each contaminant,
along with dilutions at approximately 10, 100, 1,000, and 10,000 times less than the lethal dose.
The contaminants were added individually to each spiked sample. 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.(u) Human oral LD50 data were not available for aldicarb,
so rat oral LDso data were used instead.(3) Each concentration level for the PT samples was
analyzed in triplicate.
In addition to the contaminant-only PT samples described above, a second type of PT sample
was a potential interferent sample. Three replicates of each interferent PT sample were analyzed
to determine the susceptibility of the Neuro-IQ Tox Test Kit™ to these commonly found
interferents in DW. One interferent PT sample contained calcium (Ca) and magnesium (Mg)
from carbonates spiked into ASTM Type II DI water, and the other contained humic and fulvic
acids isolated from the Elliot River (obtained from the International Humic Substances Society)
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). These spiked interferent levels were
confirmed through analysis of aliquots by ATEL. Also, each contaminant was added to these
samples, along with the potential interferent, at a concentration consistent with a lOx dilution of
the lethal dose. The resulting samples were analyzed in triplicate. Table 3-2 lists the PT samples
analyzed in this verification test for each contaminant.
Table 3-1. Lethal Dose of Target Contaminants
Contaminant
(common name)
VX
GB (sarin)
GD (soman)
aldicarb
dicrotophos
Oral Lethal Dose
Concentration
2.1 milligrams/liter (mg/L)
20 mg/L
1.4 mg/L
260 mg/L
1400 mg/L
Contaminant Class
Chemical agent
Chemical agent
Chemical agent
Carbamate pesticide
Organophosphate pesticide
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Table 3-2. Performance Test Samples
Type of PT
Sample
Sample Characteristics
Concentrations
Contaminant-
only
Contaminants in DI water
VX: 2.1 to 0.00021 mg/L
GB:20to0.002mg/L
GD: 1.4 to 0.00014 mg/L
aldicarb: 260 to 0.026 mg/L
dicrotophos: 1400 to 0.14 mg/L
Contaminants in 1 mg/L humic and
fulvic acids
Interferent
Contaminants in 5 mg/L humic and
fulvic acids
Contaminants in 50 mg/L Ca and Mg
Contaminants in 250 mg/L Ca and Mg
VX: 0.21 mg/L
GB: 2 mg/L
GD: 0.14 mg/L
aldicarb: 26 mg/L
dicrotophos: 140 mg/L
3.2.2 DWSamples
Table 3-3 lists the DW samples analyzed for each contaminant in this test. DW samples were
collected from four geographically distributed municipal sources (Ohio, New York, California,
and Florida) to evaluate the performance of the Neuro-IQ Tox Test Kit™ with various DW
matrices. These samples varied in their source, treatment, and disinfection process. All samples
had undergone either chlorination or chloramination disinfection prior to receipt. Samples were
collected from water utility systems with the following treatment and source characteristics:
• Chlorinated filtered surface water source
• Chlorinated unfiltered surface water source
• Chlorinated filtered groundwater source
• Chloraminated filtered surface water source
Approximately 175 liters (L) of each of the DW samples were collected in pre-cleaned,
translucent, low-density polyethylene containers. After sample collection, an aliquot of each
DW sample was sent to ATEL to determine the following water quality parameters:
concentration of trihalomethanes, haloacetic acids, total organic halides, Ca and Mg, pH,
conductivity, alkalinity, turbidity, organic carbon, and hardness. All DW samples were
dechlorinated prior to their use with sodium thiosulfate pentahydrate to prevent the degradation
of the target contaminants by chlorine. The dechlorination of the DW was qualitatively
confirmed by adding a diethyl-p-phenylene diamine (DPD) tablet to an aliquot of DW. If the
water did not turn pink, the dechlorination process was successful. If the water did turn pink,
additional dechlorinating reagent was added and the dechlorination confirmation procedure
repeated. Each DW sample was analyzed before addition of contaminant, as well as after
fortification with each individual contaminant at a single concentration level (lOx dilution of the
lethal dose). Aliquots of each contaminant stock solution were diluted with DW samples to the
appropriate concentration. Each sample was tested in triplicate.
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Table 3-3. Drinking Water Samples
Drinking Water Sample Description
Water
Utility
Columbus, Ohio
(OH DW)
New York City, New
York (NY DW)
Orlando, Florida
(FL DW)
Metropolitan Water
District of Southern
California (CA DW)
Water
Treatment
chlorinated
filtered
chlorinated
unfiltered
chlorinated
filtered
chloraminated
filtered
Source
Type
surface
surface
ground
surface
Contaminant Concentrations
VX: 0.21 mg/L
GB: 2.0 mg/L
GD: 0.14 mg/L
aldicarb: 26 mg/L
dicrotophos: 140 mg/L
3.2.3 QCSamples
QC samples included method blank (MB) samples consisting of ASTM Type IIDI water and
control water samples, as indicated by the vendor. Control water samples were simply an
unspiked version of the sample matrix being tested. For example, when the OH DW samples
were tested, the control water was unspiked OH DW. All MB QC samples were exposed to
sample preparation and analysis procedures identical to the test samples. Control water samples
were prepared and used according to the protocol provided by the vendor. The MB samples were
used to ensure that no sources of contamination were introduced in the sample handling and
analysis procedures. At least 10% of the test samples (seven samples for each contaminant) were
MB samples. For samples involving chemical agents, only five MB samples were run with each
chemical agent. One control water sample was run with every set of three to four test samples of
the same matrix. The test samples and MB samples were analyzed blindly by the operator in that
the samples used for analysis were prepared by someone other than the operator and were
marked with non-identifying numbers.
3.2.4 Operational Factors
3.2.4.1 Technical Operator
All of the test samples were analyzed by a technical operator who was trained by the vendor.
Operational factors such as ease of use and sample throughput were evaluated based on
observations recorded by the technical operator and the Verification Test Coordinator.
Operational factors were noted during the laboratory portions of the verification test. These
observations were summarized to describe the operational performance of the Neuro-IQ Tox
Test Kit™ in this verification.
3.2.4.2 Non-Technical Operator
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
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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. Because many of the
contaminants being tested are highly toxic and unsafe to be handled outside of a special facility,
MB samples and non-toxic control water samples were analyzed as part of the operational factors
assessment. The control water samples were provided by the vendor or prepared and used
according to the vendor's protocol as described in the previous section. Because no samples
spiked with the contaminants of interest were used, only the operational aspects of the Neuro-IQ
Tox Test Kit™ were evaluated with the non-technical operator. 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.
