September 2009
Environmental Technology
Verification Report
ABRAXIS ECOLOGENIA
ETHYNYLESTRADIOL (EE2) MICROPLATE
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
TEST KITS
Prepared by
Battelle
Battelle
7nc Business of Innovation
Under a cooperative agreement with
CfTr\ U.S. Environmental Protection Agency
ET1/ET1/ET1/
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September 2009
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
ABRAXIS ECOLOGENIA®
ETHYNYLESTRADIOL (EE2) MICROPLATE
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
TEST KITS
By
Stephanie Buehler, Zachary Willenberg, Amy Dindal, Battelle
Eric Kleiner, Michelle Henderson, and John McKernan, U.S. EPA
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Notice
The U.S. Environmental Protection Agency, through its Office of Research and Development,
funded and managed, or partially funded and collaborated in, the research described herein. It
has been subjected to the Agency's peer and administrative review and has been approved for
publication. Any opinions expressed in this report are those of the author (s) and do not
necessarily reflect the views of the Agency, therefore, no official endorsement should be inferred.
Any mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Foreword
The 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. We sincerely appreciate the
involvement and support of all staff from the participating laboratories who conducted testing as
part of this verification test. In particular, we would like to thank the following staff for their
contribution in conducting this verification at their respective laboratories: Mark Mills and Scott
Jacobs, U.S. EPANRMRL, Cincinnati, OH; Jennifer Gundersen, Dave Russell, Ronald Landy,
Annie Hilliard, John Curry, and Martin Lazarus, U.S. EPA Region 3 Fort Meade, MD; Dennis
Wesolowski, Larry Zintek, and Charles Steiner, U.S. EPA Region 5 Chicago, IL; Mike Meyer,
Keith Loftin, Larry Barber, and James Gray, USGS, Kansas; Jim Lazorchak, Tirumuru Reddy,
and Dan Bender, U.S. EPANERL, Cincinnati, OH; and Jeanette Van Emon, U.S. EPA NERL
Las Vegas, NV. Finally, we would like to thank Lisa Olsen, USGS; Paul Pennington, NOAA,
and Marion Kelly, U.S. EPA, for their review of 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
Chapter 3 Test Design and Procedures 4
3.1 Introduction 4
3.2 Test Facilities 6
3.3 Test Procedures 6
3.3.1 Test Sample Collection and Preparation 7
3.3.2 Test Sample Analysis Procedure 8
Chapter 4 Quality Assurance/Quality Control 10
4.1 Quality Control Samples 10
4.1.1 GC-MS Blank and Surrogate Spike Results 10
4.1.2 Method Blanks 11
4.2 Audits 11
4.2.1 Performance Evaluation Audit 11
4.2.2 Technical Systems Audit 12
4.2.3 Data Quality Audit 12
4.3 QA/QC Reporting 13
4.4 Data Review 13
Chapters Statistical Methods 15
5.1 Precision 15
5.2 Percent Bias 16
5.3 Matrix Effects 16
5.4 Operational Factors 17
Chapter 6 Test Results 18
6.1 Precision 18
6.2 Percent Bias 19
6.3 Matrix Effects 20
6.4 Operational Factors 22
Chapter 7 Performance Summary 23
Chapters References 24
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Figures
Figure 2-1. Abraxis Ecologenia® Ethynylestradiol (EE2) Microplate ELISA Test Kit 2
Tables
Table 3-1. Target Analytes 5
Table 3-2. ELISA Test Kit Evaluation Responsibilities for Each Participating Laboratory 5
Table 4-1. PE Audit Sample Results 12
Table 4-2. Summary of Data Recording Process 14
Table 6-1. ELISA Test Kit Average Concentration and Relative Standard Deviation (RSD)
Results 19
Table 6-2. ELISA Test Kit Percent Bias vs. GC-MS 20
Table 6-3. GC-MS Average Concentration, RSD, and Percent Bias Results 20
Table 6-4. ELISA Test Kit Percent Bias vs. Expected Spike Concentration 20
Table 6-5. Percent Recovery 21
VI
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List of Abbreviations
AMS
°C
coc
DI
El
E2
E3
EDC
EE2
ELISA
EPA
ETV
GC-MS
OFF
HPLC
L
LC-MS
MB
MDL
NERL
ng
nm
NP
NRMRL
ORD
PE
ppb
QA
QC
QMP
rpm
RSD
S
Advanced Monitoring Systems
degrees Celsius
chain of custody
deionized
estrone
17-p-estradiol
estriol
endocrine-disrupting compound
17-a-ethynylestradiol
enzyme-linked immunosorbent assay
U.S. Environmental Protection Agency
Environmental Technology Verification
gas chromatography-mass spectrometry
glass fiber filter
high performance liquid chromatography
liter
liquid chromatography-mass spectrometry
method blank
method detection limit
microliter
micron
EPA ORD National Exposure Research Laboratory
nanogram
nanometer
nonylphenol
EPA ORD National Risk Management Research Laboratory
EPA Office of Research and Development
performance evaluation
parts per billion
quality assurance
quality control
quality management plan
revolutions per minute
relative standard deviation
standard deviation
vn
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SOP standard operating procedure
SPE solid phase extraction
TSA technical systems audit
USGS United States Geological Survey
v/v volume/volume
WWTP wastewater treatment plant
Vlll
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification Program (ETV) to facilitate the deployment of innovative environmental
technologies through performance verification and dissemination of information. The goal of
ETV 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 Risk Management Research Laboratory (NRMRL) and its verification
organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under
ETV. The AMS Center recently evaluated the performance of the Abraxis Ecologenia®
Ethynylestradiol (EE2) Microplate Enzyme-Linked Immunosorbent Assay (ELIS A) test kit for
determining endocrine-disrupting compounds (EDCs) in water.
