»EPA
United States Office of Research and EPA/540/R-05/005
Environmental Protection Development March 2005
Agency Washington, DC 20460
Innovative Technology
Verification Report
Technologies for Monitoring
and Measurement of Dioxin
and Dioxin-like Compounds
in Soil and Sediment
Hybrizyme Corporation
AhRC PCR™ Kit
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EPA/540/R-05/005
March 2005
Innovative Technology
Verification Report
Hybrizyme Corporation AhRC PCR™ Kit
Prepared by
Battelle
505 King Avenue
Columbus, Ohio 43201
Contract No. 68-C-00-185
Stephen Billets
Environmental Sciences Division
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, NV 89119
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Notice
This document was prepared for the U.S. Environmental Protection Agency (EPA) Superfund Innovative Technology
Evaluation Program under Contract No. 68-C-00-185. The document has met the EPA's requirements for peer and
administrative review and has been approved for publication. Mention of corporation names, trade names, or
commercial products does not constitute endorsement or recommendation for use.
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the nation's natural
resources. Under the 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 (ORD) provides data and scientific
support that can be used to solve environmental problems, build the scientific knowledge base needed to manage
ecological resources wisely, understand how pollutants affect public health, and prevent or reduce environmental
risks.
The National Exposure Research Laboratory is the Agency's center for investigation of technical and management
approaches for identifying and quantifying risks to human health and the environment. Goals of the Laboratory's
research program are to (1) develop and evaluate methods and technologies for characterizing and monitoring air, soil,
and water; (2) support regulatory and policy decisions; and (3) provide the scientific support needed to ensure
effective implementation of environmental regulations and strategies.
The EPA's Superfund Innovative Technology Evaluation (SITE) Program evaluates technologies designed for
characterization and remediation of contaminated Superfund and Resource Conservation and Recovery Act sites. The
SITE Program was created to provide reliable cost and performance data in order to speed the acceptance and use of
innovative remediation, characterization, and monitoring technologies by the regulatory and user community.
Effective monitoring and measurement technologies are needed to assess the degree of contamination at a site,
provide data that can be used to determine the risk to public health or the environment, and monitor the success or
failure of a remediation process. One component of the EPA SITE Program, the Monitoring and Measurement
Technology (MMT) Program, demonstrates and evaluates innovative technologies to meet these needs.
Candidate technologies can originate within the federal government or the private sector. Through the SITE Program,
developers are given the opportunity to conduct a rigorous demonstration of their technologies under actual field
conditions. By completing the demonstration and distributing the results, the Agency establishes a baseline for
acceptance and use of these technologies. The MMT Program is managed by the ORD's Environmental Sciences
Division in Las Vegas, Nevada.
Gary Foley, Ph.D.
Director
National Exposure Research Laboratory
Office of Research and Development
in
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Abstract
A demonstration of technologies for determining the presence of dioxin and dioxin-like compounds in soil and
sediment was conducted under the U.S. Environmental Protection Agency's (EPA's) Superfund Innovative
Technology Evaluation Program in Saginaw, Michigan, at Green Point Environmental Learning Center from April 26
to May 5, 2004. This innovative technology verification report describes the objectives and the results of that
demonstration, and serves to verify the performance and cost of the Hybrizyme Corporation AhRC PCR™ Kit. Four
other technologies were evaluated as part of this demonstration, and separate reports have been prepared for each
technology. The objectives of the demonstration included evaluating the technology's accuracy, precision, sensitivity,
sample throughput, tendency for matrix effects, and cost. The test also included an assessment of how well the
technology's results compared to those generated by established laboratory methods using high-resolution mass
spectrometry (HRMS). The demonstration objectives were accomplished by evaluating the results generated by the
technology from 209 soil, sediment, and extract samples. The test samples included performance evaluation (PE)
samples (i.e., contaminant concentrations were certified or the samples were spiked with known contaminants) and
environmental samples collected from 10 different sampling locations.
The Hybrizyme Corporation AhRC PCR™ Kit is a technology that reports the concentration of aryl hydrocarbon (Ah)
receptor binding compounds in a sample, and the units are reported as Ah Receptor Binding Units (AhRBU). At the
time of the demonstration, this particular test was intended for use as a screening tool to rank samples from those
inducing the greatest Ah receptor (AhR) activity to those inducing the least AhR activity rather than to provide highly
accurate toxicity equivalents (TEQ). The developer's goal is a highly portable screening technology that can help to
determine areas of greatest concern for cleanup at a site and can help to minimize the number of more expensive
analyses needed for specific analytes. It has been suggested that correlation between the Hybrizyme AhRBU results
and HRMS TEQ results could be established by first characterizing a site and calibrating the Hybrizyme results to
HRMS results. This approach was not evaluated during this demonstration. Since the technology measures an actual
biological response, it is possible that the technology may give a better representation of the true toxicity from a risk
assessment standpoint. Therefore, the technology's results were compared to the HRMS D/F and PCB data as well as
polynuclear aromatic hydrocarbon (PAH) data in terms of ranking sample concentrations from low to high, rather
than in a quantitative fashion of AhRBU vs TEQ. PAH concentrations were included in the comparison because
Hybrizyme's kit responds to these compounds. The suite of PAHs that were quantified in the samples using gas
chromatograph/mass spectrometry. The PAHs were a selected target list for this demonstration and likely do not
include all of the PAHs that are responsive to this kit. The HRMS reference D/F and PCB data were generated by
AXYS Analytical Services, using EPA Methods 1613B and 1668A.
Sample concentrations that were ranked by Hybrizyme from low to high were compared to the PE certified
concentration and reference laboratory data, including contributions from PAHs where PAH data were available. The
Hybrizyme ranking agreed with the certified values for higher concentration samples, but was inconsistent for lower
concentration samples. The Hybrizyme technology's concentration ranking was consistent with the reference
laboratory ranking for the environmental samples 70 to 90% of the time. The technology's calculated estimated
method detection limit was 71 AhRBU. A significant effect was not observed for the reproducibility of Hybrizyme
results by matrix type (soil, sediment, extract) or by PAH concentration, but a significant effect was observed for
sample type (PE vs. environmental vs. extract) with the PE samples having a significantly higher mean RSD value
(44%) compared to the environmental (19%) and the extract (14%) samples. The data generated and evaluated during
this demonstration showed that the Hybrizyme technology could be used as an effective tool to rank sample
concentrations from low to high AhR activity within a particular environmental site, particularly considering that the
cost ($35,023 vs. $398,029) and the time (< two weeks vs. eight months) to analyze the 209 demonstration samples
was significantly less than that of the reference laboratory.
iv
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Contents
Chapter Page
Notice ii
Foreword iii
Abstract iv
Abbreviations, Acronyms, and Symbols ix
Acknowledgments xii
1 Introduction 1
1.1 Description of the SITE MMT Program 1
1.2 Scope of This Demonstration 3
1.2.1 Organization of Demonstration 4
1.2.2 Sample Descriptions and Experimental Design 5
1.2.3 Overview of Field Demonstration 5
2 Description of Hybrizyme Corporation AhRC PCR™ Kit 6
2.1 Company History 6
2.2 Product History 6
2.3 Technology Description 7
2.4 Developer Contact Information 8
3 Demonstration and Environmental Site Descriptions 9
3.1 Demonstration Site Description and Selection Process 9
3.2 Description of Sampling Locations 10
3.2.1 Soil Sampling Locations 10
3.2.2 Sediment Sampling Sites 12
4 Demonstration Approach 14
4.1 Demonstration Objectives 14
4.2 Toxicity Equivalents 14
4.3 Overview of Demonstration Samples 16
4.3.1 PE Samples 16
4.3.2 Environmental Samples 20
4.3.3 Extracts 21
4.4 Sample Handling 23
4.5 Pre-Demonstration Study 24
4.6 Execution of Field Demonstration 24
4.7 Assessment of Primary and Secondary Objectives 25
4.7.1 Primary Objective PI: Accuracy 25
4.7.2 Primary Objective P2: Precision 25
4.7.3 Primary Objective P3: Comparability 25
4.7.4 Primary Objective P4: Estimated Method Detection Limit 26
4.7.5 Primary Objective P5: False Positive/False Negative Results 26
4.7.6 Primary Objective P6: Matrix Effects 26
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Contents (continued)
Page
4.7.7 Primary Objective P7: Technology Costs 27
4.7.8 Secondary Objective SI: Skill Level of Operator 27
4.7.9 Secondary Objective S2: Health and Safety Aspects 27
4.7.10 Secondary Objective S3: Portability 27
4.7.11 Secondary Objective S4: Sample Throughput 27
5 Confirmatory Process 28
5.1 Traditional Methods for Measurement of Dioxin and Dioxin-Like
Compounds in Soil and Sediment 28
5.1.1 High-Resolution Mass Spectrometry 28
5.1.2 Low-Resolution Mass Spectrometry 28
5.1.3 PCB Methods 29
5.1.4 Reference Method Selection 29
5.2 Characterization of Environmental Samples 29
5.2.1 Dioxins and Furans 29
5.2.2 PCBs 30
5.2.3 PAHs 30
5.3 Reference Laboratory Selection 30
5.4 Reference Laboratory Sample Preparation and Analytical Methods 31
5.4.1 Dioxin/Furan Analysis 31
5.4.2 PCB Analysis 31
5.4.3 TEQ Calculations 32
6 Assessment of Reference Method Data Quality 33
6.1 QA Audits 33
6.2 QC Results 34
6.2.1 Holding Times and Storage Conditions 34
6.2.2 Chain of Custody 34
6.2.3 Standard Concentrations 34
6.2.4 Initial and Continuing Calibration 34
6.2.5 Column Performance and Instrument Resolution 35
6.2.6 Method Blanks 35
6.2.7 Internal Standard Recovery 35
6.2.8 Laboratory Control Spikes 35
6.2.9 Sample Batch Duplicates 35
6.3 Evaluation of Primary Objective PI: Accuracy 35
6.4 Evaluation of Primary Objective P2: Precision 36
6.5 Comparability to Characterization Data 37
6.6 Performance Summary 39
7 Performance of Hybrizyme Corporation AhRC PCR™ Kit 40
7.1 Evaluation of Hybrizyme Corporation AhRC PCR™ Kit Performance 40
7.1.1 Evaluation of Primary Objective PI: Accuracy 40
7.1.2 Evaluation of Primary Objective P2: Precision 41
7.1.3 Evaluation of Primary Objective P3: Comparability 41
7.1.4 Evaluation of Primary Objective P4: Estimated Method Detection Limit 44
7.1.5 Evaluation of Primary Objective P5: False Positive/False Negative Results 44
7.1.6 Evaluation of Primary Objective P6: Matrix Effects 44
7.1.7 Evaluation of Primary Objective P7: Technology Costs 45
VI
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Contents (continued)
Page
7.2 Observer Report: Evaluation of Secondary Objectives 45
7.2.1 Evaluation of Secondary Objective SI: Skill Level of Operator 46
7.2.2 Evaluation of Secondary Objective S2: Health and Safety Aspects 46
7.2.3 Evaluation of Secondary Objective S3: Portability 47
7.2.4 Evaluation of Secondary Objective S4: Throughput 47
7.2.5 Miscellaneous Observer Notes 47
8 Economic Analysis 49
8.1 Issues and Assumptions 49
8.1.1 Capital Equipment Cost 49
8.1.2 Cost of Supplies 49
8.1.3 Support Equipment Cost 49
8.1.4 Labor Cost 50
8.1.5 Investigation-Derived Waste Disposal Cost 50
8.1.6 Costs Not Included 50
8.2 AhRC PCR™ Kit Costs 51
8.2.1. Capital Equipment Cost 51
8.2.2 Cost of Supplies 51
8.2.3 Support Equipment Cost 51
8.2.4 Labor Cost 52
8.2.5 Investigation-Derived Waste Disposal Cost 52
8.2.6 Summary of AhRC PCR™ Kit Costs 53
8.3 Reference Method Costs 53
8.4 Comparison of Economic Analysis Results 53
9 Technology Performance Summary 55
10 References 58
Appendix A SITE Monitoring and Measurement Technology Program Verification Statement A-l
Appendix B Supplemental Information Supplied by Developer B-l
Appendix C Reference Laboratory Method Blank and Duplicate Results Summary C-l
Appendix D Summary of Developer and Reference Laboratory Data D-l
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Contents (continued)
Pas
Figures
1-1 Representative dioxin, furan, and polychlorinated biphenyl structure 3
2-1 Hybrizyme's AhRC PCR™ Test Kit 7
2-2 ABI Prism 7000 8
2-3 Cepheid Smart Cycler 8
2-4 Hybrizyme's AhRC-PCR™ Test Kit in operation during the field demonstration 8
6-1 Comparison of reference laboratory and characterization D/F data for environmental samples 38
Tables
3-1 Summary of Environmental Sampling Locations 11
4-1 World Health Organization Toxicity Equivalency Factor Values 15
4-2 Distribution of Samples for the Evaluation of Performance Objectives 17
4-3 Number and Type of Samples Analyzed in the Demonstration 17
4-4 Summary of Performance Evaluation Samples 18
4-5 Characterization and Homogenization Analysis Results for Environmental Samples 22
4-6 Distribution of Extract Samples 23
5-1 Calibration Range of HRMS Dioxin/Furan Method 28
5-2 Calibration Range of LRMS Dioxin/Furan Method 28
6-1 Objective PI Accuracy - Percent Recovery 36
6-2 Evaluation of Interferences 36
6-3a Objective P2 Precision - Relative Standard Deviation 37
6-3b Objective P2 Precision - Relative Standard Deviation (By Sample Type) 38
6-4 Reference Method Performance Summary - Primary Objectives 39
7-1 Objective PI Accuracy - Ranking of PE Samples According to the
Concentration of AhR Binding Compound 41
7-2a Objective P2 Precision - Relative Standard Deviation 42
7-2b Objective P2 Precision - Relative Standard Deviation (By Sample Type) 43
7-3 Objective P3 - Comparability by Ranking Environmental Samples from Low to High Concentration
Within an Environmental Site 43
7-4 Objective P3 - Summary of Environmental Sample Ranking Comparisons 44
7-5 Objective P4 - Estimated Method Detection Limit 44
7-6 Objective P6 - Matrix Effects Using RSD as a Description of Precision by Matrix Type 45
7-7 Objective P6 - Matrix Effects Using RSD as a Description of Precision by PAH Concentration Levels
(Environmental Samples Only) 45
8-1 Cost Summary 52
8-2 Reference Method Cost Summary 53
9-1 Hybrizyme Corporation AhRC PCR™ Kit Performance Summary - Primary Objectives 56
9-2 Hybrizyme Corporation AhRC PCR™ Kit Performance Summary - Secondary Objectives 57
Vlll
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Abbreviations, Acronyms, and Symbols
Ah
ANOVA
AhR
AhRBU
ATSDR
CIL
CoA
COC
CRM
DER
D/F
DNA
DNR
D/QAPP
ORE
ELC
EMDL
EMPC
EPA
ERA
g
GC
HPLC/GPC
HRGC
HRMS
i.d.
IDW
ITVR
kg
aryl hydrocarbon
analysis of variance
aryl hydrocarbon receptor
Ah-receptor binding units
Agency for Toxic Substances and Disease Registry
Cambridge Isotope Laboratories
Certificate of Analysis
chain of custody
certified reference material
data evaluation report
dioxin/furan
deoxyribonucleic acid
Department of Natural Resources
demonstration and quality assurance project plan
dioxin-responsive element
Environmental Learning Center
estimated method detection limit
estimated maximum possible concentration
Environmental Protection Agency
Environmental Resource Associates
gram
gas chromatography
high-performance liquid chromatography/gel permeation chromatography
high-resolution capillary gas chromatography
high-resolution mass spectrometry
internal diameter
investigation-derived waste
innovative technology verification report
kilogram
IX
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Abbreviations, Acronyms, and Symbols (Continued)
L
LRMS
m
MDEQ
MDL
mg
mL
mm
MMT
MS
NERL
ng
NIST
NOAA
ORD
PAH
PCB
PCDD/F
PCP
PCR
PE
Pg
ppb
ppm
ppt
QA/QC
RM
RPD
RSD
SDL
liter
low-resolution mass spectrometry
microliter
micrometer
meter
Michigan Department of Environmental Quality
method detection limit
milligram
milliliter
millimeter
Monitoring and Measurement Technology
mass spectrometry
National Exposure Research Laboratory
nanogram
National Institute for Standards and Technology
National Oceanic and Atmospheric Administration
Office of Research and Development
polynuclear aromatic hydrocarbon
polychlorinated biphenyl
polychlorinated dibenzo-/?-dioxin/dibenzofuran
pentachlorophenol
polymerase chain reaction
performance evaluation
picogram
parts per billion; nanogram/g; ng/g
parts per million; microgram/g; |ig/g
parts per trillion; picogram/g; pg/g
quality assurance/quality control
reference method
relative percent difference
relative standard deviation
sample-specific detection limit
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SIM
SITE
SOP
SRM
TCDD
TEF
TEQ
TEQD/F
TOC
total TEQ
WHO
Abbreviations, Acronyms, and Symbols (Continued)
selected ion monitoring
Superfund Innovative Technology Evaluation
standard operating procedure
Standard Reference Material®
tetrachlorodibenzo-/>-dioxin
toxicity equivalency factor
toxicity equivalent
total toxicity equivalents of dioxins/furans
total toxicity equivalents of World Health Organization dioxin-like polychlorinated
biphenyls
total organic carbon
total toxicity equivalents including the sum of the dioxin/furan and World Health
Organization dioxin-like polychlorinated biphenyls
World Health Organization
XI
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Acknowledgments
This report was prepared for the U.S. Environmental Protection Agency (EPA) Superfund Innovative Technology
Evaluation (SITE) Program under the direction and coordination of Stephen Billets of the EPA's National Exposure
Research Laboratory (NERL)—Environmental Sciences Division in Las Vegas, Nevada. George Brilis and
Brian Schumacher of the EPA NERL reviewed and commented on the report. The EPA NERL thanks Michael Jury,
Al Taylor, and Sue Kaelber-Matlock of the Michigan Department of Environmental Quality (MDEQ) and Becky
Goche and Doug Spencer of the U.S. Fish and Wildlife Service for their support in conducting the field
demonstration. We appreciate the support of the Dioxin SITE Demonstration Panel for their technical input to the
demonstration/quality assurance project plan. In particular, we recognize Andy Beliveau, Nardina Turner,
Greg Rudloff, Allen Debus, Craig Smith, David Williams, Dwain Winters, Jon Josephs, Bob Mouringhan,
Terry Smith, and Joe Ferrario of the U.S. EPA. Thanks also go to EPA Region 2, EPA Region 3, EPA Region 4, EPA
Region 5, EPA Region 7, and the MDEQ for collecting and supplying environmental samples for inclusion in the
demonstration. Andy Beliveau, Allen Debus, and Nardina Turner, and Dwain Winters served as EPA reviewers of
this report. Michael Jury (MDEQ), Sue Kaelber-Matlock (MDEQ), Jim Sanborn (California-EPA), and Jeffrey Archer
(U.S. Food and Drug Administration) served as additional reviewers of this report. Computer Sciences Corporation
provided a technical editing review of the report. This report was prepared for the EPA by Battelle. Special
acknowledgment is given to Amy Dindal, who was the Battelle Project Manager, and to Josh Finegold,
Nicole Iroz-Elardo, Mark Misita, Tim Pivetz, Mary Schrock, Rachel Sell, Bea Weaver, and Zack Willenberg for their
contributions to the preparation of this report.
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Chapter 1
Introduction
The U.S. Environmental Protection Agency (EPA),
Office of Research and Development (ORD), National
Exposure Research Laboratory (NERL) contracted with
Battelle (Columbus, Ohio) to conduct a demonstration of
monitoring and measurement technologies for dioxin
and dioxin-like compounds in soil and sediment. A field
demonstration was conducted as part of the EPA
Superfund Innovative Technology Evaluation (SITE)
Monitoring and Measurement Technology (MMT)
Program. The purpose of this demonstration was to
obtain reliable performance and cost data on the
technologies to provide (1) potential users with a better
understanding of the technologies' performance and
operating costs under well-defined field conditions and
(2) the technology developers with documented results
that will help promote the acceptance and use of their
technologies.
This innovative technology verification report (ITVR)
describes the SITE MMT Program and the scope of this
demonstration (Chapter 1); a description of the
Hybrizyme Corporation AhRC PCR™ Kit (Chapter 2);
the demonstration site and the sampling locations
(Chapter 3); the demonstration approach (Chapter 4); the
confirmatory process (Chapter 5); the assessment of
reference method data quality (Chapter 6); the
performance of the technology (Chapter 7); the
economic analysis for the technology and reference
method (Chapter 8); the demonstration results in
summary form (Chapter 9); and the references used to
prepare this report (Chapter 10). Appendix A contains a
verification statement; Appendix B contains
supplemental information provided by the developer;
Appendix C is a summary of method blank and batch
duplicate data by the reference laboratory; and
Appendix D contains a one-to-one matching of the
developer and reference laboratory data.
1.1 Description of the SITE MMT Program
Performance verification of innovative environmental
technologies is an integral part of the regulatory and
research mission of the EPA. The SITE Program was
established by the EPA Office of Solid Waste and
Emergency Response and ORD under the Superfund
Amendments and Reauthorization Act of 1986. The
overall goal of the Program is to conduct performance
verification studies and to promote the acceptance of
innovative technologies that may be used to achieve
long-term protection of human health and the
environment. The program is designed to meet three
primary objectives: (1) identify and remove obstacles to
the development and commercial use of innovative
technologies, (2) demonstrate promising technologies
and gather reliable performance and cost information to
support site characterization and remediation activities,
and (3) develop procedures and policies that encourage
use of innovative technologies at Superfund sites as well
as at other waste sites or commercial facilities. The SITE
Program includes the following elements:
MMT Program—Evaluates technologies that
sample, detect, monitor, or measure hazardous and
toxic substances. These technologies are expected to
provide better, faster, or more cost-effective methods
for producing real-time data during site characteriza-
tion and remediation efforts than conventional
laboratory technologies.
• Remediation Technology Program—Conducts
demonstrations of innovative treatment technologies
to provide reliable performance, cost, and
applicability data for site cleanups.
Technology Transfer Program—Provides and
disseminates technical information in the form of
updates, brochures, and other publications that
promote the SITE Program and participating
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technologies. It also supports the technologies by
offering technical assistance, training, and
workshops.
The MMT Program's technology verification process is
designed to conduct demonstrations that will generate
high-quality data so that potential users have reliable
information regarding the technology performance and
cost. Four steps are inherent in the process: (1) needs
identification and technology selection, (2) demon-
stration planning and implementation, (3) report
preparation, and (4) information distribution. The first
step of the technology verification process begins with
identifying technology needs of the EPA and regulated
community. The EPA Regional offices, the U.S.
Department of Energy, the U.S. Department of Defense,
industry, and state environmental regulatory agencies are
asked to identify technology needs for sampling,
measurement, and monitoring of environmental media.
Once a need is identified, a search is conducted to
identify suitable technologies that will address the need.
The technology search and identification process
consists of examining industry and trade publications,
attending related conferences, and exploring leads from
technology developers and industry experts. Selection of
technologies for field testing includes evaluation of the
candidate technologies based on several criteria. A
suitable technology for field testing
is designed for use in the field or in a mobile
laboratory,
• is applicable to a variety of environmentally
contaminated sites,
has potential for solving problems that current
methods cannot satisfactorily address,
• has estimated costs that are lower than those of
conventional methods,
is likely to achieve equivalent or better results than
current methods in areas such as data quality and
turnaround time,
• uses techniques that are easier or safer than current
methods, and
is commercially available.
Once candidate technologies are identified, developers
are asked to participate in a developer conference. This
conference gives the developers an opportunity to
describe their technologies' performance and to learn
about the MMT Program.
The second step of the technology verification process is
to plan and implement a demonstration that will generate
representative, high-quality data to assist potential users
in selecting a technology. Demonstration planning
activities include a pre-demonstration sampling and
analysis investigation that assesses existing conditions at
the proposed demonstration site or sites. The objectives
of the pre-demonstration investigation are to (1) confirm
available information on applicable physical, chemical,
and biological characteristics of contaminated media at
the sites to justify selection of site areas for the
demonstration; (2) provide the technology developers
with an opportunity to evaluate the areas, analyze
representative samples, and identify logistical
requirements; (3) assess the overall logistical and quality
assurance requirements for conducting the demon-
stration; and (4) select and provide the reference
laboratory with an opportunity to identify any matrix-
specific analytical problems associated with the
contaminated media and to propose appropriate
solutions. Information generated through the pre-
demonstration investigation is used to develop the final
demonstration design and to confirm the nature and
source of samples that will be used in the demonstration.
Demonstration planning activities also include
preparation of a demonstration plan that describes the
procedures to verify the performance and cost of each
technology. The demonstration plan incorporates
information generated during the pre-demonstration
investigation as well as input from technology
developers, demonstration site representatives, and
technical peer reviewers. The demonstration plan also
incorporates the quality assurance (QA)/quality control
(QC) elements needed to produce data of sufficient
quality to document the performance and cost of each
technology.
During the demonstration, each technology is evaluated
independently and, when possible and appropriate, is
compared to a reference technology. The performance
and cost of one technology are not compared to those of
another technology evaluated in the demonstration.
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Rather, demonstration data are used to evaluate
theindividual performance, cost, advantages, limitations,
and field applicability of each technology.
As part of the third step of the technology verification
process, the EPA publishes a verification statement
(Appendix A) and a detailed evaluation of each
technology in an ITVR. To ensure its quality, the ITVR
is published only after comments from the technology
developer and external peer reviewers are satisfactorily
addressed. All demonstration data used to evaluate each
technology are summarized in a data evaluation report
(DER) that constitutes a complete record of the
demonstration. The DER includes audit reports, observer
reports, completed data validation checklists, certificates
of analysis, and the data packages (i.e., raw data) from
the reference laboratory. The DER is not published as an
EPA document, but a copy may be obtained from the
EPA project manager.
The fourth step of the verification process is to distribute
demonstration information. To benefit technology
developers and potential technology users, the EPA
makes presentations, publishes and distributes fact
sheets, newsletters, bulletins, and ITVRs through direct
mailings and on the Internet. Information on the SITE
Program is available on the EPA ORD Web site
(http://www.epa.gov/ORD/SITE). Additionally, a
Visitor's Day, which is held in conjunction with the
demonstration, allows the developers to showcase their
technologies and gives potential users the opportunity to
have a firsthand look at the technologies in operation.
1.2 Scope of This Demonstration
Polychlorinated dibenzo-/>-dioxins and polychlorinated
dibenzofurans, commonly referred to collectively as
"dioxins," are of significant concern in site remediation
projects and human health assessments because they are
highly toxic. Dioxins and furans are halogenated
aromatic hydrocarbons and are similar in structure as
shown in Figure 1-1. They have similar chemical and
physical properties. Chlorinated dioxins and furans are
technically referred to as polychlorinated dibenzo-/>-
dioxins (PCDD) and polychlorinated dibenzofurans
(PCDF). For the purposes of this document, they will be
referred to simply as "dioxins," "PCDD/F," or "D/F."
Dioxins and furans are not intentionally produced in
most chemical processes. However, they can be
synthesized directly and are commonly generated as by-
products of various combustion and chemical
processes.(1) They are colorless crystals or solids with
high melting points, very low water solubility, high fat
solubility, and low volatility. Dioxins and furans are
extremely stable under most environmental conditions,
making them persistent once released in the
environment. Because they are fat soluble, they also tend
to bioaccumulate.
There are 75 individual chlorinated dioxins and 135
individual chlorinated furans. Each individual dioxin and
furan is referred to as a congener. The properties of each
congener vary according to the number of chlorine
atoms present and the position where the chlorines are
attached. The congeners with chlorines attached at a
minimum in the 2,3,1, and 8 positions are considered
most toxic. A total of seven dioxin and ten furan
congeners contain chlorines in the 2, 3, 7, 8 positions
and, of these, 2,3,7,8-tetrachlorodibenzo-/>-dioxin
(2,3,7,8-TCDD) is one of the most toxic and serves as
the marker compound for this class.
Certain polychlorinated biphenyls (PCBs) have
structural and conformational similarities to dioxin
compounds (Figure 1-1) and are therefore expected to
exhibit toxicological similarities to dioxins as well.
Cl
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin
Cl
Cl
2,3,7,8-Tetrachlorodibenzofuran
Cl
Cl Cl
3,3',4,4',5,5'-Hexachlorobiphenyl
Figure 1-1. Representative dioxin, furan,
and polychlorinated biphenyl structure.
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Currently only twelve of the total 209 PCB congeners
are thought to have "dioxin-like" toxicity. These twelve
are PCBs with four or more chlorines with just one or no
substitution in the ortho position, and which assume a
flat configuration with rings in the same plane. These
"dioxin-like" PCBs are often refered to as non-ortho and
mono-ortho substituted coplanar PCBs.