3.3 Verification Schedule
The verification test of the Neuro-IQ Tox Test Kit™ took place from November 2005 through
February 2006 at Battelle facilities in Columbus and West Jefferson, Ohio.
3.4 Test Procedure
3.4.1 Test Sample Preparation and Storage
All testing for this verification test was conducted within Battelle laboratories. Aldicarb and
dicrotophos samples were tested at Battelle's Columbus laboratories, while VX, GB, and GD
samples were tested at Battelle's Hazardous Materials Research Center (HMRC) facility in West
Jefferson, OH. Appropriate safety guidelines associated with each laboratory were followed
throughout the verification test. Samples were prepared fresh each day from stock solutions in
either DI water, an interferent matrix, or a DW matrix. Sample solutions were prepared to the
specified concentration based on the concentration of the stock solution, which was confirmed
through reference analysis. Test solutions were prepared in 1L quantities such that appropriate
aliquots (10 mL) of the sample preparation could be used for each test sample. Triplicate samples
of 10 mL each were taken from the same sample preparation. Each sample was placed in its own
container and labeled only with a sample identification number that was also recorded in a
laboratory record book (LRB) along with details of the sample preparation.
3.4.2 Test Sample Analysis Procedure
The Neuro-IQ Tox Test Kit™ is intended to be used by a technician with basic laboratory skills.
To test a water sample using the Neuro-IQ Tox Test Kit™, two different solutions had to be
assayed: first a control water sample and then a test water sample. The control water sample is
simply a sample of the same matrix being tested, only uncontaminated. According to the
manufacturer, all chlorinated water samples should be dechlorinated prior to testing. This had
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been done for all DW samples prior to testing (see Section 3.2.2). The steps for testing the
control water and test water samples are described below.
First, the testing materials including the water samples and Reagents A and B (supplied with the
test kit) were brought to room temperature. Then, the pH meter was calibrated using a 2-point
calibration curve based on buffers provided with the test kit. Reagent B was prepared for use by
adding 6 mL of DI water to the Reagent B vial and stirring. Reagent A was used as-is.
Next, the control water sample was tested. One control water sample was tested with each set of
three to four samples. The test samples and the control water were of the same matrix. To test
the control water sample, 40 |jL of Reagent A were added to 10 mL of the control water sample
and the solution was then slowly stirred on a magnetic stir plate. After five minutes, 0.02M
sodium hydroxide (NaOH) was added in 10 |jL increments until the pH of the solution was 8.30
± 0.05 pH units. The pH did not need to stabilize, only reach the specified level. While still
stirring on the magnetic stir plate, 200 |jL of Reagent B were added. Three minutes after adding
Reagent B, the pH of the solution was recorded. The test water sample was tested following the
same procedure as with the control water sample. The final pH for the test water sample, taken
three minutes after adding Reagent B, was recorded for the sample.
To determine if a sample was positive or negative, the difference between the control water
sample's final pH and the test water sample's final pH was calculated. If the difference was
> 0.2 pH units, then the test sample was considered positive. If the difference in pH was
< 0.2 pH units, then the sample was considered negative or not detected. 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, not four, samples were tested with each control water
sample since each type of sample need only to be tested in triplicate. These changes were
recommended by the vendor.
3.4.3 Drinking Water Characterization
An aliquot of each DW sample, collected as described in Section 3.2.2, was sent to ATEL to
determine the following water quality parameters: turbidity; concentration of dissolved and total
organic carbon; conductivity; alkalinity; pH; concentration of Ca and Mg; hardness; and
concentration of total organic halides, trihalomethanes, and haloacetic acids. Table 3-4 lists the
characterization data from the four water sample types used in this verification test. Water
samples were collected and water quality parameters were measured by ATEL in June 2005,
while verification testing was tested with the DW between November 2005 and February 2006.
The time delay between collection and testing was due to the fact that the water samples were
collected for use during a separate ETV test conducted prior to this one. Because of this, an
aliquot of each DW was tested by ATEL again in January 2006 to verify some of the parameters
with the most potential to change over time. Note that dissolved organic carbon was not retested
as this result was verified by the total organic carbon results, additionally the total organic
halides and calcium and magnesium were not verified as there was no reason to expect a change
in these parameters. The concentrations of most water quality parameters were similar; however,
there was a decrease in levels of volatile compounds such as trihalomethanes and haloacetic
acids over this time-period.
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Table 3-4. ATEL Water Quality Characterization of Drinking Water Samples
Parameter
Turbidity
Dissolved
Organic Carbon
Total Organic
Carbon
Specific
Conductivity
Alkalinity
PH
Calcium
Magnesium
Hardness
Total Organic
Halides
Trihalomethanes
Haloacetic Acids
Unit
NTU(a)
mg/L
mg/L
uMHO(c)
mg/L
mg/L
mg/L
mg/L
ug/L
ug/L/
analyte
ug/L/
analyte
Method
EPA180.1(4)
SM5310(5)
SM5310(5)
SM2510(5)
SM 2320(5)
EPA 150.1(6)
EPA200.8(7)
EPA200.8(7)
EPA 130.2(8)
SM 5320(5)
EPA 524.2(9)
EPA 552.2(10)
Columbus,
OH
(OH DW)
2005
0.1
2.1
2.1
572
40
7.6
33
7.7
118
220
74.9
32.8
2006
0.6
NA
2.3
602
44
7.4
NA
NA
107
NA
16.6
<6.0
New York
City, NY
(NY DW)
2005
1.1
1.1
1.6
84
14
6.9
5.6
1.3
20
82
39.0
39.0
2006
1.3
NA
4.1
78
12
6.8
NA
NA
26
NA
23.1
<6.0
Orlando, FL
(FL DW)
2005
0.5
1.6
1.7
322
142
8.5
8.8
43
143
300
56.4
34.6
2006
0.1
NA
2.1
325
125
7.6
NA
NA
130
NA
41.8
<6.0
MWD (b), CA
(CA DW)
2005
0.1
2.9
2.5
807
71
8.0
45
20
192
170
39.2
17.4
2006
0.2
NA
2.7
812
97
7.9
NA
NA
182
NA
24.1
<6.0
(a) NTU = Nephelometric turbidity unit.
(b) MWD = Metropolitan Water District of Southern California
(iMHO = micromho
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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center(11) and the test/QA plan(12) for this verification test.
