EFW
United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/R-04/036
April 2004
Demonstration and Quality
Assurance Project Plan
Technologies for the
Monitoring and Measurement
of Dioxin and Dioxin-like
Compounds in Soil and
Sediment
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EPA/600/R-04/036
April 2004
Demonstration and Quality
Assurance Project Plan
Technologies for the Monitoring and
Measurement of Dioxin and Dioxin-like
Compounds in Soil and Sediment
Prepared by
Battelle Memorial Institute
505 King Avenue
Columbus, Ohio 43201
Contract No. 68-C-OO-l85
Dr. 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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Concurrence Signatures
The primary purpose of the demonstration is to evaluate measurement technologies for dioxin in soil and
sediment based on their performance and cost as compared to conventional, off-site laboratory analytical
methods. The demonstration will take place under the sponsorship of the U.S. Environmental Protection
Agency Superfund Innovative Technology Evaluation Program.
This document is intended to ensure that all aspects of the demonstration are documented and
scientifically sound and that operational procedures are conducted in accordance with quality assurance
and quality control specifications and health and safety regulations.
The signatures of the individuals specified below indicate their concurrence and agreement to operate in
compliance with the procedures specified in this document.
Bille
Stephen Billets
EPA
Project Manager
Date
eorae'.
Geoi'ge Brilis
EPA
Quality Assurance Manager
Date
Brian Schumacher
EPA
Branch Chief
Date
Amy Dindal
Battelle
Project Manager
Date
f 7 I
Randy'Allen
Hybrizyme Corporation
Developer
¥^r
Date
Gordon
raobiotic Detection Systems, Inc.
Developer
Date
Ffernando Rubio
Abraxis, LLC
Developer
Date
Noriyoshi Inoue
Paracelsian, Inc.
Developer
Date
C//V/ J
Y7/H-
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Tpchlry Willenrjerg
ESatt/lle
lity Assurance Manager
Date
Battelle
ES&H Representative
Sue Kaelber-Matlock Date
Michigan Department of Environmental Quality
Demonstration Site Coordinator
Dale Hoover
AXYS Analytical Services Ltd.
QA Vice President
-
TDate
o!-( /C/gft
Bob Harrison
CAPE Technologies L.L.C.
Developer
Date
Masako Hayakawa
Wako Pure Chemical Industries, Ltd.
Developer
Date
Becky Goche
U.S. Fish and Wildlife Service
Demonstration Site Coordinator
Date
in
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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.
IV
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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 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 (RCRA) 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 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 Monitoring and
Measurement Technology Program is managed by the Office of Research and Development's
Environmental Sciences Division in Las Vegas, Nevada.
Gary Foley, Ph.D.
Director
National Exposure Research Laboratory
Office of Research and Development
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Abstract
A demonstration of technologies for determining the presence of dioxin and dioxin-like compounds in
soil and sediment will be conducted under the U.S. Environmental Protection Agency's Superfund
Innovative Technology Evaluation Program in Saginaw, Michigan, at Green Point Environmental
Learning Center from April 26 to May 5, 2004. The primary purpose of the demonstration is to evaluate
innovative monitoring and measurement technologies. The technologies listed below will be
demonstrated.
• AhRC PCR™ Kit, Hybrizyme Corporation
• Ah-IMMUNOASSAY® Kit, Paracelsian, Inc.
Coplanar PCB Immunoassay Kit, Abraxis LLC
• DF-1 Dioxin/Furan Immunoassay Kit, CAPE Technologies L.L.C.
• CALUX® by Xenobiotic Detection Systems, Inc.
• Dioxin ELISA Kit, Wako Pure Chemical Industries, Ltd.
This demonstration plan describes the procedures that will be used to verify the performance and cost of
these technologies. The plan incorporates the quality assurance and quality control elements needed to
generate data of sufficient quality to document each technology's performance and cost. A separate
innovative technology verification report (ITVR) will be prepared for each technology. The ITVRs will
present the demonstration findings associated with the demonstration objectives.
VI
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Contents
Chapter Page
Concurrence Signatures ii
Notice iv
Foreword v
Abstract vi
Abbreviations, Acronyms, and Symbols xii
Acknowledgments xvi
Executive Summary xvii
1 Introduction 1
1.1 Description of the SITE Program 1
1.2 Scope of Demonstration 4
1.3 Definition of Dioxin and Dioxin-Like Compounds and Toxicity Equivalents 5
1.4 Sources of Dioxins and Furans 8
1.5 Traditional Methods for Measurement of Dioxin and Dioxin-Like Compounds in
Soil and Sediment 8
1.5.1 High-Resolution Mass Spectrometry 8
1.5.2 Low-Resolution Mass Spectrometry 8
1.5.3 PCB Methods 9
1.5.4 Summary of Analytical Methods for Dioxins and Dioxin-like Compounds 9
2 Demonstration Organization and Responsibilities 10
2.1 EPA Project Personnel 10
2.2 Battelle Project Personnel 10
2.3 Developer Personnel 14
2.4 Demonstration Site Representatives 15
2.5 Reference Laboratory Personnel 15
2.6 Suppliers of Performance Evaluation Samples 15
3 Developer Technology Descriptions 16
3.1 CAPE Technologies DF1 Dioxin/Furan Immunoassay Kit 16
3.1.1 Technology Description 16
3.1.2 Operating Procedure 18
3.1.3 Advantages and Limitations 19
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Contents (Continued)
Chapter Page
3.2 Hybrizyme Corporation AhRC PCR™ Kit 20
3.2.1 Technology Description 20
3.2.2 Operating Procedure 21
3.2.3 Advantages and Limitations 22
3.3 Paracelsian, Inc., Ah-IMMUNOASSAY® 23
3.3.1 Technology Description 23
3.3.2 Operating Procedure 24
3.3.3 Advantages and Limitations 26
3.4 Abraxis LLC Coplanar PCB ELISA Kit 27
3.4.1 Technology Description 27
3.4.2 Operating Procedure 28
3.4.3 Advantages and Limitations 30
3.5 Wako Dioxin ELISA Kit 30
3.5.1 Technology Description 30
3.5.2 Operating Procedure 31
3.5.3 Advantages and Limitations 33
3.6 Xenobiotic Detection Systems, Inc., CALUX® 33
3.6.1 Technology Description 34
3.6.2 Operating Procedure 34
3.6.3 Advantages and Limitations 35
4 Sample Collection, Sample Homogenization, and Sample Characterization 36
4.1 Sample Collection 36
4.1.1 Procedure 36
4.1.2 Sample Shipping to and Archival at Battelle 37
4.2 Homogenization of Environmental Samples 37
4.2.1 Criteria for Determining Adequate Homogenization 37
4.3 Characterization of Environmental Samples 38
4.3.1 Dioxins and furans 38
4.3.2 PCBs 39
4.3.3 PAHs 39
4.4 Sample Handling, Sample Tracking, and Sample Management 39
5 Descriptions of Demonstration Site and Sampling Locations 41
5.1 Demonstration Site Description 41
5.2 Description of Sampling Locations 42
5.2.1 Soil Sampling Locations 42
5.2.
5.2.
5.2.
5.2.
5.2.
. 1 Warren County, North Carolina 42
.2 Tittabawassee River Flood Plain 43
.3 Midland, Michigan 43
.4 Winona Post 43
.5 Solutia 44
Vlll
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Contents (Continued)
Chapter Page
5.2.2 Sediment Sampling Sites 44
5.2.2.1 New York/New Jersey Harbors 44
5.2.2.2 Tittabawassee River Sediments 45
5.2.2.3 Saginaw River Sediments 45
5.2.2.4 Brunswick Wood Preserving Site 46
6 Demonstration Approach 47
6.1 Demonstration Objectives 47
6.2 Overview of Demonstration Samples 48
6.2.1 PE Samples 49
6.2.2 Environmental Samples 50
6.2.3 Extracts 50
6.3 Pre-Demonstration Study 51
6.4 Demonstration Schedule 52
6.5 Demonstration Design 53
6.6 Assessment of Primary and Secondary Objectives 57
6.6.1 Primary Objective PI: Accuracy 58
6.6.2 Primary Objective P2: Precision 59
6.6.3 Primary Objective P3: Comparability 59
6.6.4 Primary Objective P4: Method Detection Limit 60
6.6.5 Primary Objective P5: False Positive/False Negative Results 60
6.6.6 Primary Objective P6: Matrix Effects 61
6.6.7 Primary Objective P7: Technology Costs 61
6.6.8 Secondary Objective S1: Skills and Training Required to Properly Operate
the Technology 61
6.6.9 Secondary Objective S2: Document Health and Safety Aspects Associated with
the Technology 62
6.6.10 Secondary Objective S3: Documentthe Portability of the Technology 62
6.6.11 Secondary Objective S4: Evaluate Sample Throughput 62
6.7 Schedule of Events 62
7 Confirmatory Process 64
7.1 Reference Method Selection 64
7.2 Reference Laboratory Selection 64
7.3 Reference Laboratory Sample Preparation and Analytical Methods 65
7.3.1 Dioxin/Furan Analysis 65
7.3.2 PCB Analysis 68
7.3.3 Options for High-Level Samples 74
8 Data Management 75
8.1 Data Reduction 75
8.2 Data Review 75
IX
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Contents (Continued)
Chapter Page
8.2.1 Data Review by Developers 75
8.2.2 Data Review by Reference Laboratory 76
8.2.3 Data Review by Battelle 76
8.3 Data Reporting 76
8.3.1 Developer Data Packages 76
8.3.2 Reference Laboratory Data Packages 77
8.3.3 Innovative Technology Verification Reports 77
8.4 Data Evaluation Report 77
9 QA/QC Procedures 79
9.1 QA/QC Objectives 79
9.2 Internal QC Checks 79
9.2.1 Reference Method QC Checks 79
9.2.2 Developer Technology QC Checks 83
9.3 Audits, Corrective Actions, and QA Reports 83
9.3.1 Technical Systems Audits 83
9.3.2 Corrective Action Procedures 84
9.3.3 QA Reports 84
10 Health and Safety Plan 86
11 References 108
Addendum
Appendix A. List of Demonstration Panel Members
Appendix B. Request for and Obtaining of Special Use Permit
Appendix C. Example Cover Letter for Sample Collection
Appendix D. Example Chain of Custody/Results Form
Appendix E. Observer Checklists
Appendix F. AXYS Analytical Services Statement of Work
Appendix G. Questionnaire for Reference Laboratory Selection
Appendix H. Method 1613b and Method 1668a Tables
Appendix I. Reference Laboratory Procedures for Data Review
Appendix J. Data Validation Checklists for Reference Laboratory Data Review
Appendix K. Reference Laboratory Procedures for Data Package Preparation
Appendix L. Work Plan for Reference Laboratory In-Process Audit
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Contents (Continued)
Figures
1-1 Structure of representative dioxin, furan, and PCB 6
2-1 Organization chart for the dioxin demonstration 11
3-1 CAPE Technologies DF1 Dioxin/Furan Immunoassay Kit 16
3-2 DPI Immunoassay Kit analytical options 18
3-3 Hybrizyme's AhRC-PCR Test Kit 21
3-4a ABI Prism 7000 22
3-4b Cepheid Smart Cycler 22
3-5 Paracelsian, Inc., Ah-IMMUNOASSAY® 23
3-6 Abraxis PCB ELISA Kit 27
3-7 Wako Dioxin ELISA Kit procedure 31
3-8 Xenobiotic Detection Systems parented sample processing procedure 35
3-9 Luminescence produced when CALUX® cells are exposed to dioxin and
dioxin-like chemicals 35
6-1. Approximate distribution of environmental sample concentrations 49
6-2. Approximate range of pre-demonstration soil/sediment sample concentrations 51
6-3. Estimated participant schedule for field component of demonstration 52
6-4. Estimated participant schedule for laboratory component of demonstration 53
Tables
1 -1. Toxicity Equivalency Factor Values 7
1-2. Calibration Range of HRMS Dioxin/Furan Method 8
1-3. Calibration Range of LRMS Dioxin/Furan Method 9
2-1. Demonstration Participants 12
5-1. Summary of Environmental Sampling Locations 46
6-1. Distribution of Samples for the Evaluation of Performance Objectives 48
6-2. Proposed Number and Type of Samples to be Analyzed in the Demonstration 49
6-3. Designation for Generic Data Qualifiers 54
6-4. Distribution of Performance Evaluation Samples 54
6-5. Distribution of Environmental Samples 55
6-6. Distribution of Extract Samples 56
6-7. Comparison Between Developer and Reference Laboratory Data 58
6-8. Schedule of Demonstration Events 63
9-1. Data Quality Indicator Objectives for Reference Laboratory and Developer Data 79
9-2. QC Acceptance Criteria for EPA Method 1613B 80
9-3. Method 1613B Specifications for QC Samples, Instrumental Analysis, and Analyte
Quantification 81
9-4. Method 1668A QC Acceptance Criteria for Chlorinated Biphyenyls in CAL/VER,
IPR, OPR, and Samples 81
9-5. Method 1668A Specifications for QC Samples, Instrumental Analysis, and
Analyte Quantification 83
XI
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Abbreviations, Acronyms, and Symbols
AhR
ARNT
ASE
ATSDR
CALUX®
CAL/VER
coc
DEQ
DER
D/F
DMF
DMSO
DNA
DNR
ORE
EIA
ELC
ELISA
EMPC
EPA
fp
fn
g
GC
aryl hydrocarbon receptor
aryl hydrocarbon receptor nuclear translocator
accelerated solvent extraction
Agency for Toxic Substances and Disease Registry
Chemical-Activated Luciferase Expression®
Calibration verification test run
chain of custody
dioxin equivalent
data evaluation report
dioxin/furan
dimethylformamide
dimethyl sulfoxide
deoxyribonucleic acid
Department of Natural Resources
dioxin responsive elements
enzyme immunoassay
Environmental Learning Center
enzyme-linked immunosorbent assay
estimated maximum possible concentration
Environmental Protection Agency
false positive
false negative
gram
gas chromatography
Xll
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HOPE
HPLC/GPC
HRGC
HRMS
HRP
H&S
IPR
I-TEF
ITVR
LDPE
LRMS
HL
mL
MDEQ
mm
MDL
MMT
MS
ND
NERL
nm
NOAA
OD
OPR
ORD
PAH
PAR
PC
PCB
high-density polyethylene
high-performance liquid chromatography/gel permeation chromatography
high-resolution capillary gas chromatography
high-resolution mass spectrometry
horseradish peroxidase
health and safety
initial precision and recovery
International Toxicity Equivalency Factor
innovative technology verification report
low-density polyethylene
low-resolution mass spectrometry
microliter
milliliter
Michigan Department of Environmental Quality
millimeter
method detection limit
Monitoring and Measurement Technology
mass spectrometry
non-detect
National Exposure Research Laboratory
nanometer
National Oceanic and Atmospheric Administration
optical density
ongoing precision and recovery
Office of Research and Development
polycyclic aromatic hydrocarbons
Precision and Recovery Standard
positive control
polychlorinated biphenyl
Xlll
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PCDD/F
PCDH
PCP
PCR
PE
Pg
POD-conjugate
ppm
ppb
ppt
QA/QC
RPD
rpm
RSD
SDL
SIM
SITE
S:N
SOP
TCDD
TEF
TEG
TEQ
TOC
total TEQD/F
total TEQPCB
total TEQ
TSA
polychlorinated dibenzo-p-dioxin/dibenzofuran
polychlorinated diaromatic hydrocarbon
pentachlorophenol
poplymerase chain reaction
performance evaluation
picogram
peroxides conjugated with a dioxin analog
parts per million; microgram/g; |ig/g
parts per billion; nanogram/g; ng/g
parts per trillion; picogram/g; pg/g
quality assurance/quality control
relative percent difference
revolution per minute
relative standard deviation
sample-specific detection limit
selected ion monitoring
Superfund Innovative Technology Evaluation
signal to noise ratio
standard operating procedure
tetrachlorodibenzo-p-dioxin
toxicity equivalency factor
tetraethylene glycol
toxicity equivalent
total organic carbon
total toxicity equivalents of dioxins/furans
total toxicity equivalents of World Health Organization polychlorinated
biphenyls
total toxicity equivalents including the sum of the dioxin/furan and World Health
Organization polychlorinated biphenyls
technical systems audit
xiv
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WHO World Health Organization
XDS Xenobiotic Detection Systems
xv
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Acknowledgments
Battelle acknowledges the advice and support of the following individuals in preparing this document:
Stephen Billets, George Brilis, and Brian Schumacher of the U.S. Environmental Protection Agency's
National Exposure Research Laboratory; Randy Allen of Hybrizyme Corporation; Noriyoshi Inoue of
Paracelsian, Inc.; Fernando Rubio, Abraxis, LLC; Gary Hinshaw of Environmental Assurance
Monitoring; Bob Harrison of CAPE Technologies L.L.C.; Andrew Chu, Jeff Sturkey, George Clark, and
John Gordon of Xenobiotic Detection Systems, Inc.; and Hiroyuki Hayashi, Masako Hayakawa, and
Emmy Leung of Wako Pure Chemical Industries, Ltd. Battelle acknowledges the support of the Dioxin
SITE Demonstration Panel for their technical input to the plan and their review of this document. In
particular, we recognize Michael Jury, Sue Kaelber-Matlock, and Al Taylor from the Michigan
Department of Environmental Quality; Becky Goche and Doug Spencer of the U.S. Fish and Wildlife
Service; and 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. Environmental
Protection Agency.
xvi
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Executive Summary
Performance verification of innovative environmental technologies is an integral part of the regulatory
and research mission of the U.S. Environmental Protection Agency (EPA). The Superfund Innovative
Technology Evaluation (SITE) Program was established by the EPA Office of Solid Waste and
Emergency Response and the Office of Research and Development under the Superfund Amendments
and Reauthorization Act of 1986. 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 innovative technologies and gather reliable performance and cost information to support site
characterization and cleanup activities, and (3) develop procedures and policies that encourage use of
innovative technologies at Superfund sites as well as other waste sites or commercial facilities. The intent
of a SITE demonstration is to obtain representative, high-quality performance and cost data on innovative
technologies so that potential users can assess a given technology's suitability for a specific application.