Conventional analytical methods for determining
concentrations of dioxin and dioxin-like compounds are
time-consuming and costly. For example, EPA standard
methods require solvent extraction of the sample,
processing the extract through multiple cleanup
columns, and analyzing the cleaned fraction by gas
chromatography (GC)/high-resolution mass
spectrometry (HRMS). The use of a simple, rapid, cost-
effective analytical method would allow field personnel
to quickly assess the extent of contamination at a site
and could be used to direct or monitor remediation or
risk assessment activities. This data could be used to
provide immediate feedback on potential health risks
associated with the site and permit the development of a
more focused and cost-effective sampling strategy. At
this time, more affordable and quicker analytical
techniques will not replace HRMS. However, before
adopting an alternative to traditional laboratory-based
methods, a thorough assessment of how commercially
available technologies compare to conventional
laboratory-based analytical methods using certified,
spiked, and environmental samples is warranted. A
summary of the demonstration activities to evaluate
measurement technologies for dioxin and dioxin-like
compounds in soil and sediment is provided below. The
experimental design and demonstration approach are
described in greater detail in Chapter 4 and was
published in the Demonstration and Quality Assurance
Project Plan (D/QAPP).(2)
1.2.1 Organization of Demonstration
The key organizations and personnel involved in the
demonstration, including the roles and responsibilities of
each, are fully described in the D/QAPP.(2) EPA NERL
had overall responsibility for this project. The EPA
reviewed and concurred with all project deliverables
including the D/QAPP and the ITVRs, provided
oversight during the demonstration, and participated in
the Visitor's Day. Battelle served as the verification
testing organization for EPA/NERL. Battelle's
responsibilities included developing and implementing
all elements of the D/QAPP; scheduling and
coordinating the activities of all demonstration
participants; coordinating the collection of
environmental samples; serving as the characterization
laboratory by performing the homogenization of the
environmental samples and confirming the efficacy of
the homogenization and approximate sample
concentrations; conducting the demonstration by
implementing the D/QAPP; summarizing, evaluating,
interpreting, and documenting demonstration data for
inclusion in this report; and preparing draft and final
versions of each developer's ITVR. The developers were
five companies who submitted technologies for
evaluation during this demonstration. The
responsibilities of the developers included providing
input to, reviewing, and concurring with the D/QAPP;
providing personnel and supplies as needed for the
demonstration; operating their technologies during the
demonstration; and reviewing and commenting on their
technologies' ITVRs. AXYS Analytical Services, Ltd.
was selected to serve as the reference analytical
laboratory. AXYS analyzed each demonstration sample
by EPA Method 1613B(3) and EPA Method 1668A(4)
according to the statement of work provided in the
D/QAPP. The Michigan Department of Environmental
Quality (MDEQ) hosted the demonstration, coordinated
the activities of and participated in Visitor's Day, and
collected and provided some of the environmental
samples that were used in the demonstration. The Dioxin
SITE Demonstration Panel served as technical advisors
and observers of the demonstration activities. Panel
membership, which is outlined in the D/QAPP, included
representation from EPA Regions 1, 2, 3, 4, 5, 7, and 9;
EPA Program Offices; the MDEQ; and the U.S. Fish and
Wildlife Services. Members, of the panel participated in
five conference calls with the EPA, Battelle, AXYS, and
the developers. The panel contributed to the
experimental design and D/QAPP development; logistics
for the demonstration, including site selection; sample
collection, reference laboratory selection, and data
analysis and technology evaluation procedures. As an
example of the significant impact the panel had on the
demonstration, it was the EPA members of the panel
who suggested expanding the scope of the project from
focusing exclusively on dioxins and furans, to also
include PCBs and the generation of characterization data
for polynuclear aromatic hydrocarbons (PAHs).
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1.2.2 Sample Descriptions and Experimental
Design
Soil and sediment samples with a variety of
distinguishing characteristics such as high levels of
PCBs and PAHs were analyzed by each participant.
Samples were collected from a variety of dioxin-
contaminated soil and sediment sampling locations
around the country. Samples were identified and
supplied through EPA Regions 2, 3, 4, 5, and 7 and the
MDEQ. The samples were homogenized and
characterized by the characterization laboratory prior to
use in the demonstration to ensure a variety of
homogeneous, environmentally derived samples with
concentrations over a large dynamic range (< 50 to
> 10,000 picogram/gram [pg/g]) were included. The
environmental samples comprised 128 of the 209
samples included in the demonstration (61%).
Performance evaluation (PE) samples were obtained
from five commercial sources. PE samples consisted of
known quantities of dioxin and dioxin-like compounds.
Fifty-eight of the 209 demonstration samples (28%)
were PE samples. A suite of solvent extracts was
included in the demonstration to minimize the impact of
sample homogenization and to provide a uniform matrix
for evaluation. A total of 23 extracts (11% of the total
number of samples) was included in the demonstration.
The demonstration samples are described in greater
detail in Section 4.3.
1.2.3 Overview of Field Demonstration
All technology developers participated in a pre-
demonstration study where a representative subset of the
demonstration samples was analyzed. The pre-
demonstration results indicated that the Hybrizyme
technology was suitable for participation in the
demonstration. The demonstration of technologies for
the measurement of dioxin and dioxin-like compounds
was conducted at the Green Point Environmental
Learning Center (ELC) in Saginaw, Michigan, from
April 26 to May 5, 2004. Five technologies, including
immunoassay test kits and aryl hydrocarbon (Ah)
receptor-binding technologies, participated in the
demonstration. The operating procedures for the
participating technologies are described in the D/QAPP.
The technologies were operated by the developers.
Because the sample throughput of the technologies
varied widely, it was at the discretion of the developers
how many of the 209 demonstration samples were
analyzed in the field. Results from the demonstration
samples, in comparison with results generated by AXYS
using standard analytical methods, were used to evaluate
the analytical performance of the technologies, including
the parameters of accuracy, precision, and comparability.
Observations from the field demonstration were used to
assess sample throughput, ease of use, health and safety
aspects, and the field portability of each technology. The
performance evaluation of the Hybrizyme Corporation
AhRC PCR™ Kit is presented in this ITVR. Separate
ITVRs have been published for the other four
participating technologies.
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Chapter 2
Description of Hybrizyme Corporation AhRC PCR™ Kit
This technology description is based on information
provided by Hybrizyme and only editorial changes were
made to ensure document consistency. Actual cost and
performance data, as reported and observed during the
demonstration, will be provided later in this document.
The AhRC PCR™ assay couples the aryl hydrocarbon
receptor (AhR) with polymerase chain reaction (PCR)
technology to produce a method for analyzing dioxins
and related compounds in environmental or food
samples. The AhRC PCR™ kit can be shipped
worldwide and yields results within hours. It is ideally
suited for stationary or mobile laboratories.
2.1 Company History
In 1995, Hybrizyme Corporation licensed the worldwide
rights to the gene encoding the AhR from Northwestern
University to develop a rapid and cost-effective test for
dioxins. The AhR was found to mediate most, if not all,
of the harmful effects associated with dioxin-like
compounds. Hybrizyme pioneered the development of a
dioxin assay that utilized the specificity of the AhR with
the detection capabilities of PCR.
In 1998, Hybrizyme entered into a licensing and OEM
agreement with PerkinElmer Life Sciences to produce
environmental and food immunoassay kits that would
compliment the company's receptor-based technology.
The company's product line includes complementary
immunoassay products consisting of the DELFIA PCB
Food Kit, DELFIA PCB Soil Kit, and DELFIA TCDD
kit. The PCB food test has been validated for use in
Europe, and the efficacy of the PCB soil test was
demonstrated through the EPA's Environmental
Technology Verification program.
2.2 Product History
Hybrizyme's goal was to develop a quick and
inexpensive screen for dioxin-like compounds based on
the molecular events responsible for their toxicity.
Although Hybrizyme has a patented yeast-based system
for screening dioxins, the company's aim was to develop
an assay that did not require a living organism. This
allows the test to be shipped throughout the world and
used immediately upon arrival or as needed.
When the AhR binds to a dioxin-like molecule, it is
irreversibly transformed into a protein that attaches to a
specific sequence of deoxyribonucleic acid (DNA)
called the dioxin-responsive element (DRE). Binding of
the AhR to the DRE initiates a cascade of biochemical
effects in humans and animals that lead to toxicological
consequences.
The AhRC PCR assay is based on the ability of the
transformed AhR to bind to a DNA-probe containing the
DRE sequence. The advantages of the AhRC PCR assay
result, in part, from the high degree of specificity that is
required for the transformed receptor to bind the
DRE-probe and the unmatched sensitivity of PCR to
measure the bound DRE-probe. The amount of
DRE-probe measured by PCR is directly proportional to
the amount of dioxin in the sample.
The AhRC PCR™ kit has been validated in Japan for the
quantitative determination of dioxins and furans in
exhaust gas, fly ash, and sediment. This method utilizes
accelerated solvent extraction and chromatographic
clean up procedures.
A more rapid version of the AhRC PCR assay has been
developed that requires limited sample cleanup. The test
detects not only dioxin and furans but also coplanar
Information was provided by the developer and does not necesarily reflect the opinion of the EPA.
6
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PCBs and carcinogenic PAHs. This method is designed
as a cost-effective tool for quickly mapping large areas
and establishing a relative concentration gradient for
these toxicants. In addition, the method profiles the
collected samples from nondetects to highly contamin-
ated, offering a more effective use of expensive and
time-consuming HRMS high-resolution capillary gas
chromatography (HRGC).
2.3 Technology Description
This procedure uses Hybrizyme's AhRC PCR™ kit
(Figure 2-1) to detect molecules in a test sample that
bind to the AhR and is reported in Ah-receptor binding
units (AhRBU). At the time of the demonstration, this
particular test was intended for use as a screening tool to
rank samples from those inducing the greatest AhR
activity to those inducing the least AhR activity rather
than to provide highly accurate toxicity equivalents
(TEQs). Since the technology measures an actual
biological response, it is possible that the technology
may give a better representation of the true toxicity from
a risk assessment standpoint. The developer's goal is a
highly portable screening technology that can help to
determine areas of greatest concern for cleanup at a site
and can help to minimize the number of more expensive
analyses needed for specific analytes.
The AhR mediates most, if not all, of the harmful effects
associated with exposure to 2,3,7,8-substituted D/F.
How tightly or loosely these compounds bind to the AhR
is one of the determining factors of their toxicity. The
AhR also binds to certain coplanar PCBs and
carcinogenic PAHs, such as benzo-[a]-pyrene. Sample
cleanup procedures can be employed so that all or a
subset of these AhR-reactive compounds are detected by
the assay.
For this demonstration, minimal cleanup procedures
were performed on sample extracts and so the results
obtained were expected to reflect all AhR-reactive
compounds in the samples.
Samples were prepared using an extraction method
designed for speed and simplicity while maintaining
sample concentration. Two grams (g) of each sample
were placed in a 40-milliliter (mL) vial and 20 mL of an
extraction cocktail was added. The vials were placed in
an ultrasonic bath for 10 minutes, followed by brief
centrifugation to remove solids. The extract was
decanted into a new vial and 20 mL of water added.
Immediately following the addition of water, approxi-
mately 2 mL of hexane (present in the extraction
cocktail) is partitioned from the solution. Because of
their hydrophobic nature, most of the dioxin-like
molecules originally in the 2 g of soil were now present
in the 2 mL of hexane. A portion of the hexane was
removed to a disposable glass tube and dried for
analysis. Depending on the condition of the sample
being analyzed, an acid wash was added as an additional
step. The acid wash consisted of re suspending the dried
extract in 1 mL of hexane, adding 2 mL of concentrated
sulfuric acid, and vortexing for two minutes. The phases
were allowed to separate for 10 minutes, and a portion of
the hexane was removed to a disposable glass tube and
dried for analysis. The dried sample extract was sus-
pended in methanol for analysis by the AhRC PCR™
kit.
The AhRC PCR test was based on an assay format
commonly used in research and in clinical applications
ensuring that pipettes, tips, and other disposables would
be commonly available and inexpensive. Sample extracts
were added to 0.5-mL glass vials containing the assay
Figure 2-1. Hybrizyme's AhRC PCR™ Test
Kit.
Information was provided by the developer and does not necesarily reflect the opinion of the EPA.
-------
buffer. The glass vials were provided with each kit and
come racked in a convenient frame for ease of use.
After all of the sample extracts were added, activation
solution containing the AhR and DRE-probe was also
added to the glass vials and shaken at room temperature
for one hour. The reaction mix was transferred from the
glass vials to capture strips using a multichannel
pipettor, and the capture strips were shaken at room
temperature for an additional 30 minutes. During this
time, the AhR/DRE-probe complexes were trapped onto
the wells of the capture strip. The capture strips were
washed to remove free DRE-probe, and PCR master mix
was added. The strips were placed in a real-time
thermocycler, and the amount of the DRE-probe was
measured. The signal was directly proportional to the
amount of dioxin in the samples. A user guide for the
AhRC PCR™ kit can be found at www.hybrizyme.com.
The AhRC PCR™ assay was developed for real-time
PCR systems such as the ABI PRISM 7000 (Figure 2-2),
which can generate up to 96 results per run, or the
Cepheid Smart Cycler (Figure 2-3), designed to be
totally transportable.
This is the developer method that was implemented
during the field demonstration. This procedure is
different from the one presented in the demonstration
plan (2) where the developer originally intended to
demonstrate a technology that reported TEQ results. A
photo of the technology in operation during the demon-
stration is presented in Figure 2-4. Hybrizyme provided
supplemental information about the performance of its
technology during the demonstration and it is presented
in Appendix B.
2.4 Developer Contact Information
Additional information about this technology can be
obtained by contacting:
Hybrizyme
Randy Allen
Suite G-70
2801 Blue Ridge Road
Raleigh, North Carolina 27607
Phone:(919)783-9595
E-mail: rallen@hybrizyme.com
Web site: www.hybrizyme.com
Figure 2-2. ABI Prism 7000.
Figure 2-3. Cepheid Smart
Cycler.
Figure 2-4. Hybrizyme's AhRC PCR™ Test Kit in
operation during the field demonstration.
Information was provided by the developer and does not necesarily reflect the opinion of the EPA.
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Chapter 3
Demonstration and Environmental Site Descriptions
This chapter describes the demonstration site, the
sampling locations, and why each was selected.
3.1 Demonstration Site Description and
Selection Process
This section describes the site selected for hosting the
demonstration, along with the selection rationale and
criteria. Several candidate host sites were considered.
The candidate sites were required to meet certain
selection criteria, including necessary approvals,
support, and access to the demonstration site; enough
space and power to host the technology developers, the
technical support team, and other participants; and
various levels of dioxin-contaminated soil and/or
sediment that could be analyzed as part of the
demonstration. Historically, these demonstrations are
conducted at sites known to be contaminated with the
analytes of interest. The visibility afforded the sites is a
valuable way of keeping the local community informed
of new technologies and to help promote the EPA's
commitment to promote and advance science and
communication.
After review of the information available, the site
selected for the demonstration was the Green Point ELC
site, located within the city of Saginaw, Michigan. The
Saginaw city-owned, 76-acre Green Point ELC, formerly
known as the Green Point Nature Center, is managed by
the Shiawassee National Wildlife Refuge. The Green
Point ELC is situated within the Tittabawassee River
flood plain. The MDEQ found higher than normal levels
of dioxins in soil and sediment samples taken from the
flood plain of the Tittabawassee River. The flood plain is
not heavily laden with PCBs; however, low levels of
PCBs have been detected in some areas. Soil samples
taken from areas outside the flood plain were at typical
background levels. The source of the contamination was
speculated to be attributed to legacy contamination from
chemical manufacturing.
To summarize, Green Point ELC was selected as the
demonstration site based on the following criteria:
• Access and Cooperation of the State and Local
Community—Representatives from the MDEQ,
EPA Region 5, and the local U.S. Fish and Wildlife
Services supported the demonstration, providing site
access for the demonstration, logistical support for
the demonstration, and supported a Visitor's Day
during the demonstration.
• Space Requirements and Feasibility—The demon-
stration took place in the parking lot adjacent to the
Green Point ELC, not directly on an area of
contamination. The site had electrical power and
adequate space to house the trailers and mobile labs
that were used for the demonstration. Furthermore,
the site was close to an international airport. The
weather in Michigan at the time of the demonstration
was unpredictable; however, all participants were
provided heated containment (a mobile laboratory or
construction trailer).
• Site Diversity—The area encompassing the Green
Point site had different levels and types of dioxin
contamination in both the soil and sediment that
were used to evaluate the performance of the
technologies.
The demonstration was conducted at the Green Point
ELC over a 10-day period from April 26 to May 5, 2004.
All technologies were operated inside trailers equipped
with fume hoods or inside mobile laboratories. As such,
the ambient weather conditions during the demonstration
had little impact on the operation of the technologies,
since all of the work spaces were climate-controlled with
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heat and air conditioning. The outdoor weather
conditions were generally cool and rainy, but the
developers kept their working environment at
comfortable temperatures (16 to 18°C). The low
temperature over the 10-day demonstration period was
2°C, the high temperature was 26°C, and the average
temperature was 9°C. Precipitation fell on eight of the
10 days, usually in the form of rain, but occasionally as
sleet or snow flurries, depending on the temperature. The
largest amount of precipitation on a given demonstration
day was 0.50 inches.
3.2 Description of Sampling Locations
This section provides an overview of the ten sampling
sites and methods of selection. Table 3-1 summarizes
each of the locations, what type of sample (soil or
sediment) was provided, the number of samples
submitted from each location, and the number of
samples included in the demonstration from each
location. Samples were collected from multiple
sampling sites so that a wide variety of matrix conditions
could be used to evaluate the performance of the
technologies in addressing monitoring needs at a diverse
range of Superfund sites.
Samples consisted of either soil or sediment and are
described below based on this distinction. It should be
noted that it was not an objective of the demonstration to
accurately characterize the concentration of dioxins,
furans, and PCBs from a specific sampling site. It was,
however, an objective to ensure comparability between
technology samples and the reference laboratory
samples. This was accomplished by homogenizing each
matrix, such that all sub-samples of a given matrix had
consistent contaminant concentrations. As a result,
homogenized samples were not necessarily
representative of original concentrations at the site.
3.2.1 Soil Sampling Locations
This section provides descriptions of each of the soil
sampling locations, including how the sites became
contaminated and approximate dioxin concentrations, as
well as the type and concentrations of other major
constituents, where known (such as PCBs,
pentachlorophenol (PCP), and PAHs). This information
was provided by the site owners/sample providers (e.g.,
the EPA, the EPA contractors, and the MDEQ).
3.2.1.1 Warren County, North Carolina
Five areas of the Warren County PCB Landfill in North
Carolina, a site with both PCB and dioxin contamina-
tion, were sampled. Dioxin concentrations in the landfill
soils range approximately from 475 to 700 pg/g, and
PCB concentrations are greater than 100 parts per
million (ppm). The Warren County PCB Landfill
contains soil that was contaminated by the illegal
spraying of waste transformer oil containing PCBs from
over 210 miles of highway shoulders. Over
30,000 gallons of contaminated oil were disposed of in
14 North Carolina counties. The landfill is located on a
142-acre tract of land. The EPA permitted the landfill
under the Toxic Substances Control Act. Between
September and November 1982, approximately 40,000
cubic yards (equivalent to 60,000 tons) of PCB-
contaminated soil were removed and hauled to the newly
constructed landfill located in Warren County, North
Carolina. The landfill is equipped with both polyvinyl
chloride and clay caps and liners. It also has a dual
leachate collection system. The material in the landfill is
solely from the contaminated roadsides. The landfill was
never operated as a commercial facility. The remedial
action was funded by the EPA and the State of North
Carolina. The site was deleted from the National
Priorities List on March 7, 1986.
3.2.1.2 Tittabawassee River Flood Plain
The MDEQ sampled the Tittabawassee River flood plain
soils from three sites in the flood plain. The source of the
contamination was speculated to be attributed to legacy
contamination from chemical manufacturing. Two
samples were collected from two locations at Imerman
Park in Saginaw Township. The first sample was taken
near the boat launch, and the second sample was taken in
a grassy area near the river bank. Previous analysis from
these areas of this park indicated a range of PCDD/F
concentrations from 600 to 2,500 pg/g. Total PCBs from
these previous measurements were in the low parts-per-
trillion (ppt) range. Two samples were collected from
two locations at Freeland Festival Park in Freeland, MI.
The first sample was taken above the river bank, and the
second sample was taken near a brushy forested area
10
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Table 3-1. Summary of Environmental Sampling Locations
Sample Type
Soil
Sediment
Sampling Location
Warren County, North Carolina
Tittabawassee River, Michigan
Midland, Michigan
Winona Post, Missouri
Solutia, West Virginia
Newark Bay, New Jersey
Raritan Bay, New Jersey
Tittabawassee River, Michigan
Saginaw River, Michigan
Brunswick, Georgia
Total
Number of Samples
Submitted for Consideration
5
6
6
6
6
6
6
6
6
5
58
Included in Demonstration
3
o
J
4
3
3
4
3
o
3
o
3
3
32
within the park complex. Previous PCDD/F
concentrations were from 300 to 3,400 pg/g, and total
PCBs were in the low ppt range. The final two samples
were collected from Department of Natural Resources
(DNR)-owned property in Saginaw, which was formerly
a farming area located almost at the end of the
Tittabawassee River where it meets the Shiawassee
River to form the Saginaw River. Previous PCDD/F
concentrations ranged from 450 to 1,150 pg/g. Total
PCBs were not previously analyzed, but concentrations
were expected to be less than 1 ppm. The DNR property
is approximately a 10-minute walk from where the
demonstration was conducted at the Green Point ELC.
ditch and sinkhole. Areas of contaminant deposition
(approximately 8,500 cubic yards of soils/sludge) were
excavated in late 200I/early 2002. This material was
placed into an approximate 2!/2-acre treatment cell
located on facility property. During 2002/2003, material
at the treatment cell was treated through addition of
amendments (high-ammonia fertilizer and manure) and
tilling. Final concentrations achieved in the treatment
cell averaged 26 milligrams per kilogram (mg/kg) for
PCP and from 8,000 to 10,000 for pg/g dioxin
equivalents. Samples obtained for this study from this
site were obtained from the treatment cell after these
concentrations had been achieved.
3.2.1.3 Midland, Michigan
Soil samples were collected by the MDEQ from various
locations in Midland, Michigan. The soil type and nature
of dioxin contamination are different in the Midland
residential area than it is on the Tittabawassee River
flood plain, but it is from the same suspected source
(legacy contamination from chemical manufacturing).
Samples were collected in various locations around
Midland. Estimated TEQ concentrations ranged from 10
pg/g to 1,000 pg/g.
3.2.1.4 Winona Post
The Winona Post site in Winona, Missouri, was a
Superfund cleanup of a wood treatment facility.
Contaminants at the site included PCP, dioxin, diesel
fuel, and PAHs. Over a period of at least 40 years, these
contaminants were deposited into an on-site drainage
3.2.1.5 Solutia
The chemical production facility at the Solutia site in
Nitro, West Virginia, is located along the eastern bank of
the Kanawha River, in Putnam County, West Virginia.
The site has been used for chemical production since the
early 1910s. The initial production facility was
developed by the U.S. government for the production of
military munitions during the World War I era between
1918 and 1921. The facility was then purchased by a
small private chemical company, which began manu-
facturing chloride, phosphate, and phenol compounds at
the site. A major chemical manufacturer purchased the
facility in 1929 from Rubber Services Company. The
company continued to expand operations and accelerated
its growth in the 1940s. A variety of raw materials has
been used at the facility over the years, including
inorganic compounds, organic solvents, and other
11
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organic compounds, including Agent Orange. Agent
Orange is a mixture of chemicals containing equal
amounts of two herbicides: 2,4-D
(2,4 dichlorophenoxyacetic acid) and 2,4,5-T
(2,4,5 trichlorophenoxyacetic acid). Manufacture of this
chemical herbicide began at the site in 1948 and ceased
in 1969. The source of the dioxin contamination in the
site soils was associated with the manufacture of 2,4,5-T,
where dioxins are an unintentional by-product. The site
has a dioxin profile from ppt to low parts per billion
(ppb) range. No PCBs or PAHs were identified in the
soil.
3.2.2 Sediment Sampling Sites
This section provides descriptions of each of the
sediment sites that includes how the sites became
contaminated and approximate dioxin concentrations, as
well as the type and concentrations of other major
constituents (such as PCBs, PCP, and PAHs). This infor-
mation was provided from site owners/samples providers
(e.g., the EPA, EPA contractors, and the MDEQ).
3.2.2.1 New York/New Jersey Harbors
Dredged materials from the New York and New Jersey
harbors were provided as samples for the demonstration.
The U.S. Army Corps of Engineers, New York District,
and EPA Region 2 are responsible for managing dredged
materials from the New York and New Jersey harbors.
Dioxin levels affect the disposal options for dredged
material. Dredged materials are naturally occurring
bottom sediments, but some in this area have been
contaminated with dioxins and other compounds by
municipal or industrial wastes or by runoff from
terrestrial sources such as urban areas or agricultural
lands.
3.2.2.1.1 Newark Bay
Surrounded by manufacturing industries, Newark Bay is
a highly contaminated area with numerous sources
(sewage treatment plants, National Pollutant Discharge
Elimination System discharges, and nonpoint sources).
This bay is downstream from a dioxin Superfund site
that contains some of the highest dioxin concentrations
in the United States and also is downstream from a
mercury Superfund site. The dioxin concentration in the
area sampled for this demonstration was approximately
450 pg/g. Average PCB concentrations ranged from 300
to 740 ppb. Fine-grained sediments make up 50% to
90% of the dredged material. Average total organic
carbon (TOC) was about 4%.
3.2.2.1.2 Raritan Bay
Surrounded by industry and residential discharges,
Raritan Bay has dioxin contamination in the area, but it
is not to the degree of Newark Bay. No major Superfund
sites are located in the vicinity. Dioxin concentration
should be significantly less than in Newark Bay. PCB
concentrations are around 250 ppb. The fine-grained
sediment and TOC values were similar to percentages in
Newark Bay.
3.2.2.2 Tittabawassee River
The first Tittabawassee River location was
approximately %-mile upstream of the Bob Caldwell
Boat launch in Midland, Michigan. The sediments are
dark gray, fine sand with some silt. The estimated TEQ
concentration was 260 pg/g; however, concentrations as
high as 2,100 pg/g TEQ have been found in this area.
The second site was on the Tittabawassee River
approximately 100 yards downstream from old Smith's
Crossing Bridge in Midland, Michigan. The sediment
was brown and sandy with organic material. The
estimated TEQ concentration was 870 pg/g; but, again,
concentrations as high as 2,100 pg/g TEQ are possible in
the area. The third site was on Tittabawassee River at the
Emerson Park Golfside Boat Launch. The sediment was
gray black silty sand, with many leaves and high organic
matter. The estimated TEQ concentration was < 5 pg/g.
The fourth site was on the Tittabawassee River adjacent
to Imerman Park in Saginaw County across from the
fishing dock. The sediment was sand with some silt. The
estimated TEQ concentration was between 100 and
2,000 pg/g TEQ. The fifth site was on the Tittabawassee
River approximately 1 mile downstream of Center Road
Boat Launch in Saginaw Township. The sediment
consisted of sand and gravel with some shells and not
much organic matter. The estimated TEQ concentration
was between 100 and 1,000 pg/g TEQ. The sixth site
also was on the Tittabawassee River across from the
Center Road Boat Launch. The sediment was fine sand
with high organic matter. The estimated TEQ
concentration was 1,000 pg/g TEQ. The source of the
contamination was speculated to be attributed to legacy
contamination from chemical manufacturing.
12
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3.2.2.3 Saginaw River
Saginaw River samples were collected at six locations.
The first sampling location was in the Saginaw River
just downstream of Green Point Island. Samples were
collected near the middle of the river in about 21 feet of
water. The sample was granular with some organic
material. The estimated TEQ concentration was 100 ppt.
Another Saginaw River sample was taken upstream of
Genesee Bridge on the right side of the river. The sample
was a brown fine sand from about 15 feet of water. The
estimated TEQ concentration was 100 ppt. The third
location was in the Saginaw River downstream of the
Saginaw wastewater treatment plant in about eight feet
of water. The sample was gray silty clay with an
unknown TEQ concentration. The fourth location was in
the Saginaw River in about eight feet of water. The
sample was a black sandy material. The estimated TEQ
concentration for this location was unknown. The fifth
location was downstream of a petroleum pipeline
crossing upstream of the Detroit and Mackinaw railroad
bridge crossing. This location was selected because of its
proximity to a former PCB dredging location. The
sediment sample consisted of dark black silt with some
sand. The estimated TEQ concentration was unknown,
but PCB concentrations are expected to be high. The
sixth and final sampling location was near the mouth of
the Saginaw River in about five feet of water. The
sediment was a mix of fine black silt and layers of sand
and shells. The estimated TEQ concentration for this
location was also unknown.
3.2.2.4 Brunswick Wood Preserving Site
The Brunswick Wood Preserving Superfund site is
located in Glynn County, Georgia, north of the city of
Brunswick. The site was originally located in the city of
Brunswick, but moved to its present location around
1958. The site is approximately 84 acres and is about
two-thirds of a mile long. Burnett Creek, a tidally
influenced stream, is located at the western corner of the
site. At several points, most, if not all, of the drainage
from the site flows into Burnett Creek. The site was first
operated by American Creosote Company, which
constructed the facility sometime between 1958 and
1960. The site was acquired by Escambia Treating
Company in 1969 from Georgia Creosoting Company
and the Brunswick Creosoting Company. In 1985, a
corporate reorganization resulted in the purchase of the
facility by the Brunswick Wood Preserving Company,
which operated the site until it closed in early 1991.
Each of the three major wood-treating operations was
carried out at the facility: PCP, creosote, and
chromium-copper-arsenic (CCA). The site was listed on
the EPA's National Priorities List on April 1, 1997.
Sediment samples from the Brunswick Wood Preserving
site in Brunswick, Georgia, were collected from six
locations on the site, including areas thought to have
lower (< 300 pg/g TEQ) and higher (> 10,000 pg/g
TEQ) D/F concentrations. Due to the processes that
occurred on this site, the samples also contain varying
levels of PAHs and PCP, but they were not expected to
contain PCBs.