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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 report provides results for
the verification testing of the Abraxis Ecologenia® Ethynylestradiol (EE2) Microplate ELISA
test kit. The following is a description of the test kit, based on information provided by the
vendor. The EE2 microplate ELISA test kit applies the principle of ELISA to determine EE2 in
water samples. The EE2 microplate ELISA kit uses a colorimetric procedure to detect EE2.
The standards, samples, and an enzyme-labeled EE2 conjugate are added to a disposable
microtiter plate (uncoated) and mixed. 100 microliter (|jL) aliquots of the mixture are then
added to antibody (monoclonal anti-EE2) coated wells in a 96-well microplate. At this point a
competitive reaction occurs between the ethynylestradiol which may be in the sample and the
enzyme-labeled ethynylestradiol for a finite number of antibody binding sites. The reaction is
allowed to continue for sixty (60) minutes. At the end of the incubation period, the plate
contents are decanted to remove the unbound reagent from the ethynylestradiol and labeled
ethynylestradiol that remain bound to the antibodies on the plate, in proportion to their original
concentrations. After decanting, the plate is washed with Washing Solution. A substrate is then
added and enzymatically converted from a colorless to a blue solution. After an incubation
period, the reaction is stopped by the addition of diluted acid. The ethynylestradiol concentration
is determined by measuring the absorbance of the sample solution with a photometer (450 nm)
and comparing it to the absorbance of standards.
The EE2 microplate ELISA test kit (Figure 2-1)
contains a 96-well microplate coated with
ethynylestradiol antibody (mouse anti-
ethynylestradiol), two vials of lyophilized
horseradish peroxidase-labeled ethynylestradiol
analog, two 7 mL vials of buffer solution, five
1.5 mL vials of ethynylestradiol standard
concentrations (0, 0.05, 0.15, 0.5, 3.0 parts per
billion (ppb)) with preservatives and stabilizers, a
15 mL bottle of hydrogen peroxide and 3,3',5,5'-
tetramethylbenzidine solution in an organic base, a
15 mL bottle of diluted acid, a 50 mL bottle of 6X
concentrated Washing Buffer, one uncoated
microtiter plate, and one adhesive plate cover.
Figure 2-1. Abraxis Ecologenia®
Ethynylestradiol (EE2) Microplate ELISA
Test Kit
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The EE2 microplate ELISA test kit measures 7 by 5 by 4 1A inches. Final results and calibration
curves are printed from the photometric analyzer or sent directly to a laboratory computer. List
price is $699 for a 96-test kit. Other materials that are required but are not provided with the EE2
microplate ELISA test kit are pipettes, and a plate photometer capable of reading at 450
nanometer (nm). These materials can be purchased separately or rented.
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Chapter 3
Test Design and Procedures
3.1 Introduction
This verification test was conducted according to procedures specified in the Test/QA Plan for
Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits for the Quantitative
Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples1
Deviations to the test/QA plan were made due to unanticipated circumstances. As such, the test
procedures described in this chapter are a complete description of the actual test conditions.
Because of their potential to interfere with human, domestic animal, and wildlife reproduction,
EDCs are of increasing concern throughout the country. Several EPA Regions have undertaken
activities to monitor for these compounds, and several states are considering including
monitoring for EDCs in their regulatory programs. Presently, gas chromatography-mass
spectrometry (GC-MS), high performance liquid chromatography (HPLC), and liquid
chromatography-mass spectrometry (LC-MS) are being used for detecting these compounds.
However, immunoassay techniques, particularly ELISA, are becoming increasingly popular in
the field of environmental analysis due to their high sensitivity, ease of use, short analysis time,
and cost-effectiveness.
Immunoassay analytical detection is based on the capability of antibodies to specifically
recognize and form stable complexes with antigens. Immunoassays employ antibodies as
analytical reagents. In ELISA test kits, an enzyme conjugate competes with the chemical in the
sample for a limited number of binding sites on the antibody coated plate or particle. The extent
of color development is inversely proportional to the amount of chemical in the sample or
standard. The higher the concentration of a specific steroid or other EDC in the sample, the less
color reaction produced and recorded using a plate reader or tube photometer.
Testing was conducted with multiple collaborating laboratories, specifically the EPA Office of
Research and Development (ORD) National Risk Management Research Laboratory (NRMRL),
EPA ORD National Exposure Research Laboratory (NERL), and EPA Region 3. The laboratory
participation was coordinated by EPA NRMRL, in collaboration with Battelle. Laboratory
names are removed, and simply stated as "Laboratory (or Lab) 1, 2, and 3" in the test results
section, since inter-laboratory comparison was not an objective of this report.
This verification test evaluated the Abraxis EE2 microplate ELISA test kit to quantitate 17-oc-
ethynylestradiol (EE2) in four different water matrices, per the manufacturer protocols. More
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detailed information on the uses of the EDC tested is provided in Table 3-1. EPA laboratories
used the Abraxis Ecologenia® EE2 microplate ELISA kit (according to Table 3-2) to quantitate
triplicate spiked samples for hormones (EE2), which were prepared and shipped to Region 3 by
EPA NRMRL. The test was conducted in four phases from June to September 2008, with each
phase being a different aqueous matrix. As the more established method for detecting these
compounds, GC-MS served as the reference method for this test.
Table 3-1. Target Analytes
Analyte
Synonyms
CAS#
Use
17a-Ethynyl-
1,3,5(10)-estratriene-
3,17b-diol
17-a-Ethynylestradiol (EE2) 57-63-6
Synthetic estrogen
found in birth control
Table 3-2. ELISA Test Kit Evaluation Responsibilities for Each Participating Laboratory
Responsibility
Sample Collection, Processing and Distribution
Test Kit Evaluation - EE2 96 well ELISA
Reference Measurement - EE2 GC-MS
NRMRL
A/
A/
A/
NERL
A/
Region 3
A/
The Abraxis EE2 microplate ELISA test kit was verified by evaluating the following parameters:
• Precision
• Percent bias
• Matrix effects
• Operational factors.