This plan summarizes the demonstration activities that will be conducted during the SITE Demonstration
of monitoring and measurement technologies for dioxin and dioxin-like compounds. The demonstration
will be conducted in Saginaw, Michigan, from April 26 to May 5, 2004. The demonstration is being
conducted under the Monitoring and Measurement Technology Program, which is administered by the
Environmental Sciences Division of the EPA's National Exposure Research Laboratory in Las Vegas,
Nevada. The following six technologies will be demonstrated:
• AhRC PCR™ Kit, Hybrizyme Corporation
• Ah-IMMUNOASSAY® Kit, Paracelsian, Inc.
Coplanar PCB Immunoassay Kit, Abraxis LLC
• DF-1 Dioxin/Furan Immunoassay Kit, CAPE Technologies L.L.C.
CALUX® by Xenobiotic Detection Systems, Inc.
• Dioxin ELISA Kit, Wako Pure Chemical Industries, Ltd.
The performance and cost of each technology will be compared to those of conventional, off-site
laboratory analytical methods. The performance and cost characteristics of one technology will not be
compared to those of another technology. A separate innovative technology verification report (ITVR)
will be prepared for each technology.
The demonstration has both primary and secondary objectives. The primary objectives are critical
to the technology evaluation and require use of quantitative results to draw conclusions regarding
technology performance. The secondary objectives pertain to information that is useful but does not
necessarily require use of quantitative results to draw conclusions regarding technology performance.
The primary objectives for the demonstration of the participating technologies are as follows:
P1. Determine the accuracy.
P2. Determine the precision.
xvii
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P3. Determine the comparability of the technology to the reference laboratory methods.
P4. Determine the method detection limit (MDL).
P5. Determine the frequency of false positive and false negative results.
P6. Evaluate the impact of matrix effects.
P7. Estimate costs associated with the technology.
The secondary objectives for the demonstration of the participating technologies are as follows:
S1. Document the skills and training required to properly operate the technology.
S2. Document health and safety aspects associated with operating the technology.
S3. Document the portability of the technology.
S4. Evaluate sample throughput.
To address the demonstration objectives, both environmental and performance evaluation (PE)
samples will be analyzed during the demonstration. The environmental samples will be collected
from multiple sampling locations around the country so that a diverse population of environmental
samples, with varying sources and contaminant concentrations, are represented. The PE samples will
include certified, spiked, and blank samples that will be obtained from commercial providers. Upon
completion of the demonstration, the technology and reference method results will be compared to
evaluate the performance and associated cost of each technology. The ITVRs for the six technologies will
be submitted to EPA for publication in December 2004.
xvin
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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) has contracted with the Battelle Memorial Institute
(Battelle, Columbus, Ohio) to conduct a demonstration of monitoring and measurement technologies for
dioxin and dioxin-like compounds in soil and sediment. The demonstration is being conducted as part of
the EPA Superfund Innovative Technology Evaluation (SITE) Monitoring and Measurement Technology
(MMT) Program from April 26 to May 5, 2004, in Saginaw, Michigan. The purpose of this demonstration
is to obtain reliable performance and cost data on the technologies in order 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 demonstration plan describes the procedures that will be used to verify the performance of each
measurement technology. The plan also incorporates a site health and safety plan and the quality
assurance and quality control (QA/QC) elements needed to ensure that data of sufficient quality is
generated to document each technology's performance. This plan has been prepared using the NERL's "A
Guidance Manual for the Preparation of Site Characterization and Monitoring Technology Demonstration
Plans"0' and in accordance with the EPA National Risk Management Research Laboratory's "Quality
Assurance Project Plan Requirements for Applied Research Projects."(2)
This demonstration plan describes the SITE Program, the scope of the demonstration, and the definition
of dioxin and dioxin-like compounds (Chapter 1); the demonstration organization and responsibilities of
the participants (Chapter 2); the six technologies that will be demonstrated (Chapter 3); sample collection,
sample homogenization, and sample handling procedures (Chapter 4); the demonstration site and the
sampling locations (Chapter 5); the demonstration approach, including the objectives, experimental
design, data analysis procedures, and the demonstration schedule (Chapter 6); the confirmatory process,
including the reference methods and the reference laboratory that will be used during the demonstration
(Chapter 7); the data management procedures (Chapter 8); the QA/QC procedures (Chapter 9); the health
and safety plan (Chapter 10); and references (Chapter 11).
1.1 Description of the SITE 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 SITE 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
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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
innovative technologies and gather reliable performance and cost information to support site
characterization and cleanup 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 intent of a SITE demonstration is to obtain representative, high-quality performance and cost data on
one or more innovative technologies so that potential users can assess a given technology's suitability for
a specific application. 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 characterization and remediation efforts than other conventional
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
technologies. The Technology Transfer Program also supports the technologies by offering technical
assistance, training, and workshops.
The demonstration of monitoring and measurement technologies for dioxin and dioxin-like compounds is
being conducted as part of the MMT Program, which provides developers of innovative sampling,
monitoring, and measurement technologies with an opportunity to demonstrate their technology's
performance under actual field conditions. These technologies may be used to sample, detect, monitor, or
measure hazardous and toxic substances in water, soil, soil gas, and sediment. The technologies include
chemical sensors for in situ (in place) measurements, groundwater, soil, and sediment samplers, field-
portable analytical equipment, and other systems that support field sampling and analysis.
The MMT Program promotes acceptance of technologies that can be used to (1) accurately assess the
degree of contamination at a site, (2) provide data to evaluate potential effects on human health and the
environment, (3) apply data to assist in selecting the most appropriate cleanup action, and (4) monitor the
effectiveness of a remediation process. The program places a high priority on innovative technologies that
provide more cost-effective, faster, or safer methods for producing real-time or near-real-time data than
conventional, laboratory-based technologies. These innovative technologies are demonstrated under field
conditions, and the results are compiled, evaluated, published, and disseminated by the ORD. The
primary objectives of the MMT Program are as follows:
• Test and verify the performance of field sampling and analytical technologies that enhance sampling,
monitoring, and site characterization capabilities
• Identify performance attributes of innovative technologies to address field sampling, monitoring, and
characterization problems in a more cost-effective and efficient manner
• Prepare fact sheets, brochures, bulletins, newsletters, and other technical publications that enhance
acceptance of these technologies for routine use.
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The MMT Program is administered by the Environmental Sciences Division of the NERL in Las Vegas,
Nevada. The NERL is the EPA's center for investigation of technical and management approaches for
identifying and quantifying risks to human health and the environment. The NERL's mission components
include (1) developing and evaluating methods and technologies for sampling, monitoring, and
characterizing water, air, soil, and sediment; (2) supporting regulatory and policy decisions; and
(3) providing the technical support needed to ensure effective implementation of environmental
regulations and strategies. By demonstrating selected innovative field measurement technologies for
dioxin, the MMT Program is supporting the development and evaluation of methods and technologies for
field measurement of dioxin and dioxin-like compounds in a variety of soil and sediment matrices.
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) demonstration 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, exploring leads from technology developers and industry experts, and
reviewing responses to Commerce Business Daily announcements. 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, their 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,
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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 requirements
for conducting the demonstration; and (4) select and provide the reference laboratory involved 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 QA/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. Rather, demonstration data are
used to evaluate the performance, cost, advantages, limitations, and field applicability of each technology.
As part of the third step of the technology verification process, EPA publishes a verification statement
and a detailed evaluation of each technology in an innovative technology verification report (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 is not published as an EPA document, but an unpublished 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 distributes fact sheets, newsletters,
brochures, bulletins and ITVRs through direct mailings, at conferences, and on the Internet. Information
on the SITE Program are available on the EPA ORD web site (http://www.epa.gov/ORD/SITE).
Additionally, a Visitor's Day is held in conjunction with the demonstration so that potential users can
have a first-hand look at the technologies in operation.
1.2 Scope of Demonstration
Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, commonly referred to collectively
as "dioxins," are of significant concern in site cleanup projects and human health assessments because
they are highly toxic. Conventional analytical methods for determining dioxin concentrations 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 dioxin
contamination at a site and could be used to direct or monitor cleanup activities. More rapidly acquired
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. More affordable and
-------
quicker analytical techniques will not replace HRMS, but will complement an enhanced sampling design.
However, before adopting an innovative alternative to traditional laboratory-based methods, an
assessment of how commercially available technologies compare to conventional laboratory-based
analytical methods using certified, spiked, and environmental samples is warranted.
The purpose of the demonstration is to evaluate measurement technologies for dioxin and dioxin-like
compounds in soil and sediment in order to provide (1) potential users with a better understanding of each
technology's performance and cost under well-defined field conditions and (2) developers with
documented results that will assist them in promoting acceptance and use of their technologies. To meet
these demonstration objectives, samples will be collected from a variety of dioxin-contaminated soil and
sediment sampling locations around the country. Samples will be identified and supplied through several
EPA Regional offices. The samples will be homogenized and characterized prior to use in the
demonstration so that a variety of environmentally derived dioxin-contaminated samples with
concentrations over a large dynamic range (< 50 to > 10,000 picogram/gram [pg/g]) can be analyzed. Six
measurement technologies for dioxin and dioxin-like compounds will participate in a demonstration at a
field site in Saginaw, Michigan, in April 2004. The technologies will be operated by the developers in
mobile laboratories or construction trailers equipped with fume hoods at the site. Draft ITVRs are planned
to be available for developer review in September 2004, for peer review in October 2004, and final
ITVRs will be submitted to the EPA for publication in December 2004.
1.3 Definition of Dioxin and Dioxin-Like Compounds and Toxicity Equivalents
Dioxins and furans are halogenated polynuclear aromatic hydrocarbons that are considered toxic. Dioxins
and furans are similar in structure as shown in Figure 1-1 and 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. 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, 7, and 8 positions are considered most harmful. A total of seven dioxin
and 10 furan congeners contain chlorines in the 2, 3, 7, 8 positions and, of these, 2,3,7,8-tetrachlorodi-
benzo-/?-dioxin (2,3,7,8-TCDD) is the most toxic and serves as the marker compound for this class.
For risk assessment purposes, estimates of the toxicity of samples that contain a mixture of dioxin and
furan congeners are often expressed as toxicity equivalents (TEQ). TEQ is calculated by multiplying the
concentration of each congener with a toxicity equivalency factor (TEF), according to the equation:
TEQ = Cc * TEF
where Cc is the concentration of the congener. The TEF (see Table 1-1) provides an equivalency factor
for each congener's toxicity relative to the toxicity of 2,3,7,8-TCDD. In the 1990s, the most widely
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accepted system of TEQs, known as the International Toxicity Equivalency Factor (I-TEF) system, was
proposed by the North Atlantic Treaty Organization Committee on Challenges to Modern Society.(3) In
1997, a World Health Organization (WHO) Expert Group reassessed TEFs using a tiered approach, in
which the results of animal toxicity studies, especially
those involving chronic or sub-chronic exposure, were
given more weight than the results of in vitro or
biochemical studies.(4) In addition to re-evaluating the
TEFs for humans, the group also determined separate
TEFs for birds and fish. The WHO TEFs presented in
Table 1-1 were determined for mammalian species that
are applicable for human risk assessment purposes. The
total TEQ from dioxin and furans (Total TEQD/F) in a
sample is calculated by adding up all of the TEQ values
from the individual dioxin and furan congeners.
2,3,7,8-TctrachlorodRwnzo-fMiioxln
ct
2^,?,V-T«trachlorodibtticafuran
ci
3,3>,4,4',«,y-H«xachloroblph«ny1
The close toxicological similarity of certain coplanar
and mono-ortho substituted polychlorinated biphenyls
(PCBs, see Figure 1-1) to dioxins has led to the
extension of the TEF system to these "dioxin-like"
PCBs. The dioxin-like PCBs, commonly referred to as,
"WHO PCBs", that exhibit toxicity are coplanar or
mono-ortho substituted and have structural and
conformational similarities to dioxin compounds.
Currently only 12 of the total 209 PCB congeners are FiSure 1'1' structure of representative dioxin,
thought to have dioxin-like toxicity; these are PCBs with furan' and PCB<
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 12 PCBs have been assigned TEF values and are
routinely included in the calculation of TEQs in toxicity assessments. The total TEQ contribution from
PCBs (referred to as total TEQPCB) is calculated by summing up the individual PCB TEQ values. The total
TEQ in a sample is the sum of the total TEQD/F and total TEQPCB values. TEQ concentrations for soils
and sediments are typically reported in picogram per gram (pg/g) which is equivalent to parts per trillion.
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 Group noted that the TEF indicates an order
of magnitude estimate of the toxicity of a compound relative to 2,3,7,8-TCDD.(4) The WHO Group
reported that 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 non-additive effects when the congeners are
present in complex mixtures. The WHO Group 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
aryl hydrocarbon (Ah) receptor binding compounds, such as polycyclic aromatic hydrocarbons (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 contribute to the total TEQ. This
potentially can result in an underestimation of TEQs in environmental samples using the TEF approach.(4)
This demonstration was designed with the limitation of the TEQ concept in mind. The samples chosen
contain a variety of combinations of dioxins, furans, and PCBs and at a wide range of concentration
levels. Some samples are high in analytes with better understood TEFs, some are high in analytes with
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TEFs that have more uncertainty. Some are high in other Ah receptor binding compounds such as
polycyclic aromatic hydrocarbons (PAHs), while others are free of these possible TEQ contributing
compounds. (Complete descriptions of the samples analyzed in this demonstration are in Chapter 5.) The
purpose is to evaluate each of the technologies directly to the TEQDF and TEQPCB and assess how other
factors which may contribute to a sample TEQ affect the technology's determination of TEQDF and
TEQPCB. Each technology will be evaluated independently, and the statistical tests used will be described
in that technology's ITVR.
Table 1-1. 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
555555 f
OCDD
Dioxin-like PCBs
Coplanar
3,3',4,4'-TCB (PCB 77)
3,4,4',5-TCB (PCB 81)
3,3',4,4',5-PeCB (PCB 126)
3,3',4,4',5,5'-HxCB (PCB
169)
TEF Value
I-TEF WHO-
TEQ (b)
1 1
0.5 1
0.1 0.1
0.1 0.1
0.1 0.1
0.01 0.01
0.001 0.0001
0.0005 0.0001
— 0.0001
0.1 0.1
0.01 0.01
Compound
PCDFs
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,7,8,9-HxCDF
1,2,3,6,7,8-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
555555 f
1,2,3,4,7,8,9-HpCDF
555555 f
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)
TEF
I-TEF
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.001
0.0001
0.0005
0.0001
0.0001
0.0005
0.0005
0.00001
0.0001
Value
WHO-
TEQ
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
(a) T = Tetra, Pe = Penta, Hx = Hexa, Hp = Hepta, O = Octa, CDD = chlorinated dibenzo-^-dioxin, CDF =
chlorinated dibenzofuran, CB = chlorinated biphenyl
^ TEFs for human and mammals
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1.4 Sources of Dioxins and Furans
Dioxins and furans enter the environment primarily by releases in the air by incineration that are subject
to long-range transport and atmospheric deposition, by releases directly to land, by releases to water, and
by occurrence in commercial products. The U.S. EPA has compiled a database of sources of dioxin-like
compounds in the United States.(5) In this database, releases in air are by far the greatest contributors of
dioxins and furans to the environment. Air releases come from municipal solid waste incinerators,
backyard trash burning, medical waste incineration, secondary copper smelting, and cement kilns among
others. Releases to land come from land application of sewage sludge and certain pesticides contaminated
with dioxins and furans. Releases in water come largely from bleached pulp and paper mill processes. In
addition, certain chemical products such as pentachlorophenol and various pesticides are contaminated
with dioxin-like compounds as by-products or impurities.
1.5 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 from approximately $500 to $1,100 per sample per method, depending on the method selected, the
level of QA/QC incorporated into the analyses, and reporting requirements.
7.5.7 High-Resolution Mass Spectrometry
EPA Method 1613, Revision B(6) and SW846 Method 8290 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 high-resolution capillary gas chromatography (HRGC)/HRMS analysis. The main
differences between the two methods are that EPA Method 1613 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 atypical 10-gram (g) sample and
20-microliter (\\L) final sample volume are presented in Table 1-2.
Table 1-2. Calibration Range of HRMS Dioxin/Furan Method
Compound
Tetra Compounds
Penta-Hepta Compounds
Octa Compounds
EPA Method 1613, Revision B
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
7.5.2 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/low-resolution mass
-------
spectrometry (LRMS) analysis. The calibration ranges in Table 1-3 are based on atypical 10-g sample
size and 100-(iL final volume.
Table 1-3. Calibration Range of LRMS Dioxin/Furan Method
Compound SW846 Method 8280
Tetra-Penta Compounds 1,000 - 20,000 pg/g
Hexa-Hepta Compounds 2,500 - 50,000 pg/g
Octa Compounds 5,000 - 100,000 pg/g
1.5.3 PCBMethods
There are more options for analysis of dioxin-like compounds such as PCBs. EPA Method 1668,
Revision A(7) 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 co-eluting
congeners. The calibration range for PCBs based on a typical 10-g sample and 50-(iL final sample volume
is 1 to 10,000 pg/g. PCBs also can be determined as specific congeners by GC/LRMS or as Aroclors1 by
GC/electron capture detection.
1.5.4 Summary ofAnalytical Methods for Dioxins and Dioxin-like Compounds
The analytical methods described in this section were all considered for use in this demonstration. Based
on the data needs of this demonstration, EPA Method 1613B was selected as the reference method for
dioxins and furans, and EPA Method 1668A was selected as the reference method for the WHO PCBs.
Total TEQD/F concentrations will be generated by Method 1613B and total TEQPCB concentrations will be
generated by Method 1668A. These data will be summed to derive a total TEQ value for each sample.
Prior to the start of the demonstration, characterization analysis by Method 1613B and LRMS modified
Method 1668A will be used to determine which samples will be included in the study design. Additional
discussion of the analytical methods will be presented in subsequent sections of this demonstration plan.
1 Monsanto Corporation marketed products that were mixtures of 20 to 60 PCB congeners under the trade name
Aroclor. Aroclor mixtures are identified by a number (e.g., Aroclor 1260) that represents the mixture's chlorine
composition as a percentage (e.g., 60%).
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Chapter 2
Demonstration Organization and Responsibilities
This chapter identifies key project personnel and summarizes their responsibilities in planning and
executing the demonstration. Figure 2-1 is an organization chart that shows key project personnel and the
lines of communication among them. Table 2-1 presents the key demonstration participants. During the
demonstration, the participants will be asked to follow the health and safety procedures outlined in
Chapter 10. However, each organization is directly and fully responsible for the health and safety of its
own employees.