13
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Chapter 4
Demonstration Approach
This chapter discusses the demonstration objectives,
sample collection, sample homogenization, and
demonstration design.
4.1 Demonstration Objectives
The primary goal of the SITE MMT Program is to
develop reliable performance and cost data on innovative,
commercial-ready technologies. A SITE demonstration
must provide detailed and reliable performance and cost
data so that technology users have adequate information
to make sound decisions regarding comparability to
conventional methods. The demonstration had both
primary and secondary objectives. Primary objectives
were critical to the technology evaluation and required
the use of quantitative results to draw conclusions
regarding a technology's performance. Secondary
objectives pertained to information that is useful to know
about the technology but did not require the use of
quantitative results to draw conclusions regarding a
technology's performance.
The primary objectives for the demonstration of the
participating technologies were as follows:
P1. Determine the accuracy.
P2. Determine the precision.
P3. Determine the comparability of the technology to
EPA standard methods.
P4. Determine the estimated method detection limit
(EMDL).
P5. Determine the frequency of false positive and
false negative results.
P6. Evaluate the impact of matrix effects on
technology performance.
P7. Estimate costs associated with the operation of
the technology.
The secondary objectives for the demonstration of the
participating technologies were as follows:
S1. Assess the skills and training required to
properly operate the technology.
S2. Document health and safety aspects associated
with the technology.
S3. Evaluate the portability of the technology.
S4. Determine the sample throughput.
Application of these objectives to the demonstration was
addressed based on input from the Dioxin SITE
Demonstration Panel members,(2) general user
expectations of field measurement technologies, the time
available to complete the demonstration, technology
capabilities that the developers participating in the
demonstration intend to highlight, and the historical
experimental components of former SITE Program
demonstrations to maintain consistency.
Note that this demonstration does not assess all
parameters that can affect performance of the
technologies in comparison to the reference methods
(i.e., not all Ah-receptor inducing compounds have been
characterized in the test samples, calibration of
technologies results to HRMS results on site-by-site
basis was not evaluated, etc.). However, the
demonstration as outlined below was agreed upon by the
Dioxin SITE Demonstration Panel members to provide a
reasonable evaluation of the technologies.
4.2 Toxicity Equivalents
For risk assessment purposes, estimates of the toxicity of
samples that contain a mixture of dioxin, furan, and PCB
congeners are often expressed as TEQs. TEQs are
calculated by multiplying the concentration of each
congener with a toxicity equivalency factor (TEF),
according to the equation:
14
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TEQ = CC*TEF
where Cc is the concentration of the congener. The TEF
(see Table 4-1) provides an equivalency factor for each
congener's toxicity relative to the toxicity of 2,3,7,8-
TCDD. The TEFs used in this demonstration were
determined by the World Health Organization (WHO) for
mammalian species.(5) The total TEQ from dioxin and
furans (TEQD/F) in a sample is calculated by adding up all
of the TEQ values from the individual dioxin and furan
congeners. The total TEQ contribution from PCBs
(referred to as TEQPCB) is calculated by summing up the
individual PCB TEQ values. The total TEQ in a sample is
the sum of the TEQD/F and TEQPCB values. TEQ
concentrations for soils and sediments are typically
reported in pg/g, which is equivalent to ppt.
Concentrations of dioxins, furans, and PCBs, represented
as total TEQ concentration, provide a quantitative
estimate of toxicity for all congeners expressed as if the
mixture were a TEQ mass of 2,3,7,8-TCDD only. While
the TEQ concept provides a way to estimate potential
health or ecological effects, the limitations of this
approach should be understood. The WHO report noted
that the TEF indicates an order of magnitude estimate of
the toxicity of a compound relative to 2,3,7,8-TCDD.(5)
Therefore, the accuracy of the TEF factors could be
affected by differences in species, in the functional
responses elicited by the compounds, and in additive and
nonadditive effects when the congeners are present in
complex mixtures. The WHO report(5) concluded,
however, that it is unlikely that a significant error would
be observed due to these differences. The larger impact
to the TEF concept is the presence of AhR binding
compounds, such as PAHs (including naphthalenes,
anthracenes, and fluorenes) and brominated and
chloro/bromo-substituted analogues of PCDD/Fs that
have not been assigned TEF values but which may
Table 4-1. World Health Organization Toxicity Equivalency Factor Values
Compound'3'
PCDDs
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
Dioxin-like PCBs
Coplanar
3,3',4,4'-TCB (PCB 77)
3,4,4',5-TCB(PCB81)
3,3',4,4',5-PeCB (PCB 126)
3,3',4,4',5,5'-HxCB (PCB 169)
WHO TEF
1
1
0.1
0.1
0.1
0.01
0.0001
0.0001
0.0001
0.1
0.01
Compound
PCDFs
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
,2,3,4,7,8-HxCDF
,2,3,7,8,9-HxCDF
,2,3,6,7,8-HxCDF
2,3,4,6,7,8-HxCDF
,2,3,4,6,7,8-HpCDF
,2,3,4,7,8,9-HpCDF
OCDF
mono-ortho
2,3,3',4,4'-PeCB (PCB 105)
2,3,4,4',5-PeCB (PCB 114)
2,3',4,4',5-PeCB (PCB 118)
2,3,4,4',5-PeCB (PCB 123)
2,3,3',4,4',5-HxCB (PCB 156)
2,3,3',4,4',5-HxCB (PCB 157)
2,3',4,4',5,5'-HxCB (PCB 167)
2,3,3',4,4'5,5'-HpCB (PCB 189)
WHO TEF
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.0001
0.0001
0.0005
0.0001
0.0001
0.0005
0.0005
0.00001
0.0001
1 T = Tetra, Pe = Penta, Hx = Hexa, Hp = Hepta, O = Octa, CDD = chlorinated dibenzo-/>-dioxin, CDF = chlorinated
dibenzofuran, CB = chlorinated biphenyl
15
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contribute to the total TEQ. This potentially can result
in an underestimation of TEQs in environmental samples
using the TEF approach.(5)
This demonstration was designed with these limitations
of the TEQ concept in mind. The samples chosen
contained a variety of combinations of dioxins, furans,
and PCBs and at a wide range of concentration levels.
Some samples were high in analytes with better
understood TEFs, while others were high in analytes
with TEFs that have more uncertainty. Some were high
in other AhR binding compounds such as PAHs, while
still others were free of these possible TEQ contributing
compounds. The purpose was to evaluate each of the
technologies and assess the comparability of the TEQD/F
and TEQPCB values determined by the reference
laboratory.
4.3 Overview of Demonstration Samples
The goal of the demonstration was to perform a detailed
evaluation of the overall performance of each
technology for use in the field or mobile environment.
The demonstration objectives were centered around
providing performance data that support action levels for
dioxin at contaminated sites. The Centers for Disease
Control's Agency for Toxic Substances and Disease
Registry (ATSDR) has established a decision framework
for sites that are contaminated with dioxin and dioxin-
like compounds.(6) If samples are determined to have
dioxin TEQ levels between 50 and 1,000 pg/g, the site
should be further evaluated; action is recommended for
levels above 1,000 pg/g (i.e., 1 ppb) TEQ. A mix of PE
samples, environmentally contaminated ("real-world")
samples, and extracts were evaluated that bracket the
ATSDR guidance levels. Table 4-2 lists the primary and
secondary performance objectives for this demonstration
and which sample types were used in each evaluation.
The PE samples were used primarily to determine the
accuracy of the technology and consisted of purchased
soil and sediment standard reference materials with
certified concentrations of known contaminants and
newly prepared spiked samples. The PE samples also
were used to evaluate precision, comparability, EMDL,
false positive/negative results, and matrix effects.
Environmentally contaminated samples were collected
from dioxin-contaminated sites around the country and
were used to evaluate the precision, comparability, false
positive/negative results, and matrix effects. Extracts,
prepared in toluene, which was the solvent used by the
reference laboratory, were used to evaluate precision,
EMDL, and matrix effects. All samples were used to
evaluate qualitative performance objectives such as
technology cost, the required skill level of the operator,
health and safety aspects, portability, and sample
throughput. Table 4-3 shows the number of each sample
type included in the experimental design. The following
sections describe each sample type in greater detail.
4.3.1 PE Samples
PE standard reference materials are available through
Cambridge Isotope Laboratories (CIL) (Andover,
Massachusetts), LGC Promochem (United Kingdom),
Wellington Laboratories (U.S. distributor TerraChem,
Shawnee Mission, Kansas) the National Institute of
Standards and Technology (NIST) (Gaithersburg,
Maryland), and Environmental Resource Associates
(ERA, Arvada, Colorado). All of these sources were
utilized to obtain PE samples for use in this demon-
stration, and Table 4-4 summarizes the PE samples that
were included. PE samples consisted of three types of
samples: (1) reference materials (RMs) or certified
samples, which included soil and/or sediment samples
with certified concentrations of dioxin, furan, and/or
PCBs; (2) spiked samples, which included a certified
dioxin, furan, PCB, and PAH-clean matrix spiked with
known levels of dioxin and/or other contaminants; and
(3) blank samples that were certified to have levels of
dioxins, furans, WHO PCBs, and PAHs that were
non-detectable or were considerably lower than the
detection capabilities of developer technologies. The PE
samples were selected based on availability and on the
correlation of the PE composition as it related to the
environmental samples that were chosen for the
demonstration (e.g., the PE sample had a similar
congener pattern to one or more of the environmental
sites).
16
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Table 4-2. Distribution of Samples for the Evaluation of Performance Objectives
Performance Objective
P 1 : Accuracy
P2: Precision
P3: Comparability
P4: EMDL
P5: False positive/negative results
P6: Matrix effects
P7: Cost
SI : Skill level of operator
S2: Health and safety
S3: Portability
S4: Sample throughput
Sample Type Used in Evaluation
PE
PE, environmental, extracts
PE, environmental, extracts
PE, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
Table 4-3. Number and Type of Samples Analyzed in the Demonstration
Sample Type
PE
Environmental
Extracts
Total number of samples per technology
No. of Samples
58
128
23
209
Table 4-4 indicates a correlation between the
composition of the PE sample and the samples from the
environmental sites, where applicable. The certified
samples only required transfer from the original jar to
the demonstration sample jar. The spiked samples were
shipped to the characterization laboratory in bulk
quantities so each had to be aliquoted in 50-g quantities.
Additional details about each source of PE sample are
provided in this section.
4.3.1.1 Cambridge Isotopes Laboratories
Two RMs were obtained from CIL for use in this
demonstration. RM 5183 is a soil sample that was
collected from a location in Texas with the intended
purpose of serving as an uncontaminated soil for use as a
spiking material. The soil was sieved to achieve uniform
particle size and homogenized to within 5% using a
disodium fluorescein indicator. Samples were then
sterilized three times for 2 hours at 121°C and 15 pounds
per square inch (psi). Analytical results indicated that the
soil had low levels of D/F and PCBs.
RM 5184 is a heavily contaminated soil sample with
relatively high levels of D/F and PCBs. According to
the Certificate of Analysis (CoA), approximately 75 kg
of contaminated sediment were obtained from an EPA
Superfund site in Massachusetts that was known to
contain considerable contamination from PCBs and
other chemical pollutants. The sediment was sieved to
achieve uniform particle size and homogenized to within
5% using a disodium fluorescein indicator. Samples
were then sterilized three times for 2 hours at 121°C and
15 psi.
Both 5183 and 5184 are newly available RMs from CIL.
For both RM 5183 and RM 5184, certified analytical
values are provided for the D/F and the 12 WHO PCB
congeners. The samples were included in an inter-
national interlaboratory study conducted by CIL and
Cerilliant Corporation. More than 20 laboratories
participated in analysis of the D/Fs; up to 20 laboratories
participated in the analysis of the PCBs. Participating
laboratories used a variety of sample preparation and
analytical techniques.
17
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Table 4-4. Summary of Performance Evaluation Samples
Sample
Type
ID
PE#1
PE#2
PE#3
PE#4
PE#5
PE#6
PE#7
PE#8
PE#9
PE#10
PE#11
PE#12
Source
CIL
LGC
Promochem
Wellington
CIL
NIST
ERA
ERA
ERA
ERA
ERA
ERA
ERA
PE Type
Certified
Certified
Certified
Certified
Certified
Spiked
Spiked
Spiked
Spiked
Spiked
Spiked
Organic,
Semivolatile,
Blank Soil
Product No.
RM5183
CRM 529
WMS-01
RM5184
SRM 1944
custom
custom
custom
custom
custom
custom
056
(lot 56011)
Certified Concentration
TEQD/F
(Pg/g)
3.9
6,583
62
171
251
11
33
NS
NS
NS
11
0.046
TEQPCB
(Pg/g)
5.0
424C
10.5
941
41°
NSf
NS
NS
11
1,121
3,760C
0.01
PAH
(mg/kg)
0.18
NAd
NA
27
2.4e
0.33
<0.33
61g
<0.33
<0.33
<0.33
<0.33
Total Number ofPE samples
Correlation
to Environ.
Sample
Type D)a
6
5
6
2,8,9
3,4
10
10
5,7
1
1
1
not
applicable
No. of
Replicates
Per
Sample
7b
4
7b
4
4
4
4
4
4
4
4
8
58
Environmental Sample IDs are provided in Table 4-5.
Seven replicates were analyzed for EMDL evaluation.
Little or no certified PCB data were available; mean of reference laboratory measurements was used.
NA = no data available.
Approximate concentration of 2-methyl naphthalene, acenaphthene, and fluorene, which were the only PAHs that were included in the
analysis.
NS = not spiked.
Each of the 18 target PAHs was spiked at levels that ranged from 1 to 10 mg/kg. (See Section 5.2.3 for the list of 18 PAHs.)
4.3.1.2 LGC Promochem
Certified reference material (CRM) 529 was obtained
from LGC Promochem. The following description is
taken from the reference material report that
accompanied CRM 529. The soil for CRM 529 was
collected in Europe from a site where chloro-organic and
other compounds had been in large-scale production for
several decades, but where production had ceased more
than five years before sampling. The site had been
contaminated during long-term production of
trichlorophenoxyacetic acid. An area of sandy soil was
excavated to a depth of several meters. Several hundred
kilograms of this mixed soil were air-dried at about 15°C
for 3 months. After removal of stones and other foreign
matter by sieving, the remaining material was sterilized
in air at 120 °C for 2 hours, thoroughly mixed, and
ground in an Alpine air jet mill to a particle size of
< 63 micrometers (j-im). The material was homogenized
once more in a Turbula mixer and packaged in 50-g
quantities. The final mean moisture content at the time
of bottling was found to be 1.5%. According to the CoA,
certified values are provided for five dioxin congeners,
seven furan congeners, three chlorobenzene compounds,
and three chlorophenol compounds. No PCBs were
reported with certified values on the CoA, so the mean
concentration determined by the reference laboratory
was used as the certified value.
4.3.1.3 Wellington
PE sample WMS-01 was obtained from TerraChem, the
U.S. distributor for Wellington, an Ontario-based
company. As described in the CoA, WMS-0 lisa
homogeneous lake sediment that was naturally
contaminated (and not fortified). The crude, untreated
sediment used to prepare WMS-01 was collected from
Lake Ontario. The sediment obtained was subsequently
air-dried; crushed to break up agglomerates; air-dried
again; and then sieved, milled, and re-sieved (100%
< 75 |im). The sediment was then subsampled into 25-g
aliquots. The demonstration samples for only the
Wellington PE samples were 25 g rather than 50 g based
on the package size available from Wellington. Certified
18
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values for the 17 D/F congeners and the 12 WHO PCB
congeners are provided on the CoA.
4.3.1.4 National Institute for Standards and
Technology
Standard Reference Material® (SRM) 1944 was
purchased through NIST. As described in the CoA,
SRM 1944 is a mixture of marine sediment collected
from six sites in the vicinity of New York Bay and
Newark Bay in October 1994. Site selection was based
on contaminant levels measured in previous samples
from these sites and was intended to provide relatively
high concentrations for a variety of chemical classes of
contaminants. The sediment was collected using an
epoxy-coated modified Van Veen-type grab sampler
designed to sample the sediment to a depth of
10 centimeters. A total of approximately 2,100 kg of
wet sediment was collected from the six sites. The
sediment was freeze-dried, sieved (nominally 61 to
250 |im), homogenized in a cone blender, radiation
sterilized, then packaged in 50-g quantities. Certified
values are provided on the COA for the 17 D/F
congeners, 30 PCB congeners, 24 PAHs, four
chlorinated pesticides, 36 metals, and TOC. Since only
three WHO PCBs were reported out of the 30 PCB
congeners, the mean concentration of the reference
laboratory measurements was used as the certified value
so that the TEQPCB concentration would not be
underestimated when compared to the developer
technologies.
4.3.1.5 Environmental Resource Associates
ERA synthesized PE samples for this demonstration.
ERA spiked blank, uncontaminated soil to pre-
determined levels of D/Fs, PCBs, and/or PAHs. Spiked
PE samples were prepared to include additional
concentration ranges and compositions that were not
covered with the commercially available certified
materials. The organic semivolatile soil blank (ERA
Product #056, Lot 56011) is atopsoil that was obtained
from a nursery and processed according to ERA
specifications by a geochemical laboratory. The particle
size distribution of the soil was -20/+60 mesh. The soil
was processed and blended with a sandy loam soil to
create a blank soil with the following make-up: 4.1%
clay, 4.5% silt, 91.2% sand, and 0.2% organic material.
Initially, ERA was required to certify that the blank soil
matrix to be used as the blank and for the preparation of
the spiked PE samples was "clean" relative to the list of
required target analytes. This was accomplished through
a combination of ERA-conducted analyses (PAHs,
pesticides, semivolatile organic compounds, Aroclors
that are trade mixtures of PCB congeners) and
subcontracted analytical verification (D/F and PCBs).
The subcontracted analyses were performed by Alta
Analytical Perspectives, LLC, in Wilmington, North
Carolina. The Alta Analytical Certificate of Results and
the ERA Certification sheets for the organic semivolatile
soil blank indicated that trace levels of the octa-dioxins
and several WHO PCB congeners were detected, but the
total TEQ (combined D/F and PCBs) was less than 0.06
pg/g. The level of PAHs, pesticides, Aroclors, and
semivolatile organic compounds in the soil was
determined to be < 0.33 pg/g. The TEQ level was
considerably below the detection capabilities of the
participating technologies, so the organic semivolatile
soil blank was considered adequately clean for use in
this demonstration.
The manufacturing techniques that ERA used to prepare
the PE samples for this demonstration were consistent
with those used for typical semivolatile soil products by
ERA. These techniques have been validated through
hundreds of round robin performance test studies over
ERA's more than 25 years in business. The D/F stock
solutions used in the manufacture of these PE samples
were purchases from CIL. The PCB and PAH stock
solutions were purchased from ChemService. For each
PE sample, a spiking concentrate was prepared by
combining appropriate weight/volume aliquots of stock
materials required for that PE sample. Typically,
additional solvent was added to this concentrate to yield
sufficient volume of solution, appropriate for the mass of
soil to be spiked. Based on a soil mass of 1,600 g, the
volume of spike concentrate was approximately 10 to
30 mL. For each PE sample, the blank soil matrix was
weighed into a 2-liter (L) wide mouth glass jar, the spike
concentrate was distributed onto the soil, and the soil
was allowed to air-dry for 30 to 60 minutes. The PE
samples were then capped and mixed in a rotary tumbler
for 30 minutes. Each PE sample was certified as the
concentration of target analytes present in the blank
matrix, plus the amount added during manufacture,
based on volumetric and gravimetric measurements.
CoAs were provided by ERA for all six ERA-provided
PE samples. The certified values provided by ERA were
different from the commercially available certified
samples since the data were not based on analytically
19
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derived results. Further confirmation of the concentra-
tions was conducted by the reference laboratory.
4.3.2 Environmental Samples
Handling of the environmental samples is described in
this section. Note that once the environmental samples
were collected, they were dried and homogenized as best
as possible to eliminate variability introduced by sample
homogeneity. As such, the effect of moisture on the
sample analysis was not investigated.
4.3.2.1 Environmental Sample Collection
Samples were collected by the EPA, an EPA contractor,
or the MDEQ and shipped to the characterization
laboratory. When determining whether a soil or sediment
site had appropriate dioxin contamination, a guideline
concentration range of < 50 pg/g to 5,000 pg/g was used.
Once necessary approvals and sampling locations had
been secured, sample containers were shipped to site
personnel. Each site providing samples received
1-gallon containers [Environmental Sampling Supply,
Oakland, California, Part number 3785-1051, wide-
mouth, 128-ounce high-density polyethylene round
packer] for collecting five or six samples.
Instructions for sample collection, as well as how the
containers were to be labeled and returned, were
included in a cover letter with the sample containers that
were shipped to each site. Personnel collecting the
samples were instructed to label two containers
containing the same sample as "1 of 2" and "2 of 2" and
to attach a description or label to each container with a
description of the sample, including where the sample
was collected and the estimated concentrations of dioxin
and any other anticipated contamination (e.g., PCBs,
PAHs, PCP). Final instructions to sample providers
indicated that collected samples were to be shipped back
to the characterization laboratory using the provided
coolers. Federal Express labels that included an account
number and the shipping address were enclosed in each
shipment.
Sample providers also were asked to provide any
information about the possible source of contamination
or any historical data and other information, such as
descriptions of the sites, for inclusion in the D/QAPP.(2)
4.3.2.2 Homogenization of Environmental Samples
If the material had very high moisture content, the jar
contents were allowed to settle, and the water was
poured off. Extremely wet material was poured through
fine mesh nylon material to remove water. After water
removal, the material was transferred to a Pyrex™ pan
and mixed. After thorough mixing, an aliquot was stored
in a pre-cleaned jar as a sample of "unhomogenized"
material and was frozen.1 The remaining bulk sample
was mixed and folded bottom to top three times. This
material was split equally among multiple pans. In each
pan, the material was spread out to cover the entire
bottom of the pan to an equal depth of approximately
0.5 inches. The pans were placed in an oven at 35°C and
held there until the samples were visibly dry. This
process took from 24 to 72 hours, depending on the
sample moisture. The trays were removed from the oven
and allowed to rise to room temperature by sitting in a
fume hood for approximately 2 hours. Approximately
500 g of material were put in a blender and blended for
2 minutes. The blender sides were scraped with a spatula
and the sample blended for a second 2-minute period.
The sample was sieved [USA Standard testing, No. 10,
2.00-millimeter (mm) opening] and the fine material
placed in a tray. Rocks and particles that were retained
on the sieve were placed in a pan. This process was
repeated until all of the sediment or soil were blended
and sieved. The blended and sieved sediment or soil in
the tray was mixed well, and four aliquots of 100- to
300-g each were put into clean jars (short, wide-mouth
4-ounce, Environmental Sampling Supply, Oakland,
California, Part number 0125-0055) to be used for the
characterization analyses. The remaining sediment or
soil was placed in a clean jar, and the particles that were
retained on the sieve were disposed of. The jars of
homogenized sediment and soil were stored frozen
(approximately -20°C), unless the samples were being
used over a period of several days, at which time they
were temporarily stored at room temperature.
Ideally, the samples would have been stored at 4° ± 2°C;
but, due to the large volume of buckets and jars that needed
to be stored, the most adequate available storage at the
characterization laboratory was a walk-in freezer that was at
approximately minus 20°C.
20
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4.3.2.3 Selection of Environmental Samples
Once homogenized, the environmental samples were
characterized for D/Fs [EPA Method 1613B(3)), PCBs,
low-resolution mass spectrometry (LRMS) modified
EPA Method 1668A(4), and 18 target PAHs [National
Oceanic and Atmospheric Administration (NOAA)
method(7)] to establish the basic composition of the
samples. (Characterization analyses are described in
Chapter 5.) Because the soil and sediment samples were
dried and homogenized, they were indistinguishable. As
such, the soil and sediment samples were jointly referred
to as "environmental" samples, with no distinction made
between soil or sediment, other than during the matrix
effects evaluations, as described in Section 4.7.6.
Environmental samples were selected for inclusion in the
demonstration based on the preliminary characterization
data. The number and type of samples from each
sampling location included in the demonstration are
presented in Table 4-5.
Four aliquots of the homogenized material and one
aliquot of unhomogenized material were analyzed. Two
criteria had to be met for the environmental sample to be
considered for inclusion in the demonstration. The first
criterion was that the relative standard deviation (RSD)
of the total D/F TEQ values from the four aliquots had to
be less than 20% for samples with total TEQ values
> 50 pg/g; RSD values up to 30% were considered
acceptable if the concentration was < 50 pg/g TEQ. The
second criterion was that no single RSD for an
individual congener could be greater than 30%. If both
of these criteria were met, the sample met the
homogenization criteria and was considered for
inclusion in the demonstration. If either of these criteria
was not met, options for the sample included
(a) discarding it and not considering it for use in the
demonstration, (b) reanalyzing it to determine if the data
outside the homogenization criteria were due to
analytical issues, or (c) rehomogenizing and reanalyzing
it. Of these options, (a) and (b) were utilized, but (c) was
not because an adequate number of environmental
samples were selected using criteria (a) and (b). The
average D/F concentration and RSDs for the
homogenization analyses of environmental samples are
shown in Table 4-5. The composition of two particular
Saginaw River samples were of interest for inclusion in
the demonstration because of their concentration and
unique congener pattern, but the homogenization criteria
were slightly exceeded (i.e., 28% and 34% RSD, for
Saginaw River Sample #2 and Saginaw River Sample
#3, respectively). Since multiple replicates of every
sample were analyzed, those samples were included in
the study because of their unique nature but are flagged
as slightly exceeding the homogenization criteria. A
correlation of environmental samples to PE samples,
similar to that presented in Table 4-4, is presented in
Table 4-5.
4.3.3 Extracts
A summary of the extract samples is provided in
Table 4-6. The purpose of the extract samples was to
evaluate detection and measurement performance
independent of the sample extraction method. As shown
in Table 4-6, two environmental samples (both
sediments) were extracted using Soxhlet extraction with
toluene. These extractions were performed by AXYS
Analytical Services consistent with the procedures to
extract the demonstration samples for reference
analyses.(2) The environmental sample extracts repre-
sented a 10-g sediment sample extraction and were
reported in pg/mL, which was calculated by the
following equation:
pg/mL = (p^gsamples)x(lOgaUquot) y ^ Dp)
(300 mL extraction volume )
where DF = dilution factor.
Total extract volume per 10-g aliquot was 300 mL, but
the sample extracts were concentrated and provided to
the developers as 10-mL extracts, so a 30x dilution
factor is included. The extracts were not processed
through any cleanup steps, but they were derived from
sediment samples that also were included in the suite of
environmental samples. All environmental sample
extractions were prepared in the same solvent (toluene).
The extract samples also included three toluene-spiked
solutions that were not extractions of actual environ-
mental samples. Because adequate homogenization at
trace quantities was difficult to achieve, one set of
extract samples was spiked at low levels (approximately
0.5 pg/mL of 2,3,7,8-TCDD) and used as part of the
method detection limit (MDL) evaluation.
21
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Table 4-5. Characterization and Homogenization Analysis Results for Environmental Samples
Sample
Type ID
Env Site #1
Env Site #2
Env Site #3
Env Site #4
Env Site #5
Env Site #6
Env Site #7
Env Site #8
Env Site #9
Env Site
#10
Environmental
Site Location
Warren County,
North Carolina
Tittabawassee
River, Michigan
Newark Bay,
New Jersey
Raritan Bay, New
Jersey
Winona Post,
Missouri
Tittabawassee
River, Michigan
Brunswick,
Georgia
Saginaw River,
Michigan
Midland,
Michigan
Solutia, West
Virginia
Soil or
Sediment
soil
soil
sediment
sediment
soil
sediment
sediment
sediment
soil
soil
Sample
No.
1
2
3
1
2
3
1
2
3
4
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
4
1
2
3
Average Total
TEQD/F
Concentration
(Pg/g)
274
5,065
11,789
42
435
808
16
62
45
32
12
14
13
3,831
11,071
11,739
1
55
16
69
65
14,500
921
1,083
204
239
184
149
25
48
1,833
3,257
RSD (%)
11
7
3
23b
5
10
26a
14
26b
6
2
3
7
1
2
1
23b
7
26b
8
1
2
9
28C
34C
5
5
7
10
10
19
11
Average RSD for all environmental samples used in demonstration
Total number of environmental samples
No. of Replicates
Per Sample
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Correlation
with PE
Sample Type
roa
9, 10, 11
4
5
5
2,8
1,3
8
4
4
6,7
77%
72S
1 PE Sample IDs are provided in Table 4-4.
b RSD values up to 30% were allowed for samples where the characterization analyses determined concentration to be < 50 pg/g total TEQD/F.
c RSD value slightly exceeded the homogeneity criteria, but samples were included in the demonstration because they were samples of interest.
22
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Table 4-6. Distribution of Extract Samples
Sample Type ID
Extract #1
Extract #2
Extract #3
Extract #4
Extract# 5
Sample ID
Environmental #6, Sample
#2
Environmental #7,
Sample #1
Spike #la
Spike #2a
Spike#3a
Sample Description
Soxhlet extraction in toluene; no
cleanup
Soxhlet extraction in toluene; no
cleanup
0.5pg/mL
2,3,7,8-TCDD
lOOpg/mL 2,3,7,8-TCDD
1,000 pg/mL each WHO PCB
(TEQ-11)
10,000 pg/mL each WHO PCB
(TEQ~1,000)C
Total number of extracts
No. of Replicates per Sample
4
4
7b
4
4
23
a Prepared in toluene.
b Seven replicates were analyzed for EMDL evaluation.