Precision was determined by measuring the relative standard deviation of average concentration
values as reported by the test kit. Percent bias was determined as positive or negative, with
positive values indicating that ELISA concentration was higher than the reference method and
negative values indicating that it was lower. Matrix effects were determined by comparing the
percent bias measurements for Phase I deionized (DI) water samples to the percent bias
measurements for the Phase II through IV matrix-water samples.
Operational factors were determined based on documented observations of the testing staff and
the Verification Test Coordinator. Operational factors were described qualitatively, not
quantitatively; therefore, no statistical approaches were applied to the operational factors.
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3.2 Test Facilities
Laboratory analyses of the EE2 microplate ELISA test kit were conducted in three different
collaborating laboratories by the laboratory staff. These laboratories were: EPA Region 3; EPA
NRMRL, Cincinnati, OH; and EPA NERL, Cincinnati, OH.
3.3 Test Procedures
This verification test was conducted in four phases. Phase I consisted of a clean water sample
(DI water) spiked with a single concentration of EE2, split into single samples, and submitted to
the ELISA kit users in each collaborating laboratory to measure the concentration, in triplicate.
The split sample, as well as the un-spiked, matrix background sample, were also simultaneously
sent for reference GC-MS analysis of EE2 and for various compounds which are known to cause
cross-reactivity with some ELISA kits. Phase II consisted of environmental surface water
samples subjected to the same spiking and splitting process as Phase I. Phase III consisted of a
complex matrix of wastewater treatment plant (WWTP) effluent samples subjected to the same
spiking and splitting process as Phase I and II. Phase IV consisted of a complex matrix of
WWTP influent samples, spiked and split as in previous phases. Details on the sample matrices,
spiking levels, and spiking procedures for each Phase are provided in Section 3.3.1. All EE2
spike concentrations used in each phase of this verification test were based on real-world
concentrations found in environmental samples, per the procedure described in the test/QA plan.1
Background concentrations of EE2 were taken for each matrix for each phase. These GC-MS
measurements were made to determine if any measureable amounts of EE2 might exist in the
sample matrix prior to the addition of any sample spikes. If a detectable concentration was
found, this concentration was then added to the spiked amount of EE2 to calculate the total
concentration for all spiked samples of a particular phase. Specific concentrations of EE2, as
presented in Section 3.3.1, were spiked into the sample matrix for each phase, regardless of any
background concentrations of these compounds that may have been present in the collected
water. For Phase III, 1.47 ng/L of EE2 was found in the background matrix sample. The
nominal concentration of each sample was then calculated using the measured background
concentration and the expected spiked concentrations for each phase. The ELISA kit results
from the various laboratories for the EE2 microplate test kit were compared to each other and
compared to GC-MS results.
The Abraxis EE2 microplate ELISA test kit was tested only under laboratory controlled
conditions, as opposed to field conditions which would have been more variable. The analyses
were performed according to the vendor's recommended procedures as described in the user's
manual. Simple cleanup procedures, as directed by the manufacturer of the test kit, were used
for the four different matrices. Each sample was analyzed after solid phase extraction (SPE)
cleanup using the procedure detailed in the kit instructions and provided in Section 3.3.2. Each
sample for ELISA analysis was filtered through a 1 micron (um) glass fiber filter prior to SPE.
Calibration and maintenance of the technology reader (i.e., microplate reader) was performed as
specified by the vendor.
A US EPA NRMRL GC-MS standard operating procedure (SOP) was followed for reference
measurements.2 The GC-MS methods for estrone (El), E2, estriol (E3), EE2, testosterone,
dihydrotestosterone, androstenedione, and progesterone operated within a concentration range of
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2-50 ng/L. Samples for the GC-MS methods went through an extraction step to concentrate (or
dilute, depending upon the samples) to ensure the samples were within the method's analytical
range.2 The procedures for preparing, storing, and analyzing the test samples are provided
below.
3.3.1 Test Sample Collection and Preparation
All sample bottles and glassware associated with hormone samples, including the glass carboy,
were cleaned and silanized using a procedure included in the test/QA plan.l All samples were
thoroughly mixed and were thus assumed to contain the same concentration. Samples were
spiked with EE2 as one large stock solution and then split into smaller sub-samples in bottles.
All sample bottles were amber glass to prevent photodegradation of the analytes. All samples
were prepared and shipped by NRMRL, immediately after being made, in coolers on ice or
freezer packs to maintain a 4 degrees Celsius (°C) temperature. When samples were received by
each laboratory, the condition of the samples, i.e., temperature, broken bottles etc., was noted by
the receiving laboratory operator and the samples were then immediately placed in a refrigerator
at 4°C until analyzed. Holding times of hormone samples are currently unknown; therefore, all
samples were either analyzed or solvent exchanged within 24 hours of receipt to reduce error
associated with analyte degradation during sample holding. All laboratories performing
quantitative analysis, ELISA or GC-MS, received split samples from the same bulk sample.
Each laboratory that participated in the ELISA analysis received one 2.5 L spiked sample plus
one 500 mL DI water method blank. The laboratory that performed the reference analyses
received one 4 L spiked sample and one 1 L DI water method blank to be processed by the GC-
MS method.
3.3.1.1 Phase I Samples
A sample of DI water was collected in a cleaned, 20 L, glass carboy from the USEPA laboratory
in Cincinnati, Ohio. The water was spiked with EE2 to a concentration of 10 ng/L of EE2. This
concentration was selected because it is on the higher end of the range of concentrations
expected to be encountered in a real-world situation and is representative of the anticipated mid-
range of the test kit. The carboy was thoroughly mixed, by inserting a stir bar and stirring on a
stir plate at 300 revolutions per minute (rpm) for 2 hours to ensure homogeneous concentrations
of the analyte throughout the carboy. One 2.5 L spiked sample was collected for each
participating laboratory as well as one 4L sample for each reference laboratory. DI water blanks
were also prepared and shipped in separate 500 mL bottles. The blank samples were analyzed
after SPE but only in two wells (or test tubes) on the kits as opposed to three wells (or test tubes)
for all other samples. Before spiking, the DI water was sampled and analyzed by GC-MS to
confirm the background levels of EE2. Samples of the spiked mixtures were taken and the
concentrations of these samples and blank were determined using the Abraxis EE2 microplate
ELISA test kit and GC-MS.