2.1 EPA Project Personnel
The EPA program manager, Stephen Billets, has overall responsibility for the project. Dr. Billets will
review and concur with the project deliverables, including the demonstration plan, ITVRs, and DER. The
EPA QA officer at the EPA NERL, George Brilis, is responsible for reviewing and concurring with the
demonstration and quality assurance project plan. The roles for EPA in this demonstration include:
Review and approve the demonstration plan.
Review and approve the DER and ITVRs.
• Be present at the demonstration.
Participate in Visitor's Day.
Coordinate activities with the Battelle project manager.
2.2 Battelle Project Personnel
The Battelle project manager, Amy Dindal, is responsible for conducting day-to-day management of
Battelle project personnel, maintaining direct communication with the EPA and the developers, and
ensuring that all Battelle personnel involved in the demonstration understand and comply with the
demonstration plan. Ms. Dindal is also responsible for distributing the draft and final demonstration plans
to all key project personnel and for reviewing measurement and analytical data obtained during the
demonstration. Battelle project personnel will assist Ms. Dindal in preparing project deliverables and in
performing day-to-day project activities. In consultation with the EPA, Battelle project personnel are
responsible for the following elements of the demonstration:
• Developing and implementing all elements of this demonstration plan.
• Scheduling and coordinating the activities of all demonstration participants.
10
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EPA project manager
Stephen Billets
EPA NERL
QA manager
George Brilis
Demonstration site
representatives
Becky Goche
(U.S. Fish and Wildlife
Service)
Sue Kaelber-Madock
Michael Jury
(Michigan Dept. of
Environmental Quality)
PE sample suppliers
Various sources
Battelle ES&ll
representative
Gary Carlin
Technology developers
Randy Allen
(Jlybrizyme Corporation)
Noriyoshi Inoue
(Paracelfdan, Inc.)
Fernando Rubio
(Abraxis, LLC)
Bob Harrison
(CAPE TechnologiesL.L.C.)
John Gordon
(Xenobiotic Detection Systems, Inc.)
Masako Hayakawa
(Wako Pure Chemical Industries, Ltd.)
Battelle
project manager
Amy Dindal
AXYS
project manager
Georgina Brooks
AXYS
QA manager
Date Hoover
Battelle
QA stall
Zachary WUlenberg
Rosanna Buhl
Battelle observers
Josh Finegold
Mark Misita
Marv Schrock
Rattelle sample custodian
Robyn Kroeger
Rattelle
infrastructure and
logistics coordinator
Rachel Sell
Battelle Visitors Day
coordinator
Helen Latham
.Notes:
EPA = U.S. Environmental Protection Agency
NERL = National Exposure Research Laboratory
QA = quality7 assurance
Figure 2-1. Organization chart for the dioxin demonstration.
ES&H = Environmental Safety and Health
PE = performance evaluation
11
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Table 2-1. Demonstration Participants
Organization
Point of Contact
Contact Information
U. S. Environmental Protection Agency Stephen Billets
George Brilis
Battelle
Michigan Department of
Environmental Quality
U.S. Fish and Wildlife Service
Abraxis LLC
CAPE Technologies L.L.C.
Hybrizyme Corporation
Amy Dindal
Sue Kaelber-Matlock
Michael Jury
Becky Goche
Fernando Rubio
Bob Harrison
Randy Allen
Xenobiotic Detection Systems, Inc. John Gordon
Wako Pure Chemical Industries, Ltd. Masako Hayakawa
Paracelsian, Inc.
AXYS Analytical Services
Noriyoshi Inoue
Georgina Brooks
National Exposure Research Laboratory
944 East Harmon Avenue
Las Vegas, Nevada 89119
Telephone: (702) 798-2232
Fax:(702)798-2261
E-mail: billets.stephen(g),epa.gov
505 King Avenue
Columbus, Ohio 43201-2693
Telephone: (561) 422-0113
Fax:(561)258-0777
E-mail: DindalA(g),battelle.org
Remediation and Redevelopment Division
503 N. Euclid Avenue
Bay City, Michigan 48706
Telephone: 989-686-8025, X 8303
Fax: 989-684-9799
E-mail: matlocks(g),michigan. gov
Green Point Environmental Learning Center
3010 Maple Street
Saginaw, Michigan 48602
Telephone: (989) 759-1669
E-mail: becky goche@fws.gov
54 Steamwhistle Drive
Warminster, Pennsylvania 18974
Telephone: (215)357-3911
E-mail: frubio(g),abraxiskits.com
3 Adams Street
South Portland, Maine 04106-1604
Telephone: (207) 741-2995
E-mail: cape-tech(g),ceemaine.org
Suite G-70
2801 Blue Ridge Road
Raleigh, North Carolina 27607
Telephone: (919) 783-9595
E-mail: rallen(g)hvbrizvme.com
1601 E. Geer Street, Suite S
Durham, North Carolina 27704
Telephone: (919) 688-4804
E-mail: iohngordonfajdioxins.com
1600 Bellwood Road
Richmond, Virginia 23237-1326
Telephone: (877) 714-1920
E-mail:
hay akawa.masako(5),wako-chem.co.jp
72 Hampton Road
Scarsdale, New York 10583
Telephone: (914) 472-5152
E-mail: inomakfSiearthlink.net
2045 Mills Road
Sidney, British Columbia, Canada V8L358
Phone: 250-655-5800
E-mail: gbrooksfg)axys.com
12
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• Coordinating the collection of samples; performing sample homogenization; performing
characterization analyses for dioxin/furans, PCBs, and PAHs; and sample aliquoting.
• Coordinating activities with the PE sample suppliers.
• Developing and maintaining sample control process and distributing samples during the
demonstration.
• Auditing the reference laboratory (AXYS Analytical Services) to determine whether the operations
are properly performed.
Overseeing the operation of the developer technologies and documenting the operation of each
technology during the demonstration.
Summarizing, evaluating, interpreting, and documenting demonstration data for inclusion in the
ITVRs and DER.
Evaluating and reporting on the performance and cost of each technology.
Preparing draft and final versions of six ITVRs (one for each technology).
• Preparing draft and final versions of the DER, consistent with the format and content of historical
documents.
• Coordinating meetings among the EPA, the developers, and the demonstration panel (see
Appendix A).
• Providing required planning, scheduling, cost control, documentation, and data management for field
activities.
Managing demobilization activities, including proper waste disposal.
• Immediately communicating any deviation from the demonstration plan during field activities to the
EPA program manager and discussing appropriate resolutions of the deviation.
Interfacing with the demonstration site representatives and making logistical preparations for the
demonstration.
Tasks for specific Battelle staff will include:
Battelle's ES&H representative, Gary Carlin, or his designee, will review the site-specific health and
safety procedures and will audit field procedures during the demonstration to ensure compliance with
the health and safety procedures presented in Chapter 10 of the demonstration plan. Mr. Carlin will
also train the participants to the site safety and health plan on the first day of the demonstration.
• Battelle's QA manager, Zachary Willenberg, is responsible for overall project QA. Mr. Willenberg
will be available to resolve any project-specific QA issues and will conduct an in-field technical
systems audit (TSA) to assess whether Battelle is performing the demonstration activities in
accordance with this demonstration plan. Mr. Willenberg, or his designee, will also review the
13
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reference laboratory data packages. Battelle QA Auditor Rosanna Buhl will lead the on-site TSA at
the reference laboratory during sample analysis.
Rachel Sell will be Battelle's infrastructure and logistics coordinator for the demonstration. Ms. Sell
will be responsible for coordinating details with the site representatives and for securing all of the
equipment (such as rental of the mobile laboratories) and supplies (such as rental of gas cylinders) for
the demonstration.
Three Battelle observers will each be assigned to two technologies. The observers will be responsible
for observing the technologies in operation during the demonstration and understanding the technical
and operational features of the technology which is assigned to them. Josh Finegold will be
responsible for observing Abraxis and CAPE Technologies. Mary Schrock will be responsible for
observing Hybrizyme and Xenobiotic Detection Systems. Mark Misita will be responsible for
observing Wako and Paracelsian.
Robyn Kroegerwill be Battelle's Sample Custodian. Ms. Kroegerwill be responsible for filling and
labeling all of the soil, sediment, and extract samples prior to the demonstration and for distributing
the samples during the demonstration. Ms. Kroegerwill also be responsible for archiving the samples
after the demonstration activities have concluded.
Helen Latham will coordinate the Visitor's Day activities. Ms. Latham will be responsible for
ensuring that all guests during the Visitor's Day have signed in and received a name badge. Ms.
Latham will also be responsible for distribution of handouts, managing of media organizations who
attend the Visitor's Day, and photo-documentation of Visitor's Day activities.
2.3 Developer Personnel
The developers of the six technologies are responsible for providing, mobilizing, operating, and
demobilizing their respective technologies at the demonstration site. The developer responsibilities
include the following:
• Provide Battelle with information on the technologies.
• Review and concur with the demonstration plan.
Notify Battelle in writing of technology-specific requirements, such as the type of power supply and
the amount of work space needed, so that proper arrangements can be made for field demonstration of
the technologies.
• Provide the personnel and all supplies needed for demonstration of the technologies unless otherwise
arranged in advance with Battelle.
Analyze the samples specified in the demonstration plan.
• Analyze developer-specified QC samples (for example, blanks or standards) in accordance with the
technology specifications.
• Provide technology-specific demonstration results to Battelle at the end of the demonstration.
14
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Review and comment on the technology-specific ITVRs.
Conduct all activities in accordance with the schedule to ensure a timely completion of the final
reports.
2.4 Demonstration Site Representatives
The representatives for the demonstration site are Becky Goche, U.S. Fish and Wildlife Service and
Sue Kaelber-Matlock and Michael Jury from Michigan Department of Environmental Quality (MDEQ).
All work performed at each demonstration site will be scheduled, coordinated, and conducted with the
permission of the demonstration site representatives, who will be the primary contacts for Battelle. Ms.
Goche is responsible for obtaining the site access and submitting the request for the special use permit
which is necessary for performing the demonstration at the site (see Appendix B). Mr. Jury is responsible
for establishing adequate electrical power at the demonstration site and coordinating other logistics details
with Ms. Sell. Ms. Kaelber-Matlock is responsible for participating in Visitor's Day. The demonstration
site representatives are also responsible for reviewing and concurring with the demonstration plan.
2.5 Reference Laboratory Personnel
The reference laboratory for the project, AXYS Analytical Services, Ltd. (Sidney, British Columbia,
Canada) will perform laboratory analyses. The laboratory project manager, Georgina Brooks, is
responsible for overall planning, scheduling, budgeting, and reporting of laboratory activities. Mr. Dale
Hoover is the AXYS QA manager. All work will be conducted under the direct supervision of Ms.
Brooks. AXYS is also responsible for reviewing and concurring with the demonstration plan, and Ms.
Brooks will immediately discuss appropriate resolutions of any deviation from the reference laboratory
activities specified in the plan with the Battelle project manager. The impact of any deviations will be
discussed with the EPA program manager.
2.6 Suppliers of Performance Evaluation Samples
The performance evaluation (PE) samples will be supplied from various sources (see Section 6.2.1). This
will include purchasing standard reference materials and preparation of spiked samples. All activities,
including purchasing standard reference materials and spiked sample preparation, will be conducted under
the direct supervision of the Battelle project manager.
15
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Chapter 3
Developer Technology Descriptions
This chapter contains technology descriptions for each of the six technologies that are participating in the
demonstration. This information was provided by the developers with only editorial changes made by
Battelle to ensure consistency and the needs of this document.
3.1 CAPE Technologies DF1 Dioxin/Furan Immunoassay Kit
The DF1 Dioxin/Furan Immunoassay Kit from CAPE Technologies is an enzyme immunoassay (EIA)
test kit containing a polyclonal antibody specific for PCDD/Fs. Both semiquantitative screening and
quantitative analysis are possible with the DPI Dioxin/Furan Immunoassay Kit. Samples can be prepared
for analysis by EIA using a variety of methods. Extracts of soil, sediment, food, water, fly ash, stack gas,
tissue, or other samples that have been
prepared by conventional extraction
methods can be exchanged to a
water-miscible solvent system for
analysis using the CAPE immunoassay
kit. The technology description,
operating procedure, and advantages
and limitations presented below are
based on information provided by
CAPE Technologies L.L.C.
3.1.1 Technology Description
Figure 3-1. CAPE Technologies DF1 Dioxin/Furan Immunoassay
Kit.
The DF1 Dioxin/Furan Immunoassay Kit (Figure 3-1) is designed to analyze samples according to their
TEQ concentrations by responding to the toxic PCDD/F congeners in approximate correlation with their
TEFs. The test is capable of multiple congener recognition and preferentially targets congeners with high
TEF values, i.e., those with the highest toxicity relative to 2,3,7,8-TCDD. The specificity of the test is
predominantly for PCDD/Fs that contain 3 to 6 chlorines, with a strong preference for the 2,3,7,8
chlorinated congeners. This specificity roughly parallels the TEF values of the individual PCDD/F
congeners. PCDD/Fs are typically extracted with organic solvents that are incompatible with EIA;
therefore, a solvent exchange is required. PCDD/Fs have very low volatility and are retained during this
solvent exchange in a small volume of a keeper solution (Triton X-100 detergent in tetraethylene glycol
[TEG]) after evaporation of the original solvent. Methanol is added to dilute this solution, and the
methanol-TEG-Triton mixture is added directly to the EIA tubes. The solubility of PCDD/Fs in methanol
is augmented significantly by adding TEG and Triton X-100.
16
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During the first EIA incubation, PCDD/Fs are specifically bound by the anti-dioxin antibodies, which
have been immobilized on the EIA tube surface. After washing away the unbound material, the bound
PCDD/Fs remain, and a competitor-horseradish peroxidase (HRP) conjugate is added. Bound PCDD/Fs
occupy the dioxin binding sites of the antibodies in proportion to the PCDD/F content of the sample and
prevent binding of the competitor-HRP conjugate. After a short incubation, unbound conjugate is
removed, and the test tubes are washed thoroughly. The amount of conjugate bound by the anti-dioxin
antibody is inversely related to the amount of PCDD/Fs originally present in the sample. Finally, a
solution of chromogenic FIRP substrate and hydrogen peroxide is added to the test tubes. Color
development is directly proportional to enzyme concentration and inversely related to the PCDD/F
concentration in the original sample. The test tubes are analyzed using a tube reader or spectrophotometer
to measure the optical density (OD). The OD values of unknown samples are compared to the OD values
of standards to determine the level of PCDD/Fs in the samples.
The final measured EIA response is the sum of the individual congener responses, which correlates with
TEQ because the immunoassay cross-reaction profile for PCDD/Fs correlates with TEF values. Accuracy
among samples may vary solely because of the variability of congener composition. To maximize
accuracy, the variability of congener composition in the target sample population should be known. The
best performance is achieved when all samples are from a single group that shares as many properties as
possible (common source of contamination, similar congener composition, similar sample matrix, etc.).
The limit of detection for the DF1 Dioxin/Furan Immunoassay Kit claimed by CAPE Technologies is
4 pg of dioxin, which is sufficient for analysis at 500 parts per trillion (pg/g) using extract equivalent to
20 milligrams of sample. Sensitivity can be increased by adding more sample extract to the cleanup
procedure and subsequently to the EIA. For example, by loading the EIA tube with the equivalent of 1 g
of prepared sample, analysis can be performed at approximately 10 pg/g. When using increased sample
loads, the manufacturer's recommendations for extract cleanup must be followed closely. Results must be
related to the original sample concentration by back calculation using the proper dilution and volume
factors. Matrix detection limits will vary according to matrix, sample size, and dilution factor. Up to 40
samples a day can be analyzed using the procedure described below.
DF1 immunoassay kit starter packages include the following:
• DF1-ST-A, a small starter package containing two DF1-12 kits (40 antibody-coated tubes and
matching liquid reagents), one Grip-Rack, and one set of dioxin standards, plus two check samples of
dioxin in toluene made by Wellington Labs.
DF1-ST-B, a large starter package containing one DF1-60 kit (100 antibody-coated tubes and
matching liquid reagents), one Grip-Rack, and one set of dioxin standards, plus two check samples of
dioxin in toluene made by Wellington Labs.
After the purchase of one starter package, subsequent purchases are either the DF1-12 orthe DF1-60
(described below). These kits do not include dioxin standards and check samples, which must be ordered
separately. The DF1-12 kit for screening analysis of 12 samples includes 20 antibody-coated tubes and
matching liquid reagents. The DF1-60 kit for screening analysis of 60 samples includes 100 antibody-
coated tubes and matching liquid reagents. Figure 3-2 is a matrix describing the use of the DF-1
immunoassay kit.
17
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ANALYTICAL OPTIONS MATRIX
CAPE
Technologies
Analytical
Options
cost
turnaround time
method specific
training
experience and
expertise
shipping of kits to
customer
shipping of samples
to CAPE
Technologies
Option 1- customer performs
extraction, cleanup, and analysis
lowest, in the $70-90 per sample
range for immunoassay and
sample prep kits; final number
depends on overhead and amount
of QA performed
fastest; on-site use is possible;
same or next day results, totally
dependent on customer
training by PowerPoint file or in
person; kit starter package for
training and proficiency demo
chemist preferred, some analytical
experience essential,
Immunoassay experience helpful
immunoassay and sample prep
kits ship by commercial courier In
days
none required
Option 2- customer performs
extraction, CAPE Technologies
performs cleanup and analysis
higher than option 1. typically in
the $125-250 per sample range,
but quoted on an individual job
basis
customer extraction plus shipping
plus CAPE turnaround (standard =
10 business days, rush - 5 days,
priority = 2 days from receipt of
sain pies)
none, but need to follow extraction
Instructions
little required
extraction kits ship by commercial
courier in days
organic solvent extracts reduced
to small volume solid keeper can
travel by commercial courier with
no restrictions
Option 3- CAPE Technologies
performs extraction, cleanup and
analysis
higher than option 2, typically In
the $150-300 per sample range,
but quoted on an individual job
basis
shipping plus CAPE turnaround
(standard - 10 business days,
rush = 5 days, priority = 2 days
from receipt of samples)
none required
none required
none required
possible import restrictions
Figure 3-2. DF1 immunoassay kit analytical options. Various options exist for using the DPI Immunoassay Kit,
including having samples analyzed by the customer or by CAPE Technologies. It should be noted that the customer
should have technical experience with immunoassay techniques and must perform this analysis in an analytical
laboratory or some other location that can provide the necessary equipment and infrastructure (i.e., fume hood,
vortex mixer, sample evaporation system, etc.).