0 This sample was spiked with only PCBs, but a low-level (approximately 0.3 pg/mL) 2,3,7,8-TCDD contamination was
confirmed by the reference laboratory.
4.4 Sample Handling
In preparation for the demonstration, the bulk
homogenized samples were split into jars for
distribution. Each 4-ounce, amber, wide-mouth glass
sample jar (Environmental Sampling Supply, Oakland,
California, Part number 0125-0055) contained approxi-
mately 50 g of sample. Seven sets of samples were
prepared for five developers, the reference laboratory,
and one archived set. A minimum of four replicate splits
of each sample was prepared for each participant, for a
total of at least 28 aliquots prepared for each sample.
The purchased PE samples (i.e., standard reference
materials and spiked materials) were transferred from
their original packaging to the jars to be used in the
demonstration for the environmental samples, making
the environmental and PE samples visually
indistinguishable.
The samples were randomized in two ways. First, the
order in which the filled jars were distributed was
randomized. All jars had two labels. The label on the top
of the jar was the analysis order and contained sample
numbers 1 through 209. A second label placed on the
side of the jar contained a coded identifier including a
series often numbers coded to include the site, replicate,
developer, and matrix. All samples believed to have at
least one D/F or PCB congener greater than 10,000 pg/g
were marked with an asterisk for safety purposes. This
was consistent for both the developer and reference
laboratory samples. The developer was given the option
of knowing which environmental site the samples came
from and whether the sample was a soil or sediment.
Hybrizyme elected to just analyze the soil samples in the
field, so soil and sediment samples were identified to
Hybrizyme. As described in the D/QAPP, AXYS was
informed of which environmental site that the samples
came from so it could use congener profiles and dilution
schemes determined during the pre-demonstration phase
as a guide, along with the concentration range data that
was provided in the D/QAPP. This information was
supplied to the reference laboratory with the samples,
along with which samples contained high (i.e., a sample
with at least one congener with concentration
> 120,000 pg/g) or ultrahigh (i.e., a sample with at least
one congener with concentration > 1,200,000 pg/g) PCB
levels. Using this information, AXYS regrouped the
samples in batches so that, to the extent possible,
samples from the same site would be analyzed within the
same analytical batch. Because an analytical laboratory
might know at least what site samples came from, and
because it is reasonable from an analytical standpoint to
group samples that might require similar dilution
schemes and which have similar congener patterns in an
analytical batch, this approach was an acceptable
deviation from the original intention of having the
samples run by the reference laboratory completely blind
23
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and in the prescribed analytical order. Hybrizyme
analyzed the samples in the prescribed order, but only
analyzed the soils in the field (extracts and sediments
were analyzed in its laboratories).
The environmental samples were stored at room
temperature until homogenized. After homogenization
and prior to distribution during the demonstration, the
samples were stored in a walk-in freezer (approximately
-20°C) at the characterization laboratory. At the
demonstration site, the samples were stored at ambient
temperature. After the demonstration analyses were
completed, the samples were stored at the character-
ization laboratory in the walk-in freezer until the
conclusion of the project.
4.5 Pre-Demonstration Study
Prior to the demonstration, pre-demonstration samples
were sent to Hybrizyme for evaluation in its laboratory.
The pre-demonstration study comprised 15 samples,
including PE samples, environmental samples, and
extracts. The samples selected for the pre-demonstration
study covered a wide range of concentrations and
included a representative of each environmental site
analyzed during the demonstration.
The pre-demonstration study was conducted in two
phases. In Phase 1, Hybrizyme was sent six soil/
sediment samples with the corresponding D/F, PCB, and
PAH characterization data to perform a self-evaluation
of its technology. In Phase 2, seven additional soil/
sediment samples and two extracts were sent to
Hybrizyme for blind evaluation. AXYS analyzed all 15
pre-demonstration samples blindly. The Hybrizyme pre-
demonstration results were paired with the AXYS results
and returned to Hybrizyme so it could use the HRMS
pre-demonstration sample data to refine the performance
of its kit prior to participating in the field demonstration.
Results for the pre-demonstration study can be found in
the DER, which can be obtained by contacting the EPA
program manager for this demonstration. The results
confirmed that Hybrizyme was a viable candidate to
continue in the demonstration process.
4.6 Execution of Field Demonstration
Hybrizyme arrived on-site on Saturday, April 24, and
spent several hours that day and the next day (Sunday,
April 25) setting up its trailer. The demonstration
officially commenced on Monday, April 26 after
1.5 hours of safety and logistical training. During this
meeting, the health and safety plan was reviewed to
ensure participants understood the safety requirements
for the demonstration. Logistics, such as how samples
would be distributed and results reported, were also
reviewed during this meeting. After the safety and site-
specific training meeting and prior to samples being
received by the developers, each trailer and mobile
laboratory was surface wipe sampled on the floor to the
entrance of the developer work area to establish the
background level of D/F and PCB contamination. The
wipe sampling procedure was followed as described in
the D/QAPP. Following demobilization by the
developers, all of the trailers and mobile laboratories
were cleaned and surface-wipesampled. Analysis of the
pre- and post-deployment wipe samples indicated that all
trailers and mobile laboratories met the acceptable
clearance criteria that were outlined in the D/QAPP.
Only one fume hood had to be re-cleaned and re-
sampled before receiving final clearance.
Ideally, all 209 demonstration samples would have been
analyzed on-site, but sample throughput of some of the
technologies participating in the demonstration would
require three weeks or more in the field to analyze 209
samples. Consequently, it was decided, as reported in
the D/QAPP, that the number of samples to be analyzed
in the field by each developer would be determined at
the discretion of the developer.
Hybrizyme received its first batch of samples by
midmorning on April 26. Hybrizyme completed analysis
of 110 soil samples in 4 working days (on April 29). It
should be noted that the morning of April 28 was
dedicated to a Visitor's Day, so minimal work on sample
analyses was performed. The remaining 99 samples
were completed by Hybrizyme in its laboratories and
were reported on August 31. Hybrizyme was also
offered the opportunity to reanalyze any samples before
reporting final results. Hybrizyme reanalyzed all 110 soil
samples that were analyzed in the field because refine-
ments were made to the analytical procedure based on
experience gained during the field demonstration. Only
data generated using the refined method (i.e., all
analyses performed in Hybrizyme's laboratory) were
used in the evaluation of the technology. Hybrizyme
24
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reported that the total analysis time once the method
refinements were completed was one week.
4.7 Assessment of Primary and Secondary
Objectives
The purpose of this section is to describe how the
primary and secondary objectives are assessed, as
presented in Chapters 6 and 7.
The Hybrizyme Corporation AhRC PCR™ Kit is a
technology that reports the concentration of AhR
binding compounds in a sample, and the units reported
as AhRBU. At the time of the demonstration, this
particular test was intended for use as a screening tool to
rank samples from those inducing the greatest AhR
activity to those inducing the least AhR activity rather
than to provide highly accurate TEQ. It has been
suggested that correlation between the Hybrizyme
AhRBU results and HRMS TEQ could be established by
first characterizing a site and calibrating the Hybrizyme
results to HRMS TEQ results. This approach was not
evaluated during this demonstration.
The developer's goal is a highly portable screening
technology which can help to determine areas of greatest
concern for cleanup at a site and can help to minimize
the number of more expensive analyses needed for
specific analytes. Given the current state of development
of the Hybrizyme technology, the technology's results
were compared to the HRMS results in terms of ranking
sample concentrations from low to high, rather than
comparing in a quantitative fashion. The results which
were compared to Hybrizyme's results also included
contributions from PAHs because PAHs are AhR
binding compounds and are included in the Hybrizyme
results. It should be noted that the suite of PAHs which
were quantified in the samples may do not include all of
the PAHs which are responsive to this kit.
4.7.1 Primary Objective PI: Accuracy
The determination of accuracy was based on ranking of
the PE samples results from low to high AhR binding
compounds (D/F + PCB + PAH) and comparing it to the
rank order reported by Hybrizyme based on AhRBU.
For the PE samples, the D/F, PCB, and PAH
concentrations were summed from the concentrations
reported on the certificate of analysis. Note that the PAH
data for the PE samples was rather limited, and it is
possible that additional contributions from PAHs were
not included in the certified data. Ideally, the rankings
would be identical.
4.7.2 Primary Objective P2: Precision
To evaluate precision, all samples (including PE,
environmental, and extract samples) were analyzed in at
least quadruplicate. Seven replicates of three different
samples were analyzed to evaluate MDLs.
Precision was evaluated at both low and high
concentration levels and across different matrices. The
statistic used to evaluate precision was RSD. The
equation used to calculate standard deviation (SD)
between replicate measurements was:
SD =
where SD (in AhRBU) is the standard deviation and C
(in AhRBU) is the average measurement.
The equation used to calculate RSD between replicate
measurements was:
RSD =
SD
C
x 100%
RSD, reported in percent, was calculated if detectable
concentrations were reported for at least three replicates.
The mean, median, minimum, and maximum RSD
values are reported as an assessment of overall precision.
Low RSD values (< 20%) indicated high precision. For a
given set of replicate samples, the RSD of results was
compared with that of the laboratory reference method's
results to determine whether the reference method is
more precise than the technology or vice versa for a
particular sample set. The mean RSD for all samples was
calculated to determine an overall precision estimate.
4.7.3 Primary Objective P3: Comparability
Technology results reported by Hybrizyme Corporation
as AhRBU in a sample and ordered from low to high
were compared to the corresponding reference
laboratory results generated from the sum of the
reference laboratory HRMS data for D/F and PCB and
the characterization PAH data (in nanogram [ng]/g)
ordered from low to high. The comparability evaluation
was only performed for the environmental samples.
Ideally, the rankings would be identical.
25
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4.7.4 Primary Objective P4: Estimated Method
Detection Limit
The MDL calculation procedure described in the
demonstration plan was 40 CFR Part 136, Appendix B,
Revision 1.11. This procedure is based on an assumption
that the replicates are homogeneous enough to allow
proper measurement of the analytical precision and that
the concentration is in the appropriate range for
evaluation of the technology's sensitivity. For this
evaluation, Hybrizyme analyzed seven aliquots each of a
low-level PE soil, PE sediment, and a toluene-spiked
extract. MDL-designated samples are indicated in Tables
4-4 and 4-6. The developer reported values for all of the
replicates (i.e., no nondetect values were reported).
Because the true detection limits of the technology were
not known by the developer, it was not known if the
sample concentrations selected for this evaluation were
appropriate, so the evaluation was considered an EMDL.
A Student's t-value and the standard deviation of seven
replicates were used to calculate the EMDL in AhRBU
is shown in the following equation:
EMDL=t
(n-l, l-oo = 0.99)
(SD)
where t.
(n-l,1-^=0.99)
= Student's t-value appropriate for a
99% confidence level and a standard deviation estimate
with n-l degrees of freedom. The lower the EMDL
value, the more sensitive the technology is at detecting
contamination.
4.7.5 Primary Objective P5: False Positive/False
Negative Results
The tendency for the AhRC PCR™ Kit to return false
positive results (e.g., results reported above a specified
level for the field technology but below a specified level
by the reference laboratory) and false negative results
(e.g., results reported below a specified level for the field
technology but above a specified level by the reference
laboratory) was not evaluated since the Hybrizyme
technology results are not directly comparable with
HRMS results.
4.7.6 Primary Objective P6: Matrix Effects
The likelihood of matrix-dependent effects on
performance was investigated by grouping the data by
matrix type (i.e., soil, sediment, extract), by sample type
(i.e., PE, environmental, and extract), and by varying
levels of PAHs. Precision (RSD) data were summarized
by soil, sediment, and extract (matrix type); by
environmental, PE, and extract (sample type); and by
PAH concentration. Analysis of variance (ANOVA)
tests were performed to determine if there was a
dependence on matrix type or sample type. Only the
environmental samples were included in the matrix
effect assessment based on PAH concentration, because
only the environmental samples were analyzed for PAHs
during the characterization analysis (described in
Section 5.2.3). Some PAH data were available for the PE
samples, but data were not available for all of the same
analytes that were determined during the
characterization analysis. The environmental samples
were segregated into four ranges of total PAH
concentrations: < 1,000 nanogram/g (ng/g), 1,000 to
10,000 ng/g, 10,000 to 100,000 ng/g, and
> 100,000 ng/g. The precision (RSD) data were
summarized for samples within these PAH concentration
ranges. ANOVA tests were used to determine if the
summary values for RSD were statistically different,
indicating performance dependent upon PAH
concentration.
This objective also evaluated if performance was
affected by measurement location (i.e., in-field versus
laboratory conducted measurements), although this is
not a traditional matrix effect. However, the effect of
measurement location was not tested because Hybrizyme
re-ran the 110 samples that it analyzed during the field
demonstration by a modified method in its laboratory, so
all 209 samples were run in its laboratories.
This objective included an environmental site
evaluation, where the comparability values from each of
the 10 environmental sites were compared to see if the
developer results were more or less comparable to the
reference laboratory for a particular site. Since
Hybrizyme did not produce a result that was directly
comparable to HRMS reference data, this could not be
evaluated. This objective also included an assessment of
known interferences where the developer's reported
results for PE samples were summarized for samples
where the PE samples did not contain the target analyte.
This parameter also could not be evaluated, since
Hybrizyme's technology responded to all AhR binding
compounds.
26
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4.7.7 Primary Objective P7: Technology Costs
The full cost of each technology was documented and
compared the cost to typical and actual costs for D/F and
PCB reference analytical methods. Cost inputs included
equipment, consumable materials, mobilization and
demobilization, and labor. The evaluation of this
objective is described in Chapter 8, Economic Analysis.
4.7.8 Secondary Objective SI: Skill Level of
Operator
Based on observations during the field demonstration,
the type of background and training required to properly
operate the AhRC PCR™ Kit was assessed and
documented. The skill required of an operator was also
evaluated. The evaluation of this secondary objective
also included user-friendliness of the technology.
4.7.9 Secondary Objective S2: Health and Safety
Aspects
Health and safety issues, as well as the amount and type
of hazardous and nonhazardous waste generated, were
evaluated based on observer notes during the field
demonstration. This also included an assessment of the
personal protective equipment required to operate the
technology.
4.7.10 Secondary Objective S3: Portability
Observers documented whether the AhRC PCR™ Kit
could be readily transported to the field and how easy it
was to operate in the field. This included an assessment
of what infrastructure requirements were provided to
Hybrizyme (e.g., a trailer), and an assessment of whether
the infrastructure was adequate (or more than adequate)
for the technology's operation. Limitations of operating
the technology in the field are also discussed.
4.7.11 Secondary Objective S4: Sample
Throughput
Sample throughput was measured based on the observer
notes, which focused on the time-limiting steps of the
procedures, as well as the documentation of sample
custody. The number of hours Hybrizyme worked in the
field was documented using attendance log sheets where
Hybrizyme recorded the time they arrived and departed
from the demonstration site. Time was removed for
training and Visitor's Day activities. The number of
operators involved in the sample analyses also was
noted. Throughput of the developer technology was
compared to that of the reference laboratory.
27
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Chapter 5
Confirmatory Process
This chapter describes the characterization analyses and
the process for selecting the reference methods and the
reference laboratory.
5.1 Traditional Methods for Measurement of
Dioxin and Dioxin-Like Compounds in
Soil and Sediment
Traditional methods for analysis of dioxin and dioxin-
like compounds involve extensive sample preparation
and analysis using expensive instrumentation resulting in
very accurate and high-quality, but costly, information.
The ability to use traditional methods for high-volume
sampling programs or screening of a contaminated site
often is limited by budgetary constraints. The cost of
these analyses can range approximately from $500 to
$1,100 per sample per method, depending on the method
selected, the level of QA/QC incorporated into the
analyses, and the reporting requirements.
5.1.1 High-Resolution Mass Spectrometry
EPA Method 1613B(3) and SW846 Method 8290(8) are
both appropriate for low and trace-level analysis of
dioxins and furans in a variety of matrices. They involve
matrix-specific extraction, analyte-specific cleanup, and
HRGC/HRMS analysis. The main differences between
the two methods are that EPA Method 1613B has an
expanded calibration range and requires use of
additional 13C12-labeled internal standards resulting in
more accurate identifications and quantitations. The
calibration ranges for the HRMS methods based on a
typical 10-g sample and 20-microliter (\\L) final sample
volume are presented in Table 5-1.
Table 5-1. Calibration Range of HRMS
Dioxin/Furan Method
Compound
Tetra
Compounds
Penta-Hepta
Compounds
Octa
Compounds
EPA Method
1613B
1-400 pg/g
5-2,000 pg/g
10-4,000 pg/g
SW846 Method
8290
2-400 pg/g
5-1, 000 pg/g
10-2,000 pg/g
5.1.2 Low-Resolution Mass Spectrometry
SW846 Method 8280 is appropriate for determining
dioxins and furans in samples with relatively high
concentrations, such as still bottoms, fuel oils, sludges,
fly ash, and contaminated soils and waters. This method
involves matrix specific extraction, analyte-specific
cleanup, and HRGC/LRMS analysis. The calibration
ranges in Table 5-2 are based on a typical 10-g sample
size and 100-(iL final volume.
Table 5-2. Calibration Range of LRMS
Dioxin/Furan Method
Compound
Tetra-Penta Compounds
Hexa-Hepta Compounds
Octa Compounds
SW846 Method 8280
1,000-20,000 pg/g
2,500-50,000 pg/g
5,000-1 00,000 pg/g
28
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5.1.3 PCB Methods
There are more options for analysis of dioxin-like
compounds such as PCBs. EPA Method 1668A(4) is for
low- and trace-level analysis of PCBs. It involves
matrix-specific extraction, analyte-specific cleanup, and
HRGC/HRMS analysis. This method provides very
accurate determination of the WHO-designated dioxin-
like PCBs and can be used to determine all 209 PCB
congeners. Not all PCBs are determined individually
with this method because some are determined as sets of
coeluting congeners. The calibration range for PCBs
based on a typical 10-g sample and 20-(iL final sample
volume is from 0.4 to 4,000 pg/g. PCBs also can be
determined as specific congeners by GC/LRMS or as
Aroclors1 by GC/electron capture detection.
5.1.4 Reference Method Selection
Three EPA analytical methods for the quantification of
dioxins and furans were available: Method 1613B,
Method 8290, and Method 8280. Method 8280 is a
LRMS method that does not have adequate sensitivity
(i.e., the detection limits reported by the developers are
less than that of the LRMS method). Methods 1613B
and 8290 are HRMS methods with lower detection
limits. Method 1613B includes more labeled internal
standards than Method 8290, which affords more
accurate congener quantification. Therefore, it was
determined that Method 1613B best met the needs of the
demonstration, and it was selected as the D/F reference
method. Reference data of equal quality needed to be
generated to determine the PCB contribution to the TEQ,
since risk assessment is often based on TEQ values that
are not class-specific. As such, the complementary
HRMS method for PCB TEQ determinations, Method
1668A,(4) was selected as the reference method for PCBs.
Total TEQD/F concentrations were generated by Method
1613B, and total TEQPCB concentrations were generated
by Method 1668A. These data were summed to derive a
total TEQ value for each sample.
5.2 Characterization of Environmental
Samples
All of the homogenized environmental samples were
analyzed by the Battelle characterization laboratory to
determine which would be included in the
demonstration. The environmental samples were
characterized for the 17 D/Fs by Method 1613B, the
12 WHO PCBs by LRMS-modified Method 1668A, and
18 target PAHs by the NOAA Status and Trends
GC/Mass Spectrometry (MS) method.(7)
5.2.1 Dioxins and Furans
Four aliquots of homogenized material and one
unhomogenized (i.e., "as received") aliquot were
prepared and analyzed for seventeen 2,3,7,8-substituted
dioxins and furans following procedures in EPA Method
1613B. The homogenized and unhomogenized aliquots
were each approximately 200 g. Depending on the
anticipated levels of dioxins from preliminary
information received from each sampling location,
approximately 1 to 10 g of material were taken for
analysis from each aliquot, spiked with 13C12-labeled
internal standards, and extracted with methylene chloride
using accelerated solvent extraction techniques. One
method blank and one laboratory control spike were
processed with the batch of material from each site. The
sample extracts were processed through various cleanup
techniques, which included gel permeation chroma-
tography or acid/base washes, as well as acid/base silica
and carbon cleanup columns. As warranted, based on
sample compositions, some samples were put through
additional acid silica cleanup prior to the carbon column
cleanup. Extracts were spiked with 13C12-labeled
recovery standards and concentrated to a final volume of
20 to 50 \\L. Dilution and reanalysis of the extracts were
performed if high levels of a particular congener were
observed in the initial analysis; however, extracts were
not rigorously evaluated to ensure that all peaks were
below the peak area of the highest calibration standard.
Each extract was analyzed by high-resolution
GC/HRMS in the selected ion monitoring (SIM) mode at
a resolution of 10,000 or greater. A DB-5 column was
used for analysis of the seventeen 2,3,7,8-PCDD/F
congeners. The instrument was calibrated for PCDD/F at
levels specified in Method 1613B with one additional
calibration standard at concentrations equivalent to one-
half the level of Method 1613B's lowest calibration
point. Using a DBS column, 2,3,7,8-TCDF is not
separated from other non2,3,7,8-TCDF isomers.
However, since the primary objective was to determine
adequacy of homogenization and not congener
quantification, it was determined that sufficient
information on precision could be obtained with the DBS
29
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analysis of 2,3,7,8-TCDF and no second column
confirmation of 2,3,7,8-TCDF was performed. PCDD/F
data were reported as both concentration (pg/g dry) and
TEQs (pg TEQ/g dry).
5.2.2 PCBs
One aliquot of material from each sampling location was
prepared and analyzed for the 12 WHO-designated
dioxin-like PCBs by GC/LRMS. The LRMS PCB
analysis method is based on key components of the PCB
congener analysis approach described in EPA Method
166 8A and the PCB homologue approach described in
EPA Method 680. Up to 30 g of sample were spiked
with surrogates and extracted with methylene chloride
using shaker table techniques. The mass of sample
extracted was determined based on information supplied
to the laboratory regarding possible contaminant concen-
trations. The extract was dried over anhydrous sodium
sulfate and concentrated. Extracts were processed
through alumina column cleanup, followed by high-
performance liquid chromatography/gel permeation
chromatography (HPLC/GPC). Additionally, sulfur was
removed using activated granular copper. The post-
FiPLC extract was concentrated and fortified with
recovery internal standards. Extracts were concentrated
to a final volume between 500 (iL and 1 mL, depending
on the anticipated concentration of PCBs in the sample,
as reported by the sample providers. PCB congeners and
PCB homologues were separated via capillary GC on a
DB5-XLB column and identified and quantified using
electron ionization MS. This method provides specific
procedures for the identification and measurement of the
selected PCBs in SIM mode.
5.2.3 PAHs
One aliquot of material from each sampling location was
analyzed for PAHs. The 18 target PAHs included:
• naphthalene
• 2-methylnaphthalene
• 2-chloronaphthalene
• acenaphthylene
• acenaphthene
• fluorene
• phenanthrene
• anthracene
• fluoranthene
• pyrene
• benzo(a)anthracene
• chrysene
• benzo(b)fluoranthene
• benzo(k)fluoranthene
• benzo(a)pyrene
• indeno(l,2,3-cd)pyrene
• dibenzo(a,h)anthracene
• benzo(g,h,i)perylene.
The method for the identification and quantification of
PAH in sediment and soil extracts by GC/MS was based
on the NOAA Status and Trends method(7) and,
therefore, certain criteria (i.e., initial calibrations and
daily verifications) are different from those defined in
traditional EPA Methods 625 and 8270C. Up to 30 g of
sample were spiked with surrogates and extracted using
methylene chloride using shaker table techniques. The
mass of sample extracted was determined based on
information supplied to the characterization laboratory
regarding possible contaminant concentrations. The
extract was dried over anhydrous sodium sulfate and
concentrated. The extract was processed through an
alumina cleanup column followed by HPLC/GPC. The
post-HPLC extract was concentrated and fortified with
recovery internal standards. Extracts were concentrated
between 500 (iL and 1 mL, depending on the anticipated
concentration of PCBs in the sample, as reported by the
sample providers. PAHs were separated by capillary GC
on a DB-5, 60-m column and were identified and
quantified using electron impact MS. Extracts were
analyzed in the SIM mode to achieve the lowest possible
detection limits.
5.3 Reference Laboratory Selection
Based on a preliminary evaluation of performance and
credibility, ten laboratories were contacted and were sent
a questionnaire geared toward understanding the
capabilities of the laboratories, their experience with
analyzing dioxin samples for EPA, and their ability to
meet the needs of this demonstration. Two laboratories
were selected for the next phase of the selection process
and were sent three blind audit samples. Each laboratory
went through a daylong audit that included a technical
systems audit and a quality systems audit. At each
laboratory, the audit consisted of a short opening
conference; a full day of observation of laboratory
procedures, records, interviews with laboratory staff; and
a brief closing meeting. Auditors submitted followup
30
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questions to each laboratory to address gaps in the
observations.
Criteria for final selection were based on the
observations of the auditors, the performance on the
audit samples, and cost. From this process, it was
determined that AXYS Analytical Services (Sidney,
British Columbia, Canada) would best meet the needs of
this demonstration.
5.4 Reference Laboratory Sample
Preparation and Analytical Methods
AXYS Analytical Services received all 209 samples on
April 27, 2004. To report final data, AXYS submitted
14 D/F and 14 PCB data packages from June 11 to
December 20, 2004. The following sections briefly
describe the reference methods performed by AXYS.
5.4.1 Dioxin/Furan Analysis
All procedures were carried out according to protocols
as described in AXYS Summary Method Doc MSU-018
Rev 2 18-Mar-2004 [AXYS detailed Standard Operating
Procedure (SOP) MLA-017 Rev 9 May-2004], which is
based on EPA Method 1613B. AXYS modifications to
the method are summarized in the D/QAPP.(2) Briefly,
samples were spiked with a suite of isotopically labeled
surrogate standards prior to extraction, solvent extracted,
and cleaned up through a series of chromatographic
columns that included silica, Florisil, carbon/Celite, and
alumina columns. The extract was concentrated and
spiked with an isotopically labeled recovery (internal)
standard. Analysis was performed using an HRMS
coupled to an FiRGC equipped with a DB-5 capillary
chromatography column [60 meters (m), 0.25-mm
internal diameter (i.d.), 0.1-|im film thickness]. A
second column, DB-225 (30 m, 0.25-mm i.d., 0.15-|im
film thickness), was used for confirmation of
2,3,7,8-TCDF identification. Samples that were known
to contain extremely high levels of PCDD/F were
extracted without the addition of the surrogate standard,
split, then spiked with the isotopically labeled surrogate
standard prior to cleanup. This approach allowed
extraction of the method-specified 10-g sample volume,
and subsequent sufficient dilution that high level
analytes were brought within the instrument calibrated
linear range. While this approach induces some
uncertainty because the actual recovery of analytes from
the extraction process is unknown, it was decided by the
demonstration panel that in general analyte recovery
through the extraction procedures are known to be quite
good and that the uncertainty introduced by this
approach would be less than the uncertainty introduced
by other approaches such as extracting a significantly
smaller sample size.
5.4.2 PCB Analysis
The method was carried out in accordance with the
protocols described in AXYS Summary Method Doc
MSU-020 Rev 3 24-Mar-2004 (AXYS detailed SOP
MLA-010 Rev 5 Sep-2003), which is based on EPA
Method 1668A, with changes through August 20, 2003.
AXYS modifications to the method are summarized in
the D/QAPP. Briefly, samples were spiked with
isotopically labeled surrogate standards, solvent
extracted, and cleaned up on a series of chromatographic
columns that included silica, Florisil, alumina, and
carbon/Celite columns. The final extract was spiked with
isotopically labeled recovery (internal) standards prior to
instrumental analysis. The extract was analyzed by
FiRMS coupled to an HRGC equipped with a DB-1
chromatography column (30 m, 0.25-mm i.d., 0.25-|im
film thickness). Because only the WHO-designated
dioxin-like PCBs were being analyzed for this program
and in order to better eliminate interferences, all samples
were analyzed using the DB-1 column, which is an
optional confirmatory column in Method 1668A rather
than the standard SPB Octyl column. Samples that were
known to contain extremely high levels of PCBs were
extracted without the addition of the surrogate standard,
split, then spiked with the isotopically labeled surrogate
standard prior to cleanup. This approach allowed
extraction of the method-specified 10-g sample volume,
and subsequent sufficient dilution that high level
analytes were brought within the instrument calibrated
linear range. While this approach induces some
uncertainty because the actual recovery of analytes from
the extraction process is unknown, it was decided by the
demonstration panel that in general analyte recovery
through the extraction procedures are known to be quite
good and that the uncertainty introduced by this
approach would be less than the uncertainty introduced
by other approaches such as extracting a significantly
smaller sample size.
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5.4.3 TEQ Calculations
For the reference laboratory data, D/F and PCB congener
concentrations were converted to TEQ and subsequently
summed to determine total TEQ, using the TEFs
established by WHO in 1998 (see Table 4-l).(5)
Detection limits were reported as sample-specific
detection limits (SDLs). SDLs were determined from
2.5 times the noise in the chromatogram for D/F and 3.0
times the noise for PCBs, converted to an area, and then
converted to a concentration using the same calculation
procedure as for detected peaks. Any value that met all
quantification criteria (> SDL and isotope ratio) was
reported as a concentration. A "J" flag was applied to
any reported value between the SDL and the lowest level
calibration. The concentration of any detected congener
that did not meet all quantification criteria (such as
isotope ratio or peak shape) were reported but given a
"K" flag to indicate estimated maximum possible
concentration (EMPC).(8) TEQs were reported in two
ways to cover the range of possible TEQ values:
(1) All nondetect and EMPC values were assigned a
zero concentration in the TEQ calculation.