3.3.1.2 Phase II Samples
Grab samples of stream water were collected in three, clean, five gallon buckets from the South
Hasha Tributary to Eastfork Lake in Clermont County, Ohio. The tributary was accessed from
where it crosses Williamsburg-Bantam Road. Before the stream water was spiked, a single
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sample of the collected stream water was taken, split into triplicate aliquots, and analyzed by
GC-MS to confirm the background levels of EE2. Background levels of EE2 measured in the
samples were added to the spiked concentration of EE2 once results were obtained. Next, a
cleaned, 20 L, glass carboy was used to collect 20 L of the stream water, which was then spiked
to contain a 10 ng/L concentration of EE2. The carboy was thoroughly mixed by inserting a stir
bar and stirring on a stir plate at 300 rpm for 2 hours, to ensure homogeneous concentration of
the analyte throughout the carboy. Split samples were taken, as noted for Phase I. DI water
method blanks were filled with DI water at the same time as the stream water in the 20 L
carboys.
3.3.1.3 Phase III Samples
Grab samples of final effluent wastewater were collected in three, clean, five gallon buckets from
the Metropolitan Sewer District of Greater Cincinnati in Hamilton County, Ohio. After the
sample was transported back to the NRMRL laboratory, the effluent was measured and then
transferred into a clean, 20 L carboy. Before spiking, a single sample of the effluent was taken,
split into triplicate aliquots, and analyzed by GC-MS to confirm the background levels of EE2.
In a cleaned, 20 L, glass carboy, 20 L of WWTP effluent was be prepared containing 10 ng/L of
EE2. The carboy was thoroughly mixed by inserting a stir bar and stirring on a stir plate at 300
rpm for 2 hours to ensure homogeneous concentration of the analyte throughout the carboy.
Split samples were collected, as noted in Phase I.
3.3.1.4 Phase IV Samples
Grab samples of influent wastewater were collected in three, clean, five gallon bucket from the
Metropolitan Sewer District of Greater Cincinnati in Hamilton County, Ohio. After the sample
was transported back to the NRMRL laboratory, the influent was measured and transferred into a
20 L carboy. Before spiking, a single sample of the influent was taken, split into triplicate
aliquots, and analyzed by GC-MS to confirm the background levels of EE2. In a cleaned, 20 L,
glass carboy, 20 L of WWTP influent was prepared containing 10 ng/L concentration of EE2.
The carboy was thoroughly mixed by inserting a stir bar and stirring on a stir plate at 300 rpm for
2 hours to ensure homogeneous concentration of the analyte throughout the carboy. Split
samples were collected, as noted in Phase I.
3.3.2 Test Sample Analysis Procedure
The ELISA test kit users followed simple cleanup procedures as directed in the vendor's
instructions. The 2.5 L sample was split into three 500 mL aliquots. Each of the three aliquots
was analyzed by utilizing glass fiber filter (GFF) cleanup and SPE. Each aliquot sample was
transferred in triplicate to the 96-well microplate for quantification, per the test kit protocols.
The cleanup procedures are described below.
Each sample for ELISA analysis was filtered through a 1 |Jm GFF. After filtering, three 500 mL
aliquots were removed from the filtered sample for SPE clean-up. These three aliquots were
treated as three independent samples. SPE directions entitled "Extractions for EE2 from Water
Sample for ELISA", which were based on the vendor's protocols and summarized by EPA
NRMRL, were followed.1 The SPE protocol consists of the following steps:
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1. Filter 500 mL of the sample, or the remainder of liquid in the sample bottle noting the volume
for later calculation, through 1 |j,m glass fiber filter.
2. Rinse a CIS cartridge with 5 mL of methanol and then 10 mL of distilled water at a flow rate
not exceeding 20 mL/min (preconditioning).
3. Pour the filtered sample through the CIS SPE cartridge at a flow rate, no faster than 20
mL/min.
4. Wash the cartridge with 5 mL of distilled water (up to 20 mL/min). Keep suctioning for about
a minute to dry the cartridge.
5. Wash the cartridge with 5 mL of hexane (up to 20 mL/min).
6. Elute the analyte with 5 mL of dichloromethane at a rate, no faster than 3mL/min.
7. Evaporate the solvent with nitrogen gas to dryness.
8. Add 1 mL of 100% methanol to the residue and stir the mixture with a vortex mixer. To
adjust the content to 10% methanol (volume/volume (v/v)) add 9 mL of distilled water for a total
volume of 10 mL.
After the SPE column, the EE2 samples were reconstituted with 10 mL of a 10% methanol
solution. For the spiked samples, this process effectively increased the overall spike
concentration 50 times to an expected level of 490 ng/L. All reconstituted samples were applied
to three wells (100 |jL for each well) according to the manufacturer's instructions. Samples were
quantified by reading their photometric responses at a wavelength of 450 nm using a plate reader
following the manufacturer's instructions. The general steps for operating the Abraxis EE2
microplate ELISA test kit that were followed during this verification test are provided below.
The Abraxis ELISA EE2 96 well microtiter plate kit assay procedure consists of the following
steps:
1. Take the kit out of the refrigerator approximately half an hour before use and let come to room
temperature (18-25°C). Filter the sample through a l|j,m glass fiber filter and add methanol to
obtain a final methanol concentration of 10% (v/v).
2. Reconstitute the antigen-enzyme conjugate powder with buffer solution.
3. Mix 100 |jL of conjugate solution and 100 |JL of EE2 standard (or sample) in each well of the
uncoated plate. Dispense the conjugate solution first then add standard solution of sample. Each
standard will be added to two wells while each sample will be added to four wells.