3.1.2 Operating Procedure
To ensure accurate and reliable results, every effort should be made to perform the dioxin/furan immuno-
assay at temperatures between 20°C (68°F) and 25°C (77°F). The following is one option for sample
preparation and was designed for same-day analysis at 500 pg/g, using a one-step cleanup. Other sample
preparation options are available for lower concentrations. All sample extraction and extract cleanup
components are in a kit form and are disposable.
1. Sodium sulfate is added to a soil sample and mixed. Dimethylformamide (DMF) is added to the soil
sample, and the soil is extracted by shaking for two hours. The supernatant DMF extract is removed.
DMF extracts are stable from weeks to months at room temperature.
2. Interferences are removed by chemical oxidation. Hexane is added to an aliquot of the DMF extract,
then treated with 15% SO3 in concentrated H2SO4 (fuming sulfuric acid). The supernatant hexane is
removed and exchanged to a water-miscible organic solvent solution. This hexane-based fuming
sulfuric acid cleanup is sufficient for most samples; but, in certain circumstances, an additional
cleanup step may be required. This is the case for samples that contain large amounts of non-volatile
18
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aliphatic oils. When the DMF extracts of such soils are cleaned using fuming sulfuric acid, the oil is
not oxidized; it remains after evaporation of the hexane, causing a biphasic system when introduced
to the EIA first incubation. Such EIA samples appear opalescent or milky, and their results are invalid
because the biphasic system prevents capture of analyte by the antibody. For these samples, a new
aliquot of DMF extract is cleaned by carbon adsorption. In this case, the final solvent in the cleanup
procedure is toluene rather than hexane.
3. The cleaned sample in hexane or toluene is exchanged to a water-miscible organic solvent solution
for EIA analysis. PCDD/Fs have very low volatility and are retained during this solvent exchange in
Triton X-100 detergent in TEG after evaporation of the original solvent. Methanol is added to dilute
this solution, and the methanol-TEG-Triton mixture is added directly to the EIA tubes.
4. An accurately measured volume of negative control, standard, or prepared sample is mixed with an
aqueous sample diluent in test tubes with anti-dioxin antibody immobilized on the surface. The
mixture is then incubated.
5. After incubation, antibody tubes are washed, and 0.5 mL of HRP competitor conjugate is added to
each tube using a repeater pipettor. Bound PCDD/Fs occupy the dioxin binding sites of the antibodies
in proportion to the PCDD/F content of the sample and prevent binding of the competitor-HRP
conjugate. After a short incubation, unbound conjugate is removed, and the test tubes are washed
thoroughly.
6. A solution of chromogenic HRP substrate and hydrogen peroxide is added to the test tubes. Color
development is directly proportional to enzyme concentration and inversely related to the PCDD/F
concentration in the original sample. Stop solution is added to each tube using a repeater pipettor to
fix the amount of color development.
7. The test tubes are analyzed using a tube reader or spectrophotometer to measure the OD at 450 nano-
meters. The test is interpreted by measuring the signal produced by a sample and determining the
concentration from a dose-response curve constructed from standards tested at the same time.
3.1.3 Advantages and Limitations
Extraction and cleanup using the DPI Dioxin/Furan Immunoassay Kit is faster and simpler than for
GC/mass spectrometry (MS). Confirmation of selected positive samples and a portion of the negative
samples by GC/MS analysis is strongly recommended. Quantitative interpretation of data is possible in
certain situations.
The low pg sensitivity of the DF1 Dioxin/Furan Immunoassay Kit is competitive with HRMS. This
sensitivity allows for greater flexibility in designing immunoassay protocols and gives excellent tolerance
of matrix interferences.
The dioxin/furan congener recognition profile correlates to the TEFs of the individual PCDD/F
congeners. Because of this specificity, the DPI Dioxin/Furan Immunoassay Kit has a proven correlation
to TEQ.
The distribution of PCDD/Fs in soil and sediment samples can be extremely heterogeneous. Adequate
sample number, distribution, and homogeneity are the responsibility of the analyst.
19
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Samples that appear heterogeneous during the first incubation are invalid due to phase separation.
Adequate sample cleanup must be assured by the analyst.
3.2 Hybrizyme Corporation AhRC PCR™ Kit
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. The technology description, operating procedure, and
advantages and limitations below are based on information provided by Hybrizyme Corporation. The
information was edited by Battelle to ensure consistency and to meet the needs of the demonstration plan.
3.2.1 Technology Description
This procedure uses Hybrizyme's AhRC PCR™ kit (Figure 3-3) to detect molecules in a test sample that
bind to the AhR. 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 certain co-planar 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.
The Hybrizyme technology, with appropriate sample processing, provides a
quantitative analysis of dioxins. The AhRC PCR™ kit has recently been
validated and approved for use in Japan for that purpose. Correlation data
from these studies with HRGC-HRMS can be found on the Hybrizyme Web
site (www.hybrizyme.com). The method for quantitative determination of
dioxins may not be optimal for field use, since it requires specialized
training and sample processing equipment to perform.
In the SITE program, Hybrizyme will use the AhRC PCR™ kit as it would
be used by a field, mobile, or fixed-based laboratory to rapidly analyze
samples for relative concentrations of AhR-reactive compounds. This
method is inexpensive, easy to learn and uses disposable labware and
relatively small quantities of solvent.
Figure 3-3. Hybrizyme's
AhRC-PCR Test Kit.
The AhRC PCR™ kit permits the determination of compounds demonstrating measurable binding affinity
with the AhR in a manner that is largely unaffected by the chemical mixture in which they reside. By
virtue of the sensitivity of the assay, many of these compounds can be directly measured in simple crude
extracts. The Hybrizyme method will provide a measure of the additive contributions in a single
measurement of any, or all, AhR-reactive compounds present in a sample extract.
The ease of use, speed of analysis, and cost of the Hybrizyme method allows it to be effectively used as a
site-mapping tool, establishing a concentration gradient for AhR-reactive compounds across a large
surface area. When employed in this manner, its operation resembles an uncalibrated thermometer. It has
all the characteristics of a more conventional calibrated thermometer in that it is capable of providing
reproducible responses for a given temperature being measured at different times, determining
20
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temperature gradients, and comparing one temperature to another on a relative basis. In this example the
units of the thermometer are arbitrarily defined.
Once a concentration grid has been established, samples already collected from predetermined "hot spots"
can be analyzed by conventional HRGC-HGMS, or more sophisticated sample processing can be
employed to obtain quantitative results from the AhRC PCR assay. When the Hybrizyme method is used
as a screening tool, the fundamental procedures for sample preparation are simple and use relatively
unsophisticated instrumentation. The results are both reproducible and reliable within the constraints of
the sample processing method used to generate the reported value.
The Hybrizyme method can be performed on as many as three sets of 20 samples each per day by a team
of two technicians. Using this technology, the boundaries of a contaminated site can be more rapidly
established. Once the initial measurements are completed, remediation efforts can begin without the need
to await final data as generated by HRGC-HRMS, saving both time and money. By eliminating the need
to use such sophisticated techniques on all of the collected samples, many of which typically contain low
to non-detectable levels, the production of useful data is expedited in all portions of a given study.
The equipment necessary to implement the Hybrizyme method can be purchased for under $45,000 U.S.
A single AhR PCR™ kit is capable of producing 96 measurements; however, a portion of these assays
must be dedicated to calibration and QA/QC procedures. The cost of the kit is $2,400 U.S. When used
optimally, the cost of consumables per sample including the Hybrizyme kit, sample processing materials,
and reagents would be under $35 per sample.
The Hybrizyme method is useful in pre-screening samples in the typical laboratory performing HRGC-
HRMS analyses on relatively large sample sets. Experienced laboratory personnel could eliminate all
samples having low to negligible contamination using the AhRC PCR kit. Simultaneously, this data
would permit organizing the remainder of the samples from the lowest to highest concentration of AhR-
binding compounds. As such, the laboratory will not spend inordinate amounts of time examining "non-
detects" nor will it accidentally cross-contaminate its laboratory equipment by unknowingly processing
samples containing elevated levels of the expected analytes.
3.2.2 Operating Procedure
1. Reconstitute PCR wash solution to 1 X with distilled or deionized water for use with an automated
plate washer. Prime the plate washer with PCR wash solution.
2. Prepare standards and unknowns in methanol.
3. Prepare the 1 X capture reagent by diluting 40 |iL of stock reagent into 600 |iL of assay buffer for
each strip used. Place desired number of strips in the strip frame and reseal the remainder in the foil
pouch. Wash the strips using the "3XWASH" program of the plate washer. Using the multichannel
pipetter, dispense 50 |iL to each well in the strip. Shake 60 to 90 minutes.
4. Thaw the activation solution and mix gently during the process. Do not allow the activation solution
to remain at room temperature for more than 20 minutes prior to use. For best results, mix the
activation solution vials together prior to dispensing when performing multiple strips.
5. Dispense 50 |iL of the assay buffer into each glass vial by using a multichannel pipette. The assay
buffer should be at room temperature prior to use. Add 5 |iL of standard or sample. The use of a
21
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pipetter (i.e., P-10 Pipetman) with a filter tip is preferred. After adding sample to the entire row, tap
gently to mix. Repeat the process for each row of glass vials.
6. Add 50 |iL of activation solution to each glass vial using a multichannel pipette. Shake for 1 hour at
room temperature.
7. Wash the strips using the "3XWASH" program of the plate washer. Using a multichannel pipetter,
transfer 30 |iL of each reaction to each well. Shake 30 minutes.
8. Wash the strips using the "PCRWash" program of the plate washer. This series of soaks and washes
takes about 15 minutes. Thaw the primer/probe solution at this time.
9. Add 100 |iL of primer/probe solution, 400 |iL of water, and 500 |iL of 2 X Universal Master Mix to
make each 1,000 |iL of 1 X Master Mix. Dispense 40 |iL into each well.
10. Seal the wells with adhesive tape and cover with two compression pads. Insert the strips into a
thermocycler and run the "PCR" template.
11. Analyze the data.
3.2.3 Advantages and Limitations
The advantages of the AhRC PCR™ assay result, in part, from the high degree of specificity that is
required for the activated receptor to bind the DNA-probe, and the unmatched sensitivity of PCR.
The AhRC PCR™ assay couples the AhR
with Nobel Prize-winning PCR technology
to produce an easy to use, sensitive method
for dioxin analysis. The AhRC PCR™ Kit
can be shipped worldwide, yielding results
from sample extracts within hours. It is
ideally suited for stationary or mobile
laboratories. The AhRC PCR™ assay takes
place in disposable microwells or
specialized PCR tubes, and the final
product is measured by a real-time
thermocycler. The
Figure 3-4a. ABI Prism 7000. thermocycler
never comes in
contact with the sample or the assay reagents. The assay system is simple to use,
requiring only limited training and technical experience.
The AhRC PCR™ assay was developed for real-time PCR systems such as the
ABI PRISM 7000 (Figure 3-4a), which can generate up to 96 results per run, or
the Cepheid Smart Cycler (Figure 3-4b), designed to be totally transportable.
Figure 3-4b. Cepheid
Smart Cycler.
22
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3.3 Paracelsian, Inc., Ah-IMMUNOASSAY®
The Ah-IMMUNOASSAY® (Figure 3-5) is an enzyme-linked immunosorbent assay (ELISA) invented at
Paracelsian, Inc. (patents pending and allowed). It is designed to screen for dioxin-like toxicity in
environmental and tissue samples. The technology description, operating procedure, and advantages and
limitations below are based on information provided by Paracelsian, Inc. and only editorial changes were
made by Battelle to ensure document consistency and meet the needs of the demonstration plan.
3.3.1 Technology Description
The Ah-IMMUNOASSAY® makes it possible to screen large numbers of samples to eliminate
non-positives before subjecting the positive samples to further analysis. The Ah-IMMUNOASSAY®
takes advantage of the correlation between the toxicity of dioxin-like compounds and their interaction
with the Ah-receptor. The Ah-IMMUNOASSAY® measures the ability of the test mixture to interact
with the Ah-receptor, thus providing data for the total toxicity of the mixture as 2,3,7,8-TCDD
equivalents. Samples to be tested in the Ah-IMMUNOASSAY® are added to a reagent mixture
containing Ah-receptor and other components in a special ELISA plate and allowed to incubate at room
temperature for two hours. At that time, any Ah-receptor transformed by dioxin-like compounds is bound
to the plate, and the remaining material is washed away. Antibodies are added to the ELISA to detect the
transformed, bound Ah-receptor, which is in turn detected colorimetrically. Color development is directly
proportional to the amount of transformed Ah-receptor.
The detection limit is one pg 2,3,7,8-TCDD equivalent per ELISA plate well. The linear range is 1 to 64
pg TCDD equivalents per ELISA plate well. About 2 |il of final sample extract is required per ELISA
plate well. The assay does not separate and identify single dioxin congeners, but measures the ability of
the test mixture to interact with the Ah-receptor, thus measuring the total toxic potential of the mixture as
2,3,7,8-TCDD equivalents. Duplicate aliquots of each reference
standard and sample dilution are recommended. Replicate
absorbance readings of OD should show a coefficient of
variation of 20% or less.
The Ah-IMMUNOASSAY® Kit is supplied as a 96- or a
4 8-well version and contains
• Cytosol
ORE Oligo
• Aryl hydrocarbon receptor nuclear translocator (ARNT)
extract Figure 3-5 . Paracelsian, Inc., Ah-
Activator EMMUNOASSAY®.
• Mixing tube (50 mL)
• Reagent reservoirs
• ELISA plate and cover
20 Wash buffer
AB 1 and AB 2 (Antibodies 1 and 2)
• AB diluent
• Detection tablets
• Detection buffer
• Positive control (a-napthoflavone or 7,8-benzoflavone).
23
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The Activated Cytosol components (Cytosol, DRE Oligo, ARNT Extract, and Activator) are shipped
separately on dry ice. These components are stable for a minimum of one month when stored at -20°C
and for three months when stored at -80°C. The remaining components are shipped at ambient
temperature but should be refrigerated once received. They are stable for a minimum of one year when
stored at 4°C.
The user is to supply calibration standards (normally 2,3,7,8-TCDD) and any materials needed to prepare
samples for analysis in dimethyl sulfoxide (DMSO).
3.3.2 Operating Procedure
The following protocol is for an assay that uses all wells of the 96-well version of the kit. Steps 18
through 21, describing the procedure for the three replicate washes following each incubation, can be
automated by a commercial plate washer. Likewise, any residual fluid remaining in the plate wells can be
completely removed by a properly adjusted plate washer, thereby eliminating the need for the manual
blotting in step 31.
1. Remove the refrigerated components of the kit from cold storage and warm to room
temperature.
2. Remove the cytosol components (cytosol, DRE Oligo, ARNT extract, activator) from frozen
storage. Thaw up to four 7.5-mL vials of cytosol in a beaker of tepid water. Steps 3 through 9 will
generate the activated cytosol. Keep all intermediate mixtures in an ice bath as much as possible.
3. Pool the thawed tubes of cytosol into the 50-mL mixing tube.
4. Add the required volume of DRE Oligo to the pooled cytosol. Return any unused DRE Oligo to
the freezer.
5. Mix by gently rocking the tube.
6. Add the required volume of ARNT to the cytosol, DRE Oligo mixture. Return any unused ARNT
to the freezer.
7. Mix by gently rocking the tube.
8. Add the required volume of activator to the cytosol, DRE Oligo, ARNT mixture. Return any
unused activator to the freezer.
9. Mix immediately (to avoid high local concentrations of salt) by gently rocking the tube. This
mixture is the activated cytosol and must be kept on ice.
10. Add 10 (iL each of one reference standard, one negative control, and up to 10 samples in DMSO
(the negative control) to 1.0-mL aliquots of the activated cytosol and gently mix. Tubes for these
preparations are supplied by the user. These mixtures compose the treated stock for each type of
determination.
11. The 96-well ELISA plate layout will accommodate one negative control, seven dilutions of the
TCDD reference standard (or a single dilution of the NAP Standard), and four dilutions of 10
24
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samples, all in duplicate. If less than 96 wells are used, return the unused ELISA strips to 4°C
storage in the foil bag with desiccant.
12. Remove the ELISA plate from the desiccated plastic bag and add 400 (iL of each treated stock to
the top row of the wells to give duplicate runs of each of the 10 samples and the single reference
standard.
13. With a multichannel pipettor, add 200 (iL of activated cytosol to all the remaining wells. One of
the kit reservoirs will facilitate loading the pipettor.
14. Beginning with the first sets of duplicate wells, make serial dilutions (seven dilutions for the
TCDD reference or the single dilution of the NAP, four dilutions for each sample) down the
respective plate columns. Individual dilutions are mixed by a minimum of six repetitive
aspiration and redispensing cycles of the well contents. The residual waste following the final
dilution must be collected for proper disposal.
15. Attach the lid to the plate.
16. Incubate the plate for two hours at 30°C. Incubation at room temperature (i.e., 20°C) is
satisfactory, but will result in a slightly lower response. Incubation at a higher temperature (i.e.,
37°C) will also result in a lower response.
17. Prepare the IX wash buffer by diluting the contents of the 20 X wash buffer bottle to 500 mL
with reagent-grade water (250 mL for the 48-well kit). Store unused 1 X wash buffer at 4°C.
18. After the two-hour incubation, remove the well contents by aspiration into a waste receptacle for
proper disposal.
19. Add 400 (iL IX wash buffer to all wells, loading the multichannel pipette from a kit reservoir.
20. Wait two minutes. Remove the well contents by aspiration into the waste receptacle for proper
disposal.
21. Repeat steps 19 and 20 twice for a total of three separate washes.
22. Prepare the primary antibody stock by aliquoting the required amount of AB 1 into the
appropriate volume of AB diluent. Mix gently. Store unused AB 1 and AB diluent at 4°C.
23. Deliver 200 (iL AB 1 stock to all wells of the plate, loading the multichannel pipette from a kit
reservoir.
24. Incubate the plate for one hour at 30°C.
25. After the one hour incubation, repeat steps 18 through 21.
26. Prepare the secondary antibody stock by aliquoting the required amount of AB 2 into the
appropriate volume of AB diluent. Mix gently. Store unused AB 2 and AB diluent at 4°C.
25
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27. Deliver 200 (iL AB 2 stock to all wells of the plate, loading the multichannel pipette from a kit
reservoir.