(2) Nondetects were assigned a concentration of one-
half the SDL. EMPCs were assigned a value equal
to the EMPC.
In both cases, any total TEQ value that had 10%
contribution or more from J-flagged or K-flagged data
was flagged as J or K (or both) as appropriate.
TEQs were calculated both ways for all samples. For
TEQD/F, 63% of the samples had the same TEQ value
based on the two different calculation methods, and the
average relative percent difference (RPD) was 8%
(median = 0%). For TEQPCB, 65% of the samples had the
same TEQ value based on the two different calculation
methods, and the average RPD was 9% (median = 0%).
Because overall there were little differences between the
two calculation methods, as presented in Appendix D,
TEQ values calculated by option #1 were used in
comparison with the developer technologies. On a case-
by-case basis, developer results were compared to TEQs
calculated by option #2 above, but no significant
differences in comparability results were observed so no
additional data analysis results using these TEQ values
were presented.
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Chapter 6
Assessment of Reference Method Data Quality
Ensuring reference method data quality is of paramount
importance to accurately assessing and evaluating each
of the innovative technologies. To ensure that the
reference method has generated accurate, defensible
data, a quality systems/technical audit of the reference
laboratory was performed during analysis of demon-
stration samples after the first batch of demonstration
sample analyses was complete. The quality systems/
technical audit evaluated implementation of the
demonstration plan. In addition, a full data package was
prepared by the reference laboratory for each sample
batch for both dioxin and dioxin-like PCB analyses.
Each data package was reviewed by both a QA specialist
and technical personnel with expertise in the reference
methods for agreement with the reference method as
described in the demonstration plan. Any issues
identified during the quality systems/technical audit and
the data package reviews were addressed by the
reference laboratory prior to acceptance of the data. In
this section, the reference laboratory performance on the
QC parameters is evaluated. In addition, the reference
data were statistically evaluated for the demonstration
primary objectives of accuracy and precision.
6.1 QA Audits
A quality systems/technical audit was conducted at the
reference laboratory, AXYS Analytical Services, Ltd.,
by Battelle auditors on May 26, 2004, during the
analysis of demonstration samples. The purpose of the
audit was to verify AXYS compliance with its internal
quality system and the D/QAPP.(2) The scope
specifically included a review of dioxin and PCB
congener sample processing, analysis, and data
reduction; sample receipt, handling, and tracking;
supporting laboratory systems; and followup to
observations and findings identified during the
independent laboratory assessment conducted by Battelle
on February 11, 2004, prior to contract award.
Checklists were prepared to guide the audit, which
consisted of a review of laboratory records and
documents, staff interviews, and direct observation.
The AXYS quality system is documented in a
comprehensive QA/QC manual and detailed SOPs. No
major problems or issues were noted during the audit.
Two findings were identified, one related to a backlog of
unfiled custody records and the other related to the need
for performance criteria for the DB-1 column used for
the analysis of PCB congeners by HRMS. Both issues
were addressed satisfactorily by AXYS after the audit.
One laboratory practice that required procedural
modification was identified: the laboratory did not
subject all QC samples to the most rigorous cleanup
procedures that might be required for individual samples
within a batch. The AXYS management team agreed that
this procedure was incorrect. As corrective action, the
QA manager provided written instructions regarding
cleanup of the QC samples to the staff, and the
laboratory manager conducted follow up discussion with
the staff. Other isolated issues noted by the auditors did
not reflect systemic problems and were typical of
analytical laboratories (e.g., occasional documentation
lapses or an untrackable balance weight).
The audit confirmed that the laboratory procedures
conformed to the SOPs and D/QAPP and that the quality
system was implemented effectively. Samples were
processed and analyzed according to the laboratory
SOPs and D/QAPP using the Soxhlet Dean Stark
extraction method. No substantial deviations were
noted. The audit verified the traceability of samples
within the laboratory, as well as the traceability of
standards, reagents, and solvents used in preparation,
and that the purity and reliability of the latter materials
were demonstrated through documented quality checks.
In addition, the audit confirmed that analytical
instruments and equipment were maintained and
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calibrated according to manufacturers' specifications and
laboratory SOPs. Analytical staff members were
knowledgeable in their areas of expertise. QC samples
were processed and analyzed with each batch of
authentic samples as specified by the D/QAPP. QA/QC
procedures were implemented effectively, and corrective
action was taken to address specific QC failures. Data
verification, reporting, and validation procedures were
found to be rigorous and sufficient to ensure the
accuracy of the reported data. The auditors concluded
that AXYS is in compliance with the D/QAPP and its
SOPs, and that the data generated at the laboratory are of
sufficient and known quality to be used as a reference
method for this project.
In addition, each data package was reviewed by both a
QA specialist and technical personnel with expertise in
the reference methods for agreement with the reference
method as described in the demonstration plan.
Checklists were prepared to guide the data package
review. This review included an evaluation of data
package documentation such as chain-of-custody (COC)
and record completeness, adherence to method
prescribed holding times and storage conditions,
standard spiking concentrations, initial and continuing
calibrations meeting established criteria, GC column
performance, HRMS instrument resolution, method
blanks, lab control spikes (ongoing precision and
recovery samples), sample duplicates, internal standard
recovery, transcription of raw data into the final data
spreadsheets, calculation of TEQs, and data flag
accuracy. Any issues identified during the data package
reviews were addressed by the reference laboratory prior
to acceptance of the data. All of the audit reports and
responses are included in the DER.
6.2 QC Results
Each data package was reviewed for agreement with the
reference method as described in the demonstration plan.
This section summarizes the evaluation of the reference
method quality control data.
6.2.1 Holding Times and Storage Conditions
All demonstration samples were stored frozen (< -10°C)
upon receipt and were analyzed within the method
holding time of one year.
6.2.2 Chain of Custody
All sample identifications were tracked from sample
login to preparation of record sheets, to instrument
analysis sheets, to the final report summary sheets and
found to be consistent throughout. One COC with an
incomplete signature and one discrepancy in date of
receipt between the COC and sample login were
identified during the Battelle audit and were corrected
before the data packages with these affected items were
accepted as final.
6.2.3 Standard Concentrations
The concentration of all calibration and spiking
standards was verified.
6.2.4 Initial and Continuing Calibration
All initial calibrations met the criteria for response factor
RSD and minimal signal-to-noise ratio requirements for
the lowest calibration point.
Continuing calibrations were performed at the beginning
and end of every 12-hour analysis period with one minor
exception for D/F sample batch WG13551, which
contained five samples from Environmental Site # 1
(North Carolina) and 12 samples from Environmental
Site #5 (Winona Post). On one analysis day, a high-
level sample analyzed just prior to the ending calibration
verification caused the verification to fail. In this
instance, the verification was repeated just outside of the
12-hour period. The repeat calibration verification met
the acceptance criteria and was considered to show
acceptable instrument performance in the preceding
analytical period; therefore, the data were accepted.
Continuing calibration results were within the criteria
stated in Table 9-2 (D/F) and Table 9-4 (PCB) of the
D/QAPP, with one exception. For PCB sample batch
WG12108, which contained nine samples from
Environmental Site #3 (Newark Bay) and 12 samples
from Environmental Site #4 (Raritan Bay), isotopically
labeled PCB 169 was above the acceptable range during
one calibration verification on May 15, 2004. The
acceptance range included in the D/QAPP is tighter than
the acceptance range in Method 1668A Table 6.
Because the result for labeled PCB 169 was within the
Method 166 8A acceptance limits, the data were
accepted.
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The minimum signal-to-noise criteria for analytes in the
calibration verification solution were met in all
instances.
6.2.5 Column Performance and Instrument
Resolution
Column performance was checked at the beginning of
each 12-hour analytical period and met method criteria.
Instrument resolution was documented at the beginning
and end of each 12-hour period with one exception. In
PCB sample batch WG13554, which contained five PE
samples and 15 extract samples, on one analysis day
(September 17, 2004), the ending resolution docu-
mentation was conducted at 12 hours and 54 minutes.
However, as this resolution documentation met all
criteria, it was considered representative of acceptable
instrument performance during the analytical period, and
the data were accepted.
6.2.6 Method Blanks
Method blanks were analyzed with each sample batch to
verify that laboratory procedures did not introduce
significant contamination. A summary of the method
blank data is presented in Appendix C. There were many
instances for both D/F and PCB data where analyte
concentrations in the method blank exceeded the target
criteria in the D/QAPP. Samples from this
demonstration, which had very high D/F and PCB
concentrations, contributed to the difficulty in achieving
method blank criteria in spite of steps the reference
laboratory took to minimize contamination (such as
proofing the glassware before use in each analytical
batch). In many instances, the concentrations of D/F and
PCBs in the samples exceeded 20 times the
concentrations in the blanks. For all instances, the
sample results were unaffected because the method
blank TEQ concentration was compared to the sample
TEQ concentrations to ensure that background
contamination did not significantly impact sample
results.
6.2.7 Internal Standard Recovery
Internal standard recoveries were generally within the
D/QAPP criteria. D/QAPP criteria were tighter than the
standard EPA method criteria; in instances where
internal standard recoveries were outside of the D/QAPP
criteria, but within the standard EPA method criteria,
results were accepted. In several instances, the dioxin
cleanup standard recoveries were affected by
interferences. As the cleanup standard is not used for
quantification of native analytes, these data were
accepted. Any samples affected by internal standard
recoveries outside of the D/QAPP and outside of the
EPA method criteria were evaluated for possible impact
on total TEQ and for comparability with replicates
processed during the program before being accepted.
6.2.8 Laboratory Control Spikes
One laboratory control spike (ongoing precision and
recovery sample), which consisted of native analytes
spiked into a reference matrix (sand), was processed
with each analytical batch to assess accuracy. Recovery
of spiked analytes was within the D/QAPP criteria in
Table 9-2 for all analytes in all laboratory control spike
samples.
6.2.9 Sample Batch Duplicates
A summary of the duplicate data is presented in
Appendix C. One sample was prepared in duplicate in
most sample batches; four batches were reported without
a duplicate. Three of 14 dioxin sample batches and 5 of
14 PCB sample batches did not meet criteria of <20%
RPD between duplicates. Data where duplicates did not
meet D/QAPP criteria were evaluated on an individual
basis.
6.3 Evaluation of Primary Objective PI:
Accuracy
Accuracy was assessed through the analysis of PE
samples consisting of certified standard reference
materials, certified spikes, and certified blanks. A
summary of reference method percent recovery (R)
values is presented in Table 6-1. The Rvalues are
presented for TEQPCB, TEQD/F, and total TEQ. The
minimum, maximum, mean, and median R values are
presented for each set of TEQ results. The reference
method values were in best agreement with the certified
values for the TEQPCB results, with a mean R value of
96%. The mean R values for TEQD/F and total TEQ were
125% and 94%, respectively. The mean and median R
values for the TEQPCB and total TEQ were identical. The
mean and median R values for TEQD/F were not similar
and were largely influenced by the TEQD/F recovery for
ERA Aroclor of 324%. The ERA Aroclor-certified
TEQD/F values were based on TCDD and TCDF only,
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Table 6-1. Objective PI Accuracy - Percent Recovery
PE Sample
ID
1
2
3
4
5
6
7
8
9
10
11
12
PE Sample
Description
Cambridge 5 183
LCG CRM-529
Wellington WMS-01
Cambridge 5 184
NIST 1944
ERATCDD 10
ERA TCDD 30
ERA PAH
ERAPCB 100
ERAPCB 10000
ERA Aroclor
ERA Blank
All PE Samples
% Recovery
TEQprR
81
100
93
120
102
NA
NA
NA
96
95
82
NA
NUMBER
MIN
MAX
MEDIAN
MEAN
8
81
120
96
96
TEQn/F
111
106
106
106
91
79
77
NA
NA
NA
324
NA
NUMBER
MIN
MAX
MEDIAN
MEAN
8
77
324
106
125
Total TEQ
94
106
105
118
93
79
77
NA
95
95
83
NA
NUMBER
MIN
MAX
MEDIAN
MEAN
10
77
118
94
94
NA = not applicable.
whereas the reference method TEQD/F values were
based on contributions from all 2,3,7,8-substituted D/F
analytes. The Rvalues presented in Table 6-1 indicate
that the reference method reported data that were on
average between 94 and 125% of the certified values
of the PE samples.
The effect of known interferences on reference method
TEQs is listed in Table 6-2. D/F and PCB TEQs were
not affected by PAH as evidenced through the analysis
of ERA PAH reference material. D/F and PCB TEQs
were not affected by each other as evidenced by spikes
that contained only one set of analytes having negli-
gible influence on the TEQ of the other analyte set.
6.4 Evaluation of Primary Objective P2:
Precision
The 209 samples included in the demonstration
consisted of replicates of 49 discrete samples.
There were four replicates of each sample except
for PE sample Cambridge 5183 (7 replicates),
ERA blank reference material (8 replicates),
Wellington WMS-01 standard reference material
(7 replicates), and 0.5 pg/mL 2,3,7,8-TCDD extract
(7 replicates). Reference method data were obtained
for all 209 samples; however, TEQD/F and total TEQ
data for samples Ref 197 (ERA PCB 100) and Ref 202
(LCG CRM-529) were omitted as outliers as it
appeared that these two samples were switched
Table 6-2. Evaluation of Interferences
PE Material with Known Interference
ERA PAH
ERAPCB 100
ERAPCB 10,000
ERA TCDD 10
ERA TCDD 30
Mean TEQ (pg/g)
0. 195 (D/F + PCB)
0.073 (D/F)
0.220 (D/F)
0.025 (PCB)
0.036 (PCB)
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during preparation after observing results of the
replicates and evaluating the congener profiles of these
two samples.
A summary of the reference method replicate RSD
values is presented in Tables 6-3a and 6-3b. The RSD
values are presented for TEQPCB, TEQD/F, and total TEQ
in Table 6-3a, and a summary by sample type is
presented in Table 6-3b, along with the minimum R
value, the maximum R value, and the mean R value for
each set of TEQ results and sample types. In terms of
sample type, the reference method had the most precise
data for the environmental sample TEQD/F results, with a
mean RSD value of 12%. This was followed closely by
environmental sample TEQPCB and total TEQ results,
which both had mean RSDs of 13%. In terms of TEQ
values, the reference method had the most precise data
for the total TEQ values, with a mean overall RSD of
13%. Overall RSD values ranged from l%to 119%.
Precision was significantly worse for certified blanks
and blank samples (e.g., samples that contained spikes of
only one analyte set and were blank for the other
analytes) as might be expected due to the very low levels
detected in these samples.
6.5 Comparability to Characterization Data
To assess comparability, reference laboratory D/F data
for environmental samples were plotted against the
characterization data that was generated by Battelle prior
to the demonstration. Characterization data were
obtained as part of the process to verify homogenization
of candidate soil and sediment samples as described in
Chapter 5 and reported in Table 4-5. It should be noted
that second column confirmations of 2,3,7,8-TCDF
results were not performed during characterization
analyses; therefore, characterization TEQs are biased
high for samples where a large concentrations of
non2,3,7,8-TCDF coeluted with 2,3,7,8-TCDF on the
DB-5 column. Characterization samples also were not
rigorously evaluated to ensure that high concentration
extracts were diluted sufficiently so that all peak areas
were less than the peak areas of the highest calibration
standard. In spite of these differences between reference
and characterization analyses, the results had fairly high
correlation (R2 = 0.9899) as demonstrated in Figure 6-1.
Table 6-3a. Objective P2 Precision - Relative Standard Deviation
Sample Type
Environmental
Sample ID
Brunswick #1
Brunswick #2
Brunswick #3
Midland #1
Midland #2
Midland #3
Midland #4
NC PCB Site #1
NC PCB Site #2
NC PCB Site #3
Newark Bay #1
Newark Bay #2
Newark Bay #3
Newark Bay #4
RaritanBay #1
Raritan Bay #2
Raritan Bay #3
S aginaw River #1
Saginaw River #2
Saginaw River #3
Solutiajl
Solutia #2
Solutia #3
Titta. River Soil #1
Titta. River Soil #2
RSD for TEQPCB
(%)
8
o
3
5
4
10
4
77
21
21
25
7
2
6
1
6
o
3
o
3
8
7
60
36
4
11
7
9
RSD for TEQD/F
(%)
6
16
8
9
6
6
9
15
2
12
28
22
6
12
5
2
5
25
19
19
13
7
5
6
10
RSD for Total TEQ
(%)
6
16
8
9
6
6
10
20
21
24
25
20
6
11
4
1
4
23
18
19
13
7
5
5
10
37
-------
Sample Type
Extract
PE
Sample ID
Titta. River Soil #3
Titta. River Sed #1
Titta. River Sed #2
Titta. River Sed #3
WinonaPost#l
Winona Post #2
Winona Post #3
En vir Extract #1
Envir Extract #2
Spike #1
Spike #2
Spike #3
Cambridge 5 1 83
Cambridge 5184
ERA Aroclor
ERA Blank
ERA PAH
ERAPCB 100
ERAPCB 10000
ERATCDD 10
ERA TCDD 30
LCG CRM-529
NIST 1944
Wellington WMS-01
RSD for TEQPCB
(%)
12
19
14
13
13
4
9
71
83
119
1
4
7
o
J
44
62
83
4
7
60
39
14
4
5
RSD for TEQD/F
(%)
26
27
37
9
2
9
4
50
2
6
5
13
19
4
6
65
27
65 a
91
5
6
T
9
3
RSD for Total TEQ
(%)
26
26
37
8
2
9
4
50
2
9
3
4
9
2
43
61
30
3
7
5
6
1
7
3
"Does not include sample excluded due to sample preparation error.
Table 6-3b. Objective P2 Precision - Relative Standard Deviation (By Sample Type)
Sample Type
Environmental
Extract
PE
Overall
RSD for TEQprR (%)
N
32
5
12
49
MIN
1
1
3
1
MAX
77
119
83
119
MED
8
71
11
8
MEAN
13
56
28
21
RSD for TEQn/F (%)
N
32
5
12
49
MIN
2
2
2
2
MAX
37
50
91
91
MED
9
6
7
9
MEAN
12
15
25
16
RSD for Total TEQ (%)
N
32
5
12
49
MIN
1
2
1
1
MAX
37
50
61
61
MED
10
4
7
8
MEAN
13
14
15
13
re
Q
o
5
ui
y = 0.8595x + 41.181
5000 10000 15000
Characterization Data (TEQ D/F pg/g)
20000
Figure 6-1. Comparison of reference laboratory and characterization D/F data for environmental
samples.
38
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6.6 Performance Summary
This section provides a performance summary of the
reference method by summarizing the evaluation of the
applicable primary objectives of this demonstration
(accuracy, precision, and cost) in Table 6-4. A total of
209 samples was analyzed for seventeen
2,3,7,8-substituted D/F and 12 PCBs over an eight-
month time frame (April 27 to December 20, 2004).
Valid results were obtained for all 209 PCB analyses,
while 207 valid results were obtained for D/F. The D/F
and total TEQ results for samples Ref 197 (ERA PCB
100) and Ref 202 (LCG CRM-529) were omitted as
outliers because it appeared that these two samples were
switched during preparation after observing results of
the replicates and evaluating the congener profiles of
these two samples. The demonstration sample set
provided particular challenges to the reference
laboratory in that there was a considerable range of
sample concentrations for D/F and PCB. This caused
some difficulty in striving for low MDLs in the presence
of high-level samples. The range of concentrations in the
demonstration sample set also required the laboratory to
modify standard procedures, which contributed to
increased cost and turnaround time delay. For example,
an automated sample cleanup system could not be used
due to carryover from high-level samples; instead, more
labor-intensive manual cleanup procedures were used;
glassware required extra cleaning and proofing before
being reused; cleanup columns sometimes became
overloaded from interferences and high-level samples,
causing low recoveries so that samples had to be
re-extracted or cleanup fractions had to be analyzed for
the lost analytes; and method blanks often showed trace
levels of contamination, triggering the repeat of low-
level samples.
Because the reference method was not to be altered
significantly for this demonstration, the reference
laboratory was limited in its ability to adapt the trace-
level analysis to higher level samples. In spite of these
challenges, the quality of the data generated met the
project goals. The main effect of the difficulties
associated with these samples was on schedule and cost.
Table 6-4. Reference Method Performance Summary - Primary Objectives
Objective
P 1 : Accuracy
P2: Precision
P7: Cost
Performance
Statistic
Number of data points
Median Recovery (%)
Mean Recovery (%)
Number of data points
Median RSD (%)
MeanRSD (%)
TEQPCB
8
96
96
49
8
21
TEQD/F
8
106
125
49
9
16
Total TEQ
10
94
94
49
8
13
209 samples were analyzed for 17 D/F and 12 PCBs. Total cost was $398,029.
D/F cost was $213,580 ($1,022 per sample) and PCB cost was $184,449 ($883 per sample).
39
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Chapter 7
Performance of Hybrizyme Corporation AhRC PCR™ Kit
7.1 Evaluation of AhRC PCR™ Kit
Performance
The Hybrizyme Corporation AhRC PCR™ Kit is a
technology that reports the concentration of AhR
binding compounds in units reported as AhRBU. At the
time of the demonstration, this particular test was
intended for use as a screening tool to rank samples from
those inducing the greatest AhR activity to those
inducing the least AhR activity rather than to provide
highly accurate TEQ. The developer's goal is a highly
portable screening technology that can help determine
areas of greatest concern for cleanup at a site and can
help minimize the number of more expensive analyses
needed for specific analytes. It has been suggested that
correlation between the Hybrizyme AhRBU results and
HRMS TEQ results could be established by first
characterizing a site and calibrating the Hybrizyme
results to HRMS results. This approach was not
evaluated during this demonstration. Since the
technology measures an actual biological response, it is
possible that the technology may give a better
representation of the true toxicity of a sample or site
from a risk assessment standpoint.
The following sections describe the performance of the
AhRC PCR™ Kit, according to the primary objectives
for this demonstration. The developer and reference
laboratory data are presented in Appendix D. The
statistical methods used to evaluate the primary
objectives are described in Section 4.7. Detailed data
evaluation records can be found in the DER.
7.1.1 Evaluation of Primary Objective PI:
Accuracy
Based on the current state of development of the
Hybrizyme technology, the technology's results were
compared to the HRMS results in terms of ranking
sample concentrations from low to high, rather than
comparing to HRMS TEQ in a quantitative fashion. The
determination of accuracy was based on ranking of the
PE samples results from low to high concentration and
comparing it to the rank order reported by Hybrizyme
based on AhRBU. For the PE samples, the D/F, PCB,
and PAH concentrations were summed from the
concentrations reported on the certificate of analysis.
Note that the PAH data for the PE samples was rather
limited, and it is possible that there were additional PAH
compounds present in the PE samples that were not
included in the certified data.
Table 7-1 compares the Hybrizyme AhRC PCR™ Kit
ranking of the PE samples in average AhRBU
concentration from low to high to the low to high
ranking by total concentration (D/F+PCB+PAH)
determined from the certified data. The Wellington and
LCG CRM 529 PE samples were excluded from this
evaluation because PAH data were not available on the
certificates of analysis. The Hybrizyme ranking was
identical to the certified concentrations for one of the 10
PE samples (Cambridge 5184). Of the four highest
sample concentrations according to the certified values
(NIST 1944, ERA Aroclor, Cambridge 5184, and ERA
PAH), Hybrizyme's data ranked three of the samples
(NIST 1944, Cambridge 5184, and ERA PAH) as having
the highest concentrations. The ERA Aroclor sample,
which was spiked with Aroclor 1254, was ranked as the
next lowest concentration by Hybrizyme and the next to
highest concentration by the certified data.
40
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Table 7-1. Objective PI Accuracy - Ranking of PE Samples According to the Concentration of AhR Binding
Compound
PE Sample ID
1
2
3
4
5
6
7
8
9
10
11
12
PE Sample
Description
Cambridge 5 183
LCG CRM-529
Wellington
WMS-01
Cambridge 5 1 84
NIST 1944
ERA TCDD 10
ERA TCDD 30
ERA PAH
ERAPCB 100
ERAPCB 10000
ERA Aroclor
ERA Blank
Low to High Ranking
Hybrizyme AhRBTJ
Results
5
Certified Results
(D/F+PCB+PAH)
6
not included
not included
9
10
4
7
8
3
1
2
6
9
7
o
3
4
10
2
5
8
1
7.1.2 Evaluation of Primary Objective P2:
Precision
A summary of the Hybrizyme AhRC PCR™ RSD values
is presented in Tables 7-2a and 7-2b. A minimum of
three and maximum of four replicate results were used to
calculate RSD. The RSD values are presented for
AhRBU in Table 7-2a, and a summary by sample type is
presented in Table 7-2b, with the minimum RSD value,
the maximum RSD value, the median RSD value, and
the mean RSD value presented. Low RSD values (< 20
%) indicate high precision. The Hybrizyme AhRC
PCR™ Kit values had the most precise data for the
extract analysis, with a mean RSD of 14%. The overall
mean RSD was 25%, with values ranging from 2% to
111%. The median overall RSD value was 19% and in
good agreement with the mean, indicating that the
precision values were symmetrically distributed.
7.1.3 Evaluation of Primary Objective P3:
Comparability
Given the state of development of the Hybrizyme
technology, the technology's results were compared to
the HRMS results in terms of ranking sample
concentrations of AhR-binding compounds from low to
high, rather than comparing to HRMS TEQ in a
quantitative fashion. PAH data from the characterization
were added to the HRMS D/F and PCB data generated
during the demonstration because PAHs are AhR
binding compounds and are included in the Hybrizyme
results. It should be noted that the suite of PAHs which
were quantified in the samples were a selected target list
for this demonstration and likely do not include all of the
PAHs which are responsive to this kit.
Table 7-3 compares the Hybrizyme low to high ranking
of the environmental samples to the reference laboratory
low to high ranking. The environmental samples were
the only samples included in this evaluation because the
PAH data were consistently generated for each of the
environmental sites. The environmental samples were
ordered in terms of the sample numbers provided in
Table 4-5. For this evaluation, the environmental
samples were ranked with the samples from each site
only, rather than ranking all of the environmental sites in
one ordering, because the Hybrizyme technology is
intended to rank samples within a particular site.
This evaluation demonstrated that the Hybrizyme
technology was able to rank the samples from low to
high concentration within an environmental site fairly
consistently with the reference laboratory based on
average total concentration data. Table 7-4 summarizes
the environmental sample ranking comparisons. For
seven of the 10 environmental sites (70%), Hybrizyme's
ranking was identical to the reference laboratory's
ranking. If samples that are close in average HRMS
concentration and are indistinguishable when
uncertainties are considered, the Hybrizyme rankings
41
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Table 7-2a. Objective P2 Precision - Relative Standard Deviation
Sample Type
Environmental
Extracts
PE
Sample ID
Brunswick #1
Brunswick #2
Brunswick #3
Midland #1
Midland #2
Midland #3
Midland #4
NC PCB Site #1
NC PCB Site #2
NC PCB Site #3
Newark Bay #1
Newark Bay #2
Newark Bay #3
Newark Bay #4
RaritanBay #1
Raritan Bay #2
Raritan Bay #3
S aginaw River #1
Saginaw River #2
Saginaw River #3
Solutia #1
Solutia #2
Solutia #3
Titta. River Soil #1
Titta. River Soil #2
Titta. River Soil #3
Titta. River Sed #1
Titta. River Sed #2
Titta. River Sed #3
WinonaPost#l
Winona Post #2
Winona Post #3
Envir. Extract #1
Envir. Extract #2
Spike #1
Spike #2
Spike #3
Cambridge 5183
Cambridge 5184
ERA Aroclor
ERA Blank
ERA PAH
ERA PCB 100
ERA PCB 10000
ERATCDD 10
ERA TCDD 30
LCG CRM-529
NIST 1944
Wellington WMS-01
RSD (%)a
15
16
13
16
2
14
18
20
8
17
30
28
8
14
4
29
24
25
8
6
20
31
45
16
19
15
20
33
29
19
12
21
16
11
NA
13
16
35
10
30
83
25
46
37
91
111
12
26
19
NA = not applicable; all values reported as nondetects.
1 Three or four replicate results were used to calculate the RSD values.
42
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Table 7-2b. Objective P2 Precision - Relative Standard Deviation (By Sample Type)
Statistic
Environmental
Extract
PE
Overall
RSD (%)
N
32
4
12
48
MIN
2
11
10
2
MAX
45
16
111
111
MEDIAN
17
14
33
19
MEAN
19
14
44
25
Table 7-3. Objective P3 - Comparability by Ranking Environmental Samples from Low to High Concentration
Within an Environmental Site
Environmental Site
Brunswick, GA
Midland, MI
Warren County, NC
Newark Bay, NJ
Raritan Bay, NJ
Saginaw River, MI
Solutia, WV
Tittabawassee River, MI (soil)
Tittabawassee River, MI
(sediment)
Winona Post, MO
Hybrizyme Ranking by
Average AhRBTJ"
2
1
3
4
1
3
2
1
2
3
1
4
2
3
1
2
3
o
J
2
1
1
3
2
3
2
1
3
1
2
1
2
3
Reference Laboratory HRMS
Ranking by Average Total
Concentration (ng/g) a
2
1
o
J
4
1
3
2
1
2
o
J
1
2
4
o
J
1
3
2
o
J
2
1
1
3
2
3
1
2
o
J
1
2
1
2
o
J
Did Ranking Agree?