4. Dispense 100 (jL aliquots of the standard/sample and conjugate mixture into each coated well
of the microplate. Incubate it for 60 minutes at room temperature (18-25°C).
5. Dilute wash solution (6-fold concentration) in 5 times of its volume of distilled water to
prepare a wash solution.
6. Rinse each microplate well with approximately 300 (jL of the wash solution and repeat the
step twice more. Firmly tap out the plate on a lint-free paper towel to remove solution from the
microplate.
7. Dispense 100 (jL of the color solution into each well and incubate it for 30 minutes at room
temperature (18-25°C). Then, add 100 |jL of stop solution to terminate reaction.
8. Measure the absorbance at 450 nm for each standard solution and generate a standard curve.
The quantity of EE2 in the sample is then calculated from an absorbance reading and
interpolation from the standard curve.
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Chapter 4
Quality Assurance/Quality Control
QA/Quality Control (QC) procedures were performed in accordance with the quality
management plan (QMP) for the AMS Center3 and the test/QA plan for this verification test.1
Test procedures were as stated in the test/QA plan1; however deviations to the test/QA plan were
made due to unanticipated circumstances. As such, the test procedures described in Chapter 3 are
a complete description of the actual test conditions. The statistical calculations intended for
analysis of the test kit results were also changed. This deviation is further described in Chapter
5. This change had no impact on the quality of the results. QA/QC procedures and results are
described below.
4.1 Quality Control Samples
Steps taken to maintain the quality of data collected during this verification test included
analyzing specific quality control samples for both the reference method (GC-MS) and the test
kits.
4.1.1 GC-MS Method Blank and Surrogate Spike Results
This verification test included a comparison of the Abraxis EE2 microplate ELISA test kit results
to those of the GC/MS reference method for EE2. Samples analyzed for each phase included PE
samples, test samples, background samples, and blank samples. The quality of the reference
measurements was evaluated by adherence to the requirements of the GC-MS method for this
compound, including requirements for method blanks (MBs), instrument solvent blanks, and
surrogate spikes, as indicated in the test/QA plan.l Method blank samples were analyzed to
ensure that no sources of contamination were present. If the analysis of a method blank sample
indicated a concentration above five times the method detection limit, contamination was
suspected. Any contamination source(s) were corrected and samples were reanalyzed or flagged
before proceeding with the analyses. Surrogate spikes were also included in each sample.
Average acceptable recoveries for these samples were between 60 and 140%. Samples outside of
the acceptable range were generally flagged and/or reanalyzed. D4-EE2 was used as a surrogate
standard for the GC/MS analysis of EE2 in the samples. No levels of EE2 were detected in any
of the reference method blank samples.
Surrogate recoveries in Phase I - IV samples varied across phases. Phase I surrogate recoveries
ranged from 59 - 96% and averaged 85 ± 10% across 13 samples. All recoveries were
considered in the acceptable range. Phase II recoveries ranged from 77 to 155% and averaged
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132 ± 25% over 11 samples. Surrogate recoveries for six of the samples were outside of the
acceptable range. Compared to the surrogates, the peak shapes for the target analytes were good
and the baselines were clean in the chromatogram. Phase III surrogate recoveries ranged from
154% to 197% and averaged 176 ± 14% over 8 samples. Surrogate recoveries for all samples
were outside of the acceptable range for Phase III. Phase IV surrogate recoveries ranged from
61% to 93% and averaged 76 ± 10% over 11 samples. Surrogate recoveries for all Phase IV
samples were within the acceptable range.
4.1.2 Test Kit Method Blanks
Method blank samples were run in duplicate after SPE clean-up with each set of samples for all
four phases. Method blank samples were unspiked DI water. Because concentrations for
samples analyzed with the test kit are calculated based on the interpolations from a curve
constructed from the standards run with each batch of samples, it is possible to obtain
concentration values for all samples. However, the Abraxis EE2 microplate ELISA test kit has a
stated method detection limit (MDL) of 0.05 - 3 ng/L. Based on this MDL, it is assumed that
sample concentrations lower than this level cannot be reliably determined or reported. Thus, any
samples, including method blank samples, with concentrations lower than the manufacturer's
stated MDL were considered non-detects.
The EE2 microplate test kit was evaluated by three laboratories (see Table 3-2). Concentrations
of EE2 were not detected in any of the method blank samples from two of the participating
laboratories. For the NERL - Cincinnati results, levels of EE2 above the MDL were not detected
in Phase II - IV method blanks. However, during the analysis of Phase I samples, concentrations
of EE2 above the MDL were found for one of the two replicates in one set (the method blank
analyzed after SPE) of duplicate method blanks for one test kit. Two EE2 microplate ELISA test
kits were evaluated by this laboratory for each phase of testing. The second test kit operated
during Phase I did not show any detectable levels of EE2 in any of the method blanks analyzed.
Averaging the duplicate samples for the after-SPE method blanks in the first test kit put the
overall EE2 concentration below the MDL. All method blanks for this phase came from the
same initial sample.
4.2 Audits
Three types of audits were performed during the verification test: a PE audit of the reference
method measurements (GC-MS analyses), a technical systems audit (TSA) of the verification
test performance, and a data quality audit. Audit procedures are described further below.
4.2.1 Performance Evaluation Audit
A PE audit was conducted to assess the quality of the reference method measurements (GC-MS
analyses) made in this verification test. The reference method PE audit was performed by
supplying an independent second standard solution of EE2 prepared from a different source other
than that used in verification testing. The PE audit samples were analyzed in the same manner as
all other samples and the analytical results for the PE audit samples were compared to the
nominal concentration. The target criterion for this PE audit was agreement of the analytical
result within 30% of the expected concentration. This audit was performed once during each
phase of testing. Table 4-1 shows the percent error results for the PE samples for each phase.