28. Incubate the plate for one hour at 30°C.
29. After the one-hour incubation, repeat Steps 18 through 21.
30. Prepare the detection reagent stock by dissolving the required number of detection tablets in the
appropriate volume of detection buffer with gentle mixing while protected from light. Allow a
minimum of 15 minutes for the tablets to completely dissolve. Unused detection buffer and
tablets are kept at 4°C.
31. Following the incubation of the final wash, strike the inverted plate against paper toweling to
remove residual fluid before proceeding to the next step.
32. Deliver 200 (iL of the detection reagent stock from step 30 to all wells of the plate, loading the
multichannel pipette from a kit reservoir.
33. Incubate the plate at 30°C while protected from light. Residual moisture from condensation
and/or from contact with incubator water must be carefully blotted from the plate before reading.
The plate is read at 405 nanometers (nm) after 15, 30, 45, and 60 minutes. Variations in
incubation temperatures and plate reader characteristics may dictate the best incubation times for
individual conditions.
A standard curve is used to determine the amount of TCDD equivalents present in a sample. The standard
curve is generated by plotting the average absorbance (OD) measured at 405 nm for each of the TCDD
reference standards on the vertical (Y) axis versus the corresponding quantity (pg) of TCDD on the
horizontal (X) axis. Results are calculated manually using graph paper or a curve-fitting statistical
software package. The TCDD equivalents for the samples are determined from the standard curve by
interpolating from the absorbance value (Y axis) to the quantity (pg) of TCDD (X axis). The initial
TCDD concentration in the sample is found by correcting for the sample dilution. The supplied NAP
solution, at 2 (iL per test well, may optionally be used as a single-point positive control that is equivalent
to 32 pg TCDD.
3.3.3 Advantages and Limitations
Equipment required for the test is relatively inexpensive and readily available. The
Ah-IMMUNOASSAY® is simple to perform and highly reproducible. It is very sensitive to the
respective toxicity of the dioxins and related compounds of interest. Toxicity is measured as the ability of
the sample to transform the AhR. The result, therefore, denotes total toxicity from all toxins that are
present in the sample.
The assay does not identify individual dioxin congeners. Competing enzyme immunoassay methods are
less sensitive, typically detecting only certain dioxin congeners without direct correlation to total toxicity.
Some related toxic compounds are not detected at all by direct immunoassays. False negatives results
with the Ah-IMMUNOASSAY® are rare. Positive results can be further characterized by selective
pretreatment steps or by specific GC/MS testing.
26
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3.4 Abraxis LLC Coplanar PCB ELISA Kit
The Abraxis coplanar PCB ELISA kit applies the principle of enzyme immunoassays for the qualitative
or semi-quantitative analysis of coplanar PCBs in a variety of sample extracts. Extracts from soil,
sediment, fish tissue, and others matrices can be exchanged to methanol for ELISA analysis. Water
samples can be diluted 1:1 in methanol and analyzed directly in the assay. The technology description,
operating procedure, and advantages and limitations below are based on information provided by Abraxis
LLC. and only editorial changes were made by Battelle to ensure document consistency and meet the
needs of the demonstration plan.
3.4.1 Technology Description
The Abraxis coplanar PCB ELISA kit (Figure 3-6) can screen samples according to their TEQ
concentration. The specificity of the test is predominantly for those congeners with high TEF values; i.e.
congeners 126 and 169. Samples extracted with organic solvents that are incompatible with ELISA can be
evaporated and re-dissolved in methanol. For a quick screen of soil and sediment samples, the samples
can be extracted in 20% acetone in hexane, diluted 1:10 in
the provided diluent, and run directly in the assay.
A solution containing a primary antibody (rabbit) that reacts
with coplanar PCBs is added to a microplate containing a
secondary antibody that captures the primary antibody.
Calibrators (congener 126) and samples are added and
allowed to incubate, followed by the addition of a coplanar . __„ rT
D/-D tron •+ A i D/-D +u 4. Figure 3-6. Abraxis PCB ELISA Kit
PCB-HRP enzyme conjugate. Any coplanar PCBs that may
be in the sample competes with the coplanar PCBs enzyme label conjugate for a finite number of
antibody binding sites. At the end of the incubation period, the unbound conjugate is removed, and the
plate is washed. A substrate/chromogen solution is then added and enzymatically converted from a
colorless to a blue solution by the captured coplanar PCB-HRP conjugate on the plate. The reaction is
then terminated by acidification. The coplanar PCBs concentration is determined by measuring the
absorbance (at 450 nm) of the sample solution using a microplate reader and comparing it to the
absorbance of the calibrators. The amount of color produced is inversely proportional to the amount of
coplanar PCBs present in the sample.
The final value measured by ELISA is the sum of the various congeners responses, this value
approximates TEQPCB because of the immunoassay kit cross-reaction profile for coplanar PCBs
approximates TEF values. Accuracy among samples may vary solely because of the variability of
congener composition. To help maximize accuracy, the variability of congener composition in the target
sample should be known.
The primary use of the Abraxis coplanar PCB ELISA kit is to screen samples that have low coplanar PCB
concentrations. The sensitivity of the test in water samples is claimed by Abraxis to be 4 parts per trillion
(pg/mL). This value must be related to the original sample concentration by using the appropriate dilution
and volume factors. Detection levels depend on how much sample is evaporated and the volume of
solvent used to resuspend the sample. Matrix detection limits will vary according to the matrix being
analyzed, sample size, and dilution factor. Up to 100 samples per day can be analyzed using the
procedure described below.
27
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The Abraxis coplanar PCB ELISA Kit consists of
1. Microtiter Plate coated with Goat-Anti Rabbit Antibody
96-testkit: 8X 12 strips
2. Coplanar PCB Antibody Solution
Rabbit anti-coplanar PCB solution in a colored buffered saline solution with preservative and
stabilizers.
96-test kit: one 6-mL vial
3. Coplanar PCB Standards (Congener 126)
Seven concentrations (0, 25, 50, 100, 250, 500, 1,000 ppt) in 50%methanol.
96-testkit: one 1-mLvial
4. Coplanar PCB-HRP Enzyme Conjugate
Coplanar PCB labeled with HRP diluted in colored buffered solution with preservative and
stabilizers.
96-test kit: one 6-mL vial
5. Diluent/Zero Standard
50% methanol in distilled water (v/v) without any detectable PCB.
96-testkit: one 30-mLvial
6. Color Solution
A solution of hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine in an organic base.
96-testkit: one 16-mL vial
7. Stopping Solution
A solution of diluted acid.
96-test kit: one 6-mL vial
8. Washing Buffer 5X Concentrate
Buffer salts with detergent and preservatives.
96-testkit: one 100-mLvial
3.4.2 Operating Procedure
To ensure accurate and reliable results, every effort should be made to perform the coplanar PCB ELISA
at temperatures between 20°C and 25°C and to allow the reagents to be at the same temperature. The
following sample preparation was designed for a quick screen at 625 pg/g. Other sample preparation
options are available for lower concentration.
3.4.2.1 Preparation of Sample Extracts From Soil
1. Label soil collection bottles and extract collection vials.
2. Remove the screw cap from the soil collector bottle (containing dispersion device) and collect soil by
weight using a digital balance. Place the bottle in an upright position on the balance and tare weight.
28
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3. Weigh 2 g of soil in a 30-mL high-density polyethylene (HDPE) bottle.
4. Add 6 g of anhydrous sodium sulfate and mix until sample is free flowing.
5. Add 1 steel mixing ball.
6. Add 10 mL of 20% acetone in hexane. Rotate for one hour.
7. Remove the organic extract from the soil particulates by filtration, sedimentation, or centrifugation.
8. Transfer extract to a 40 mL screw cap extraction tube and oxidize using concentrated sulfuric acid
(4 mL). Mix by agitation for a minute and allow phases to separate.
9. Remove the organic phase (top layer), transfer to a fresh extraction tube and add 4 mL of
concentrated sulfuric acid. Mix by agitation for a minute and allow phases to separate.
10. Repeat step 9 until the acid phase is colorless.
11. Evaporate 1 mL of the organic phase using a nitrogen stream.
12. Redissolve in 0.25 mL of methanol.
13. Add 0.25 mL of water. If cloudy, centrifuge.
NOTE: This extraction procedure dilutes the sample by a factor of 2.5. Therefore, assay results need to
be multiplied by 2.5 to obtain the final coplanar PCB concentration in the sample.
3.4.2.2 Dilution of Sample Extracts
Dilute sample (1:10) by adding 50 |iL of extract to 450 |iL of diluent/zero standard (provided).
3.4.2.3 Assay Procedure
1. Add 50 |iL of anti-coplanar PCB antibody solution successively to each well.
2. Add 50 |iL of the appropriate standard, control, or sample. Using duplicates or triplicates is recom-
mended. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a
circular motion on the benchtop. Be careful not to spill the contents. Incubate at room temperature for
30 minutes.
3. After the incubation, remove the covering and add 50 |il of enzyme conjugate solution to the
individual wells. Cover the wells with parafilm or tape and mix the contents by moving the strip
holder in a circular motion on the benchtop. Be careful not to spill the contents. Incubate at room
temperature for 90 minutes.
4. After the incubation, remove the covering and vigorously shake the contents of the wells into a waste
container. Wash the strips 3 times using the 1 X wash solution with a volume of at least 250 uL for
each wash step. Any remaining buffer in the wells should be removed by patting the plate on a dry
stack of paper towels.
29
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5. Add 150 uL of color solution successively to each well. Incubate for 20 to 30 minutes.
6. Add 50 uL of stopping solution to each well in the same sequence as for the other reagents.
7. Read absorbance using a microplate reader at 450 nm within 15 minutes after adding the stopping
solution.
8. Construct a standard curve by plotting the %B/B0 for each standard on a vertical logit (y axis) versus
the corresponding PCB 126 standard concentration on the horizontal logarithmic axis (x) on a graph
paper. Alternatively, commercial ELISA programs can be used (4-parameter or logit-log).
9. To obtain the total coplanar PCB TEQ in a sample, multiply sample assay results by the cross-
reactivity factor 0.01.
3.4.3 Advantages and Limitations
The Abraxis coplanar PCB ELISA kit can detect the total coplanar PCBs at various levels and eliminates
the complex extraction and clean-up steps used with GC/MS.
The coplanar PCB kit can be used in the field or laboratory. The low part per trillion (ppt) sensitivity of
the kit is competitive with HRMS.
The distribution of coplanar PCBs can be heterogeneous. Adequate number (statistically significant),
distribution, and homogeneity are the responsibility of the analyst.
Samples that appear heterogeneous during assay incubations (cloudiness) are invalid. Adequate sample
cleanup must be assured by the analyst.
The Abraxis coplanar PCB kit is cost-effective compared to other methods; however, it is a screening
method only. The method does not provide quantitative results because of the variability of
concentrations of individual congeners in real samples. The relationship between immunoassay response
and TEQ may vary significantly for different samples. This ELISA kit provides an approximation.
Forty samples can be analyzed in less than 4 hours from sample extraction to results.
3.5 Wako Dioxin ELISA Kit
The Wako Dioxin ELISA Kit from Wako Pure Chemical Industries, Ltd. was developed to screen minute
amounts of dioxin. With a microplate reader, samples can be assayed simultaneously for PCDD/Fs. The
technology description, operating procedure, and advantages and limitations below are based on
information provided by Wako Pure Chemical Industries, Ltd. and editorial changes were made by
Battelle to ensure consistency and to meet the needs of the demonstration plan.
3.5.1 Technology Description
A monoclonal antibody specific to dioxin is mixed with a sample solution or the positive control (PC)
provided with the Dioxin ELISA Kit. Peroxidase conjugated with a dioxin analog (POD-conjugate) is
then added, reacting with a primary antibody to dioxin in the sample. The mixture is added to a
30
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microplate coated with a secondary antibody that captures the antibody-POD-conjugate and incubated at
2° to 8°C for 18 to 20 hours. After washing the resultant microplate with a buffer, the
antibody-POD-conjugate complex formed on the plate is reacted with substrate for peroxidase. The
reaction is stopped by adding stop solution, and the microplate reader reads the signal.
The Dioxin ELISA Kit contains the secondary antibody microplate, the PC, buffers A and B, the primary
antibody, peroxidase conjugate (lyophilized), sample solubilizer, wash solution concentrate, substrate,
citrate buffer, stop solution, and a plate seal.
The monoclonal antibodies used for the Dioxin ELISA Kit indicate cross-reactivity nearly equal to the
positive control (2,7,8-trichlorodibenzo [1,4] dioxin-1-yl) acrylic acid) and 2,3,7,8-TCDD. It is possible
to find dioxin concentrations as the amount equivalent to 2,3,7,8-TCDD TEQ.
The Dioxin ELISA Kit sensitivity is claimed by Wako to be 1.6 to 100 pg per assay, and 96 samples can
be assayed in two days. The procedure is summarized in Figure 3-7.
3. 5. 2 Operating Procedure
The Dioxin ELISA Kit should be used at
room temperature (20° to 25°C) and stored at
2°tolO°C.
1 . To prepare the PC solution
(5,000 pg/mL), add 2.0 mL of methanol to
the tube containing the positive control
(PC). Then, gently stir the tube four or
five times, and leave it at room
temperature for 10 minutes. Gentle
stirring is repeated once more before use.
Do not vigorously shake or vortex the
solution to avoid PC material adhering to
the tube wall. The solution can be used
within four weeks at 2° to 10°C after
reconstitution.
2. Prepare the dilution solution by
dispensing 2 mL of sample solubilizer in a
tube and adding 2 mL of methanol.
POD-Conjugate
Sample or PC
Reaction of POD-
conjugate and
un reacted "*
antibody
\ Add to microplate well
, with secondary
A Antibody fixation plate
Y I V V I I Y V I
Fix reactants to
microplate well
3 .
Prepare 1/10-fold diluted PC solution
(500 nanograms/mL) by adding 900 |iL of
dilution solution to a disposable culture
tube rinsed with acetone and dried. Then
add 100 |iL of concentrated PC solution
and gently stir the tube two or three times.
Leave the tube at room temperature.
React the POD with the substrate for coloring. Then
measure the color density with the microolate reader.
Figure 3-7. Wako Dioxin ELISA Kit procedure.
4.
Prepare PC dilution solutions for the standard curve using the 1/10-fold diluted PC solution described
above as follows:
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Tube Number:
Concentration
Dilution solution
Concentrated PC
solution
1/1 0-fold diluted
PC solution
Total volume
1
0 pg/mL
500 |iL
—
0|iL
500 |JL
2
40 pg/mL
460 |iL
—
40|iL
500 |JL
3
lOOpg/mL
400 |iL
—
100 |iL
500 ML
4
250 pg/mL
250 |iL
—
250 |iL
500 ML
5
500 pg/mL
450 |iL
50|iL
—
500 ML
6
l,OOOpg/mL
400 |iL
100 |iL
—
500 ML
7
2,500 pg/mL
250 |iL
250 |iL
—
500 ML
5. Dry the contents of each tube.
6. To prepare a dilution series of PC solutions, add 500 |iL of Buffer B to each tube, lightly stir it with a
vortex mixer, and centrifuge at 2,000 revolutions per minute (rpm) at room temperature to collect the
liquid at the bottom.
7. To prepare the primary antibody stock solution, add 200 |iL of purified water to the tube and gently
stir, then leave the tube for 10 minutes at room temperature. The solution can be used within four
weeks.
8. Rinse a beaker or a 5- to 10-mL vial with acetone and air dry on clean paper towels.
9. Prepare the primary antibody working solution using a 100-fold dilution of the primary antibody
stock solution described above with Buffer A according to assay sample numbers and volumes. Stir
the diluted solution gently several times, and leave it at room temperature for 10 minutes. Stir the
solution several times just before use. This solution cannot be stored prior to use.
10. Prepare the POD-conjugate solution by adding 4 mL of Buffer A to the tube and gently stir several
times. Leave the tube at room temperature for 10 minutes. Gently stir the tube just before use. The
solution can be used within two weeks when stored at 2° to 10°C.
11. Fix the necessary number of microplate wells (coated with the secondary antibody) to a microplate
holder with Scotch® tape. Place the holder in a closed box, and store it in a refrigerator for at least
30 minutes before use.
12. Add 100 |iL of Buffer B to the tube containing the material obtained by sample pretreatment and
gently stir the solution along the inner surface of the tube several times with vortex mixer. Briefly
centrifuge the tube at 2,000 rpm for five minutes to collect the solution at the bottom.
13. Line up the tubes containing the dilution series of PC solutions and the samples dissolved in Buffer B
in a tube rack. Dispense an equal volume of the primary antibody working solution to each tube.
14. Immediately agitate the solutions several times, then seal the tubes with Parafilm and leave them at
room temperature for 30 minutes.
15. During the incubation, prepare chilled water containing ice in a water bath and immerse the tubes in
the bath. After the 30 minute-incubation at room temperature, add 250 |iL and 50 |iL of
POD-conjugate solution to the tubes containing the diluted PC solutions and those containing sample
material, respectively.
16. Gently agitate the tubes several times and leave them for 10 minutes. Avoid vigorous shaking with
the vortex because dioxins may adsorb to the tube wall.
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17. Take the microplate holder from the box stored in a refrigerator. Dispense 100 |iL of the reaction
mixture to the wells. Briefly agitate the solutions in the wells. Seal the wells with Parafilm, return the
holder to the box, and store the box in a refrigerator at 2° to 10°C for 18 to 20 hours.
18. On the second day, prepare the wash solution by diluting the wash solution concentrate (X6) with five
volumes of purified water. The solution volume prepared can be adjusted depending upon the number
of wells used. Usually, one well needs 1.2 mL of the wash solution. The solution can be used within
two weeks after dilution when stored at 2° to 10°C.
19. Prepare the necessary volume of color-developing solution by mixing the substrate and the citrate
buffer in a ratio of 1:50 just before use. (The tube used in this preparation should be cleaned with
purified water.) Add the citrate buffer to the tube, then the substrate. Avoid vigorous agitation of the
mixed solution because it may cause crystalization.
20. Take the well holder from the box stored in a refrigerator and remove the seal from the wells.
Remove the reaction mixture from the wells, being careful not to spill the solution to the holder.
Lightly tap the holder upside down on sheets of paper to remove the residual mixture in the wells.
Dispense approximately 0.35 mL of wash solution to each well and then drain the solution, being
careful not to spill the solution. Remove the solution from the well walls by tapping the holder upside
down on the papers. Repeat washing twice. At the last washing, remove the solution by tapping as
much as possible.