Yes
Yes
Yes
No
No
Yes
Yes
No
Yes
Yes
a Ranking of sample numbers within a site from low to high. See Table 4-5 for sample numbers.
43
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Table 7-4. Objective P3 - Summary of Environmental Sample Ranking Comparisons
Evaluation Parameter
Hybrizyme ranking agreed with reference laboratory
within the environmental site
Hybrizyme ranking when uncertainly around reference
laboratory values was considered
Hybrizyme individual rankings which agreed with the
reference laboratory
Performance
Number
7 out of 1 0 environmental
sites
9 out of 1 0 environmental
sites
26 out of 32 individual
rankings
Percentage
70
90
81
agree with the reference laboratory's ranking for nine of
10 sites (90%). On an individual ranking basis, the
Hybrizyme and reference laboratory rankings agreed
81% of the time (26 of 32 rankings).
7.1.4 Evaluation of Primary Objective P4:
Estimated Method Detection Limit
The EMDL of the Hybrizyme AhRC PCR™ Kit was
determined using Cambridge 5183 and Wellington
WMS-01. Extract Spike #1 was prepared with 0.5 pg/mL
of 2,3,7,8-TCDD only, which was reported as not
detected by Hybrizyme for all seven replicates. As
shown in Table 7-5, the Hybrizyme AhRC PCR™ Kit
EMDL was determined to be 71 AhRBU for the
Cambridge 5183. The calculated EMDL for Wellington
WMS-01 sample (576 AhRBU) was considerably
higher, due to the higher concentration of analytes in the
sample, so it was not included in the evaluation of
EMDL. Hybrizyme noted that their estimated detection
limits were on the order of 100 AhRBU, so the
calculated EMDL appears to be a reasonable estimation.
Table 7-5. Objective P4 - Estimated Method
Detection Limit
Statistic
Degrees of Freedom
Standard Deviation
(AhRBU)
EMDL (AhRBU)
Cambridge 5183
6
23
71
7.1.5 Evaluation of Primary Objective P5: False
Positive/False Negative Results
This parameter was not evaluated. See Section 4.7.5 for
further explanation.
7.1.6 Evaluation of Primary Objective P6:
Matrix Effects
Six types of potential matrix effects were investigated:
(1) measurement location (field vs. laboratory),
(2) matrix type (soil vs. sediment vs. extract), (3) sample
type (PE vs. environmental vs. extract), (4) PAH
concentration, (5) environmental sites, and (6) known
interferences. A summary of the matrix effects is
provided in the bullets below, followed by a detailed
discussion:
• Measurement location: not evaluated (all results
generated in the laboratory)
• Matrix type: none (according to statistical evaluation
of mean RSD values)
• Sample type: Significant effect
• PAH concentration: none
• Environmental site: not evaluated since AhRBU
results weren't directly comparable to TEQ
• Known interferences: not evaluated since assay reacts
to AhR binding compounds
In Table 7-6, precision summary values are presented by
matrix type. A one-way ANOVA model was used to test
the effect of soil vs. sediment vs. extract on RSD. These
tests showed no significant effect on mean RSD
although the range of RSD values was much greater for
soil than for sediment and extracts. In Table 7-7,
precision summary values are presented by PAH
concentrations for environmental samples only. A one-
way ANOVA model was used to test the effect of PAH
44
-------
Table 7-6. Objective P6 - Matrix Effects Using RSD as a Description of Precision by Matrix Type
Matrix Type
Soil
Sediment
Extract
Overall
RSD for AhRBU (%)
N
26
18
4
48
MIN
2
4
11
2
MAX
111
33
16
111
MEAN
30
19
14
25
MEDIAN
20
19
14
19
Table 7-7. Objective P6 - Matrix Effects Using RSD as a Description of Precision by PAH Concentration Levels
(Environmental Samples Only)
PAH Concentration Level
(ng/g)
> 100,000
10,000-100,000
1,000-10,000
< 1,000
Overall
(Environmental Samples Only)
RSD for AhRBU (%)
N
3
4
16
9
32
MIN
8
12
2
6
2
MAX
17
21
45
33
45
MED
13
17
20
19
19
MEAN
13
17
18
19
17
concentration on RSD. These tests showed no effect.
The summary of RSD values segregated by sample type
is presented in Table 7-2b. A one-way ANOVA model
was used to test the effect of sample type (PE vs.
environmental vs. extract) on RSD. These tests showed a
significant effect on RSD. This effect is visually
noticeable, as the environmental (mean 19%) and extract
(mean 14%) RSD values were half as much as the PE
RSD (mean 44%). An evaluation of effect on
performance based on measurement location was not
performed because all sample results used in this
evaluation were performed in Hybrizyme's laboratory.
The comparability to the HRMS values for the assess-
ment of environmental sites was not performed because
the Hybrizyme results are not quantitatively comparable
to the HRMS TEQ data.
7.1.7 Evaluation of Primary Objective P7:
Technology Costs
Evaluation of this objective is fully described in
Chapter 8, Economic Analysis.
7.2 Observer Report: Evaluation of
Secondary Objectives
The toxicity of a compound can be indicated by how
strongly it binds to the AhR. Therefore, in a sample
extract, how well the extract activates the AhR gives an
indication of the toxicity of compounds in the extract.
Dioxins and furans are known to activate the AhR. The
AhRC PCR™ test kit detects compounds that activate
the AhR in a sample extract in the following manner:
toxic components in a sample extract activate the AhR,
the activated AhR binds to a DNA-probe, and the AhR
bound DNA probe is amplified and measured by PCR.
The AhR specificity to dioxin-like compounds is similar
to the TEQ approach for estimating toxicity of a mixture
of dioxin-like compounds, i.e., 2,3,7,8-TCDD is strongly
bound to the AhR, OCDD is significantly less strongly
bound. Therefore, this assay can assess the toxicity of a
sample in a manner similar to the TEQ approach of
traditional dioxin and furan analysis. However, it should
be noted that the AhR response to various dioxin-like
compounds, while similar, is not identical to the TEF
used to determine TEQ, and AhR activity can be induced
by compounds other than the dioxins and furans. While
the AhR activity can be calibrated to TCDD to generate
a TEQ value, a highly accurate TEQ value is not the goal
of this technology due to the differences in AhR activity
vs. TEFs and the responses induced by other toxic
compounds. At the time of the demonstration, this
particular test was intended for use as a screening tool to
rank samples from those inducing the greatest AhR
45
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activity to those inducing the least AhR activity rather
than to provide a highly accurate TEQ. The developer's
goal is a highly portable screening technology that can
help to determine areas of greatest concern for cleanup at
a site and can help to minimize the number of more
expensive analyses needed for specific analytes.
The following activities were observed during the
demonstration: sample weighing and extraction, addition
of extract to the activation solution, plate washing,
addition of primer/probe solution, and instrument
read-out of results. The sample extraction procedure
was not included in the demonstration plan; otherwise,
instructions in the demonstration plan were generally
followed. The developer intends to add more specific
sample extraction instructions to the kit instructions.
Note that the extraction procedure observed during the
demonstration was not the procedure used to generate
the data submitted for this evaluation. Hybrizyme
refined its extraction procedure after the field
demonstration and reanalyzed all samples with its new
procedure in its laboratory. The refined procedure is
described in Section 2.3. Hybrizyme had no knowledge
of reference sample results at this point in the process,
but they requested to reanalyze the samples because they
were not satisfied with their data based on their quality
control procedures. Hybrizyme's request was granted to
ensure that the most representative data would be
evaluated during this demonstration. The refined
analytical procedure and field demonstration procedure
were very similar, differing only in the type and quantity
of reagents and chemicals that were used. The type of
activities (extraction by sonication, clean-up, PCR assay)
were the same in both procedures.
7.2.1 Evaluation of Secondary Objective SI:
Skill Level of Operator
During the demonstration, sample weighing and
extraction activities were carried out by Dr. Terry
Nestrick, who has a Ph.D. in chemistry and 26 years of
experience at Dow Chemical Co. in the analytical
laboratories located in Midland, Michigan. His primary
experience was trace and ultra-trace determination of
organic species in environmental, product, and process
matrices specializing in dioxins/furans. Dr. Randy Allen
processed the extracts with the AhRC PCR™ kit and
performed the data collection and reduction. Dr. Allen
has a Ph.D. in molecular biology/biochemistry and
17 years of experience in developing environmental
bioassays.
For successful operation of this technology, Hybrizyme
recommends that users have a minimum of a high school
degree, have good work skills, and be trainable. Based
on observation of the technology, the recommended
level of experience and education seemed reasonable.
Decent laboratory skills, respect for safety, and having
reasonable attention to detail would also be useful
attributes for successful technology operation.
Information needs to be added to instructions on how to
extract samples. Otherwise, instructions for kit use were
reasonably clear. A day of training would help to ensure
that someone who already possessed good laboratory
skills would know how to properly use the kit. For the
most part, this technology seemed very forgiving.
Temperatures were not critical as long as there were no
extremes, samples needed to be weighed only to 0.1 g
(as a screening tool, it is more important for the weights
to be consistent between samples than to be highly
accurate), and times for shaking and incubation were not
critical. There are several places where the technology
can be stopped and stored (e.g., after sonication, when
exchanged to hexane, when loaded onto the strips and
ready to analyze, etc.). The activation solution and the
primer/probe require freezing and the capture reagent
and PCR wash concentrate require refrigeration;
otherwise, the reagents can be kept at room temperature.
Only 5 |iL of the 200-|iL sample extract is used for
analysis so there is plenty of extract available should
repeat analyses be warranted. The assay is relatively
quick (~5 hours from sample weigh-out to first read of
results) and so repeat analyses can be performed without
significant time delay. In addition to purchasing the kits,
samples can be sent to the developer for processing in
the developer's laboratory. The developer will also
consider coming to the client's laboratory or the field.
There are no particular health or safety concerns with
this technology beyond what is common for laboratory
sample processing.
7.2.2 Evaluation of Secondary Objective S2:
Health and Safety Aspects
A complete inventory of the waste generated was
performed after processing 110 samples by Hybrizyme.
46
-------
None of the containers were verified as full. Note that
this summary does not include the samples which were
analyzed in the Hybrizyme laboratories.
(1) One 5-gallon container marked "low
concentration" with 80 polypropylene weigh
pans; 80 plastic weigh spoons; 160
polypropylene vial caps; four 10-mL glass
pipettes; and 90 glass vials (0.5 mL).
(2) One 5-gallon container marked "high
concentration" with 31 polypropylene weigh
pans; 31 plastic weigh spoons; 62 polypropylene
vial caps; and 31 glass vials (0.5 mL).
(3) One 5-gallon container with 2 L buffer salts.
(4) Three 5-gallon containers containing among
them: 168 glass vials (42 mL) each filled with
2 g of soil plus 10 mLof 50%HC1; 168 used
10-mL glass pipettes; 120 vials with 10 mL of
50% HC1 and 800 |iL of hexane; 168 pipette
tips; and 96 glass test tubes with 150 |iL MeOH.
The reader should be advised that, although no
difficulties were encountered during this project,
difficulties could arise with disposal of dioxin-
contaminated waste.
7.2.3 Evaluation of Secondary Objective S3:
Portability
The developer intends for this technology to become a
highly portable field kit. However, as used in the
demonstration, this technology required at a minimum a
trailer to protect from the environment and to house
equipment such as a centrifuge, sonicator, and the PCR
thermocycler analyzer. Electricity was also a necessity.
The developer ultimately intends for this technology to
be usable in a minimally controlled environment and is
even considering having an option of leasing out
miniaturized equipment that will make the technology
more field friendly. As tested in the demonstration, this
technology required approximately 2 hours of setup to
get the trailer ready to begin analyses. Operation in a
fume hood is not a critical requirement for this
technology because there are no lengthy solvent
extractions or cleanup steps with particular ventilation
requirements.
7.2.4 Evaluation of Secondary Objective 84:
Throughput
Sample throughput during the demonstration was
approximately 28 samples per day. A total of
110 samples was processed in the field during four days.
Out of the four days, several hours of the first day were
not active working hours due to start-up meetings for the
program, and several hours were lost on the third day for
participation in Visitor's Day. Two people processed the
samples. One person focused on the sample weighing
and extraction, and the second person focused on
analyzing the extracts.
According to the developer, if operating in a production
mode, 40 to 60 samples could be processed in one day
with two people operating the technology. Forty
samples would appear to be the more reasonable/
comfortable goal. One person would focus on the
sample weighing and extraction and the second person
would focus on the analysis of the extracts. The
developer felt this throughput goal would be about the
same regardless of whether the operators were
experienced or novice kit users. If looking for a rapid
turnaround on a specific set of samples (i.e., a small
number, fewer than 10 samples), the quickest results
could be delivered on such a set in about 5 hours. It
takes roughly 2 to 3 hours to get through the sample
weighing and extraction and 2 to 3 hours to get the
extract prepared and analyzed. Based on the
observations of this technology in the field, two trained,
but not necessarily highly experienced people could
reasonably process 30 to 40 samples per day. This pace
would likely be sustainable for several days. Sample
processing would proceed as quickly in the field as in
the lab; however, analysis may be somewhat more rapid
in a laboratory setting due to more comfortable
conditions. Laboratory analysis instrumentation is set up
to read an entire 96-well plate at once, whereas a field
portable unit is only able to read 16 wells at a time.
Each kit contains materials to process one 96-well plate.
The number of samples processed with each plate will
vary based on the number of wells used for calibration
and quality control standards and whether the samples
are analyzed in duplicate.
7.2.5 Miscellaneous Observer Notes
Hybrizyme is a U.S.-based company. Its test kit comes
with a set of instructions with reasonable details. At the
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time of the demonstration, the instructions were going to
be revised with additional information on sample
extraction procedures implemented as a result of this
demonstration. Hybrizyme provides phone support to its
customers during business hours. Training by
Hybrizyme staff is available and quotes for this service
can be provided. According to Hybrizyme, effective
training could be accomplished in one day. Hybrizyme
did not have training videos at the time of the
demonstration; however, this option may be available in
the future.
This kit comes with supplies to process one 96-well
plate. At this time, kits are available off-the-shelf for
orders of less than 10 kits. Orders of more than 10 kits
are filled within a two-week period. The kit includes
activation solution, capture strips, assay buffer, capture
reagent, primer/probe, PCR wash concentrate, glass vials
(rack with 96 vials), and a set of instructions. The user
needs to supply a mini-balance and supplies for
weighing out samples (such as weighing boats and
spatulas or disposable spoons), vials to extract the
sample (such as 4-dram, polytetrafluoroethylene-lined
vials), methanol, hydrochloric acid, distilled water, a
multichannel pipettor, a sample shaker, a centrifuge, an
incubator, a dioxin standard in methanol (commercially
available from vendors such as CIL), and a
thermocycler. The developer notes that if a user did not
have a thermocycler, sample preparation could be
stopped at the stage of drying the strips immediately
prior to analysis and the strips sent to Hybrizyme for the
final instrument read-out. Also, leasing field portable
analysis equipment may become an option from this
developer in the future. Most of the equipment and
materials necessary to use this technology are common
laboratory items. The developer notes that the PCR
analysis is clinical in origin making replacement parts
easily accessible since most hospitals use this type of
equipment and could be contacted if there were
emergency equipment needs in the field. This
technology has been developed for use with both
laboratory-based and fie Id-based PCR systems.
At the time of the demonstration, the developer was still
refining recommendations for quality control samples to
be processed with a sample batch. For the
demonstration, one blank and one sample extracted in
duplicate were prepared with each set of 20 test samples.
Future recommendations may also include a lab control
spike with each sample batch. For this technology, the
need for confirmation or verification of results by
conventional HRMS methods depends on project-
specific goals and action levels and may need to be
applied on a project-specific basis.
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Chapter 8
Economic Analysis
During the demonstration, the AhRC PCR™ kit and the
reference laboratory analytical methods were each used
to perform more than 200 analyses of dioxin-
contaminated samples, including samples with a variety
of distinguishing characteristics such as high levels of
polychlorinated biphenyls and PAHs. The purpose of the
economic analysis was to estimate the total cost of
generating results by using the AhRC PCR™ kit and
then comparing this cost to the reference method. This
cost estimate also is provided so that potential users can
understand the costs involved with using this
technology.
This chapter provides information on the issues and
assumptions involved in the economic analysis
(Section 8.1), discusses the costs associated with using
the AhRC PCR™ kit (Section 8.2), discusses the costs
associated with using the reference method (Section 8.3),
and presents a comparison of the economic analysis
results for the AhRC PCR™ kit and the reference
laboratory (Section 8.4).
8.1 Issues and Assumptions
Several factors affect sample measurement costs.
Wherever possible in this chapter, these factors are
identified in such a way that decision-makers can
independently complete a project-specific economic
analysis. The following five cost categories were
included in the economic analysis for the demonstration:
capital equipment, supplies, support equipment, labor,
and investigation-derived waste (IDW) disposal. The
issues and assumptions associated with these categories
and the costs not included in the analysis are briefly
discussed below. The issues and assumptions discussed
below only apply to the AhRC PCR™ kit unless
otherwise stated.
8.1.1 Capital Equipment Cost
The capital equipment cost was the cost associated with
the purchase of the AhRC PCR™ kit. Components of
the AhRC PCR™ kit are presented in detail in
Chapters 2 and 7. Hybrizyme offers a lease option for
the PCR thermocycler, but the thermocycler can also be
purchased. The purchase price information was obtained
from a standard price list provided by Hybrizyme.
8.1.2 Cost of Supplies
The cost of supplies was estimated based on the supplies
required to analyze all demonstration samples using the
AhRC PCR™ kit that were not included in the capital
equipment cost category. Examples of such supplies
include filters, cleanup columns, gas cylinders, solvents,
and distilled water. The supplies that Hybrizyme used
during the demonstration fall into two general
categories: consumable (or expendable) and reusable.
Examples of expendable supplies utilized by Hybrizyme
during the demonstration include methanol, pipette tips,
and extraction vials. Examples of reusable supplies
include a PCR thermocycler, microplate shaker and
washer, ultrasonic bath, centrifuge, and pipettors. It
should be noted that this type of equipment may or may
not be already owned by a potential AhRC PCR™ kit
user; however, for this economic analysis, an assumption
was made that the user does not possess these items.
The purchase price of these supplies was either obtained
from a standard price list provided by Hybrizyme, or it
was estimated based on price quotes from independent
sources.
8.1.3 Support Equipment Cost
This section details the equipment used at the
demonstration such as the construction trailer, fume
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hood, and laptop computer required by the technology.
Costs for these items will be reported per actual costs for
the demonstration.
8.1.4 Labor Cost
The labor cost was estimated based on the time required
for work space setup, sample preparation, sample
analysis, and reporting. For the demonstration,
developers reported results by submitting a COC/result
form. The measurement of the time required for
Hybrizyme to complete all analyses (74 labor-hours) was
estimated by the sign-in log sheets that recorded the time
the Hybrizyme operators were on-site. Time was
removed for site-specific training activities and Visitor's
Day. Time estimates were rounded to the nearest hour.
During the demonstration, the skill level required for the
operators to complete analyses and report results was
evaluated. As stated in Section 7.2.1, based on the field
observations, the recommended skill level for operation
of this technology includes a minimum of a high school
degree, have good work skills, and be trainable. Decent
laboratory skills, respect for safety, and having
reasonable attention to detail would also be useful
attributes for successful technology operation. This
information was corroborated by Hybrizyme.
Education levels of the actual field operators included
Ph.D. degrees for both operators. For the economic
analysis, costs were estimated using both actual and
projected necessary skill levels for operators.
8.1.5 Investigation-Derived Waste Disposal Cost
During the demonstration, Hybrizyme was provided
with 5-gallon containers for collecting wastes generated
during the demonstration. Sample by-products such as
used samples, aqueous and solvent-based effluents
generated from analytical processes, and used glassware
were disposed of in the containers. The total cost to
dispose of these wastes generated during the demon-
stration is included in the economic analysis. Items such
as coffee cups, food waste, and office waste were
disposed of in regular public refuse containers and were
not included as IDW and, therefore, not discussed in this
economic analysis.
8.1.6 Costs Not Included
Items whose costs were not included in the economic
analysis are identified below along with a rationale for
the exclusion of each.
Electricity. During the demonstration, some of the
capital equipment was operated using AC power. The
costs associated with providing the power supply were
not included in the economic analysis as it is difficult to
estimate the electricity used solely by the Hybrizyme
technology. The total cost for electricity usage over the
10-day demonstration was $288. With seven mobile
labs/trailers and miscellaneous equipment being operated
continuously during the course of the demonstration, the
cost of Hybrizyme electricity usage would be no more
than $41. There was significantly more cost
(approximately $13,000) to install an electrical board
and additional power at the demonstration site, but this
was a function of the demonstration site and not the
responsibility of the individual developers, so this cost
was not included in the economic analysis.
Oversight of Demonstration Activities. Atypical user
of the AhRC PCR™ kit would not be required to pay
for customer oversight of sample analysis. The EPA, the
MDEQ, and Battelle representatives were present during
the field demonstration, but costs for oversight were not
included in the economic analysis because these
activities were project-specific. For these same reasons,
cost for auditing activities (i.e., technical systems audits
at the reference laboratory and during the field
demonstration) were also not included.
Travel and Per Diem for Operators. Operators may be
available locally. Because the availability of operators is
primarily a function of the location of the project site,
travel and per diem costs for operators were not included
in the economic analysis.
Sample Collection and Management. Costs for sample
collection and management activities, including sample
homogenization and labeling, were not included in the
economic analysis because these activities were project-
specific and were not dependent upon the selected
reference method or developer technology. Additionally,
sample shipping, COC activities, preservation of
50
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samples, and distribution of samples were specific
requirements of this project that applied to all developer
technologies and may vary from site to site. None of
these costs were included in the economic analysis.
Shipping. Costs for (1) shipping equipment and supplies
to the demonstration site and (2) sample coolers to the
reference laboratory were not included in the economic
analysis because such costs vary depending on the
shipping distance and the service used (for example, a
courier or overnight shipping versus economy shipping).
Items Costing Less Than $10. The cost of inexpensive
items was not included in the economic analysis when
the estimated cost was less than $10. Items where it is
estimated that the cost was less than $10 included:
• Distilled water
• Personal protective equipment
• Waste containers
• Lab stools
8.2 AhRC PCR™ Kit Costs
This section presents information on the individual costs
of capital equipment, supplies, support equipment, labor,
and IDW disposal for the AhRC PCR™ kit as well as a
summary of these costs. Additionally, Table 8-1
summarizes the AhRC PCR™ kit costs. As described in
Section 4.6, Hybrizyme analyzed 110 samples during the
field demonstration and then re-analyzed the 110
samples in its laboratory in addition to the 99 samples
remaining in the total 209 demonstration samples. It is
important to note that costs estimated in this section are
based on actual costs to analyze the 110 samples during
the field demonstration. Cost estimates for analyzing the
entire set of 209 demonstration samples were then
determined based on the field demonstration costs.
8.2.1 Capital Equipment Cost
The capital equipment cost was the cost associated with
the purchase of the technology in order to perform
sample preparation and analysis. Capital equipment
includes the kit itself and purchase of a real-time PCR
thermocycler ($40,000). For the purposes of this cost
estimate, a lease price of $1,200 per week was used
because it is common practice for Hybrizyme to lease
the PCR thermocycler to customers. The lease of the
thermocycler also includes lease of a laptop computer,
microplate shaker, microplate washer, ultrasonic bath,
centrifuge, all pipettors, and bottle top dispensers.
The AhRC PCR™ kit can be purchased from
Hybrizyme for $2,350. One kit contains enough supplies
for 30-60 samples to be analyzed. During the field
demonstration, Hybrizyme utilized three AhRC PCR™
kits for approximately four days to analyze 110 samples.
It is estimated that four additional kits were used in
Hybrizyme's laboratory to complete the sample
analyses. It is possible that fewer kits could be used for
209 sample analyses since 30-60 samples can be
analyzed with each kit, but this reflects the number of
kits used during this demonstration.
8.2.2 Cost of Supplies
The supplies that Hybrizyme used during the
demonstration fall into two general categories:
expendable or reusable. Table 8-1 lists all expendable
supplies that Hybrizyme used during the demonstration
and the corresponding costs. The cost of each item was
rounded to the nearest $1. Expendable supplies are ones
that are consumed during the preparation or analysis.
Reusable costs are items that must be used during the
analysis but ones that can be repeatedly reused. Resuable
supplies (microplate shaker, microplate washer,
ultrasonic bath, centrifuge, pipettors, and bottle top
dispensers) are included as part of the lease of the PCR
thermocycler, so these items were not costed separately.
If the resuable items were purchased, the approximate
cost would be $13,600. The total cost of the supplies
employed by Hybrizyme during the demonstration was
$676. Supplies have to be purchased from a retail vendor
of laboratory supplies. Reusable items listed in Table 8-1
can be substituted with other models that operate under
the same specifications; thereby, modifying the cost of
supplies to the potential kit user.
8.2.3 Support Equipment Cost
Hybrizyme analyzed demonstration samples in a 32-foot
construction trailer equipped with a fume hood. The
rental cost for the construction trailer for use during
sample extraction and sample analysis was $1,919. The
minimum rental rate for the construction trailer was
one month. Hybrizyme only used the mobile laboratory
for four days. Since weekly or daily rental rates for the
mobile lab were not an option, the entire cost is reported.
As determined by the observers, a trailer with a fume
hood is necessary for operation of this technology in the
field, although the developer hopes to make the tech-
nology infrastructure less dependent in the future. A
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Table 8-1. Cost Summary
Quantity Used
Item
Capital Equipment
Purchase of AhRC PCR™ Kit
PCR Real-time Thermocycler and Reusable
Supplies (one-week lease)
Supplies
Expendable0
Extraction Cocktail
(Acetic acid/Hexane/Acetone)
Methanol
Pipette Tips (Filter 10)
Pipette Tips (Filter 200)
Pipette Tips (Non-Filter 200)
Pipette Tips (Non-Filter 1000)
Extraction Vials (I-Chem 100, 40 mL)
Extract Vials (16x100 mm)
Support Equipment
Construction Trailer
Fume Hood
Labor
Operator
IDW Disposal6
Total Cost
During Field
2
1
2
2
2
2
6
2
2
2
1
1
74
1
Demo
kits
unit
unit
unit
unit
unit
unit
unit
unit
unit
unit
unit
labor hours
unit
Unit Cost ($)
2,350
1,200
40
1
7
5
3
3
110
3
1,919
1,100
8()d
334
Itemized
110 Samples
4,700
1,200
80
2
14
10
18
6
220
6
1,919
1,100
5,920
334
$15,529
Cost3 ($)
209 Samples
17,045b
2,400
152
4
27
19
34
11
418
11
1,919
1,100
11,248
635
$35,023
Itemized costs were rounded to the nearest $1.
It is possible that fewer kits could be used for 209 sample analyses, since the number of samples that can be
analyzed per kit is 30-60 samples, but this is the number used in this demonstration.
Hybrizyme is preferred vendor of all expendable supplies except the extraction and extract vials.
Labor rate for field technicians to operate technology rather than research scientists was $50.75 an hour,
$3,756 for 110 samples and $7,135 for 209 samples.
Further discussion about waste generated during demonstration can be found in Chapter 7.
laptop computer is necessary for efficiently operating
this technology, but it was not costed separately because
it is included in the thermocycler lease.
8.2.4 Labor Cost
As described in Section 8.1.4, 74 labor-hours were spent
in the field to analyze 110 samples. An hourly rate of
$32.10 was used for a research scientist performing.
sample extractions and sample analysis, and a
multiplication factor of 2.5 was applied to labor costs in
order to account for overhead costs.(9) Based on this
hourly rate and multiplication factor, a labor rate of
$5,920 was determined for the analysis of the 110
samples during the field demonstration. It was estimated
that the labor cost for the total 209 samples was $ 11,248.
Based on observation, it is anticipated that lower-cost
field technicians, with proper training and skill levels,
could have analyzed the samples in a similar amount of
time. As such, the labor rate for the analysis of 110
samples during the field demonstration could have been
as low as $3,756 (hourly rate of $20.30 with 2.5
multiplication factor for 74 labor-hours), and $7,135 for
all 209 demonstration samples.
8.2.5 Investigation-Derived Waste Disposal Cost
As discussed in Chapter 7, Hybrizyme was provided
with 5-gallon containers for collecting wastes generated
during the demonstration. Chapter 7 discusses the type
and amount of waste generated by the technology during
the field demonstration in more detail.
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During the demonstration, Hybrizyme analyzed 110
samples. The total cost to dispose of the waste generated
for these samples was $334. The cost to dispose of waste
for all 209 samples is estimated at $635.
8.2.6 Summary ofAhRC PCRrM Kit Costs
The total cost for performing the AhRBU analyses using
the AhRC PCR™ kit was $35,023. The analyses were
performed for 58 soil and sediment PE samples, 128 soil
and sediment environmental samples, and 23 extracts.
When Hybrizyme performed multiple dilutions or
reanalyses for a sample, these were not included in the
number of samples analyzed.
The total cost of $35,023 for analyzing the
demonstration samples under the AhRC PCR™ kit
included $19,445 for capital equipment; $676 for
supplies; $3,019 for support equipment; $11,248 for
labor; and $635 for IDW disposal. Of these five costs,
the largest cost was for the capital equipment (56% of
the total cost).