11
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The percent error was calculated based on the difference between the actual and expected E2
concentrations divided by the expected concentration. The EE2 PE audit samples were within 30
% of the expected concentration for Phases I and II but were outside of this range for Phases III
and IV (112% more and 43% less, respectively). The PE audit sample results for Phase III were
similar to the GC-MS sample results for Phase III. This could have impacted the comparison of
the ELISA test kit results to the GC-MS data for Phase III. No adjustments were made to the
standards nor were PE audit samples reanalyzed based on these results.
Table 4-1. PE Audit Sample Results
Phase
1
II
III
IV
Expected
Concentration (ng/L)
10
10
10
10
Actual
Concentration (ng/L)
8.27
9.47
21.2
5.73
% Error
-17
-5
112
-43
4.2.2 Technical Systems Audit
The Battelle Quality Manager performed a TSA twice during this verification test. Battelle
conducted TSAs at the Cincinnati, OH facilities on July 23-24, 2008 and at the Fort Meade, MD
facility on July 31, 2008. Both laboratories participating in Cincinnati, OH, were audited. All
TSA findings were reported to the Verification Test Coordinator.
The purpose of this audit was to ensure that the verification test was being performed in
accordance with the AMS Center QMP,3 the test/QA plan,1 and the GC-MS SOP2 used in the
verification test. In the TSA, the Battelle Quality Manager reviewed the reference methods used,
compared actual test procedures to those specified or referenced in test/QA plan, and reviewed
data acquisition and handling procedures. The Battelle Quality Manager also toured the
laboratory where verification and reference testing were taking place,1 inspected sample chain of
custody (COC) documentation, reviewed technology-specific record books, checked standard
certifications and technology data acquisition procedures, and conferred with technical staff. A
TSA report was prepared, including a statement of findings and the actions taken to address any
adverse findings, and a copy of Battelle's TSA report was sent to the EPA AMS Center QA
Manager. No adverse findings were reported. The TSA findings were communicated to
technical staff at the time of the audit.
4.2.3 Data Quality Audit
At least 10% of the data acquired during the verification test were audited. Battelle's Quality
Manager traced the data from the initial acquisition, through reduction and statistical analysis, to
final reporting to ensure the integrity of the reported results. All calculations performed on the
data undergoing the audit were checked. Minor transcription errors and errors due to rounding
were identified and corrected before the results were used for the calculations described in
Chapter 5.
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4.3 QA/QC Reporting
Each audit was documented in accordance with Section 3.3.4 of the AMS Center QMP.3 Once
the audit reports were prepared, the Battelle Verification Test Coordinator ensured that a
response was provided for each adverse finding or 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 submitted to the EPA.
4.4 Data Review
Records generated in the verification test received an independent internal review before these
records were used to calculate, evaluate, or report verification results. Table 4-2 summarizes the
types of data recorded. Data were reviewed by a Battelle technical staff member involved in the
verification test. The person performing the review added his/her initials and the date to a hard
copy of the record being reviewed.
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Table 4-2. Summary of Data Recording Process
Data Recorded
Dates times and
details of test
events
Technology
calibration
information
Technology
readings
Sample collection
and reference
method analysis
procedures,
calibrations, etc.
Reference method
results
Where Recorded
Laboratory record
books or data
recording forms, or
electronically
Laboratory record
books, data
recording forms, or
electronically
Recorded
electronically or
manually by the
operator or
electronically by
the technology
reader, as
appropriate
Laboratory record
books, chain-of-
custody,
electronically, or
other data
recording forms
Electronically from
reference
measurement
technology
How Often
Recorded
Start/end of test
procedure, and at
each change of a
test parameter or
change of
technology status
At technology
reader calibration
or recalibration, as
applicable
Each sample and
QC analysis
Throughout
sampling and
analysis processes
Every sample or
QC analysis
By Whom
Participating
laboratories
Participating
laboratories
Participating
laboratories
Participating
laboratories
Participating
laboratories
Disposition of
Data
Used to organize
and check test
results; manually
incorporated into
data spreadsheets
as necessary
Incorporated into
verification report
as necessary
Converted to or
manually entered
into spreadsheets
for statistical
analysis or
comparisons
Retained as
documentation of
sample collection
or reference
method
performance
Transferred to
spreadsheets for
calculation of
results and
statistical analysis
or comparisons
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Chapter 5
Statistical Methods
The statistical methods used to evaluate the quantitative performance factors listed in Section 3.1
are presented in this chapter. Qualitative observations were also used to evaluate verification test
data.
Per the test/QA plan1, repeatability and reproducibility were intended to be calculated as
performance parameters for this verification test. However, after further discussion with EPA,
and in agreement with EPA, it was determined that higher level summary statistics provided a
better synopsis of the test kit results. Thus, the mean and relative standard deviations (precision)
were calculated for the test kit results.
5.1 Precision
The standard deviation (S) of the results for the replicate analyses of the same sample was
calculated as follows:
n — 1
(1)
where n is the number of replicate samples, M is the ELISA test kit measurement for the k
K.
sample, and Mis the average ELISA test kit measurement of the replicate samples. The precision
for each sample is reported in terms of the relative standard deviation (RSD), which was
calculated as follows:
RSD(%) =
S
M
xlOO
(2)
The RSD was calculated for each laboratory that participated in the verification test and for each
test kit that was tested. The RSD was also calculated across all laboratories and test kits for each
phase of testing.
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5.2 Percent Bias
Percent bias was calculated as a percentage for each measurement in each phase for each kit
using Equation 3 :
%Bias = [ — -1] xlOO (3)
where y = 1, 2, 3 denotes the laboratory, / = 1, 2 denotes the ELISA test kit within laboratory, n =
1, 2 denotes the reference method, Xjt is the ELISA concentration for the/ laboratory and the i'
test kit, yn is the concentration of the reference method GC-MS or the concentration of the spike.
Ideally percent bias results will be within ±25%.