QC procedures as noted in the reference methods or laboratory's operating procedures were
followed in confirming analyses of stock or reference solutions of contaminants and interfering
compounds and in characterizing the DW. The reference methods for this verification test are
listed in Table 4-1. A summary of the QC samples and acceptance criteria associated with each
method is presented in Table 7 in the test/QA plan.(12)
4.1 Sample Chain-of Custody Procedures
Sample custody was documented throughout collection, shipping, and analysis of the samples.
Sample chain-of-custody procedures were in accordance with ASAT.I-009-DRAFT, Standard
Operating Procedure for Sample Chain of Custody. The chain-of-custody forms summarized the
samples collected and analyses requested and were signed by the person relinquishing samples
once that person had verified that the custody forms were accurate. The original sample custody
forms accompanied the samples; the shipper kept a copy. Upon receipt at the sample destination,
sample custody forms were signed by the person receiving the samples once that person had
verified that all samples identified on the custody forms were present in the shipping container.
4.2 QC Samples
The QC measures for the reference methods included the analysis of a MB sample with the
analyses of the reference or stock solution. MB samples were analyzed to ensure that no sources
of contamination were present. If the analysis of an MB sample indicated a concentration above
the minimum detection limit for the confirmatory instrument, contamination was suspected. Any
contamination source(s) were corrected, and proper blank readings were achieved, before
proceeding with the analyses. In general, a matrix spike or laboratory fortified spike sample was
also analyzed. Average acceptable recoveries for these samples were between 70 and 150%.
Samples outside of the acceptable range were generally flagged and rerun once the QC
acceptance criteria had been met. QC samples were run with every batch of 1 to 20 samples.
Specific QC samples and acceptance criteria associated with each method can be found in the
appropriate reference (Table 4-1).
10
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Table 4-1. Reference Methods for Target Contaminants and Interferents
Target
Analyte/Interferent
VX
GB (sarin)
GD (soman)
aldicarb
dicrotophos
calcium (Ca)
magnesium (Mg)
Humic and Mvic
acids
Reference Method
(Instrumentation)
Battelle Internally
Developed Method (LC-MS)
HMRC-IV-1 18-05 (13)
(GC-MS)
HMRC-IV-1 18-05 (13)
(GC-MS)
SOP for Analysis of Water
Sample Extracts for Type 1
Analytes by Liquid
Chromatography/Mass
Spectrometry (14) (LC-MS)
SOP for Extracting and
Preparing Water Samples for
Analysis of Dicrotophos,
Mevinphos, and
Dichlorovos (15) (GC-MS)
EPA 200.8 (7)(ICP-MS)
EPA 200.8 (7)(ICP-MS)
Standard Method 53 10 (5)
Combustion Infrared NDR
Number of
Observations
10
4
4
2
2
4
1
1
1
1
Expected
Concentrations
(mg/L)
2.1
20.0
1.4
26.0
260
140
1400
125
125
1.0
Average
Measured
Concentration
(mg/L) ± SD
2.1±0.1
17.0 ±1.4
1.7 ±0.05
34
303
157 ± 24
1326
140
130
0.9
Recovery
(%R) ± SD
101 ±5
85 ±7
121 ±4
123 ±7 (a)
108 ± 17 (a)
112
104
90
a> Average of two concentration levels.
QC samples as provided with the Neuro-IQ Tox Test Kit™ were also run per the vendor's
instructions, and MB samples were run as part of the verification test (Section 3.2.3). Seven MB
samples were run with each set of pesticide samples. Only five MB samples were run with each
set of chemical agent samples. Of the 15 MB samples run across VX, GB, and GD samples, four
positive responses were obtained, one with GB samples and three with GD samples. There was
no indication of contamination despite the positive MB results on days when those samples were
run. For ease of testing, at least seven sets of triplicate MB samples (21 total MB samples) were
run for each pesticide, instead of seven total MB samples. All MB samples analyzed with
aldicarb and dicrotophos samples were negative.
11
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4.3 Equipment/Calibration
The instruments used for the reference analyses were calibrated per the standard reference
methods being used to make each measurement or the standard operating procedures (SOPs) of
the analysis laboratory. Instruments used in the reference analyses for this test included gas
chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-
MS), pH electrodes, inductively coupled plasma-mass spectrometry (ICP-MS), and gas
chromatography with electron capture detector (GC-ECD). All calibrations were documented by
Battelle in the project LRB. Calibration of mass spectrometers involved a 4- to 8-point
calibration curve covering the range of concentrations of the reference solutions to be analyzed.
Calibration of each reference instrument was performed as frequently as required by the
reference method guidelines.
The vendor provided the Battelle technical operator with instructions on how to properly
maintain components of the Neuro-IQ Tox Test Kit™ requiring calibration, namely the pH
probe. The pH probe was calibrated at the beginning of each day of testing using at least a
2-point calibration curve based on buffer solutions provided by the vendor.
Pipettes used during solution preparation were maintained and calibrated as required by Battelle
SOPs (i.e., minimum of every 6 months). Pipettes were checked and either recalibrated or
replaced if they were dropped over the course of testing.
4.4 Characterization of Stock Solutions
During testing, aliquots of the stock solutions used for sample preparation were submitted for
concentration confirmation via the respective methods. The results, along with the reference
methods, are listed in Table 4-1. Averages and associated standard deviations are given in cases
where more than two samples were tested. Recovery (%R) is calculated by the following
equation:
%R = — xlOO (1)
where C is the measured concentration (or average measured concentration if more than one
sample was tested) and^4 is the expected concentration of the contaminant or interferent in
solution. For aldicarb and dicrotophos, aliquots at two different concentration levels were
confirmed through reference analysis. The %R, listed in Table 4-1, represents the average of the
%R across both concentration levels for those compounds. Table 4-1 shows that %R values
ranged from 85% to 123% across all analytes and interferents.
Contaminant stock solutions were prepared and tested individually. Interferent stock solutions
contained multiple analytes in the same solution (e.g., calcium and magnesium or humic and
fulvic acids together). Up to four aliquots of each stock solution were analyzed over the course
of the verification test. In the case of VX, extra aliquots were analyzed and all were reported in
Table 4-1. Aliquots were preserved or extracted on the day of preparation and stored as
prescribed by the standard method.
12
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4.5 Audits
4.5.1 Performance Evaluation Audit
The concentration of the standards used to prepare the samples fortified with contaminants and
potential interfering compounds was confirmed by analyzing standards prepared in ASTM
Type IIDI water from two separate commercial vendors using the reference methods noted in
Table 4-1. The standards from one vendor were used during the verification test, while the
standards from the second vendor were used exclusively to confirm the accuracy of the standards
from the first vendor.