8.3 Reference Method Costs
This section presents the costs associated with the
reference method used to analyze the 209 demonstration
samples for dioxin and dioxin-like PCBs. Typical costs
of these analyses can range from $800 to $1,100 per
sample, depending on the method selected, the level of
quality assurance/quality control incorporated into the
analyses, and reporting requirements. The reference
laboratory utilized EPA Method 1613B for D/F analysis
and EPA Method 1668A for coplanar PCB analysis for
Table 8-2. Reference Method Cost Summary
all soil and sediment samples for comparison with the
CALUX® system. The reference method costs were
calculated using cost information from the reference
laboratory invoices.
Table 8-2 summarizes the projected and actual reference
method costs. At the start of the demonstration, the
reference laboratory's projected cost per sample was
$785 for D/F analysis and $885 for PCB analysis. This
cost covered the preparation and analysis of the
demonstration samples, required method QC samples,
electronic data deliverable, and the data package for
each. The actual cost for the 209 demonstration analyses
was $213,580 for D/F and $184,449 for PCBs, and a
total of $398,029. This was higher than the projected
($321,380) due to reanalysis, re-extractions, dilutions
and additional cleanups that were above the 30% repeats
allowable by the original quote. The turnaround time by
the reference laboratory for reporting all 209 samples
was approximately eight months (171 business days).
The quoted turnaround time was three months.
8.4 Comparison of Economic Analysis
Results
The total costs for the AhRC PCR™ kit to analyze all
209 demonstration samples ($35,023) and the reference
method ($398,029) are listed in Tables 8-1 and 8-2,
respectively. The total cost for the AhRC PCR™ kit
purchase was $363,006 less than the reference method.
After the demonstration, Hybrizyme refined its sample
preparation method, analyzed all 209 samples in its
Analyses Performed
D/F, EPA Method 161 3B,
GC/HRMS
WHO PCBs EPA Method 1668A,
GC/HRMS
1668 Optional Carbon Column
DB1
Total Cost
Number of
Samples
Analyzed
23 extracts
186
soil/sediment
23 extracts
186
soil/sediment
40
209 samples
Cost per sample
Quotation ($)
735
785
685
735
150
Itemized Cost ($)
Quotation3
16,905
146,010
15,755
136,710
6,000
321,380
Actual
213,580
184,449
398,029
Price includes up to 30% of samples requiring additional work of some kind (dilutions or extra cleanup).
Greater than that would require additional work with further charges associated to them ($150 to $180 per
sample per procedure).
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laboratory, and reported results. The developer reported
that the total analysis time was approximately one week
once the method refinements were completed. By
comparison, the reference laboratory took eight months
to analyze all 209 samples.
Use of the AhRC PCR™ kit in the field will likely
produce additional cost savings because the results will
be available within a few hours of sample collection;
therefore, critical decisions regarding sampling and
analysis can be made in the field, resulting in a more
complete data set. Additional possible advantages to
using field technologies include reduction of multiple
crew and equipment mobilization-demobilization cycles
to a single cycle, dramatically increased spatial
resolution mapping for higher statistical confidence,
leading to reduced insurance costs and reduced disposal
costs, and compression of total project time to reduce
administrative overhead. However, these savings cannot
be accurately estimated and thus were not included in the
economic analysis. Project-specific costs associated with
the use of the technology, such as the cost for HRMS
confirmation analyses and training costs to be proficient
in the use of the technology, were also not accounted for
in this analysis.
The Hybrizyme AhRC PCR™ kit is a screening method
that reports the amount of AhR binding compounds in
the sample unlike the reference method which reports
TEQ results and concentrations for individual congeners.
The AhRC PCR™ kit provided data which resulted in
ranking samples from low to high concentration of AhR-
binding compounds identically to ranking generated by
reference laboratory data 70-90% of the time.
Additionally, the AhRC PCR™ kit can provide AhRBU
results on-site at significant cost and time savings
compared to the reference laboratory.
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Chapter 9
Technology Performance Summary
The purpose of this chapter is to provide a performance
summary of the Hybrizyme Corporation AhRC PCR™
Kit by summarizing the evaluation of the primary and
secondary objectives of this demonstration in Tables 9-1
and 9-2, respectively. Detailed information about these
evaluations, including a complete evaluation of the
reference laboratory data, can be found in previous
sections of this report.
At the time of the demonstration, this particular test was
intended for use as a screening tool to rank samples from
those inducing the greatest AhR activity to those
inducing the least AhR activity rather than to provide
highly accurate TEQ. The developer's goal is a highly
portable screening technology which can help to
determine areas of greatest concern for cleanup at a site
and can help to minimize the number of more expensive
analyses needed for specific analytes. It has been
suggested that correlation between the Hybrizyme
AhRBU results and HRMS results could be established
by first characterizing a site and calibrating the
Hybrizyme results to HRMS results. This approach was
not evaluated during this demonstration. Since the
technology measures an actual biological response, it is
possible that the technology may give a better
representation of the true toxicity from a risk assessment
standpoint.
The data generated and evaluated during this
demonstration showed that the Hybrizyme technology
could be used as an effective tool to rank sample
concentrations from low to high within a particular
environmental site, particularly considering that both the
cost ($35,023 vs. $398,029) and the time (less than two
weeks vs. eight months) to analyze the 209 demon-
stration samples were significantly less than that of the
reference laboratory.
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Table 9-1. Hybrizyme Corporation AhRC PCR™ Kit Performance Summary - Primary Objectives
Objective
P 1 : Accuracy : Ranking versus PE certified
values
P2: Precision
P3: Comparability: Ranking versus reference
laboratory average total concentration for
environmental samples
P4: Estimated method detection limit
P5: False Positive/False Negative Rate
P6: Matrix Effects
P7: Cost
Performance
• The Hybrizyme ranking was identical to the certified concentrations for
one of the 10 PE samples (Cambridge 5184).
• Of the four highest sample concentrations according to the certified values
(NIST 1944, ERA Aroclor, Cambridge 5184, and ERA PAH),
Hybrizyme's data ranked three of the samples as having the highest
concentrations (NIST 1944, Cambridge 5184, and ERA PAH).
• The ERA Aroclor sample, which was spiked with Aroclor 1254, was
ranked as the next lowest concentration by Hybrizyme and the next to
highest concentration by the certified data.
Number of data points 48
Median RSD (%) 19
MeanRSD (%) 25
• Hybrizyme ranking agreed with reference laboratory within the
environmental site for 7 of the 10 sites (70%)
• Hybrizyme ranking agreed with the reference laboratory for 9 of the
1 0 sites when uncertainly around reference laboratory values was
considered (90%)
• Hybrizyme individual rankings agreed with the reference laboratory for 26
of the 32 individual rankings (81%)
EMDL=71 AhRBU
not evaluated
• Measurement location: not evaluated (all results generated in the
laboratory)
• Matrix type: none
• Sample type: Significant effect
• PAH concentration: none
• Environmental site: not evaluated since AhRBU results weren't directly
comparable to TEQ
• Known interferences: not evaluated since assay reacts to AhR binding
compounds
110 samples during field demonstration: $15,529
Projected if all 209 demonstration samples were analyzed in field: $35,023
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Table 9-2. Hybrizyme Corporation AhRC PCR™ Kit Performance Summary - Secondary Objectives
Objective
Performance
SI: Skill level of Operator
Based on observation during the field demonstration, the recommended skill level for
operation of this technology includes a minimum of a high school degree, having good work
skills, and being trainable. Decent laboratory skills, respect for safely, and having reasonable
attention to detail would also be useful attributes for successful technology operation.
S2: Health and Safely Aspects
The majority of this technology's waste was generated during the sample extraction. While
hydrochloric acid was used for this demonstration, the process normally uses hexane, acetic
acid, acetone, and, optionally, sulfuric acid. A fume hood is recommended for solvent
extraction.
S3: Portability
As used in the demonstration, this technology required at a minimum a trailer to protect from
the environment and to house some equipment such as a centrifuge, sonicator, and the PCR
thermocycler analyzer. Electricity was also a necessity, so a trailer with a fume hood would
be the minimum required for successful field operation. The developer intends for this
technology ultimately to be usable in a minimally controlled environment.
S4: Sample Throughput
During the field demonstration, 110 samples were processed by Hybrizyme, equating to a
sample throughput rate of 28 samples per day. This was accomplished in about four full
working days (74 labor-hours), with two operators (one doing sample preparation and one
performing the analysis) performing the work. Hybrizyme reported that, once the method
refinements were completed, the total analysis time in its laboratory to complete the sample
analysis and repeat analysis of the 110 samples that were analyzed during the field
demonstration was one week.
57
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Chapter 10
References
1. EPA. 2001. Database of Sources of Environmental
Release of Dioxin-like Compounds in the United
States, EPA/600/C-01/012, March.
2. EPA. 2004."Technologies for the Monitoring and
Measurement of Dioxin and Dioxin-like Compounds
in Soil and Sediment," Demonstration and Quality
Assurance Project Plan, U.S. EPA/600/R-04/036,
April.
3. EPA Method 1613B. 1994. Dioxins, Tetra-thru
Octa-(CDDs) and Furans (CDFs), EPA/821/B-94-
005, 40 Code of Federal Regulations Part 136,
Appendix A, October.
4. EPA Method 1668A. 1999. Chlorinated biphenyl
congeners byHRGC/HRMS, EPA/821/R-00-002,
December.
5. van den Berg, M., Birnbaum, L., Bosveld, A. T. C.,
Brunstrom, B., Cook, P., Feeley, M., Giesy, J. P.,
Hanberg, A., Hasagawa, R., Kennedy, S. W.,
Kubiak, T., Larsen, J. C., van Leeuwen, F. X. R.,
Liem, A. K. D., Nolt, C., Peterson, R. E., Poellinger,
L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M.,
Younes, M., Waern, F., and Zacharewski, T. 1998.
Toxic equivalency factors (TEFs) for PCBs, PCDDs,
PCDFs for humans and wildlife. Environmental
Health Perspectives 106: 775-792.
6. De Rosa, Christopher T., et al. 1997. Dioxin and
dioxin-like compounds in soil, Part 1: ATSDR
Interim Policy Guideline. Toxicology and Industrial
Health, Vol. 13, No. 6, pp. 759-768.
7. NOAA. 1998. Sampling and analytical methods of
the national status and trends program mussel watch
project: 1993-1996 update. NOAA Technical
Memorandum NOS ORCA 130. Silver Spring,
Maryland.
8. EPA SW-846 Method 8290. 1994. Polychlorinated
dibenzodioxins (PCDDs) and polychlorinated
dibenzofurans (PCDFs) by high-resolution gas
chromatography/high-resolution mass spectrometry
(HRGC/HRMS), September.
9. U.S. Bureau of Labor Statistics, National
Compensation Survey. Accessed on 7/26/04.
Available at:
http://data.bls.gov/labjava/outside jsp?survey=nc
58
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Appendix A
SITE Monitoring and Measurement Technology Program
Verification Statement
-------
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Washington, DC 20460
xvEPA
SITE Monitoring and Measurement Technology Program
Verification Statement
TECHNOLOGY TYPE: Ah Receptor - Polymerase Chain Reaction
APPLICATION: MEASUREMENT OF DIOXIN AND DIOXIN-LIKE
COMPOUNDS
TECHNOLOGY NAME: AhRC PCR™ Kit
COMPANY: Hybrizyme Corporation
ADDRESS: Suite G-70
2801 Blue Ridge Road
Raleigh, North Carolina 27607
PHONE: (919) 783-9595
WEB SITE: www.hybrizyme.com
E-MAIL: rallen@hybrizyme.com
VERIFICATION PROGRAM DESCRIPTION
The U.S. Environmental Protection Agency (EPA) created the Superfund Innovative Technology Evaluation (SITE)
Monitoring and Measurement Technology (MMT) Program to facilitate deployment of innovative technologies
through performance verification and information dissemination. The goal of this program is to further environmental
protection by substantially accelerating the acceptance and use of improved and cost-effective technologies. The
program assists and informs those involved in designing, distributing, permitting, and purchasing environmental
technologies. This document summarizes results of a demonstration of the Hybrizyme Corporation AhRC PCR™ Kit.
PROGRAM OPERATION
Under the SITE MMT Program, with the full participation of the technology developers, the EPA evaluates and
documents the performance of innovative technologies by developing demonstration plans, conducting field tests,
collecting and analyzing demonstration data, and preparing reports. The technologies are evaluated under rigorous
quality assurance protocols to produce well-documented data of known quality. The EPA's National Exposure
Research Laboratory, which demonstrates field sampling, monitoring, and measurement technologies, selected
Battelle as the verification organization to assist in field testing technologies for measuring dioxin and dioxin-like
compounds in soil and sediment.
A-l
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DEMONSTRATION DESCRIPTION
The demonstration of technologies for the measurement of dioxin and dioxin-like compounds was conducted at the
Green Point Environmental Learning Center in Saginaw, Michigan, from April 26 to May 5, 2004. The primary
objectives for the demonstration were as follows:
P1. Determine the accuracy.
P2. Determine the precision.
P3. Determine the comparability of the technology to EPA standard methods.
P4. Determine the estimated method detection limit (EMDL).
P5. Determine the frequency of false positive and false negative results.
P6. Evaluate the impact of matrix effects on technology performance.
P7. Estimate costs associated with the operation of the technology.
The secondary objectives for the demonstration were as follows:
S1. Assess the skills and training required to properly operate the technology.
S2. Document health and safety aspects associated with the technology.
S3. Evaluate the portability of the technology.
S4. Determine the sample throughput.
A total of 209 samples was analyzed by each technology, including a mix of performance evaluation (PE) samples,
environmentally contaminated samples, and extracts. Hybrizyme analyzed 110 samples in the field, but refined its
method after the demonstration and analyzed all 209 samples in its laboratory. The PE samples were used primarily to
determine the accuracy of the technology and consisted of purchased reference materials with certified concentrations.
The PE samples also were used to evaluate precision, comparability, EMDL, false positive/negative results, and
matrix effects. Dioxin-contaminated samples from Warren County, North Carolina; the Saginaw River, Michigan;
Tittabawassee River, Michigan; Midland, Michigan; Winona Post, Missouri; Nitro, West Virginia; Newark Bay, New
Jersey; Raritan Bay, New Jersey; and Brunswick, Georgia were used to evaluate precision, comparability, false
positive/negative results, and matrix effects. Extracts prepared in toluene were used to evaluate precision, EMDL, and
matrix effects. All samples were used to evaluate qualitative performance objectives such as technology cost, the
required skill level of the operator, health and safety aspects, portability, and sample throughput. AXYS Analytical
Services (Sidney, British Columbia) was contracted to perform the reference analyses by high-resolution mass
spectrometry (HRMS) (EPA Method 1613B and EPA Method 1668A). The purpose of the verification statement is to
provide a summary of the demonstration and its results; detailed information is available in Technologies for
Monitoring and Measurement of Dioxin and Dioxin-like Compounds in Soil and Sediment — Hybrizyme Corporation
AhRC PCR™ Kit (EPA/54Q/R-Q5/QQ5).
TECHNOLOGY DESCRIPTION
The technology description and operating procedure below are based on information provided by Hybrizyme
Corporation.
The Hybrizyme AhRC PCR™ kit detects molecules in a test sample that bind to the aryl hydrocarbon receptor (AhR).
The AhR mediates most, if not all, of the harmful effects associated with exposure to 2,3,7,8-substituted dioxin/furan
(D/F). How tightly or loosely these compounds bind to the AhR is one of the determining factors of their toxicity. The
AhR also binds certain coplanar polychlorinated biphenyls and carcinogenic polynuclear aromatic hydrocarbons
(PAHs), such as benzo-[a]-pyrene. Sample cleanup procedures can be employed so that all or a subset of these
AhR-reactive compounds are detected by the assay. Sample results are reported in Ah-receptor binding units
(AhRBU).
Samples are prepared using an extraction method designed for speed and simplicity while maintaining sample
concentration. Two grams of each sample are placed in a vial, and an extraction cocktail is added. The samples are
extracted using an ultrasonic bath followed by brief centrifugation to remove solids. Water is added to the sample,
forcing a small amount of hexane to partition from the extraction cocktail. A portion of the hexane is removed and
dried for analysis. Depending on the condition of the sample being analyzed, an acid wash may be added as an
additional step. The dried sample extracts are suspended in methanol for analysis by the AhRC PCR™ Kit.
A-2
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Extracts in methanol are added to glass vials, followed by the addition of activation solution containing the AhR and
dioxin-responsive element (DRE) probe and incubated at room temperature for one hour. This reaction mix is
transferred from the glass vials to capture strips using a multichannel pipettor, and the capture strips are incubated at
room temperature for an additional 30 minutes. During this time, the AhR/DRE-probe complexes are trapped onto the
wells of the capture strip. The capture strips are washed to remove free DRE-probe, and a polymerase chain reaction
(PCR) master mix is added. The strips are placed in a real-time thermocycler, and the amount of the DRE-probe is
measured. The signal generated from the DRE-probe is directly proportional to the amount of dioxin in the samples.
VERIFICATION OF PERFORMANCE
At the time of the demonstration, this particular test was intended for use as a screening tool to rank samples from
those inducing the greatest AhR activity to those inducing the least AhR activity rather than to provide highly accurate
toxicity equivalent (TEQ). The developer's goal is a highly portable screening technology that can help to determine
areas of greatest concern for cleanup at a site and can help to minimize the number of more expensive analyses needed
for specific analytes. It has been suggested that correlation between the Hybrizyme AhRBU results and HRMS results
could be established by first characterizing a site and calibrating the Hybrizyme results to HRMS results. This
approach was not evaluated during this demonstration. Since the technology measures an actual biological response, it
is possible that the technology may give a better representation of the true toxicity from a risk assessment standpoint.
Accuracy: The determination of accuracy was based on ranking of the PE samples results from low to high
concentration and comparing it to the rank order reported by Hybrizyme based on AhRBU. The Hybrizyme ranking
was identical to the certified concentrations for one of the 10 PE samples (Cambridge 5184). Of the four highest
sample concentrations according to the certified values, Hybrizyme's data ranked three of the samples as having the
highest concentrations. One PE sample that was spiked with Aroclor 1254 was ranked as the next lowest concentration
by Hybrizyme and the next to highest concentration by the certified data.
Precision: Replicates were incorporated for all samples (PE, environmental, and extracts) included in the 209 samples
analyzed in the demonstration. Three samples had seven replicates in the experimental design, one sample had eight
replicates, and all other samples had four replicates. Precision was determined by calculating the standard deviation of
the replicates, dividing by the average concentration of the replicates, and multiplying by 100%. Ideal relative
standard deviation (RSD) values are less than 20%. The overall RSD values were 25% (mean), 19% (median), 2%
(minimum), and 111% (maximum).
Comparability: The reference laboratory average total cocnentration results were compared to Hybrizyme's AhRBU
results including contributions from PAHs because PAHs are AhR binding compounds and are included in the
Hybrizyme results. For this evaluation, the environmental samples were ranked with the samples from that site only,
rather than ranking all of the environmental sites in one ordering, because the Hybrizyme technology is intended to
rank samples within a particular site. This evaluation demonstrated that the Hybrizyme technology was able to rank the
samples from low to high concentration within an environmental site fairly consistently with the reference laboratory
based on average total concentration data. For seven of the 10 environmental sites (70%), Hybrizyme's ranking was
identical to the reference laboratory's ranking. For samples that were close in average HRMS concentration and were
indistinguishable when uncertainties were considered, the Hybrizyme rankings agreed with the reference laboratory's
ranking for nine of 10 sites (90%). On an individual ranking basis, the Hybrizyme and reference laboratory rankings
agreed 81% of the time (26 of 32 rankings). These data suggest that the Hybrizyme technology could be an effective
screening tool for ranking samples within an environmental site from low to high concentration.
Estimated method detection limit: EMDL was calculated according to the procedure described in 40 CRF Part 136,
Appendix B, Revision 1.11. Lower EMDL values indicate better sensitivity. The calculated EMDL was 71 AhRBU.
False positive/negative results: This parameter was not evaluated for this technology because quantitative
comparisons to HRMS results are not appropriate.
A-3
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Matrix effects: The likelihood of matrix-dependent effects on performance was investigated by evaluating results in a
variety of ways. No significant effect was observed for the reproducibility of Hybrizyme results by matrix type (soil,
sediment, and extract) or by PAH concentration, but a significant effect was observed for sample type (PE vs.
environmental vs. extract).
Cost: The full cost of the technology was documented and compared to the cost of the reference analyses. The total
cost for the Hybrizyme kit to analyze all 209 samples was $35,023. The total cost for the reference laboratory to
analyze all 209 samples by EPA Method 1613B and EPA Method 1668A was $398,029. The total cost for the use of
the Hybrizyme kit was $363,006 less than the reference method.
Skills and training required: Based on observation during the field demonstration, the recommended skill level for
operation of this technology includes a minimum of a high school degree, having good work skills, and being trainable.
Decent laboratory skills, respect for safety, and having reasonable attention to detail would also be useful attributes for
successful technology operation.
Health and safety aspects: The majority of this technology's waste was generated during the sample extraction. While
hydrochloric acid was used for this demonstration, the process normally uses hexane, acetic acid, acetone, and,
optionally, sulfuric acid. A fume hood is recommended for solvent extraction.
Portability: As used in the demonstration, this technology required at a minimum a trailer to protect from the
environment and to house equipment such as a centrifuge, sonicator, and the PCR thermocycler analyzer. Electricity
was also a necessity, so a trailer with a fume hood would be the minimum required for successful field operation. The
developer intends for this technology ultimately to be useable in a minimally controlled environment.
Sample throughput: During the field demonstration, 110 samples were processed by Hybrizyme, equating to a sample
throughput rate of 28 samples per day. This was accomplished in about four full working days (74 labor-hours), with
two operators (one doing sample preparation and one performing the analysis) performing the work. After the
demonstration, Hybrizyme refined its sample preparation method, analyzed all 209 samples in its laboratory.
Hybrizyme reported that the total analysis time for the samples was approximately one week once the method
refinements were completed. By comparison, the reference laboratory took eight months to analyze all 209 samples.
NOTICE: Verifications are based on an evaluation of technology performance under specific, predetermined
criteria and the appropriate quality assurance procedures. The EPA makes no expressed or implied warranties as
to the performance of the technology and does not certify that a technology will always operate as verified. The
end user is solely responsible for complying with any and all applicable federal, state, and local requirements.
A-4
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Appendix B
Supplemental Information Supplied by the Developer
The purpose of this section is for the developer to provide additional information about the technology. This can
include updates/changes/modifications planned for the technology or which have occurred since the technology
was tested. The developers can also use this section to comment and expand on the findings of the report.
Information was provided by the developer and does not necesarily reflect the opinion of the EPA.
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Hybrizyme Comments
Versatility of the AhRC PCR assay system
The AhRC PCR assay can be used in a variety of modes depending on the sample preparation steps
employed. Typically, the more labor intensive the cleanup the more specific the test results reported. For
example, the AhRC PCR assay has been accepted in Japan as a TEQ method that can be used in lieu of
HRGC-HRMS for fly ash analysis (see below). Minimal sample cleanup was used in this study to maximize
field portability, ease-of-use, and speed. The assay detects toxicants other than dioxins and furans including
carcinogenic PAHs and, as such, reports in arbitrarily defined Ah-receptor binding units (AhRBUs).
Straightforward sample cleanup in combination with the ease-of-use of the AhRC PCR assay makes the
method rapid, inexpensive, and precise (something that few, if any, trace organic analytical technologies can
claim). This method provides a tool to quickly rank samples at a given site according to their reactivity with
the Ah receptor. Because the assay may recognize additional hazardous compounds other than dioxins and
furans the technology is best used in an environmentally site-specific manner. Environmental samples from
a site with known concentrations of targeted contaminants can be used to calibrate the response of the AhRC
PCR assay providing a quantitative measurement (please call for additional information).
Generating a Concentration Map
The ability to rapidly generate relative concentration data makes it feasible to produce a concentration map
of a large environmental site in a reasonably short period of time. With such a concentration map in hand,
the most relevant collected matrices can be subjected to more sophisticated techniques capable of accurately
identifying the contaminants present. By eliminating the need to use sophisticated and expensive techniques
on all of the collected samples, many of which typically contain low to non-detectable levels, one expedites
the production of useful data in all areas of a given study. As a result, site personnel can begin remediation
efforts faster saving both time and money.
The ability for two field technicians to determine site boundaries beyond which there is no measurable
contamination using a field executable methodology capable of producing up to 40 to 60 data points per day
is also beyond the current state of the art.
A Sample Pre-Screening Procedure for the HRGC-HRMS Laboratory
The method also has a pre-screening application in the typical laboratory performing HRGC-HRMS analyses
of extended sets of environmental samples. In such a role, the assay system is used by experienced
laboratory personnel to eliminate all samples demonstrating low to negligible response at a rate of perhaps
greater than 60 samples per day in a fully equipped laboratory setting. Simultaneously, this data would also
permit organizing the remainder of the samples from lowest to highest concentration of AhR reactive
molecules. As such, when they are finally processed for HRGC-HRMS examination, the laboratory will not
spend inordinate amounts of time examining "non-detects" nor will they accidentally cross-contaminate their
laboratory equipment by processing samples containing elevated levels of the expected analytes.
The described benefits for using the Hybrizyme Method to pre-screen large sample sets as a regular function
of sample processing and analysis in the HRGC-HRMS laboratory will likely decrease overall time and
expense for these critical analyses. Data quality will also improve while permitting a much greater fraction
of usable information to be generated on the first pass through the system. Reanalysis due to circumstances
Information was provided by the developer and does not necesarily reflect the opinion of the EPA.
B-l
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arising from cross-contamination caused by the presence of only a few very high concentration samples in a
batch of otherwise low to negligible concentration samples will ultimately become a non-issue.
EQ Analysis
The AhRC PCR assay kit has been validated for use in Japan. Both Daiichi Fine Chemicals and the National
Institute for Environmental Studies (NIES) analyzed the samples below for the Japanese Ministry of the
Environment using AhRC PCR. Daiichi and NIES generated R2 values of 0.99 and 0.92, respectively. A
similar study was presented at DIOXIN 2004 comparing a number of quantitative bioassays. The authors
indicated that the AhRC PCR demonstrated superior reproducibility over other bioassays tested (Ota, S. t
a/., Comparison of Various Bioassays for Dioxins Measurements in Fuel gas, Fly ash and Bottom ash.
Organohalogen Compounds, Vol. 66, pp 682-689, 2004; http://dioxin2004.abstract-
management.de/pdf/p528.pdf). Hybrizyme, in collaboration with Daiichi, is in the process of developing a
similar method for use in the US for quantitative TEQ analysis.
Information was provided by the developer and does not necesarily reflect the opinion of the EPA.
B-2
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Daiichi Fine Chemical Co. LTD.
Sample 2A
Sample 2B
Sample 2C
Sample 2D
Sample 2E
Sample 2F
Sample 2G
Sample 2H
Sample 21
Ex. Gas
Ex. Gas
Ex. Gas
Fly Ash
Fly Ash
Fly Ash
Bottom Ash
Bottom Ash
Bottom Ash
AhR PCR
Conversion
TEQ
0.18
0.23
0.058
0.00089
0.014
1.0
0.049
0.0022
0.010
GC/MS
TEQ
0.10
0.12
0.015
0.0046
0.000056
0.44
0.000034
0.000035
0.0067
Unit
ng - TEQ /m3N
ng - TEQ /m3N
ng - TEQ /m3N
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
Daiichi
O^n
.ou
0 4^
U.HvJ
0 40
U.HU
m n ^
(/) U.OO
t* n ^n
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0
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x n 1 ^
•*" U. I O
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n n^
U.UvJ
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y = 0.4469X - 0.0008
R2 = 0.991 */*
/^
/^
^
/^
/^
+ ./r
/^
-------
National Institute for Environmental Studies
Sample 2A
Sample 2B
Sample 2C
Sample 2D
Sample 2E
Sample 2F
Sample 2G
Sample 2H
Sample 21
Ex. Gas
Ex. Gas
Ex. Gas
Fly Ash
Fly Ash
Fly Ash
Bottom Ash
Bottom Ash
Bottom Ash
AhR PCR
Conversion
TEQ
0.49
0.33
0.070
0.0055
0.0017
0.93
0.030
0.0010
0.013
GC/MS
TEQ
0.10
0.12
0.015
0.0046
0.000056
0.44
0.000034
0.000035
0.0067
Unit
ng - TEQ /m3N
ng - TEQ /m3N
ng - TEQ /m3N
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
ng - TEQ /g
NIES
O^n
.ou
0 4^
U.HvJ
0 40
U.HU
0 ^
(/) U-'30
^ n ^n
^ u.ou
^ 09^
6 u^°
® n 90
£ U.ZU
^ n 1 ^
U. I O
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. I U
On^
.uo
Onn
•
y = 04259x -0 011
R2 = 0.919£^^
^^
^^
^^
^^
-***+
^^
+^*
.UU r I I I II
0.00 0.20 0.40 0.60 0.80 1.00
AhRC PCR (TEQ)
Information was provided by the developer and does not necesarily reflect the opinion of the EPA.
B-4
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Appendix C
Reference Laboratory Method Blank and Duplicate Results Summary
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Table C-1. Summary of Method Blank Performance
Sample Batch
Number
D/FWG12107
D/F WG12148
D/F WG 12264
D/F WG12534
D/F WG 12641
D/F WG12737
D/F WG 12804
D/F WG 13 547
Criteria
Met
Y
N
N
N
N
N
N
N
Method
Blank TEQa
(Pg/g)
0.000812
0.133
0.0437
0.610
0.0475
0.348
0.0153
0.0553
Sample TEQ Range3 (pg/g)
26. 1-74. 1 (Newark Bay)
9.93-13.3(RantanBay)
13.5-50.4 (Newark Bay)
49.5-15,200 (Brunswick)
1.0-94.1 (Titta. River sediment)
0.237-6,900 (PE)
25. 3-7,100 (PE)
3 1-269 (Midland)
72.8 (Brunswick)
123 (Titta. River sediment)
0.1 59-7,690 (PE)
25. 7-1 92 (Midland)
35.2-1,300 (Titta. River soil)
3. 89-188 (PE)
57.5-3,000 (Nitro)
37.9 (North Carolina)
122 (Saginaw River)
26.4- 222 (Midland)
Comments
Many samples had concentrations >20x
blank. Few that didn't were not significantly
affected on a total TEQ basis.