5.3 Matrix Effects
Matrix effects were examined by comparing the percent bias measurements for the Phase IDI
water samples to the percent bias measurements for the Phase II - IV samples. Percent bias was
determined as described in Section 5.2.
General observations of potential matrix effects, such as false negatives, if observed, were
documented but were not used in statistical calculations. False negatives were defined as a
negative (zero) response in a sample that is spiked with contaminant at a detectable
concentration.
General observations on potential cross-reactivity were documented. Blank samples of each
matrix were evaluated by GC-MS to determine background levels of the compounds with which
the kits have cross-reactivity, as stated by the vendor.
Percent recovery results were calculated on a per-sample and per-phase basis and were based on
the expected spiked concentration of the analyte in each sample matrix. Percent recovery was
calculated using the Equation 4:
^
% Recovery = — x 100 (4)
E
Where A is the actual ELISA test kit measurement and E is the expected concentration. The
expected concentration includes the known spike concentration as well as any detected
background levels of EE2 in the matrix water (see Section 3.3). Percent recovery results are
presented to provide another measure of test kit performance to the end user. Ideal percent
recovery values are near 100%.
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5.4 Operational Factors
Operational factors were determined based on documented observations of the testing staff.
Operational factors are described qualitatively, not quantitatively; therefore, no statistical
approaches were applied to the operational factors.
17
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Chapter 6
Test Results
The results of the verification tests of the Abraxis EE2 microplate ELISA test kit are presented
below for each of the performance parameters.
6.1 Precision
The relative standard deviation (RSD) is used as a means of evaluating the precision of the
ELISA test kit. Three laboratories operated the EE2 microplate ELISA test kit. Two of the
laboratories (Lab 1 and Lab 2) ran identical samples on two separate test kits (kit "a" and kit
"b"). Lab 3 ran a single kit. Table 6-1 presents the resulting RSD for each participating
laboratory and test kit along with the overall average concentrations per phase of EE2 found
using the EE2 microplate ELISA test kit for all analyses. RSD values are also presented across
all results for each phase.
RSDs ranged from 6 to 72% for SPE analysis. The RSDs among all analyses were the same for
Phases II and IV (41%). For Phase I which was performed with DI water, the overall RSD was
67%. This RSD was approximately 2.5 times that for Phase III, which had the lowest RSD at
25%. There was no discernable trend in RSD when going from less (i.e., Phase I - DI water) to
presumably more challenging phases (i.e., Phase IV - wastewater influent).
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Table 6-1. ELISA Test Kit Average Concentration and Relative Standard Deviation (RSD)
Results"
Phase I
Phase II
Phase
Phase IV
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Average
Cone (ng/L)
778
499
163
467
out of rangeb
587
947
466
505
856
688
370
589
495
485
401
508
446
573
303
Overall
Average
RSD Cone (ng/L) RSD
64%
50%
14% 508 67%
15%
9%
29%
16% 672 41%
21%
39%
11%
23%
9% 525 25%
13%
22%
6%
21%
72% 437 41%
8%
35%
Expected
Phase
Cone (ng/L)
490
490
564
490
The average concentration and RSD are based on all replicates within the detectable range of the test kit.
' Test kit results were below the lower end of the test kit's range.
6.2 Percent Bias
Bias is a systematic error that causes measurements to err in one direction, either high or low.
For this section, percent bias was calculated relative to the GC-MS reference method results. A
positive percent bias indicates that the ELISA test kit concentration is higher than the reference
method, while a negative percent bias indicates that the ELISA test kit concentrations are lower
than the reference method. Table 6-2 presents the percent bias results.
Phase III percent bias results were consistently negative among the test kit analyses. For Phases
I, II, and IV, the percent bias results were mixed. Phase IV percent bias results were all positive
except for Lab 3, which was negative. For Phase II, the bias was negative for Lab 2 and positive
for Lab 3. Within these agreements, the percent bias results were different for kits a and b. Kit a
had significantly higher percent bias for both Labs 1 and 2 than Kit b in Phase I. For Phase II,
Lab 1 results indicated a positive and negative bias of approximately the same magnitude for the
two kits tested. Only Lab 2 results produced a percent bias absolute value of <5%.
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Table 6-2. ELISA Test Kit Percent Bias vs. GC-MS
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Phase I
66
7
-65
0
out of range3
Phase II
-25
20
-41
-36
9
Phase III
-28
-61
-38
-48
-49
Phase IV
8
37
20
54
-18
Test kit results were below the lower end of the test kit's range.
For comparison, average concentrations, RSD, and percent bias for the GC-MS measurements
with regard to the expected concentration are presented in Table 6-3 for each phase. RSD values
were less than 30% for all phases. Phases I and IV GC-MS results were lower than the expected
concentration, but the percent bias was within 30% of the expected value. Percent bias results
for Phases II and III were positive and more than two times higher (based on absolute values)
than for Phase IV. These results demonstrate that the GC-MS results were biased high (in
Phases II and III) and low (in Phases I and IV) as compared to the expected concentration.
Table 6-3. GC-MS Average Concentration, RSD, and Percent Bias Results
Phase
I
II
III
IV
Average
Cone (ng/L)
9.37
15.72
19.08
7.43
RSD
4%
1%
11%
6%
% Bias
(vs. Expected Cone)
-4
61
69
-24
6.3 Matrix Effects
To understand how the matrix of each phase of testing might have affected the results, percent
bias and percent recovery were calculated for the test kit results in comparison to the expected
spiked concentration of EE2. A positive percent bias indicates that the ELISA test kit
concentration is higher than the expected spike concentration, while a negative percent bias
indicates that the ELISA test kit concentrations are lower than the expected spike concentration.
Table 6-4 presents the percent bias results. No false negatives were observed during this
verification test.
Table 6-4. ELISA Test Kit Percent Bias vs. Expected Spike Concentration
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Phase I
59
2
-67
-5
out of range3
Phase II
20
93
-5
3
75
Phase III
22
-34
4
-12
-14
Phase IV
-18
4
-9
17
-38
Test kit results were below the lower end of the test kit's range.