Given the security requirements and lack of alternate sources for the chemical agents (VX, GB,
and GD) used in this verification test, PE audits were not performed for these contaminants. PE
audits were done for all remaining compounds when more than one source of the contaminant or
potential interfering compounds was available. PE audits were performed only on compounds
used to prepare test samples and not on any solutions supplied as part of the Neuro-IQ Tox Test
Kit™. Agreement of the standards within 25% (percent difference) was required for the
measurements to be considered acceptable. The percent difference (%D) between the measured
concentration of the PE sample and the nominal concentration of that sample was calculated
using the following equation:
= —xlOO
A
(2)
where Mis the absolute value of the difference between the measured and the expected
concentration, and^4 is the expected concentration. The results of the PE samples are given in
Table 4-2. All %D values calculated were within the 25% acceptable tolerance.
Table 4-2. Performance Evaluation Samples and Percent Difference
Contaminant
aldicarb
dicrotophos
Ca
Mg
Expected
Concentration
(ng/mL)
50
1000
1000
1000
Measured
Concentration
(ng/mL)
57
1103
890
990
Percent
Difference
(%)
14
10
11
1
4.5.2 Technical Systems A udit
The Battelle Quality Manager conducted technical systems audits (TSAs) in November 2005
(11/01, 11/11, 11/16, 11/18), December 2005 (12/01, 12/29), and January 2006 (01/30) to ensure
that the verification test was performed in accordance with the AMS Center QMP,(11) the test/QA
plan,(12) published reference methods, and any SOPs used by Battelle. As part of the audit, the
Battelle Quality Manager reviewed the reference methods, compared actual test procedures to
those specified or referenced in the test/QA plan, and reviewed data acquisition and handling
13
-------
procedures. The Battelle Quality Manager also observed testing in progress and the reference
method sample preparation and analysis, inspected documentation, and reviewed the LRBs used
to record testing results. The Battelle Quality Manager also checked calibration certifications and
conferred with Battelle staff. Observations and findings from this audit were documented and
submitted to the Battelle Verification Test Coordinator for response. No major findings were
reported from the audits. The records concerning the TSA are permanently stored with the
Battelle Quality Manager.
4.5.3 Audit of Data Quality
At least 10% of the data acquired during the verification test was audited. The Battelle Quality
Manager traced the data from initial acquisition, through reduction and statistical comparisons, to
final reporting. All calculations performed on the data undergoing the audit were checked.
4.6 QA/QC Reporting
Each assessment and audit was documented in accordance with Section 3.3.4 of the AMS Center
QMP.(11) Once the assessment report was prepared, the Battelle Verification Test Coordinator
responded to each potential problem and implemented any necessary follow-up corrective action.
The Battelle Quality Manager ensured that follow-up corrective action was taken. The results of
the TSA were sent to the EPA.
4.7 Data Review
Records generated in the verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-3 summarizes the types of data recorded. The
review was performed by a technical staff member involved in the verification test but not the
staff member who originally generated the record. The person performing the review added
his/her initials and the date to a hard copy of the record being reviewed.
14
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Table 4-3. Summary of Data Recording Process
Data to Be Recorded
Dates, times, and
details of test events
Sample preparation
(dates, concentrations,
etc.)
Enzymatic test kit
procedures and sample
results
Reference method
sample preparation
Reference method
procedures,
calibrations, QA, etc.
Reference method
analysis results
Responsible
Party
Battelle
Battelle
Battelle
Battelle
Battelle or
subcontract
laboratory
Battelle or
subcontract
laboratory
Where
Recorded
ETV laboratory
record book or
data recording
forms
ETV laboratory
record books
ETV data sheets
and laboratory
record book
ETV laboratory
record book
Laboratory
record book or
data recording
forms
Electronically
from reference
analytical method
How Often
Recorded
Start/end of test
procedure, and at
each change of a
test parameter
When each
solution was
prepared
Throughout test
duration
Throughout
sample
preparation
Throughout
sampling and
analysis
processes
Every sample
analysis
Disposition
of Data
Used to organize and
check test results and
manually incorporated
into data spreadsheets
as necessary
Used to confirm the
concentration and
integrity of the
samples analyzed
Manually incorporated
into data spreadsheets
for statistical analysis
and comparisons
Used to demonstrate
validity of samples
submitted for
reference
measurements
Retained as
documentation of
reference method
performance
Converted to
spreadsheets for
calculations
15
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Chapter 5
Statistical Methods and Reported Parameters
The Neuro-IQ Tox Test Kit™ was evaluated for qualitative results (i.e., positive/negative
responses to samples). All data analyses were based on these qualitative results. QC and MB
samples were not included in any of the analyses.
5.1 Accuracy
Accuracy was assessed by evaluating how often the Neuro-IQ Tox Test Kit™ result is positive in
the presence of a concentration above the limit of detection (LOD). Contaminant-only PT
samples were used for this analysis. An overall percent agreement was determined by dividing
the number of positive responses by the overall number of analyses of contaminant-only PT
samples greater than the Neuro-IQ Tox Test Kit™'s LOD (see Equation 3). If the LOD was not
known or available, then all analyzed contaminant-only PT samples greater than the
concentration level where consistent negative results were obtained were used.
Accuracy (% Agreement) = # of positive contaminant only PT samples x 100 (3)
total # of contaminant only PT samples
5.2 False Positive/False Negative Rates
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 positive rate
was reported as the number of false positive results out of the total number of unspiked samples
(Equation 4).
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 as defined above. Spiked PT (contaminant and interferent) samples and
spiked DW samples were included in the analysis. Contaminant-only PT samples above the
Neuro-IQ Tox Test Kit™'s LOD or the level at which consistent negative responses are obtained
(when the LOD was not known) were included in the analysis. A false negative rate was
evaluated as the number of false negative results out of the total number of spiked samples for a
particular contaminant (Equation 5).
False Positive Rate = # of positive results (4)
total # of unspiked samples
16
-------
False Negative Rate = # of negative results (5)
total # of spiked samples
5.3 Precision
Precision measures the repeatability and reproducibility of the Neuro-IQ Tox Test Kit™'s
responses. The precision of three replicates of each sample set was assessed. Responses were
considered inconsistent if one or more of the three replicates differed from the response of the
other samples in the replicate set. The precision for the Neuro-IQ Tox Test Kit™ was assessed
by calculating the overall number of consistent responses for all the sample sets. The results are
reported as the percentage of consistent responses out of all replicate sets (Equation 6).