Most samples had concentrations >20x
blank. Low level Tittabawassee River
sediment samples L6749-2 (Ref 48b), -9 (Ref
130), -10 (Ref 183), and -12 (Ref 207) were
evaluated based on their replication within
the demonstration analyses and comparison
to characterization results and considered
unaffected by method blank exceedances.
Low level PE samples L6760-1 (Ref 25), -3
(Ref 28), and -4 (Ref 29) were D/F blanks
with resulting TEQs sufficiently low enough
to still be distinguished as blank samples.
Sample concentrations > 20x blank.
All but PE sample Ref 177 (0. 159 TEQ) had
significantly higher total TEQ than blank.
Ref 177 was confirmed by running in
another batch and results, which agreed
within 18%. Additionally, Ref 177 was
compared to its replicates within the program
and considered acceptable.
Sample concentrations >20x blank.
A few analytes higher than criteria but no
significant contribution to total TEQ.
Several analytes exceeded criteria, but blank
total TEQ contribution to sample is relatively
small.
C-1
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Sample Batch
Number
D/FWG13548
D/FWG13549
D/FWG13551
D/FWG13552
D/FWG13984
D/F WG 14274
Criteria
Met
N
N
N
Y
N
N
Method
Blank TEQa
(Pg/g)
0.0114
0.0925
2.40
0.000969
0.0154
0.0434
Sample TEQ Range3 (pg/g)
99.6-99.7 (Sagmaw River)
32.9-36.4 (North Carolina)
0.268-100 (Extracts)
2,1 60-3,080 (Nitro)
146-1,320 (Saginaw River)
788-8,410 (North Carolina)
1,100-10,800 (North Carolina)
7,160-11,300 (Winona Post)
0.0386-9.28 (PE)
25.8 (Midland)
0.524-24.8 (PE)
10.4(RantanBay)
53. 1-444 (Extracts)
2,800 (Nitro)
35.5-8,320 (North Carolina)
0.0530-5.93 (PE)
Comments
Several analytes exceeded criteria. In
general, the blank contribution to total TEQ
was negligible and in those cases results
were accepted. Several low-level extract
samples were evaluated as follows: Extract
Spike #1 samples L6754-4 (Ref 4), -8 (Ref
8), -10 (Ref 10), -14 (Ref 14), -19 (Ref 19), -
22 (Ref 22), and -23 (Ref 23) were known
TCDD spikes at 0.5 pg/mL. Results were
compared to the known spiked TEQ and
considered unaffected by blank contribution
to TEQ. Extract Spike #3 samples L6754-1
(Ref 1), -7 (Ref 7), -12 (Ref 12), and -15
(Ref 1 5) were PCB spikes and not expected
to contain D/F. These spikes consistently
contained a D/F TEQ of ~0.3. However, this
came from a consistent ~0.3 pg/mL of
TCDD detected in these extracts that was
confirmed as a low-level TCDD
contamination by AXYS. Since TCDD was
not present in the lab blank, these results
were accepted as unaffected by any blank
contribution to TEQ.
Many analytes exceeded limits, but the blank
contribution to total TEQ is small relative to
sample TEQs.
Many analytes exceeded limits, but the blank
contribution to total TEQ is small relative to
sample TEQs.
Blank contribution to total TEQ was
negligible except for PE samples L7 179-7
(Ref 94), -8 (Ref 96), -1 1 (Ref 108), -12
(Ref 109), -17 (Ref 132), and L7182-6 (Ref
1 50). All but L7 179-8 were certified blanks.
L7 179-8 was a PAH spike with no D/F TEQ
expected. The TEQs of these samples were
considered sufficiently low enough to still be
distinguished as blank samples and were
accepted.
Sample TEQs were large enough to be
unaffected by the blank TEQ except for four
PE samples L7 179-4 (Ref 85), -16 (Ref 124)
and L7182-12 (Ref 169) and -14 (Ref 184).
These PE samples were either certified
blanks or PCB spikes with no expected D/F
TEQ. Resulting TEQs for these samples
were considered low enough to be
distinguished as blank samples and were
accepted.
C-2
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Sample Batch
Number
PCBWG12108
PCB WG12147
PCB WG12265
PCB WG12457
PCB WG12687
PCB WG12834
PCB WG12835
PCB WG12836
PCB WG1 3008
PCB WG13256
PCBWG13257
PCBWG13258
PCB WG13554
PCB WG14109
Criteria
Met
N
Y
Y
N
N
N
N
N
N
Y
Y
Y
N
N
Method
Blank TEQa
(Pg/g)
0.000137
0.000
0.0000584
0.000208
0.0183
0.000405
0.000125
0.0499
0.0221
0.000102
0.000251
0.000301
0.0000900
0.000288
Sample TEQ Range3 (pg/g)
2.63-5. 19 (Newark Bay)
2.04-2.82 (Rantan Bay)
1.21-5. 06 (Newark Bay)
0.104-0.330 (Brunswick)
0.132-0.369 (Brunswick)
0.034-0.649 (Titta. River sediment)
0.00277-1, 030 (PE)
4.20-1, 020 (PE)
0.974-2.73 (Midland)
1 0.3-1,1 80 (PE)
0.0157-62.4 (Sagmaw River)
0.181-0.203 (Brunswick)
0.986-7.57 (Titta. River Soil)
0.822-2.06 (Wmona Post)
1,060-904,000 (North Carolina)
2.38-3. 15 (Midland)
1.03-8.37 (Titta. River soil)
41. 0-1, 140 (PE)
0.00385-0.051 (PE)
0.253-0.318 (Midland)
0.1 35-2.08 (Extracts)
3.53-9.62 (PE)
1.14-1.33 (Titta. River Soil)
0.1 63-37.0 (Nitro)
29.8-73.6 (Sagmaw River)
40.1^2.1 (PE)
0.000103-1,080 (Extracts)
435-1, 160 (PE)
0.388-0.452 (Nitro)
0.0467 (Saginaw River)
0.654-1.87 (WinonaPost)
0.00300-0.0420 (PE)
Comments
PCB 77 slightly high, but all samples >20x
blank levels.
PCB 77 slightly high. Did not report any
samples where PCB 77 was <10x blank. No
significant effect on total TEQ.
PCB 77 and 156 high, but all samples >20x
blank levels.
PCB 77 slightly high. Does not affect total
TEQ.
PCB 77 slightly high. Sample TEQs much
greater than blank TEQ.
PCBs77, 123, 126, 156, 167, and 118 high,
but most samples significantly > 20x blank
levels.
PCBs 77 and 1 18 high, but all samples >20x
blank levels.
PCB 77 slightly high. Does not affect total
TEQ.
PCB 77 high. PE certified blanks Ref 85, Ref
85 duplicate, and Ref 108 were the only
samples where PCB 77 was not >20x blank.
TEQs for these certified blanks were
considered low enough to be distinguished as
blank samples and were accepted.
1 All nondetect and EMPC values were assigned a zero concentration for the TEQ calculation.
b "Ref XX" is a reference laboratory sample ID number.
C-3
-------
Table C-2. Sample Batch Duplicate Summary
Sample Batch
Number
D/FWG12107
D/F WG12148
D/F WG 12264
D/F WG12534
D/F WG 12641
D/F WG12737
D/F WG 12804
D/F WG 13 547
D/FWG13548
D/F WG13549
D/F WG13551
D/FWG13552
D/F WG 13 984
D/F WG 14274
PCBWG12108
Criteria
Met
N
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
N
N
Duplicate RPDa
(%)
23
2.1
1.2
5.7
4.6
14
none
16
5.9
3.6
0.0
20
(onU=l/2DLbasisb)
3.4
54
22
Comments
L6744-5, Ref 100 Newark Bay
Because this was above the 20% criteria, an additional
aliquot of this sample was prepared. Results for the
additional aliquot were within 1 1 % RPD from the original
results; therefore, this duplicate result was accepted.
L6744-9, Ref 122 Newark Bay
L6760-2,Ref27PE
L6760-14, RefSSPE
L6747-1, Ref 32 Midland
L6750-3, Ref 78 Tittabawassee River Soil
The duplicate processed with this batch was to be repeated
due to some analytes being <20x blank level. However, it
was reprocessed as a single sample and not a duplicate.
Samples in this set were accepted based on their agreement
with other replicates within the demonstration program.
L7 163-1, Ref 26 Nitro
L6751-14, Ref 83 North Carolina
L6751-7, Ref 135 North Carolina
L675 1 - 1 , Ref 42 North Carolina
L7179-3, Ref 74 PE. Fails on a U=0 DL basis due to
presence of "K" flagged analytes in one replicate. When
compared on U-l/2 DL basis where "K" concentrations are
included in the TEQ calculation, the duplicate passed.
L7179-14,Ref 113PE
L7179-16, Ref 124PE
This was a PCB PE sample and contained only trace levels
of D/F. Replicate precision is affected because D/F content
is so low. This is not expected to indicate any problems
with precision within this sample set. Samples in this set
were accepted based on their agreement with other
replicates within the demonstration program.
L6744-2, Ref 49 Newark Bay
This result is only slightly above the acceptance criteria of
20%. The variability was influenced by 25% RPD for
PCB 126 (which has the highest TEF of the PCBs and,
therefore, a larger influence on total TEQ). The slight
exceedance in duplicate criteria was not considered to have
any significant impact on the data reported in this sample
batch. All samples in this set were also evaluated based on
their agreement with other replicates within the
demonstration program and deemed to be acceptable.
C-4
-------
Sample Batch
Number
PCBWG12147
PCB WG12265
PCB WG12457
PCB WG12687
PCB WG12834
PCB WG12835
PCB WG12836
PCB WG1 3008
PCBWG13256
PCBWG13257
PCBWG13258
PCB WG13554
PCBWG14109
Criteria
Met
N
Y
N
Y
Y
N
Y
Y
Y
Y
Y
Y
N
Duplicate RPDa
(%)
none
2.5
none
4.3
4.2
none
2.6
5.1
1.7
(on U=1/2DL basis)
15
19
12
85
(on U=1/2DL basis)
Comments
L6748-9, Ref 129 Brunswick
The duplicate sample for this batch required reprocessing.
When reprocessed, it was not prepared in duplicate.
Samples in this set were accepted based on the RPD of site
replicates that were processed within the batch (RPDs
<10%).
L6760-5, Ref35PE
L6760-16, Ref62PE
This duplicate set was to be repeated due to low internal
standard recovery. When repeated, it was not prepared in
duplicate. Data for this set was accepted because all
samples in the set were PE samples. These PE samples met
accuracy criteria and reproducibility criteria to other
replicates of the same PE material processed within the
demonstration.
L6762-12, Ref 169PE
L6750-8, Ref 164 Tittabawassee River Soil
Duplicate sample repeated in WG13258. Results reported
with that sample set. Three sets of sample replicates within
this batch were also compared and found to have <13.5%
RPD showing acceptable precision with this sample set.
L6751-6, Ref 126 North Carolina
L6750-6, Ref 121 Tittabawassee River Soil
L6761-3, Ref 74 PE. Fails on a U=0 DL basis due to
presence of "K" flagged analytes in one replicate. When
compared on U=l/2 DL basis where "K" concentrations are
included in the TEQ calculation, the duplicate passed.
L7 187-5, Ref 92 Tittabawassee River Soil
L6743-2,Ref36Nitro
L6762-l,Ref202PE
L7179-4, PE. Fails based on both U=0 and U=l/2 DL.
This was a blank PE sample and contained only trace levels
of PCBs. Replicate precision is affected because the PCB
content is so low. This is not expected to indicate any
problems with precision within this sample set. Samples in
this set were accepted based on their agreement with other
replicates within the demonstration program.
1 Nondetects were assigned a concentration of zero unless otherwise noted and are referred to as U=0 DL values.
b U=l/2 DL indicates that nondetects were assigned a concentration equal to one-half the SDL and EMPC concentrations were assigned a value
equal to the EMPC.
C-5
-------
-------
Appendix D
Summary of Developer and Reference Laboratory Data
-------
-------
Appendix D. Hybrizyme and Reference Laboratory One-to-One Matching3
Sample Type
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Sample
Number
HYB 163
HYB 194
HYB 93
HYB 203
HYB 116
HYB 72
HYB 135
HYB 104
HYB 52
HYB 197
HYB 94
HYB 151
HYB 44
HYB 119
HYB 102
HYB 170
HYB 34
HYB 134
HYB 124
HYB 175
HYB 155
HYB 86
HYB 168
HYB 48
HYB 68
HYB 80
HYB 71
HYB 157
HYB 199
HYB 49
HYB 202
HYB 59
HYB 178
HYB 110
HYB 146
HYB 121
Measurement
Location1"
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Sample Description
Brunswick #1
Brunswick #1
Brunswick #1
Brunswick #1
Brunswick #2
Brunswick #2
Brunswick #2
Brunswick #2
Brunswick #3
Brunswick #3
Brunswick #3
Brunswick #3
Midland #1
Midland #1
Midland #1
Midland #1
Midland #2
Midland #2
Midland #2
Midland #2
Midland #3
Midland #3
Midland #3
Midland #3
Midland #4
Midland #4
Midland #4
Midland #4
NC PCB Site #1
NC PCB Site #1
NC PCB Site #1
NC PCB Site #1
NC PCB Site #2
NC PCB Site #2
NC PCB Site #2
NC PCB Site #2
REP
1
2
o
J
4
1
2
o
J
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
o
J
4
1
2
3
4
1
2
3
4
1
2
3
4
Developer0
AhRBU
1154
895
1249
977
658
567
812
778
165375
156894
209582
190299
371
457
548
493
1416
1363
1389
1429
1119
1372
1422
1089
72
89.3
109
104
18473
15485
20356
25156
115702
96224
114690
112695
Reference Laboratory11
TEQD/F
(pg/g)
67.2
71.6
61.7
67.8
49.5
72.8
56
60.4
12600
15200
13100
13600
222
241
269
268
208
179
197
192
185
174
176
161
25.7
26.4
31
25.8
788
1100
852
906
3400
3300
3430
3490
Total TEQ
(pg/g)e
67.51
71.94
62.07
68.11
49.63
72.93
56.13
60.52
12600.19
15200.18
13100.20
13600.18
224.59
243.73
271.5
270.53
210.7
181.81
199.48
195.15
187.28
176.17
178.23
163.38
25.95
26.72
31.97
26.063
53788
66400
81352
86006
314400
308300
213430
364490
Total Concentration
(ng/g)f
22513
22513
22513
22513
9818
9818
9818
9818
1210432
1210432
1210432
1210432
1150
1150
1150
1150
2531
2531
2531
2531
1821
1821
1821
1821
147
147
147
147
41960
41960
41960
41960
340394
340394
340394
340394
D-l
-------
Sample Type
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Sample
Number
HYB89
HYB81
HYB43
HYB63
HYB 128
HYB 130
HYB 142
HYB 112
HYB 141
HYB 140
HYB 164
HYB 165
HYB 91
HYB 118
HYB 162
HYB 47
HYB 132
HYB 37
HYB 182
HYB 159
HYB 145
HYB 40
HYB 35
HYB 139
HYB 183
HYB 50
HYB 136
HYB 190
HYB 166
HYB 113
HYB 147
HYB 161
HYB 171
HYB 186
HYB 67
HYB 87
HYB 201
HYB 58
Measurement
Location1"
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Sample Description
NC PCB Site #3
NC PCB Site #3
NC PCB Site #3
NC PCB Site #3
Newark Bay #1
Newark Bay #1
Newark Bay #1
Newark Bay #1
Newark Bay #2
Newark Bay #2
Newark Bay #2
Newark Bay #2
Newark Bay #3
Newark Bay #3
Newark Bay #3
Newark Bay #3
Newark Bay #4
Newark Bay #4
Newark Bay #4
Newark Bay #4
RaritanBay #1
RaritanBay #1
RaritanBay #1
RaritanBay #1
Raritan Bay #2
Raritan Bay #2
Raritan Bay #2
Raritan Bay #2
Raritan Bay #3
Raritan Bay #3
Raritan Bay #3
Raritan Bay #3
Saginaw River #1
Saginaw River #1
S aginaw River #1
Saginaw River #1
Saginaw River #2
Saginaw River #2
REP
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
Developer0
AhRBU
221536
233517
314727
235576
1930
1525
2986
2824
5486
3299
3046
3869
4318
5015
4630
4191
2640
3454
3694
3493
5698
5278
5278
5536
6964
4573
4706
8116
5977
8930
6832
5129
4666
8848
8069
7473
4048
3872
Reference Laboratory11
TEQD/F
(Pg/g)
8320
8410
9360
10800
23
14
14.5
13.5
50.6
47.4
74.1
50.4
38.9
44.9
40.2
41.9
33.6
26.1
27.6
26.8
10.2
10.3
10.4
11.4
13.3
13.1
12.8
13
10.4
11.1
10.6
9.93
1050
683
1070
694
1110
953
Total TEQ
(Pg/g)e
856320
626410
542360
914800
24.22
15.44
15.89
14.84
55.61
52.59
79.24
55.49
43.51
49.94
44.7
46.93
36.33
28.75
30.32
29.5
12.53
12.36
12.75
13.65
16
15.77
15.48
15.85
12.83
13.53
12.9
12.26
1112.4
756.6
1139.9
757.7
1140.6
984
Total Concentration
(ng/g)f
869927
869927
869927
869927
2348
2348
2348
2348
4046
4046
4046
4046
8030
8030
8030
8030
4906
4906
4906
4906
3850
3850
3850
3850
7376
7376
7376
7376
4465
4465
4465
4465
6188
6188
6188
6188
5474
5474
D-2
-------
Sample Type
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Sample
Number
HYB 200
HYB 189
HYB 174
HYB 30
HYB 126
HYB 51
HYB 193
HYB 152
HYB 154
HYB 106
HYB 46
HYB 198
HYB 96
HYB 90
HYB 187
HYB 38
HYB 185
HYB 148
HYB 73
HYB 41
HYB 53
HYB 120
HYB 208
HYB 78
HYB 97
HYB 60
HYB 123
HYB 61
HYB 184
HYB 56
HYB 99
HYB 108
HYB 33
HYB 24
HYB 153
HYB 133
HYB 158
HYB 64
Measurement
Location1"
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Sample Description
Saginaw River #2
Saginaw River #2
Saginaw River #3
Saginaw River #3
Saginaw River #3
Saginaw River #3
Solutia#l
Solutia#l
Solutia#l
Solutiajl
Solutia #2
Solutia #2
Solutia #2
Solutia #2
Solutia #3
Solutia #3
Solutia #3
Solutia #3
Titta. River Soil #1
Titta. River Soil #1
Titta. River Soil #1
Titta. River Soil #1
Titta. River Soil #2
Titta. River Soil #2
Titta. River Soil #2
Titta. River Soil #2
Titta. River Soil #3
Titta. River Soil #3
Titta. River Soil #3
Titta. River Soil #3
Titta. River Sed #1
Titta. River Sed #1
Titta. River Sed #1
Titta. River Sed #1
Titta. River Sed #2
Titta. River Sed #2
Titta. River Sed #2
Titta. River Sed #2
REP
3
4
1
2
o
5
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
o
J
4
1
2
3
4
1
2
o
J
4
1
2
3
4
1
2
o
J
4
Developer0
AhRBU
3329
3872
129
126
113
122
154
149
121
197
873
464
899
1058
790
329
430
346
501
630
728
567
354
518
357
428
110
106
144
138
206
236
213
144
185
305
139
241
Reference Laboratory11
TEQD/F
(Pg/g)
1320
864
99.7
146
122
99.6
57.5
76.9
62
61.6
2090
1950
1860
2160
2810
2800
3000
3080
35
35.2
40
35.8
420
450
523
506
1050
676
1220
1300
1.05
1.11
1
1.7
52.8
123
66.1
94.1
Total TEQ
(Pg/g)e
1346.7
893.8
99.72
146.02
122.05
99.62
57.95
77.06
62.39
61.99
2107.6
1968.8
1879.2
2178.5
2839.7
2836.9
3037
3111.5
42.32
43.46
47.57
44.17
420.99
451.2
524.03
507.06
1051.26
677.16
1221.54
1301.33
1.10
1.14
1.04
1.74
53.45
123.71
66.67
94.62
Total Concentration
(ng/g)f
5474
5474
770
770
770
770
436
436
436
436
2789
2789
2789
2789
1365
1365
1365
1365
613
613
613
613
831
831
831
831
268
268
268
268
271
271
271
271
693
693
693
693
D-3
-------
Sample Type
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Environmental
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Extract
Sample
Number
HYB88
HYB 149
HYB 101
HYB 137
HYB 42
HYB 27
HYB 156
HYB 143
HYB 195
HYB 84
HYB 179
HYB 75
HYB 65
HYB 181
HYB 205
HYB 177
HYB 13
HYB 18
HYB 22
HYB 12
HYB 4
HYB 11
HYB 17
HYB 7
HYB 5
HYB 8
HYB 21
HYB 14
HYB 9
HYB 2
HYB 10
HYB 19
HYB 15
HYB 1
HYB 20
HYB 6
HYB 3
HYB 23
Measurement
Location1"
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Sample Description
Titta. River Sed #3
Titta. River Sed #3
Titta. River Sed #3
Titta. River Sed #3
WinonaPost#l
WinonaPost#l
WinonaPost#l
WinonaPost#l
Winona Post #2
Winona Post #2
Winona Post #2
Winona Post #2
Winona Post #3
Winona Post #3
Winona Post #3
Winona Post #3
Envir. Extract #1
Envir. Extract #1
Envir. Extract #1
Envir. Extract #1
Envir. Extract #2
Envir. Extract #2
Envir. Extract #2
Envir. Extract #2
Spike #1
Spike #1
Spike #1
Spike #1
Spike #1
Spike #1
Spike #1
Spike #2
Spike #2
Spike #2
Spike #2
Spike #3
Spike #3
Spike #3
REP
1
2
o
J
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
o
J
4
1
2
3
4
1
2
o
J
4
5
6
7
1
2
o
J
4
1
2
3
Developer0
AhRBU
74
105
55
103
42535
30739
34759
27185
79297
64166
67060
82873
57214
86611
68254
90517
1408
1603
2041
1895
2480
2260
2550
2959
ND
ND
ND
ND
ND
ND
ND
1.77
2.25
2.31
2.43
10.83
9.62
13.2
Reference Laboratory11
TEQD/F
(Pg/g)
13
11.2
12.7
13.8
7290
7370
7450
7160
9720
9770
9200
11300
10300
9770
9320
9870
175
444
176
439
55.3
53.3
53.1
53.6
0.504
0.509
0.537
0.524
0.585
0.576
0.52
91.6
91.8
89.1
100
0.324
0.348
0.363
Total TEQ
(Pg/g)e
13.07
11.30
12.78
13.89
7290.65
7370.90
7450.83
7160.82
9721.2
9771.3
9201.32
11301.28
10301.68
9771.87
9321.8
9872.06
175.63
444.67
176.64
441.08
56.04
53.44
53.40
53.77
0.568
0.509
0.537
0.552
0.641
0.583
0.659
204.6
204.8
200.1
213
1060.32
1080.35
1060.36
Total Concentration
(ng/g)f
77
77
77
77
31343
31343
31343
31343
53038
53038
53038
53038
53819
53819
53819
53819
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
D-4
-------
Sample Type
Extract
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Sample
Number
HYB 16
HYB 172
HYB 191
HYB 127
HYB 160
HYB 76
HYB 176
HYB 114
HYB 85
HYB 207
HYB 169
HYB 77
HYB 196
HYB 54
HYB 100
HYB 25
HYB 29
HYB 79
HYB 95
HYB 122
HYB 144
HYB 83
HYB 206
HYB 109
HYB 188
HYB 82
HYB 69
HYB 39
HYB 209
HYB 62
HYB 28
HYB 74
HYB 150
HYB 204
HYB 66
HYB 36
HYB 103
HYB 45
Measurement
Location1"
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Sample Description
Spike #3
Cambridge 5183
Cambridge 5183
Cambridge 5183
Cambridge 5183
Cambridge 5 183
Cambridge 5 183
Cambridge 5 183
Cambridge 5 1 84
Cambridge 5 1 84
Cambridge 5 1 84
Cambridge 5 1 84
ERA Aroclor
ERA Aroclor
ERA Aroclor
ERA Aroclor
ERA Blank
ERA Blank
ERA Blank
ERA Blank
ERA Blank
ERA Blank
ERA Blank
ERA Blank
ERA PAH
ERA PAH
ERA PAH
ERA PAH
ERAPCB 100
ERAPCB 100
ERAPCB 100
ERAPCB 100
ERAPCB 10000
ERAPCB 10000
ERAPCB 10000
ERAPCB 10000
ERATCDD 10
ERATCDD10
REP
4
1
2
o
J
4
5
6
7
1
2
3
4
1
2
3
4
1
2
o
J
4
5
6
7
8
1
2
3
4
1
2
3
4
1
2
3
4
1
2
Developer0
AhRBU
9.62
82.5
80.9
46.8
93.1
33.1
46.4
72.1
18355
16597
19570
15710
24.4
52
52.2
44.9
23.2
44.1
49.6
218
40.3
110
41.3
85.7
2788
1661
2112
1739
36.2
32
77
38.2
33.8
56.9
32.3
25.5
21.7
118
Reference Laboratory11
TEQD/F
(Pg/g)
0.268
4.78
4.08
4.06
3.56
3.89
5.93
3.89
187
188
173
180
36.4
32.9
37.9
35.5
0.0942
0.0728
0.237
0.307
0.113
0.0524
0.211
0.0692
0.159
0.141
0.161
0.248
0.0386
NAg
0.053
0.127
0.204
0.507
0.105
0.0628
8.69
9.28
Total TEQ
(Pg/g)e
990.27
8.59
8.41
8.26
7.8
8.14
9.79
7.42
1267
1308
1313
1340
1096.4
3722.9
3827.9
3835.5
0.119
0.077
0.240
0.349
0.136
0.072
0.244
0.092
0.184
0.145
0.165
0.274
10.64
NAg
10.65
10.08
1030.20
1030.51
1180.11
1020.06
8.70
9.29
Total Concentration
(ng/g)f
NA
193
193
193
193
193
193
193
31879
31879
31879
31879
18893
18893
18893
18893
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
61170
61170
61170
61170
1
1
1
1
120
120
120
120
20
20
D-5
-------
Sample Type
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Performance
Sample
Number
HYB 107
HYB 173
HYB 98
HYB 105
HYB 125
HYB 1 1 1
HYB 92
HYB 180
HYB 32
HYB 117
HYB 57
HYB 70
HYB 131
HYB 167
HYB 138
HYB 115
HYB 192
HYB 55
HYB 26
HYB 129
HYB 31
Measurement
Location1"
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Laboratory
Sample Description
ERATCDD 10
ERATCDD 10
ERA TCDD 30
ERA TCDD 30
ERA TCDD 30
ERA TCDD 30
LCG CRM-529
LCG CRM-529
LCG CRM-529
LCG CRM-529
NIST 1944
NIST 1944
NIST 1944
NIST 1944
Wellington WMS-01
Wellington WMS-01
Wellington WMS-01
Wellington WMS-01
Wellington WMS-01
Wellington WMS-01
Wellington WMS-01
REP
3
4
1
2
3
4
1
2
3
4
1
2
o
5
4
1
2
3
4
5
6
7
Developer0
AhRBU
33.1
27.6
26.5
211
31.4
49.1
702
867
671
813
38927
41758
65899
59314
902
1017
777
997
754
1292
1048
Reference Laboratory11
TEQD/F
(Pg/g)
8.44
8.2
27.4
25.3
24.8
23.9
NAg
6930
6900
7190
237
206
252
219
68
65.7
61.9
66.1
68
65.7
65.4
Total TEQ
(Pg/g)e
8.50
8.24
27.45
25.32
24.84
23.94
NAg
7335
7398
7546
277.1
249.7
294.1
260
78.6
75.1
71.52
75.17
78.3
75.32
75.08
Total Concentration
(ng/g)f
20
20
60
60
60
60
NA
NA
NA
NA
2481
2481
2481
2481
NA
NA
NA
NA
NA
NA
NA
a Due to the state of development of the Hybrizyme technology, which is intended to rank samples by concentration of AhR binding compounds, the Hybrizyme results were not
compared to HRMS TEQ values in this report. The HRMS TEQ data are provided in this appendix for convenient reference and document consistency.
b 110 samples were analyzed in the field by Hybrizyme, but the sample results were repeated and reported from laboratory analysis.
0 Data listed exactly as reported by the developer.
d Qualifier flags (e.g., J and K flags) included in the raw data have been removed for the purposes of statistical analysis.
e Data calculated by summing TEQPCB and TEQD/F.
' Total concentration values for environmental samples are the sum of reference laboratory total D/F concentration, reference laboratory total PCB concentration and PAH
concentration. Total concentration values for PE samples were calculated using certified data. Samples where PAH data were not available are listed as "NA."
g Reference laboratory data was discarded due to laboratory sample preparation error.
ND = nondetect
D-6
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