20
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Percent bias results varied for the EE2 microplate ELISA test kit. There was a mixture of
positive and negative percent bias for all phases and within multiple test kits used by one
laboratory. No consistent trends were apparent for any phase or laboratory. Percent bias results
were negative for all samples in Phase IV except for that from Lab 1 kit b, which had a positive
bias. Discrepancies between the percent bias for duplicate test kits operated by the same
laboratory were apparent for both Lab 1 and 2. Bias for one kit was 2 to 22 times that for the
other kit operated by the same laboratory. The highest percent bias results were found in Phase
II samples for different laboratories. A positive percent bias, indicating that the EE2 microplate
ELISA test kit results were higher than the expected spiked concentration, of 93% and 75% were
found. The remaining percent bias results for Phase II were some of the lowest found across all
phases. In comparing the results of Table 6-4 to Table 6-2 where the test kit results were
compared to the GC-MS results, in general the test kits results were closer to the expected
concentrations than they were to the GC-MS results. However, bias results for Phase I were
similar in both cases.
As another measure of accuracy, percent recovery results, comparing the test kit results against
the expected spiked concentration, were also calculated on a per sample and per phase average
basis. Table 6-5 presents these results.
Table 6-5. Percent Recovery
% Recovery
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Average
Phase I
159
102
33
95
no data
97
Phase II
120
193
95
103
175
137
Phase III
122
66
104
88
86
93
Phase IV
82
104
91
117
62
91
Average percent recoveries for Phases I, III, and IV for the Abraxis EE2 microplate ELISA test
kit were 97%, 93%, and 91%, respectively. Phase II had an average percent recovery of 137%.
Recoveries across all phases ranged from 62% to 120% except for four samples, where the
recoveries were outside this range at 33%, 156%, 175%, and 193%. Two of these outlier
recoveries were in Phase I and two were in Phase II. There was variability in the percent
recovery across phases within a laboratory's results. None of the three analyses provided
consistently high or low percent recoveries. The average percent recoveries for all phases were
within the range of acceptable recoveries for the GC-MS reference method (60% - 130%).
Some ELISA kits will react with compounds similar to the target compound, known as cross-
reactivity. The EE2 microplate ELISA 96 well hormone kit for EE2 is compound specific with
minimal cross reactivity (<0.2% for all cross-reactive compound). Thus, an evaluation of cross-
reactivity was not performed for this test kit.
21
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6.4 Operational Factors
In general, training is needed to effectively and properly operate the test kits. The vendor trained
staff on the operation of the test kit, but these trained staff were, in some cases, not available for
the verification test because of testing delays and staff turn over. Therefore, staff that operated
the test kits during the verification test may not have been trained by the vendor.
Operational concerns or issues were not reported from any of the three participating laboratories.
The test kit instructions were readily followed by each of the operators. Preparation time (at
least half an hour) was required prior to the introduction of the sample to allow all reagents time
to come to room temperature before using them. Calibrated pipettes, disposable test tubes, and a
plate reader capable of reading at 450 nm are required for the operation of the test kit but are not
supplied with the test kit. Any OFF or SPE equipment used with the samples was also not
supplied with the test kit.
Each purchased test kit is capable of conducting 96 tests and costs $699. For comparison, GC-
MS analyses of these samples are estimated to cost between $500 and $900 per sample.l
22
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Chapter 7
Performance Summary
The ability of the Abraxis EE2 microplate ELISA test kit to detect EE2 in water was evaluated
using four different water matrices. The test kit was operated by three different laboratories with
the use of SPE cleanup. The test kit results were evaluated against the expected spike
concentrations and the reference measurements of the same samples made using GC-MS.
Relative standard deviations (RSDs) ranged from 6 to 72% for SPE analysis. The RSDs among
all analyses were the same for Phases II and IV (41%). For Phase I which was performed with
DI water, the overall RSD was 67%. This RSD was approximately 2.5 times that for Phase III,
which had the lowest RSD at 25%. There was no discernable trend in RSD when going from
less (i.e., Phase I - DI water) to presumably more challenging phases (i.e., Phase IV -
wastewater influent).
Percent bias, as compared to the GC-MS reference analysis results, for Phases I, II, and IV, was
mixed. Phase III percent bias results were consistently negative among the test kit analyses.
There was a mixture of positive and negative bias for Phases I, II, and IV, and within multiple
test kits used by one laboratory. No consistent trends were apparent among the analyses.
No false negatives were observed during this verification test. Average percent recoveries of
EE2 for Phases I, III, and IV were 91%, 93%, and 97%, respectively. Phase II had an average
percent recovery of 137%. Recoveries across all phases ranged from 62 to 120%. None of the
three analyses provided consistently high or low percent recoveries. The percent recoveries for
all phases were within the range of acceptable recoveries for the GC-MS reference method (60%
- 130%).
Operational concerns or issues were not reported from any of the three participating laboratories.
The test kit instructions were readily followed by each of the operators. Preparation time (at
least half an hour) was required prior to the introduction of the sample to allow all reagents time
to come to room temperature before using them. Calibrated pipettes, disposable test tubes, and a
plate reader capable of reading at 450 nm are required for the operation of the test kit but are not
supplied with the test kit. Any GFF or SPE equipment used with the samples was also not
supplied with the test kit. Each purchased test kit is capable of conducting 96 tests and costs
$699.
23
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Chapter 8
References
1. Test/QA Plan for Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits for
the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous
Phase Samples, Battelle, Columbus, Ohio, July 1, 2008.
2. US EPA NRMRL Standard Operating Procedure (SOP) for the Analysis of Steroid
Hormones in Aqueous Samples, QA ID 503-P3-0, 09/29/05.
3. Quality Management Plan (QMP)for the ETV Advanced Monitoring Systems Center,
Version 6.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, November 2005.
24
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