Precision (% Consistent results) = # of consistent responses of replicate sets x 100 (6)
total # of replicate sets
5.4 Potential Matrix and Interferent Effects
The potential effect of the DW matrix on the Neuro-IQ Tox Test Kit™'s performance was
evaluated qualitatively by comparing the results for the spiked and unspiked DW samples to
those for the PT samples spiked with the contaminant at 10 times less than the lethal dose.
Similarly, the potential effect of interferent PT samples was evaluated. The results indicating the
correct or incorrect reporting of the presence of a contaminant were evaluated. The findings are
reported and discussed in Section 6.4.
5.5 Operational Factors
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 are
the qualitative observations of the verification staff pertaining to the performance of the Neuro-
IQ Tox Test Kit™ from both the technical and non-technical operators' perspective.
17
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Chapter 6
Test Results
The Neuro-IQ Tox Test Kit™ did not produce distinctive "detected" or "not detected" responses.
Instead, pH values were obtained for each sample. These pH values were then converted into
qualitative results. This was done by comparing the pH value for a given sample to the pH value
for the control water sample for that particular group of samples. The difference between the
control water pH value and the sample pH value (both taken after Reagent B was added and
three minutes had elapsed) was calculated. 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 hit, 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 non-detect was recorded for that sample. All of the results
presented in this chapter were calculated using the qualitative responses determined for the
Neuro-IQ Tox Test Kit™.
6.1 Accuracy
The accuracy results for the Neuro-IQ Tox Test Kit™ using the contaminant-only PT samples
are discussed in this section. Table 6-1 presents the accuracy results for VX, GB, GD, aldicarb,
and dicrotophos. The results for the lethal dose concentration of each contaminant are given in
the table. Results are presented for all tested concentration levels; but, by definition, only those
results above the kit's LOD are included in the calculation. Because a LOD was not available
for the Neuro-IQ Tox Test Kit™, only samples above the level for each contaminant where
consistent negative responses were obtained were considered for accuracy calculations. For VX,
GB, GD, and aldicarb, consistent negative responses were not obtained from any concentration
level, so all contaminant-only PT samples were included in the accuracy calculations. For
dicrotophos, consistent negative responses were found starting at a l,000x dilution of the lethal
dose (i.e., at 1.4 mg/L), thus only three sets of replicates were included in the accuracy
calculations for that contaminant.
All concentration levels analyzed for VX generated 3 out of 3 positive responses for each set of
replicates, resulting in 100% agreement for the overall accuracy. No other contaminant tested
with the Neuro-IQ Tox Test Kit™ resulted in 100% overall accuracy. All but one concentration
level for the GB samples resulted in 3 out of 3 positive responses. The GB lethal dose level
(20 mg/L) samples generated 2 out of 3 positive responses, resulting in 93% agreement for
overall accuracy. Similarly, only one level of GD contaminant-only PT samples did not have
18
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Table 6-1. Contaminant-Only PT Sample Results
Contaminant
VX
GB
GD
aldicarb
dicrotophos
Concentration
(mg/L)
2.1 (a)
0.21
0.021
0.0021
0.00021
20(a)
2.0
0.20
0.020
0.0020
14(a)
0.14
0.014
0.0014
0.00014
260 (a)
26
2.6
0.26
0.026
1400 (a)
140
14
1.4
0.014
Positive Results
Out of
Total Replicates
o /o
3/3
3/3
3/3
3/3
3/3
2/3
o /o
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
1/3
o /o
3/3
3/3
3/3
0/3
1/3
3/3
0/3
1/3
0/3 (b)
0/3 (b)
Accuracy
100% (15/15)
93% (14/15)
87% (13/15)
67% (10/15)
44% (4/9)
^Lethal dose.
(b) Not used in accuracy calculations because samples are at or below level of consistent negative response.
three positive results; the lowest tested concentration level for GD (0.00014 mg/L) generated
only 1 out of 3 positive results. The resulting overall accuracy for GD was 87%. Aldicarb
samples resulted in 67% overall accuracy. Samples at both l,000x (0.26 mg/L) and 10,000x
(0.026 mg/L) dilution of the lethal dose had less than three positive responses (0 out of 3 and 1
out of 3, respectively). Only those nine samples with dicrotophos concentrations of 14 to
1,400 mg/L were used in the assessment of accuracy, and of these levels only the lethal dose
concentration generated 3 out of 3 positive responses, resulting in 44% overall accuracy.
19
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6.2 False Positive/False Negative Rates
Contaminant-only PT samples, interferent PT samples, and DW samples were evaluated to
determine false positive and false negative results for the Neuro-IQ Tox Test Kit™. A false
positive response was defined as a positive result when the contaminant was not spiked into the
sample. A false negative response was defined as a negative result when the sample was spiked
with a contaminant at a concentration greater than the level where consistent negative responses
were obtained (see Section 6.1). Tables 6-2a through 6-2e present the false positive and false
negative responses for VX, GB, GD, aldicarb, and dicrotophos, respectively. The number of
positive samples out of the total replicates analyzed is presented in each table.
For VX, GB, and GD, only one set of unspiked DW and PT interferent samples were run for all
three chemical agents. Thus, the unspiked DW and PT-interferent sample results shown in
Tables 6-2a through 6-2c are the same and from only one set of triplicate samples. For aldicarb
and dicrotophos, sets of unspiked DW and PT interferent samples were run separately for each
pesticide.
One false negative was found for VX; one of the three replicates for VX spiked 250 mg/L Ca and
Mg was negative. However, 13 false positives were found: three positive responses for unspiked
1 mg/L humic and fulvic acids, one for unspiked 5 mg/L humic and fulvic acids, three for
50 mg/L Ca and Mg, as well as three positive responses for both unspiked OH and FL DW.
These false positives were the same for GB and GD. GB also had one false negative response
when only two of the three replicates at the lethal dose (20 mg/L) resulted in positive responses.
For GD, 2 out of 39 samples were falsely negative. Both of these false negatives occurred at the
lowest concentration contaminant-only PT sample (0.00014 mg/L).
Both aldicarb and dicrotophos had three false positive responses. In both cases they occurred in
unspiked 5 mg/L humic and fulvic acid samples. Aldicarb also had eight false negative
responses: three at 0.26 mg/L aldicarb in DI water, two at 0.026 mg/L aldicarb in DI water, and
three in 250 mg/L Ca and Mg PT interferent samples spiked with aldicarb. For dicrotophos,
seven false negative responses were found: three at lOx less than the lethal dose (140 mg/L
dicrotophos in DI water), two at lOOx less than the lethal dose (14 mg/L dicrotophos in DI
water), and two in the 1 mg/L humic and fulvic acid samples spiked with the contaminant.