21. For the enzyme reaction, dispense 100 |iL of the color-developing solution to all the wells. Seal with
Parafilm, cover the holder with aluminum foil to block out light, and leave the holder at room
temperature for 30 minutes. Stop the reaction by adding 100 |iL of stop solution in the same well
sequence as the color-developing solution. Tap the side of the holder to mix the solution. Read the
signal with a microplate reader at 450 nm or at 450/650 nm within 15 minutes after stopping the
reaction. Construct the standard curve obtained with the diluted PC solutions and estimate the dioxin
concentrations of samples as 3,7,8-TCDD. The primary antibody used in this kit reacts with
2,3,7,8-TCDD and 3,7,8-TCDD with equal intensity.
22. In estimating the dioxin concentration, it is useful to plot the obtained values with a regression of
polynomial quadratic equation or four-parameter logic after log-logit conversion.
3.5.3 Advantages and Limitations
Highly sensitive analysis is possible with the Dioxin ELISA Kit.
A non-toxic standard (2, 7, 8-trichlorodibenzo [1,4] dioxin-1-yl) acrylic acid) is used.
The 96-microplate format makes possible a simultaneous multisample assay. The assay process takes only
two days.
3.6 Xenobiotic Detection Systems, Inc., CALUX®
CALUX® (chemical-activated luciferase expression) by Xenobiotic Detection Systems Inc. (XDS)
technology is based on a reporter gene system using a genetically engineered cell line capable of detecting
all of the WHO-recognized dioxins, furans and PCBs. Giving results for dioxins/furans and PCBs
33
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separately or together, as well as being available as a screening and/or quantitative analysis. CALUX® by
Xenobiotic Detection Systems is used to analyze soil, sediment, fly ash, stack gas emissions, food, feed,
blood, and any other substance suspected of being contaminated with dioxins/furans and PCBs. The
technology description, operating procedure, and advantages and limitations provided below for the
CALUX® are based on information provided by Xenobiotic Detection Systems, Inc. and editorial
changes were made by Battelle to ensure consistency and to meet the needs of the demonstration plan.
3.6.1 Technology Description
XDS has patented (patent # 5,854,010) a genetically engineered cell line that contains the firefly
luciferase gene under trans-activational control of the AhR. This cell line can be used for the detection
and quantification of the AhR agonists, the target receptor of dioxins, furans, and PCBs. The XDS term
for the in vitro assay is the Chemical-Activated Luciferase Expression (CALUX®) by Xenobiotic
Detection Systems assay. The most widely studied compounds that activate this system are the
polychlorinated diaromatic hydrocarbons (PCDH), such as 2,3,7,8-TCDD. Many PCDH compounds are
quantified relative to TCDD, since this is one of the most potent activators of AhR-mediated gene
transcription. These relative quantifications are known as TEQs, and the results from the CALUX® by
Xenobiotic Detection Systems assay provide a measure of TEQs in a sample. By using proprietary
cleanup methods developed by XDS, it is possible to separate PCBs from dioxins/dibenzofurans and to
determine what portion of the total TEQ in a sample is due to each of these classes of compounds. XDS
has termed this procedure the Dioxin/Furan and PCB-Specific (DIPS) or DIPS-CALUX® by Xenobiotic
Detection Systems bioassay.
Prices start at $200.00 for a dioxin screening (single) analysis and $250.00 for a dioxin and PCB analysis,
with analysis provided as a fee for service at the XDS laboratories. Field analysis is available with 96 well
plates being shipped to the site for analytical procedures to be performed by trained personnel. Costs per
96 well plates are approximately $2,400, with each plate capable of analyzing up to 40 samples along
with standard curves and quality control standards. Rental of equipment and proprietary software to
perform the CALUX® by Xenobiotic Detection Systems is also available.
3.6.2 Operating Procedure
Xenobiotic Detection Systems, Inc. has a patented genetically engineered cell line (mouse hepatoma
H1L1) that contains the gene for firefly luciferase under transactivational control of the AhR. This cell
line can be used for the detection and relative quantification of a sample's total dioxin I-TEQ. The XDS
CALUX® bioassay for dioxin-like chemicals uses a patented sample processing procedure (U.S. patent #
6720431) that allows separation of coplanar PCBs and PCDDs/PCDFs so that estimates of I-TEQ can be
made for each chemical class. This allows reporting of I-TEQ estimates for chlorinated dioxins/furans
and for the PCBs.
The samples are extracted using a modification of the EPA 8290 extraction method. Briefly, the dried
samples are ground and 1-g aliquots are placed in solvent-cleaned glass vials with
polytetrafluoroethylene-lined caps. The sample is extracted with a 20% solution of methanol in toluene
then twice with toluene. During each extraction step, the samples are incubated in an ultrasonic water
bath. The three extracts from each sample are filtered, pooled, and concentrated by vacuum
centrifugation. The sample extract is suspended in hexane and rapidly processed through a patented (U.S.
patent # 6720431) two column chromatographic procedure to produce two extracts, one containing
chlorinated dioxins/furans and one containing PCBs (see Figure 3-8). The extracts are exchanged into
34
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DMSO and used to dose the genetically engineered cells in the
CALUX® assay by XDS to provide I-TEQ estimates for PCBs
and PCDD/PCDFs.
Prior to dosing the cells, the sample extracts in DMSO are
suspended in cell culture medium. This medium is then used to
expose monolayers of the H1L1 cell line grown in 96-well
culture plates (see
Figure 3-9). In addition
to the samples, a
standard curve of
Figure 3-9. Luminescence produced
when CALTJX® cells are exposed to
dioxin and dioxin-like chemicals.
Figure 3-8. Xenobiotic Detection Systems
patented sample processing procedure.
2,3,7,8-TCDD is
assayed (161, 80.5, 40.2, 20.1, 10.1, 5.0, 2.5, 1.2, and 0.6 ppt
TCDD). The plates are incubated for a time to produce optimal
expression of the luciferase activity in a humidified CO2
incubator. Following incubation, the medium is removed and the
cells are examined microscopically for viability. The induction of
luciferase activity is quantified using the luciferase assay kit
from Promega.
3.6.3 Advantages and Limitations
CALUX® by Xenobiotic Detection Systems analyses are approximately 25% of the cost of FIRGC/MS
analyses, and sample sizes are small (10 g or less). Results can be made available within 28 hours in
emergency situations. Expenses for laboratory setup are minimal, and the proprietary cleanup method
eliminates false positives and false negatives.
CALUX® by Xenobiotic Detection Systems analyzes environmental, foodstuff, and biological samples.
CALUX® by Xenobiotic Detection Systems also analyzes all 17 lexicologically active dioxin and 12 PCB
congeners, and results are reproducible. Detection limits below 0.5 ppt TEQ scores are provided for both
dioxin and PCB analyses. CALUX® by Xenobiotic Detection Systems does not, however, identify
individual congeners.
CALUX® by Xenobiotic Detection Systems has been authenticated as a European Union-certified
screening tool for dioxin in foodstuffs by BELTEST.
35
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Chapter 4
Sample Collection, Sample Homogenization, and Sample Characterization
This chapter discusses the sample collection, sample homogenization, and sample characterization
procedures used in the demonstration.
4.1 Sample Collection
This section describes the environmental sample collection activities performed at various sites across the
country. Battelle performed an intensive search for sampling sites that had varying levels of dioxin
contamination in either soil or sediments. This diversity will make the demonstration results more
applicable to a larger user community, as well as to challenge the technologies using a variety of sample
compositions. Samples were collected by EPA, an EPA contractor, or MDEQ and shipped to Battelle.
When determining if a soil or sediment site had appropriate dioxin contamination, a guideline
concentration range was provided to sample providers of < 50 pg/g to 5,000 pg/g. It was understood that
this concentration range was to be used as a general guideline, and that those providing the sample
descriptions were to provide concentration data based on best available information.
4.1.1 Procedure
This section describes the method that was used to collect the samples by each of the site personnel. Once
necessary approvals and sampling locations had been secured, site personnel were shipped sample
containers. Each site providing samples received one-gallon containers (Environmental Sampling
Supply, Oakland, California, Part number 3785-1051, wide-mouth, 128-ounce HDPE round packer) for
the collection of five or six samples. At least 2 gallons of material (soil or sediment) for each sample were
needed; therefore, if a site was providing six samples, twelve 1-gallon containers were shipped to the site
for the collection of six 2-gallon samples. With the 12 containers provided, it was anticipated that the six
sample containers were filled in duplicate.
Shipments consisted of pre-cleaned, 1-gallon sample containers with instructions on how to fill the
containers for each sample collected. Instructions for sample collection as well as how the containers
were to be labeled and returned were included in a cover letter (see example in Appendix C) 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
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,
pentachlorophenol [PCP]). Final instructions to sample providers indicated that collected samples were to
be shipped back to Battelle using the provided coolers. Federal Express labels that included an account
number and the shipping address were enclosed in each shipment.
36
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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
demonstration plan.
4.1.2 Sample Shipping to and Archival at Battelle
The samples were received at Battelle in the same coolers that were used to send the containers to the
field. The environmental samples were stored at room temperature until homogenized.
4.2 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 frozen1. 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 inch. The pans were placed in an oven at 35 °C and held there until the
samples were visibly dry. This process took 24 to 72 hours, depending on the sample moisture. The trays
were removed from the oven and allowed to come to room temperature by sitting in a fume hood for
approximately 2 hours. Approximately 500 g of material was 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 and soil was blended and sieved. The blended and sieved
sediment/soil in the tray were 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 actively used
over a period of several days, at which time they were temporarily stored at room temperature.
It should be noted that none of the technologies participating in this demonstration require that samples be
dried prior to analysis. As such, moisture effects on technology performance that will commonly be
encountered with real-world soil and sediment samples, will not be evaluated during this demonstration.
4.2.1 Criteria for Determining Adequate Homogenization
Two criteria had to be met in order for the sample to be considered adequately homogenized. The first
criteria 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 con-
sidered acceptable. The second criteria was that no single RSD for an individual congener could be
greater than 30%. If both of these criteria were met, the sample was considered homogeneous and
considered for inclusion in the demonstration. If either of these criteria were not met, options for the
Ideally, the samples would have been stored at 4° ± 2° but due to the large volume of buckets and jars that needed
to be stored, the most adequate available storage at Battelle was a walk-in freezer that was at approximately minus
20°C.
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sample included: (a) discard it and not consider it for use in the demonstration, (b) reanalyze it to
determine if the data outside the homogenization criteria were due to analytical issues, or
(c) rehomogenize and reanalyze 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). All samples included
in the demonstration met the criteria for acceptable homogenization. In a few cases, the composition of a
particular sample was of interest for inclusion in the demonstration because of concentration or unique
congener pattern, but the homogenization criteria were slightly exceed (i.e., no more than 30% RSD for
TEQ and no more than 40% RSD for a particular congener). Since multiple replicates of every sample
will be analyzed in the design, it was the recommendation of the demonstration panel (Appendix A) to
still include that sample in the study because of the unique nature of the sample, but flag it as slightly
exceeding the homogenization criteria.
4.3 Characterization of Environmental Samples
A total of 58 environmental samples were received from ten different sampling locations (see descriptions
in Chapter 5). All of the environmental samples were homogenized, as described in Section 4.2, and
analyzed by Battelle. The environmental samples were characterized for the 17 D/Fs by Method 1613B,
the 12 WHO PCBs by LRMS Method 1668A, and 18 target PAHs by National Oceanic and Atmospheric
Administration Status and Trends GC/MS method. All characterization analyses were performed by
Battelle in their laboratories. The purpose of the characterization analyses was two-fold. First, the
analyses determined the adequacy of the homogenization. Second, the approximate concentrations of the
target analytes were determined. These two pieces of information served as the basis of sample selection
for inclusion in the demonstration.
4.3.1 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
1613, Revision B. 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 was taken for analysis from each aliquot and spiked with
13C12-labeled internal standards, and extracted with methylene chloride using accelerated solvent
extraction (ASE) 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 chromatography 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 (iL. Dilution and reanalysis of the
extracts were performed if high levels of a particular congener were observed in the initial analysis.
Each extract was analyzed by GC/FIRMS in the selected ion monitoring (SIM) mode at a resolution of
10,000 or greater. A DB5 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 1613 with one additional calibration
standard at concentrations equivalent to one-half the level of Method 1613's lowest calibration point.
Using a DB5 column, 2,3,7,8-TCDF is not separated from other non-2,3,7,8-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 DB5
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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/PCDF data were reported as both concentration (pg/g dry) and TEQs (pg TEQ/g dry).
4.3.2 PCBs
One aliquot of material from each sampling location was prepared and analyzed for the 12 WHO-
designated dioxin-like PCBs by LRMS. Battelle's LRMS PCB analysis method is based on key
components of the PCB congener analysis approach described in EPA Method 1668A and the PCB
homologue approach described in EPA Method 680. Up to 30 g of sample was 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
concentrations. 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 gas chromatography on a DB5-XLB column and identified and
quantified using electron ionization mass spectrometry. This method provides specific procedures for the
identification and measurement of the selected PCBs in SIM mode.
4.3.3 PAHs
One aliquot of material from each sampling location was analyzed for PAHs. The PAHs were selected by
the demonstration panel (see Appendix A). 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. Battelle's method for the identification and quantification of PAH in sediment and
soil extracts by GC/MS is based on the National Oceanic and Atmospheric Administration (NOAA) status
and trends method (8) and, therefore, certain criteria (i.e., initial calibrations and daily verifications) are
different than those defined in traditional EPA methods 625 and 8270C. Up to 30 g of sample was 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 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 gas chromatography on a DB-5, 60-m column
and were identified and quantified using electron impact mass spectrometry. Extracts were analyzed in
the SIM mode to achieve the lowest possible detection limits.
4.4 Sample Handling, Sample Tracking, and Sample Management
In preparation for the demonstration, the bulk homogenized samples will be split into jars for distribution.
Each 4-ounce, amber, wide mouth glass sample jar (Environmental Sampling Supply, Oakland,
California, Part number 0125-0055) will contain approximately 50 g of sample. Seven sets of samples
will be prepared for the six developers and one reference laboratory. A minimum of four replicate splits
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of each sample will be 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) will be
transferred from their original packaging to the jars to be used in the demonstration for the environmental
samples, such that the environmental and PE samples will be visually indistinguishable.
The samples will be randomized in two fashions. First, the order in which the filled jars will be
distributed will be randomized, such that the same developer will not always receive the first jar filled for
a given sample set. Second, the order of analysis will be randomized so that each developer analyzes the
same set of samples, but in a different order. PE materials will be randomized in the same manner, such
that the PE samples are indistinguishable from other samples.
All jars will have two labels. The label on the top of the jar will be the analysis order (i.e., Developer
Name 1) and contain sample numbers 1 through 209. A second label will be placed on the side of the jar
will contain a coded identifier including a series of 10 numbers. The 10 numbers will be coded to include
the site, replicate, developer, and matrix in the number series that can be readily decoded by Battelle but
will be meaningless to the developers so the analyses will occur blindly.
Battelle will be responsible for sample distribution during the demonstration. All samples will be
prepared for distribution at the start of the verification. When ready to perform analyses, each developer
will go to the sample distribution point to pick up the samples. The samples will be distributed in batches
of 10 and will be released at each developer's request. More than one batch of 10 samples can be
relinquished at a time, if desired by the developer. Completion of a chain-of-custody (COC) form will
document sample transfer and will be used to report results. An example COC is included in Appendix D.
Samples that have been analyzed by the developer will be turned in to Battelle, along with COC/results
form. Results will be entered onto the COC by the developer using black ink. Corrections will be made
with a single line/initial/date procedure.
During the demonstration, all samples not in the possession of the developer will be stored in the Battelle
Headquarters trailer. All trailers will be locked after hours and hired security guards will patrol the site
from 7 pm to 7 am.
During the demonstration, the samples will be stored at room temperature. There is no concern about
significant volatilization of the target contaminants because standard reference materials for dioxins are
routinely stored at room temperature, and the samples used in this demonstration will be prepared in a
manner similar to how SRMs are processed.
An archived set of samples will be at the demonstration site in case a sample is dropped, the integrity is
comprised, or ajar is broken during transit to the site. After the demonstration, all unused demonstration
samples will be returned to Battelle for archival in a freezer (approximately -20°C) at least until after the
reports are final. At the conclusion of the study, the archive set of samples may be provided to EPA for
additional use or as reference material.
Debris from the demonstration that can be discarded by routine waste removal services will be disposed
of from the demonstration site. Sample by-products, including unused sample, aqueous and solvent-based
effluents, and miscellaneous used supplies (e.g., glassware, pipette tips, shoe covers, and gloves) will be
returned to Battelle for confirmation and quantification of by-products generated by each of the
participating technologies. After categorizing and inventorying all of the materials, Battelle will be
responsible for final waste disposal.
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Chapter 5
Descriptions of Demonstration Site and Sampling Locations
This chapter describes the demonstration site and the sampling locations and why each was selected.
5.1 Demonstration Site Description
This section describes the site selected for hosting the demonstration, along with the selection rationale
and criteria. Several candidates 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, Battelle, 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.
After review of the information available, the site selected for the demonstration is the Green Point
Environmental Learning Center (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 Refuge is one of over 540 National Wildlife Refuges
managed by the U.S. Fish and Wildlife Service, a part of the United States government, in the Department
of the Interior. The Green Point ELC offers a variety of environmental educational programs for students
of Saginaw and surrounding communities. Furthermore, it offers over 2.5 miles of trails that wind through
bottomland hardwood forest, fields, wetlands, and along the banks of the Tittabawassee River. These
trails offer opportunities to study various habitats and the wildlife associated with them. Green Point ELC
also has a bird watching area, ponds, restored grasslands, and learning center displays.
The Green Point ELC is situated within the Tittabawassee River flood plain. The Michigan Department of
Environmental Quality (MDEQ) has 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. The MDEQ is taking more
samples in the flood plain to determine the extent of contamination. Soil samples taken from areas outside
the flood plain have been at normal background levels. The source of the contamination has not been
clearly determined and is under investigation. MDEQ is monitoring the level of dioxin in soil and
considering the regulatory levels of soil of 50 ppt as a screening level, indicating a need for further study
to 1,000 ppt (i.e., 1 ppb) as an action level.
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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 MDEQ, EPA
Region 5, and local U.S. Fish and Wildlife expressed interest in supporting the demonstration by
providing site access for the demonstration. In addition, MDEQ personnel will provide logistical
support required for the demonstration and will support a Visitor's Day during the demonstration.