20
-------
Table 6-2a. VX False Positive/Negative Results
Sample Type Matrix
DI water
DI water
Contaminant-only TJJ water
PT samples
DI water
DI water
1 mg/L humic and
fulvic acids
1 mg/L humic and
fulvic acids
5 mg/L humic and
fulvic acids
Interferent PT 5 mg/L humic and
samples (c) fulvic acids
50 mg/L Ca and Mg
50 mg/L Ca and Mg
250 mg/L Ca and Mg
250 mg/L Ca and Mg
OH
OH
CA
CA
DW samples (c)
rL
FL
NY
NY
DW
DW
DW
DW
DW
DW
DW
DW
Concentration
(mg/L)
0.21
0.021
0.0021
0.00021
Blank
0.21
Blank
0.21
Blank
0.21
Blank
0.21
Blank
0.21
Blank
0.21
Blank
0.21
Blank
0.21
False Positive Rate
False Negative Rate
(a) Boxed results indicate false positive
(b) T otTiol Anro
responses; shaded
Positive Results Out of
Total Replicates (a)
3/3
3/3
3/3
3/3
3/3
3/3
3/3
1/3
3/3
3/3
3/3
0/3
2/3
3/3
3/3
0/3
3/3
3/3
3/3
0/3
3/3
13/24
1/39
results indicate false negative responses.
Only one set of unspiked DW and PT interferent samples were run for VX, GB, and GD.
21
-------
Table 6-2b. GB False Positive/Negative Results
Sample Type
Contaminant-only
PT samples
Interferent PT
samples (G:I
DW samples (c)
M . Concentration Positive Results Out of
(mg/L) Total Replicates (a)
DI water 20 (b) 2/3
DI water 2.0 3/3
DI water 0.20 3/3
DI water 0.020 3/3
DI water 0.0020 3/3
1 mg/L humic and Blank 3/3
fulvic acids
1 mg/L humic and 2.0 3/3
fulvic acids
5 mg/L humic and Blank 1/3
fulvic acids
5 mg/L humic and 2.0 3/3
fulvic acids
50 mg/L Ca and Mg Blank 3/3
50 mg/L Ca and Mg 2.0 3/3
250 mg/L Ca and Mg Blank 0/3
250 mg/L Ca and Mg 2.0 3/3
OH DW Blank 3/3
OH DW 2.0 3/3
CA DW Blank 0/3
CA DW 2.0 3/3
FL DW Blank 3/3
FL DW 2.0 3/3
NY DW Blank 0/3
NY DW 2.0 3/3
False Positive Rate 13/24
False Negative Rate 1/39
(a) Boxed results indicate false positive responses; shaded results indicate false negative responses.
(b) Lethal dose.
(c) Only one set of unspiked DW and PT interferent samples were run for VX, GB, and GD.
22
-------
Table 6-2c. GD False Positive/Negative Results
Sample Type Matrix
DI water
DI water
Contaminant-only pjj water
PT samples
DI water
DI water
1 mg/L humic and
fulvic acids
1 mg/L humic and
fulvic acids
5 mg/L humic and
fulvic acids
Interferent PT 5 mg/L humic and
samples (c) fulvic acids
50 mg/L Ca and Mg
50 mg/L Ca and Mg
250 mg/L
250 mg/L
OH
OH
CA
CA
DW samples (c)
rL
FL
NY
NY
Ca and Mg
Ca and Mg
DW
DW
DW
DW
DW
DW
DW
DW
Concentration
(mg/L)
L4(b)
0.14
0.014
0.0014
0.00014
Blank
0.14
Blank
0.14
Blank
0.14
Blank
0.14
Blank
0.14
Blank
0.14
Blank
0.14
Blank
0.14
False Positive Rate
False Negative Rate
(a) Boxed results indicate false positive
(b) T Ptliol Hr,cp
responses; shaded
Positive Results Out of
Total Replicates (a)
3/3
3/3
3/3
3/3
1/3
3/3
3/3
1/3
3/3
3/3
3/3
0/3
3/3
3/3
3/3
0/3
3/3
3/3
3/3
0/3
3/3
13/24
2/39
results indicate false negative responses.
Only one set of unspiked DW and PT interferent samples were run for VX, GB, and GD.
23
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Table 6-2d. Aldicarb False Positive/Negative Results
Sample Type Matrix
DI water
DI water
Contaminant-only TJJ water
PT samples
DI water
DI water
1 mg/L humic and
fulvic acids
1 mg/L humic and
fulvic acids
5 mg/L humic and
fulvic acids
Interferent PT 5 mg/L humic and
samples fulvic acids
50 mg/L Ca and Mg
50 mg/L Ca and Mg
250 mg/L Ca and Mg
250 mg/L Ca and Mg
OHDW
OHDW
CADW
CADW
DW samples „ nw
FLOW
NYDW
NYDW
Concentration
(mg/L)
260 (b)
26
2.6
0.26
0.026
Blank
26
Blank
26
Blank
26
Blank
26
Blank
26
Blank
26
Blank
26
Blank
26
False Positive Rate
False Negative Rate
(a) Boxed results indicate false positive responses; shaded
Positive Results Out of
Total Replicates (a)
3/3
3/3
3/3
1/3
0/3
3/3
3/3
3/3
0/3
3/3
0/3
0/3
0/3
3/3
0/3
3/3
0/3
3/3
0/3
3/3
3/24
8/39
results indicate false negative responses.
(b) Lethal dose.
24
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Table 6-2e. Dicrotophos False Positive/Negative Results
Sample Type
Contaminant-only
PT samples
Interferent PT
samples
DW samples
Matrix
DI water
DI water
DI water
1 mg/L humic and
fulvic acids
1 mg/L humic and
fulvic acids
5 mg/L humic and
fulvic acids
5 mg/L humic and
fulvic acids
50 mg/L Ca and Mg
50 mg/L Ca and Mg
250 mg/L Ca and Mg
250 mg/L Ca and Mg
OHDW
OHDW
CADW
CADW
FLOW
FLOW
NYDW
NYDW
Concentration
(mg/L)
1400 (b)
140
14
Blank
140
Blank
140
Blank
140
Blank
140
Blank
140
Blank
140
Blank
140
Blank
140
False Positive Rate
False Negative Rate
Positive Results Out of
Total Replicates (a)
3/3
0/3
1/3
3/3
3/3
0/3
3/3
0/3
3/3
0/3
3/3
0/3
3/3
0/3
3/3
0/3
3/3
3/24
7/33
(a) Boxed results indicate false positive responses; shaded results indicate false negative responses.
(b) Lethal dose.
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6.3 Precision
The performance of the Neuro-IQ Tox Test Kit™ in measuring VX within sets of three replicate
samples was generally consistent. Only two sets of replicates were inconsistent: unspiked
5 mg/L humic and fulvic acids and spiked 250 mg/L Ca and Mg. One positive and two negative
responses were found for the unspiked humic and fulvic acid replicates while two positive and
one negative responses were found for the Ca and Mg replicates. Thus, two of the 21 sets of
replicates that were analyzed was determined to be inconsistent, indicating that 90% of the
sample sets showed consistent results among the replicates.
The Neuro-IQ Tox Test Kit™ results for GB and GD were also consistent in 19 out of 21 sets of
replicates, indicating that 90% of the sample sets showed consistent results for these two
contaminants. For GB, samples at the lethal dose of the chemical agent as well as samples in
unspiked 5 mg/L humic and fulvic acids were inconsistent. For GD, inconsistencies were found
in PT samples at 10,000x less than the lethal dose (i.e., at 0.00014 mg/L) and in unspiked 5 mg/L
humic and fulvic acid samples.
Results for samples spiked and not spiked with aldicarb were consistent 95% of the time with
results being the same in 20 out of 21 sample sets. Only the PT sample at 10,000x less than the
lethal dose (i.e., at 0.026 mg/L) were inconsistent. Of the 21 sample sets, 19 showed consistent
results for dicrotophos samples, resulting in 90% precision. Replicates at both the 14 mg/L (in
DI water) concentration level and the 1 mg/L humic and fulvic acids spiked with the pesticide
were inconsistent.
6.4 Potential Matrix and Interferent Effects
6.4.1 Interferent PT Samples
The Neuro-IQ Tox Test Kit™ was able to consistently detect VX, GB, and GD at lOx less than
the lethal dose in DI water (see Tables 6-2a - c, respectively). Across all three chemical agents
at lOx less than the lethal dose spiked into interferent PT samples, the Neuro-IQ Tox test
produced positive responses for all sample replicates except in one instance. The one exception
was that for interferent samples spiked with VX in 250 mg/L Ca and Mg, only two out of three
positive responses were achieved. Only one set of unspiked interferent PT samples provided all
negative responses, that for 250 mg/L Ca and Mg. All other unspiked interferent samples had
one or more positive responses. These results seem to indicate that the Neuro-IQ Tox Test Kit™
may have some sensitivity to the interferents used in this test.
For both aldicarb and dicrotophos samples (see Tables 6-2d and e), unspiked 5 mg/L humic and
fulvic acid samples had positive responses for all three replicates, further confirming the
potential sensitivity of the Neuro-IQ Tox Test Kit™ to this interferent. In interferent PT samples
spiked with aldicarb, results were as expected except for 250 mg/L Ca and Mg replicate. For
these spiked samples, no positive responses were found. Aldicarb spiked into DI water at lOx
less than the lethal dose was consistently detected by the Neuro-IQ Tox Test Kit™. Similarly,
for 1 mg/L humic and fulvic acids spiked with dicrotophos, only one positive response was
26
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generated. Dicrotophos spiked at lOx less than the lethal dose in DI water was not detected in
any of the contaminant-only PT sample replicates. Dicrotophos was however detected in the
next lowest contaminant-only PT sample concentration level, and at lOx less than the lethal dose
in all other spiked interferent samples.
6.4.2 DWSamples
OH and FL unspiked DW samples were positive for all three replicates when tested as part of the
chemical agent's sample set. These results indicate that there could be potential confounding
compounds in these DW samples to which the Neuro-IQ Tox Test Kit™ is sensitive. No false
positives or negatives were found for DW samples tested as part of the pesticides' sample set.
6.5 Operational Factors
6.5.1 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.
The technicians were trained by the vendor in the operation of the test kit. Training was
conducted at Battelle for one half day by the vendor. Both technicians had extensive laboratory
experience.
The combination of the stir plate and the pH meter made the Neuro-IQ Tox Test Kit™
cumbersome to use. Multiple problems were encountered with the test kit operation. At one
point, the pH probe supplied with the kit did not work properly, and the vendor had to supply
another probe for testing to continue. After Reagent B was added and three minutes had passed,
the pH was often still fluctuating, making it hard to determine the actual pH at that point in time.
Since the instructions indicate that taking the pH after exactly three minutes is critical, such an
issue was troublesome. Reaching a stable pH of 8.30 after adding NaOH was also generally
difficult. However, per the vendor's direction, the pH did not have to stabilize at that level in
order to move on with the test.
Two reagents were used to test a water sample with the Neuro-IQ Tox Test Kit™. Reagent A is
stored frozen and must come to room temperature before it can be used. Reagent B had to be
reconstituted with DI water before use. Individual vials of each reagent were provided with the
kit to make daily testing easier.
Because of the Neuro-IQ Tox Test Kit™'s design, only one sample could be analyzed at a time.
It took the operators different lengths of time to complete testing for one group of three
replicates. For one operator, it took, on average, 75 minutes (±17 minutes) to test a set of three
replicate water samples using the Neuro-IQ Tox Test Kit™. It took the other operator 52 ±
7 minutes to test one set of replicates. Overall, it took an average of 64 minutes (±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 per day.
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6.5.2 Non-Technical Operator
Unspiked DI water samples were tested on the Neuro-IQ Tox Test Kit™ by a non-technical
operator both in and not in PPE (see Section 3.2.4). The SCBA apparatus, including the mask,
was worn throughout the entire testing procedure when PPE was to be worn. However, the
operator ran the air from the SCBA only part of the time during testing to conserve the tank. The
pH meter was operated using batteries and a portable (battery-operated) magnetic stir plate was
used for this portion of the test. Figure 6-1 shows the full PPE as worn for this verification test.
Figure 6-2 shows the testing of the Neuro-IQ Tox Test Kit™ with the non-technical operator
wearing PPE. With the PPE on, two negative and one positive response were obtained. Without
the PPE, three negative responses were recorded.