• Space Requirements and Feasibility—The demonstration will take place in the parking lot adjacent to
the Green Point ELC, not directly on the area of soil and sediment contamination. The site has
electrical power and adequate space to house the trailers and mobile labs that will be used for the
demonstration. Furthermore, the site is close to an international airport and hospitals and has a
sufficient number of lodgings and restaurants. Weather in Michigan can be unpredictable in the
month of April; however, all participants will be provided heated containment (a mobile laboratory or
hard-sided trailer) so that the weather should not impact the demonstration.
Site Diversity—The area encompassing the Green Point site has different levels and types of dioxin
contamination in both the soil and sediment needed to evaluate the monitoring and measurement
technologies described in Chapter 3.
5.2 Description of Sampling Locations
This section provides an overview of the 12 sampling sites and methods of selection. Table 5-1
summarizes each of the locations, what type of sample (soil or sediment) was provided, and the number
of samples from each location. Samples provided consisted of either soil or sediment and will be
described below based on this distinction. It should be noted that it is not an objective of the
demonstration to characterize the concentration of dioxins, furans, and PCBs in material from a specific
sampling site. It is, however, necessary to ensure comparability between technology results and the
reference laboratory results. This will be accomplished by homogenizing each matrix, such that all
sub-samples of a given matrix have consistent contaminant concentrations. As a result, homogenized
samples are not necessarily representative of original concentrations at the site.
5.2.1. 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, PCP, and PAHs). This information was provided by the
site owners/sample providers (e.g., EPA, EPA contractors, and MDEQ), and only editorial changes were
made byBattelle.
5.2.1.1. Warren County, North Carolina
Five different areas of the Warren County PCB Landfill in North Carolina, a site with both PCB and
dioxin contamination, were sampled. Dioxin concentrations in the landfill soils range from
approximately 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. EPA permitted the landfill under the Toxic Substances Control Act. Between September and
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November 1982, approximately 40,000 cubic yards (equivalent to 60,000 tons) of PCB-contaminated soil
were removed and hauled to the new 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 EPA and the State of North
Carolina. The site was deleted from the National Priorities List on March 7, 1986.
5.2.1.2. Tittabawassee River Flood Plain
MDEQ sampled the Tittabawassee River flood plain soils from three sites in the flood plain. 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 range. Two
samples were collected from two locations at Freeland Festival Park in Freeland. The first sample was
taken above the river bank, and the second sample was taken near a brushy forested area 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 farmed 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 have not been analyzed, but concentrations are expected to be
less than 1 ppm. The DNR property is approximately a 10-minute walk from where the demonstration
will be conducted at the Green Point ELC.
5.2.1.3 Midland, Michigan
Soil samples were collected by 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 flood plain.
The first sample location was soil at the Chippewa Nature Center adjacent to their parking lot in the
woods. The soil was a brown sandy loam with an estimated TEQ concentration of less than 10 ppt. The
next sample location was in Midland, in the greenbelt between Lynn and Patrick Streets. The soil was
dark brown clay loam with an estimated TEQ concentration of 200 ppt. The third location sampled in
Midland was near the intersection of Swede and Patrick. Again, the estimated TEQ concentration is 200
ppt. The next soil sample was gathered from a traffic island at the intersection of Saginaw and Bay City
Roads. The sample consisted of brown sandy topsoil with an estimated TEQ concentration of 600 ppt.
The fifth and final Midland sample location was in the green space between Saginaw Road and sidewalk
just south of the Mark Putnam Road intersection. The estimated TEQ concentration is up to 1,000 ppt.
5.2.1.4 WinonaPost
The Winona Post site in Winona, Missouri was a Superfund cleanup of a woodtreater facility.
Contaminants at the site included pentachlorophenol, dioxin, diesel, and PAHs. Over a period of at least
40 years, these contaminants were apparently deposited into an onsite drainage ditch and sinkhole. Areas
of contaminant deposition (approximately 8,500 cubic yards of soils/sludge) were excavated in late
200 I/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 mg/kg for pentachlorophenol and 8,000 to 10,000 for pg/g dioxin equivalents. Samples
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obtained for this study from this site were obtained from the treatment cell after these concentrations had
been achieved.
5.2.7.5 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 manufacturing chloride,
phosphate and phenol compounds at the site. A major chemical manufacturer purchased the facility in
1929 from Rubber Services Company and continued to expand operations at the facility and accelerated
its growth in the 1940s. A variety of raw materials have been used at the facility over the years including
inorganic compounds, organic solvents, and other organic compounds. The source of the dioxin
contamination in the site soils was the manufacture of 2,4,5-T. (Agent Orange is a mixture of chemicals
containing equal amounts of two active ingredients: 2, 4-D and 2,4,5-T.) Manufacture of the chemical
herbicide began at the site in 1948 and ceased in 1969. The site has a dioxin profile from ppt to low ppb
range. No PCBs or PAHs have been identified in the soil.
5.2.2. Sediment Sampling Sites
This section provides specific 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 information was provided from site
owners/samples providers (e.g., EPA, EPA contractors, and MDEQ), and only editorial changes were
made byBattelle.
5.2.2.1 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 Corp 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.
5.2.2.1.1 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 non-
point sources). This Bay is downstream from a dioxin Superfund site that contains some of the highest
dioxin concentrations yet found in the United States, and also is downstream from a mercury Superfund
site. The dioxin concentration in the area sampled for this demonstration is approximately 450 pg/g.
Average PCB concentrations range from 300 to 740 ppb. Fine-grained sediments make up 50% to 90%.
Average total organic carbon (TOC) is about 4%.
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5.2.2.7.2 Raritan Bay
Surrounded by industry and residential discharges, Raritan Bay has dioxin contamination in the area, but
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 are similar to percentages in Newark Bay.
5.2.2.2 Tittabawassee River Sediments
MDEQ sampled six locations in the Tittabawassee River flood plain. The first location is 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 is 260 pg/g; however, concentrations as high
as 2,100 pg/g TEQ are possible in this area. The second site is on the Tittabawassee River approximately
100 yards downstream from old Smith's Crossing Bridge in Midland, Michigan. The sediment is brown
and sandy with organic material. The estimated TEQ concentration is 870 pg/g; but, again, concentrations
as high as 2,100 pg/g TEQ are possible in the area. The third site is on Tittabawassee River at the
Emerson Park Golfside Boat Launch. The sediment is gray black silty sand, with many leaves and high
organic matter. The estimated TEQ concentration is < 5 pg/g. The fourth site is on the Tittabawassee
River adjacent to Imerman Park in Saginaw County across from the fishing dock. The sediment is sand
with some silt. The estimated TEQ concentration is between 100 and 2,000 pg/g TEQ. The fifth site is on
the Tittabawassee River approximately 1 mile downstream of Center Road Boat Launch in Saginaw
Township. The sediment consists of sand and gravel with some shells and not much organic matter. The
estimated TEQ concentration is between 100 and 1,000 pg/g TEQ. The sixth site also is on the
Tittabawassee River across from the Center Road Boat Launch. The sediment is fine sand with high
organic matter. The estimated TEQ concentration is 1,000 pg/g TEQ.
5.2.2.3 Saginaw River Sediments
MDEQ sampled Saginaw River flood plain sediments with the assistance of staff from the U.S. EPA
Great Lakes National Program Office. Samples were collected at six locations in the flood plain area. The
first sampling location is 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 is granular with some organic
material. The estimated TEQ concentration is 100 ppt. The next Saginaw River sample was taken
upstream of Genesee Bridge on the right side of river. The sample is a brown fine sand from about 15 feet
of water. Again, the estimated TEQ concentration is 100 ppt. The third location is in the Saginaw River
downstream of the Saginaw wastewater treatment plant in about eight feet of water. The sample is gray
silty clay with an unknown TEQ concentration. The fourth location is in the Saginaw River in about eight
feet of water. The sample is a black sandy material. The estimated TEQ concentration for this location is
again unknown. The fifth location is 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 consists of dark black silt with some sand. The estimated
TEQ concentration is unknown, but PCB concentrations are expected to be high. The sixth and final
sampling location is near the mouth of the Saginaw River in about five feet of water. The sediment is a
mix of fine black silt and layers of sand and shells. The estimated TEQ concentration for this location is
also unknown.
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5.2.2.4 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, thought to be the same 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 were carried out at the facility: PCP,
creosote, and chromium-copper-arsenic (or CCA). The site was listed on 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) dioxin/furan concentrations. Due to the processes that occurred on this site, the
samples also contain varying levels of PAHs and PCP, but should not contain PCBs.
Table 5-1. Summary of Environmental Sampling Locations
Sample Type
Soil
Sediment
Sampling Location
Warren County, North Carolina
Tittabawassee River Flood Plain,
Michigan
Midland, Michigan
Winona Post, Missouri
Nitro, West Virginia
Newark Bay, New Jersey
Raritan Bay, New Jersey
Tittabawassee River Flood Pain,
Michigan
Saginaw River, Michigan
Brunswick, Georgia
Number of Samples
5
6
6
6
6
6
6
6
6
5
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Chapter 6
Demonstration Approach
This chapter presents the objectives, design, data analysis procedures, and schedule for this technology
demonstration.
6.1 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 judgments
regarding comparability to conventional methods. The demonstration has both primary and secondary
objectives. Primary objectives are critical to the technology evaluation and require the use of quantitative
results to draw conclusions regarding a technology's performance. Secondary objectives pertain to
information that is useful but will not necessarily require the use of quantitative results to draw
conclusions regarding a technology's performance. Each report will summarizes the findings of these
objectives and provide sufficient documentation for a user to choose an alternative to conventional
technology.
The primary objectives for the demonstration of the participating technologies are as follows:
P1. Determine the accuracy.
P2. Determine the precision.
P3. Determine the comparability of the technology to EPA standard methods.
P4. Determine the method detection limit (MDL).
P5. Determine the frequency of false positive (fp) and false negative (fn) results.
P 6. 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 are as follows:
S1. Document the skills and training required to properly operate the technology.
S2. Document health and safety aspects associated with the technology.
S3. Document the portability of the technology.
S4. Evaluate sample throughput.
The objectives for the demonstration were developed based on input from the Dioxin SITE
Demonstration Panel members (Appendix A), general user expectations of field measurement
technologies, the time available to complete the demonstration, technology capabilities that the
47
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developers participating in the demonstration intend to highlight, and the historical experimental
components of former SITE Program demonstrations to maintain consistency.
6.2 Overview of Demonstration Samples
The goal of the demonstration is to perform a detailed evaluation of the overall performance of the
technology for use in contaminated site evaluation. The demonstration objectives will be centered around
providing performance data that support action levels for 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.(9) If samples are
determined to have dioxin TEQ levels between 50 and 1000 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 will be evaluated that bracket the
ATSDR guidance levels. Table 6-1 lists the primary and secondary performance objectives for this
demonstration and which sample types will be used in each evaluation. The PE samples will be used to
determine the accuracy of the technology and will consist of purchased soil and sediment standard
reference materials with certified concentrations of known contaminants and newly-prepared spiked
samples. The PE samples will also be used to evaluate precision, comparability, MDL, and false positive/
negative results. Environmentally contaminated samples will be collected from dioxin-contaminated sites
around the country (as described in Chapters 4 and 5) and will be used to evaluate the precision,
comparability, MDL, false positive/negative results, and matrix effects. Extracts, prepared in toluene, will
be used to evaluate precision, MDL, and matrix effects. All samples will be 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 6-2 is an outline of the number of each sample type
that will be included in the experimental design, and Figure 6-1 illustrates a distribution of the
environmental sample concentrations, according to total TEQD/F as determined by the characterization
analyses (see Section 4.3). The following sections describe each sample type in greater detail.
Table 6-1. Distribution of Samples for the Evaluation of Performance Objectives
Performance Objective
P 1 : Accuracy
P2: Precision
P3: Comparability
P4: MDL
P5: False positive/negative results
P6: Matrix effects
P7: Cost
S 1 : Skill level of operator
S2: Health and safety
S3: Portability
S4: Sample throughput
Type of sample that will be evaluated
PE
PE, environmental, extracts
PE, environmental, extracts
PE, environmental extracts
PE, environmental
environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
PE, environmental, extracts
Total TEQD/F (pg/g) range of samples
< 5 to > 5,000
<1 to > 15,000
< 1 to > 15,000
< 1 to > 50
<5to > 15,000
< 1 to > 15,000
n/a
n/a
n/a
n/a
n/a
n/a - not applicable
48
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Table 6-2. Proposed Number and Type of Samples to be Analyzed in the Demonstration
Sample Type
Performance Evaluation
Environmental
Extracts
Estimated D/F Concentration Range
< 5 to 6,500 pg/g total TEQ
1 to 15,000 pg/g total TEQ
<1 to l,OOOpg/mL
Total number of samples per technology
No. of Samples
58
128
23
209
6.2.1 PE Samples
PE standard reference materials are available through the National Institute of Standards and Technology
(Gaithersburg, Maryland), Cambridge Isotope Laboratories (Andover, Massachusetts), Wellington
Laboratories (U.S. Distributor TerraChem, Shawnee Mission, Kansas), LGC Promochem (United
Kingdom), and Environmental Resource Associates (Arvada, Colorado). One or more of these sources
ntration
o
c
o
o
LJ_
Q
o
UJ
H
|5
B)
a.
16000 -,
14000 -
12000 -
10000 -
8000 -
6000 -
4000 -
2000 -
H
C
•••
•
) 5 10 15 20 25 30 35
Sample Number
Figure 6-1. Approximate distribution of environmental sample concentrations.
will be utilized to obtain PE samples for use in this demonstration. PE samples will consist of three types
of samples:
Standard reference materials or certified samples: These will include soil and/or sediment samples
with certified concentrations of dioxin, furan, and/or PCBs that are commercially available for
purchase. These samples are supplied with a certificate of analysis, including a range of acceptable
results based on analytical verification data.
Spiked samples: These will include a certified dioxin, furan, PCB, and PAH-clean matrix spiked
with known levels of dioxin and/or other contaminants. The spiked samples will be included
primarily to allow for more samples to be analyzed across the analytical range of interest, since the
49
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availability of a wide-concentration range of certified reference standards is limited. The
concentrations of the spiked samples will be verified analytically by the reference laboratory for
accuracy to within 10% of the spike concentration. The results for these samples will be compared to
the spike concentration (rather than to one analytical verification result) if the measured concentration
is within 10% of the spike concentration. These samples will also have a range of acceptable results
provided by the supplier, but this range will be based on an arithmetic calculation and will not be
based on analytical data.
• Blank samples: These samples will be certified free of dioxins (2,3,7,8-TCDD < 0.1 pg/g), furans
(2,3,7,8-TCDF < 0.1 pg/g), WHO PCBs (< 10 pg/g), and PAHs, (< 350 ng/g).
PE samples will be included at levels bracketing the ATSDR action levels (i.e., < 50 to > 1,000 pg/g
TEQ), and will cover a similar concentration range and congener patterns as the environmental samples.
6.2.2 Environmental Samples
Prior to the demonstration, samples were collected from 10 sampling locations from around the country
and shipped to Battelle. The sampling locations, which are described in Chapter 5, were identified by
EPA Regional staff and MDEQ. The EPA and MDEQ staff who identified specific sampling locations
were part of the Dioxin SITE Demonstration Panel (Appendix A) and were responsible for arranging for
sample collection and providing information about the sites (Chapter 5). Once received at Battelle, the
environmental samples were homogenized and characterized for dioxin/furans (EPA Method 1613B),
PCBs (LRMS modified EPA Method 1668A), and 18 target PAHs (NOAA method) to establish the basic
composition of the samples. Environmental samples were selected for inclusion in the demonstration
based on the preliminary characterization data. Because the soil and sediment samples were dried and
homogenized, they will essentially be indistinguishable to the developers. As such, the soil and sediment
samples will be jointly referred to as "environmental" samples, with no distinction made between soil or
sediment other than in Chapter 5 where the sampling locations are described in detail.
6.2.3 Extracts
To evaluate the detection and measurement performance of each technology independent of the sample
extraction method, soil and sediment samples will be extracted using toluene and soxhlet extraction.
These extractions will be performed by AXYS Analytical Services (the reference laboratory, see Chapter
7), consistent with the procedures to extract the demonstration samples. The extracts will represent a 10 g
soil/sediment sample extraction and will be reported in pg/mL. Total extract volume per 10 g aliquot will
be 300 mL, but the sample extracts will be concentrated and provided to the developers as 10 mL
extracts. The extracts will not be processed through any clean-up steps. The extracts will be derived from
soil/sediment samples that are also included in the suite of environmental samples, so these samples will
be analyzed by the developers starting from both the environmental matrix and the extract. All
environmental sample extractions will be prepared in the same solvent (toluene), and the developers will
be responsible for any solvent exchange steps. The extract samples will also include toluene-spiked
solutions that are not extractions of actual environmental samples. Because adequate homogenization at
trace quantities will be difficult to achieve, one set of extract samples will be spiked at low levels (< 1
pg/mL) and used as part of the MDL evaluation.
50
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6.3 Pre-Demonstration Study
The best way to predict and prevent problems from occurring during the demonstration is to perform a
"dry run" exercise. This was accomplished through a pre-demonstration study. The pre-demonstration
samples were sent to all six developers for evaluation in their laboratories, as well as to the reference
laboratory. The pre-demonstration study served as a final readiness check for the developer so that
modifications could be made to their procedure if warranted by site-specific conditions. It was also a test
of the demonstration plan to ensure a well-established process of sampling, compositing, homogenizing,
splitting, extract preparation and aliquoting, and shipping of samples to the developers and the reference
laboratory. The pre-demonstration study was comprised of 15 samples, including PE samples,
environmental samples, and extracts. A distribution of the sample concentrations, as determined by the
characterization analyses (see Section 4.3), is presented in Figure 6-2. The samples selected for the pre-
demonstration study covered a wide range of concentrations and included a representative of each
environmental site that will be analyzed during the demonstration.
The pre-demonstration study was conducted in two phases. In Phase 1, the developers were sent six soil/
sediment samples and provided the D/F, PCB, and PAH characterization data with the samples, so that
each developer could perform a self-evaluation of their technology's performance. In Phase 2, seven
10000 -,
9000
8000 -
| g- 7000 -
| ^ 6000
8 1 5000 -
c *>
o f| 4000
U- Q.
S ~ 3000 -
2000 -
1000
C
B
•
B
• "
) 2 4 6 8 10 12 14
Sample No.