During the initial test of the Neuro-IQ Tox Test Kit™ with PPE, the operator exceeded the
intended pH (8.3) when adding NaOH. Adjusting the pH to 8.3 as the kit directions indicate
proved to be slightly difficult and was easy for the operator to overshoot. The test was restarted
so that the proper pH could be obtained. Reagent A was hard to handle with the gloves on, and
the magnetic stir plate was difficult to adjust while in full PPE.
The Neuro-IQ Tox Test Kit™ instructions indicate that it is to be used by a "technician with
basic laboratory skills." Most first responders do not have any laboratory skills. 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 place the pH probe and magnetic
stirrer to obtain proper readings. This setup required patience and time from the operator and
could be problematic in the field, especially for a first-responder when time is critical. Testing of
three MB samples while in PPE took 52 minutes, while testing of three MB samples without PPE
took 40 minutes. Consequently, having the PPE on did slow the operator down a bit as it took
12 more minutes to conduct the test with PPE than without. During the portability testing, a
table-top surface was used, making the setup of the Neuro-IQ Tox Test Kit™ a bit easier. If no
such surface were available in the field, the test kit would be very difficult for the operator to set
up and use. As noted earlier in this report, a control water sample is needed for the Neuro-IQ
Tox Test Kit™ protocol. This means that water that is the same matrix as the test sample but not
contaminated would have to be obtained to use this kit. This could be problematic in the field.
Overall, the Neuro-IQ Tox Test Kit™ would be hard for a first-responder with no experience and
no laboratory skills to use if the operator is donned in the level of PPE used in this verification
test.
28
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Figure 6-1. Side View of PPE Worn by
the Non-Technical Operator
Figure 6-2. Testing of the Neuro-IQ Tox
Test Kit™ with the Non-Technical
Operator Wearing PPE
29
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Chapter 7
Performance Summary
The Neuro-IQ Tox Test Kit™ results for this verification test for samples containing VX, GB,
GD, aldicarb, and dicrotophos are presented in Tables 7-1 through 7-5. The results for each
contaminant are presented in a separate table. Qualitative responses for each set of sample
replicates as well as accuracy, false negatives and positives, and precision are presented in each
table. A summary of the other performance factors associated with the Neuro-IQ Tox Test Kit™
is presented at the end of this chapter. These performance factors apply across all contaminants.
30
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Table 7-1. VX Summary Table
Parameter
Qualitative
Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
Matrix
DI Water
Humic and Fulvic
Acids
Ca and Mg
DW
VX
Concentration
2.1mg/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.
False Positives
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.
False Negatives
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.
Precision
90% (20 out of 21) of the sample sets showed consistent
results among the individual replicates within that set.
(a)
Lethal dose.
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Table 7-2. GB Summary Table
Parameter
Qualitative
Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
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.
False Positives
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.
False Negatives
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.
Precision
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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Table 7-3. GD Summary Table
Parameter
Qualitative
Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
Matrix
DI Water
Humic and Fulvic
Acids
Ca and Mg
DW
GD
Concentration
1.4mg/L(a)
0.14mg/L
0.014mg/L
0.0014 mg/L
0.0001 4 mg/L
0.14 mg/L
0.14 mg/L
0.14 mg/L
Number
Detected/Number
of Samples
3/3
3/3
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.
False Positives
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.
False Negatives
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.
Precision
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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Table 7-4. Aldicarb Summary Table
Parameter
Qualitative
Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
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.
False Positives
Three false positive responses were obtained. Positive
responses were found for all replicates of the unspiked
5 mg/L humic and fulvic acids samples.
False Negatives
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.
Precision
95% (20 out of 21) of the sample sets showed consistent
results among the individual replicates within that set.
w Lethal dose.
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Table 7-5. Dicrotophos Summary Table
Parameter
Qualitative
Results
Contaminant-
Only PT
Samples
Interferent PT
Samples
DW Samples
Accuracy
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.
False Positives
Three false positive responses were obtained. Positive
responses were found for all replicates of the unspiked 5 mg/L
humic and fulvic acids samples.
False Negatives
Seven false negative results were obtained for spiked PT and
DW 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.
Precision
90% (19 out of 21) of the sample sets showed consistent
results among the individual replicates within that set.
W
(b)
Lethal dose.
Not used in accuracy calculations because samples are at or below level of consistent negative response.
35
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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.
36
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Chapter 8
References
1. U.S. Army Center for Health Promotion and Preventative Medicine, USACHPPM
Technical Guide 230, Chemical Exposure Guidelines for Deployed Military Personnel,
January 2002.
2. Gosselin et al., Clinical Toxicology of Commercial Products. 5th edition, Baltimore, MD,
1984.
3. World Health Organization, The WHO Recommended Classification of Pesticides by
Hazard and Guidelines to Classification: 2004, 2005.
4. EPA-600-R-93/100. EPA Method 180.1. Turbidity (Nephelometric), Methods for the
Determination of Inorganic Substances in Environmental Samples. 1993.
5. American Public Health Association, et al. Standard Methods for Examination of Water
and Wastewater. 19th Edition. 1997. Washington D.C.
6. EPA 600/4-79/020 Method 150.1. pH, Electrometric Method.. 1982.
7. EPA 600/R-94/111 Method 200.8. Determination of Trace Metals by Inductively Coupled
Plasma - Mass Spectrometry. 1994.
8. EPA 600/4-79/020 Method 130.2. Hardness, Total (mg/L as CaCO3) Titrimetric, EDTA.
1982.
9. EPA 600/R-95/131. EPA Method 524.2. Purgeable Organic Compounds by Capillary
Column GC/Mass Spectrometry. Methods for Determination of Organic Compounds in
Drinking Water, Supplement III. 1995.
10. EPA 600/R-95/131. EPA Method 552.2. Haloacetic Acids andDalapon by Liquid-Liquid
Extraction, Derivatization and GC with Electron Capture Detector. Methods for the
Determination of Organic Compounds in Drinking Water, Supplement III. 1995.
11. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, March 2004.
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12. Test/QA Plan for Verification of Enzymatic Test Kits, Battelle, Columbus, Ohio,
September 2005.
13. Battelle, SOP HMRC-IV-118-05: Standard Operating Procedure for the Determination of
CA in Wastewater.
14. Battelle, Standard Operating Procedure for Analysis of Water Extracts for Type I Analytes
by Liquid Chromatography/Mass Spectrometry, Version 1, January 2004.
15. Battelle, Standard Operating Procedure for Extracting and Preparing Water Samples for
Analysis of Dicrotophos, Mevinphos, andDichlorovos, Version 3, March 2005.
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