Figure 6-2. Approximate range of pre-demonstration soil/sediment sample concentrations.
additional soil/sediment samples and two extracts were sent to the developers for blind evaluation. The
reference laboratory analyzed all 15 pre-demonstration samples blindly. Battelle collected the pre-
demonstration results from the developers and the reference laboratory, and then returned the developer/
reference laboratory correlated data back to each developer so that the developers could use the HRMS
pre-demonstration sample data to refine the performance of their technologies prior to participating in the
field demonstration.
51
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6.4 Demonstration Schedule
The developers will analyze the demonstration samples in the mobile laboratories or trailers equipped
with fume hoods at the demonstration site in Saginaw, Michigan. The schedule for the demonstration is as
follows. Developers will begin shipping equipment and supplies to the demonstration site the week of
April 19. On Thursday, April 22, the mobile laboratories and trailers will be installed. The developers can
begin arriving as early as April 22 and will have access to the demonstration site as soon as the trailers
and mobile laboratories are installed and ready for occupancy. The developers can work at the site on
April 23, 24, and 25 setting up their oeprations and running their own quality control samples. The
demonstration will officially commence on Monday, April 26, with a safety and site-specific training
meeting at the auditorium in the Green Point ELC at 8 am. During this meeting, the health and safety plan
will be reviewed so that all participants understand the safety requirements for the demonstration. Site
health and safety (H&S) personnel, identified in Chapter 10, will be readily accessible throughout the
course of the demonstration in case H&S questions arise. During the kick-off meeting, logistics will also
be discussed, such as how samples will be distributed and results reported. It is anticipated that the
developers will analyze the demonstration samples for 3 to 9 days, depending upon the sample throughput
of each technology. Ideally, all 209 samples would be analyzed on-site, but the sample throughout of
some of these technologies would require three weeks or more in the field to analyze 209 samples.
Consequently, it was decided that the number of samples to be analyzed in the field by each developer
would be determined at the developer's discretion. The developers are requested to analyze all 23 extract
samples and at least half of the soil/sediment samples (93 samples). If a developer does not complete all
209 analyses in the field, the remaining samples will be shipped to the developer's laboratory at the
conclusion of the demonstration activities and results will be due to Battelle at a pre-determined time after
leaving the site. The time that the results generated off-site will be due to Battelle will be specified and
adhered to by the developer. The estimated schedules for each developer for the field and laboratory
portions of the demonstration are presented in Figures 6-3 and 6-4, respectively.1 The schedule for the
reference laboratory is also included.
rarticipanT
Abraxis
r*App T^rhnftlfjfii^^L
HubriTvnriA
Paracelsian
Wako
Xenobiotic Detection
Systems
AXYS Analytical Services
19
20
21
22
23
Ap
24
ril
25
-
26
27
28
29
30
1
2
M
3
ay
4
5
6
Number of samples
TO De analyzed
on-site
116
1 1 Fi
1 m
0
onq
A Q
•HO
0
Figure 6-3. Estimated participant schedule for field component of demonstration.
Please see Addendum for additional information regarding Paracelsian's participation.
52
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Participant
Abraxis
CAPE Technologies
Hybrizyme
Paracelsian
Wako
Xenobiotic Detection
Systems
AXYS Analytical Services
May
3
•
10
•
17
•
24
•
31
•
June
?
•
14
•
21
28
July
§
12
19
28
Number of samples
to be analyzed in
laboratory
116
116
99
209
0
166
209
Figure 6-4. Estimated participant schedule for laboratory portion of demonstration.
6.5 Demonstration Design
Tables 6-3 through 6-6 include a generic summary of the samples to be included in the demonstration.
The samples will be prepared, homogenized, and split into subsamples according to the processes
outlined in Chapter 4. Samples will be labeled with a unique identifier and randomized blind analysis will
be performed by the developers. Certified, spiked, uncontaminated (i.e., blank), and environmental
samples will be randomly inserted into the testing scheme. Chain-of-custody procedures will be used to
transfer samples from Battelle to the developers. Some sample information will be provided when the
samples are distributed. All samples that are believed to have at least one D/F or PCB congener greater
than 10,000 pg/g will be marked with an asterisk for safety purposes. The sites for the environmental
samples will be identified to the reference laboratory for congener pattern recognition. Wako and CAPE
Technologies requested that samples be identified as "soil" or "sediment"; none of the other developers
wished to have that information supplied. This sample information will be supplied when the first batch
of samples are released.
53
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Table 6-3 Designations for Generic Data Qualifiers
Designation
Lb
M
H
Uc
Total TEQD/F
(Pg/g)
<50
50-1,000
> 1,000
not characterized
WHO PCB a
(Pg/g)
< 1,000
1,000-10,000
> 10,000
not characterized
PAHsa
(ng/g)
< 1,000
1,000-10,000
> 10,000
not characterized
a Characterized for target list only; indicates approximate value of each target analyte
b Includes non-detects
c Applies to SRMs only where certified data not available for certain PAHs or PCBs
Table 6-4 Distribution of Performance Evaluation Samples
Sample Type
PE#1
PE#2
PE#3
PE#4
PE#5
PE#6
PE#7
PE#8
PE#9
PE#10
PE#11
PE#12
Generic Data Qualifier
D/F
L
H
M
M
M
L
L
L
L
L
L
certified
blank
PCB
L
U
L
H
H
L
L
L
L
H
M
certified
blank
PAH
U
U
U
U
L
L
L
M
L
L
L
certified
blank
Total Number ofPE samples
No. of Replicates per
Sample
7
4
7
4
4
4
4
4
4
4
4
8
58
54
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Table 6-5 Distribution of Environmental Samples
Sample Type
Environmental Site #1
Warren Co, North Carolina
Environmental Site #2
Tittabawassee River (soil),
Michigan
Environmental Site #3
Newark Bay, New Jersey
Environmental Site #4
Raritan Bay , New Jersey
Environmental Site #5
Winona, Missouri
Environmental Site #6
Tittabawassee River (sediment),
Michigan
Environmental Site #7
Brunswick, Georgia
Environmental Site #8
Saginaw River, Michigan
Environmental Site #9
Midland, Michigan
Environmental Site #10
Nitro, West Virginia
Generic Data Qualifier
D/F
M
H
H
L
M
M
L
M
L
L
L
L
L
H
H
H
L
M
L
L
L
H
M
H
M
M
M
M
L
L
H
H
PCB
H
H
H
L
L
L
L
L
L
L
L
L
L
L
M
L
L
L
L
L
L
L
M
M
L
L
L
L
L
H
H
H
PAH
L
H
H
L
L
L
L
L
L
L
L
L
L
M
M
M
L
L
L
M
L
H
L
L
H
L
L
L
L
L
L
L
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
128
55
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Table 6-6 Distribution of Extract Samples3
Sample Type
Extract* 1
Extract* 2
Extract* 3
Extract* 4
Extract* 5
Generic Data Qualifier
D/F
L
H
L
M
L
PCB
L
L
L
M
H
PAH
L
M
L
L
L
Total number of extracts
No. of Replicates
per Sample
4
4
7
4
4
23
a All developers will analyze the 23 extract samples on-site during the demonstration. Extract samples will be prepared by the
reference laboratory in their typical extraction solvent (toluene).
Independent technical observers, Battelle staff who are familiar with use of these technologies, will watch
the developer analyses as they are being performed to meet the second objectives of the demonstration to
understand the operational aspects of the technologies. It is likely that all observers will intermittently
observe each technology in operation over the course of the demonstration, but one observer will be
assigned primary responsible for each technology. The checklists that will be used to document the
observers notes and facilitate the observations are presented in Appendix E. These observations will be
summarized and reported in each developer's ITVR. Each developer will have the opportunity to review
the observer notes for their technology prior to inclusion in the report. Photo-documentation will
supplement written observations recorded by Battelle staff. However, Battelle will not photograph any
portion of the developer's technology without approval.
The amount and type of hazardous and non-hazardous waste that is generated during the demonstration
will be recorded and confirmed by Battelle. All sample by-products produced during testing will be
returned to Battelle for quantification and characterization prior to disposal.
The results of the demonstration will likely be impacted by fatigue if the developers work more than
12 hours per day, so the developers will be advised to work no more than a 12-hour day for each day of
testing (e.g., 7 am - 7 pm). The exact start and end time for each demonstration day will be decided on-
site amongst all the participants. It is requested that the developers stay on approximately the same start
time, as requests to start and end significantly later than the other participants (for example, working noon
to midnight when all of the other participants are working approximately 7 am to 7 pm) will not be
granted.
The developers will be responsible for operating their own technologies and for providing all equipment
and supplies needed for its operation during the demonstration, unless otherwise arranged with Battelle
prior to the demonstration. The developers will also be responsible for supplying their own personal
protective equipment such as gloves and lab coats.
56
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6.6 Assessment of Primary and Secondary Objectives
The term, "dioxin and dioxin-like compounds" groups several hundreds of compounds in a category
because these compounds are similar in chemical structure and biological activity. The reference
laboratory will measure individual congeners that are the most significant sources of toxicity. The
seventeen 2,3,7,8-substituted PCDD/F will be determined by Method 1613B; 12 WHO PCBs will be
reported using Method 1668A. The developer technologies will report TEQ, total TEQ or DEQ (dioxin
equivalents), but none will report data for individual congeners. As shown in Table 6-7, some
technologies will report (1) total TEQD/F(2) total TEQPCB, or (3) total TEQ, including contributions from
D/F and PCBs. For the reference laboratory data, congener concentrations will be converted to TEQ and
subsequently summed to determine total TEQ, using the TEFs established by WHO in 1998(4) (see Table
1-1).
For the reference laboratory data, non-detects will be assigned a TEQ value of zero and 1A of the detection
limit. Detection limits will be reported as SDLs (Sample-Specific Detection Limits). SDLs will be
determined from 2.5 times the noise in the chromatogram, converted to an area and then to a
concentration using the same calculation procedure as for detected peaks. Any value that meets all
quantification criteria (>SDL and ratio) will be reported as a concentration. A"J" flag will be applied to
any reported value that is between the SDL and the lowest level calibration. The concentration of any
detected congener that does not meet all quantification criteria (such as ratio or peak shape) will be
reported but given a "K" flag to indicate estimated maximum possible concentration (EMPC). TEQs will
be reported in 2 ways to cover the range of possible TEQ values:
1. All ND and EMPC values will be assigned a zero concentration in the TEQ calculation.
2. NDs will be assigned a concentration of one half the SDL. EMPCs will be assigned a value equal to
the EMPC.
In both cases, any TEQ which has = 10% contribution from J flagged or K flagged data will also be
flagged as J or K or both as appropriate.
57
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Table 6-7. Comparison Between Developer and Reference Laboratory Data
Developer
CAPE Technologies
Hybrizyme
Paracelsian
Abraxis
Wako
Xenobiotic Detection
Systems
Data will be
reported as ...
Total TEQD/F
AhR units
Total DEQb
Total TEQPCB
2,3,7,8 TCDD EQC
Pg/g
TEQD/F
TEQ PCBs
Comparison to
Reference
Laboratory Data
Total TEQD/F
Total TEQD/F
Total TEQa
Total TEQD/F
Total TEQ
Total TEQPCB
PCB 126 TEQ
2,3,7,8-TCDD TEQ
Total TEQD/F
total TEQD/F
total TEQPCB
Detection
Limit
Claimed by
Developer
(pg TEQ/g)
1
10
1
6.25
20
0.3
Reference
Data From
Method ...
1613B
1613B
1613B+1668A
1613B
1613B+1668A
1668A
1613B
1613B
1668A
aTotal TEQ is the sum of the total TEQD/F and total TEQPCB
b DEQ = equivalents to 2,3,7,8-TCDD
c EQ = equivalents
Because each technology will report data slightly differently, as described in Table 6-7, and each
technology functions and is calibrated by different procedures, specific comparisons with the reference
laboratory data will be technology-dependent and fully described in each ITVR. However, the similar
statistical principles will be applied to the evaluation of each technology, as described below.
6.6.1 Primary Objective PI: Accuracy
The determination of accuracy for each technology's measurements will be based on their agreement with
the certified or spiked levels of PE samples. For each technology, PE samples containing concentrations
from across the analytical range of interest will be analyzed. The technology measurements from the 58
PE samples will be evaluated to determine whether there is a statistically significant difference between
the technology measurements and the certified value or spiked level. Percent recovery values relative to
the certified or spiked concentrations will also be calculated. Battelle will evaluate whether a statistically
significant difference exists between a given technology's results and the reference values by performing
a two-tailed, paired, Student's t-test. The null hypothesis will be that the mean difference between the
technology results and the certified or spiked value is zero. The PE samples will also be analyzed by the
laboratory reference method for confirmation of certified and spiked values.
To evaluate accuracy, the average of replicate results from the field technology measurement will be
compared to the certified or spiked value of the PE samples to calculate percent recovery. The equation to
be used will be:
R= C /Co x 100
58
-------
where C is the average concentration value calculated from the technology replicate measurements and
CR is the certified value. For the spiked samples, if the reference laboratory's average measured value is
within 10% of the spiked concentration value, the spiked concentration value will be used as the certified
value. If the average measured value by the reference laboratory is > 10% different, the reference
laboratory's average measured value will be the certified value.
6.6.2 Primary Objective P2: Precision
A technology's precision refers to its reproducibility. Higher precision leads to less uncertainty in the
results. To evaluate each technology's precision, all samples (including PE, environmental and extract
samples) will be analyzed in at least quadruplicate. Seven replicates of three different samples will also be
analyzed to evaluate method detection limits. Replication is necessary because precision will be evaluated
at both low and high concentration levels, and across different matrices. The statistic used to evaluate
precision is RSD. The equation used to calculate standard deviation (SD) between replicate measurements
will be:
SD =
-V
- C)
k=l
1/2
where SD is the standard deviation and C is the average measurement.
The equation used to calculate RSD between replicate measurements will be:
RSD =
SD
x 100
C
Low RSD values indicate high precision. For a given set of replicate samples, the RSD of a given
technology's results will be 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.
6.6.3 Primary ObjectiveP3: Comparability
A third primary performance objective is comparability, i.e., the degree of agreement between each
technology and reference laboratory results. Battelle maximized data comparability by using the
homogenization procedures and applying criteria for acceptable results prior to a sample being included
in the demonstration, as described in Section 4.2.
For comparability, Battelle will evaluate whether a statistically significant difference exists between the
measurements provided by a given technology and the laboratory reference method by performing a two-
tailed, paired, Student's t-test. If the data are found to be non-normally distributed, a nonparametric
Wilcoxon signed-rank test will be performed to determine if the two sets of results are statistically the
same or different.
Technology results will also be compared to the corresponding reference laboratory by calculating a
relative percent difference (RPD) for the average of each paired and replicate measurement. The equation
for RPD is as follows:
59
-------
(MR - MD)
RPD = —i '-—
average(Mr, MD)
where MR is the reference laboratory measurement and MD is the developer measurement. RPD values less
than 25% will indicate good agreement between the two measurements. Because the absolute value will
not taken, negative RPD values can be obtained (which would indicate that the technology measurements
were less than the reference laboratory measurements). As such, the median RPD value will be calculated
(rather than the average RPD where the negative and positive values would be neutralized) to provide a
summary calculation of comparability between each technology's results and reference laboratory
measurements.
As described in Section 1.3, the reference laboratory concentration data will be converted to TEQ using
the WHO TEF values. TEQs will be calculated by the developer technologies in unique ways that may or
may not be directly comparable to the HRMS total TEQD/F, total TEQPCB, or total TEQ results, depending
upon the developer calibration techniques (i.e., calibration to a single congener) and/or the functionality
of the developer's technology and its relative responsiveness to the WHO-established TEFs. The
correlation of the developer results with the HRMS results will be evaluated and it will be noted if the
developer technology does or does not track the HRMS results on a relative scale. Assessments will be
made as to how well the developer results tracked the HRMS results in light of possible confounding
factors such as the sample containing other Ah Receptor binding compounds, cross-reactive compounds,
or extremely high concentrations of particular analytes.
6.6.4 Primary Objective P4: Method Detection Limit
A fourth primary performance objective is to determine the MDL for each technology. To determine the
MDLs, the developer will analyze seven aliquots of two low-level PE soil samples and seven aliquots of a
low-level spiked extract. Battelle will use these data to calculate an MDL for each technology. The
concentration of the samples will be dependent on the detection capability of each technology, but will
ideally be three to five times the reporting limit for each technology.
The MDL calculation procedure to be followed is described in 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, which will be true for the demonstration because of
the homogenization and sample preparation procedures that will be followed.
Battelle will use the Student's t-value and standard deviation to calculate the MDL for each technology in
soil and sediment as shown in the following equation:
MDL = t(n.u_0.99)(SD)
where t(n_u_^99) = Student's t-value appropriate for a 99 percent confidence level and a standard
deviation estimate with n-1 degrees of freedom.
6.6.5 Primary Objective P5: False Positive/False Negative Results
Battelle will investigate the tendency for each technology to return fp results, that is, results reported
above the reporting limits for the field technology but below the reporting limits of the reference
laboratory. The frequency of fp results will be reported as a fraction of results available for fp analysis.
Similarly, the frequency of fn results will also be examined. For this purpose, Battelle will evaluate
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results for samples reported as having concentrations above the reporting limits of the reference
laboratory method to identify cases of the reference laboratory reporting detections and a technology
reporting non-detects. As with the analysis of false positives, the statistic analyzed for each technology
will be the percentage of fn out of all samples reported as detections by the laboratory method.
A point of clarification regarding the "reporting limits" of a technology and the "method detection limits"
as described in Section 6.6.4. The reporting limits to be used in this evaluation will be the value
designated by each technology developer and the reference laboratory, as the lowest level to be reported
for each sample. This will vary from developer to developer. The MDL determination will include an
evaluation of the stated reporting limits, with a sample 3 to 5 times the reporting limit of each technology
analyzed 7 times, to assess the closeness of the technology's calculated MDL to the reporting limits used
in the demonstration.
6.6.6 Primary Objective P6: Matrix Effects
The likelihood of matrix-dependent effects on performance will be investigated by evaluating the data
sets in multiple ways. This will include evaluation of results in the following ways: samples from the ten
different environmental sampling locations individually and as a group to determine if performance was
different for environmental samples versus PE samples; grouping the data by matrix (i.e., soil, sediment,
extract); evaluating samples with similar congener patterns (e.g., samples with high hepta- and octa-
dioxins); assessing the performance with samples containing high levels of contaminants other than
dioxins (e.g., PCBs or PAHs); and evaluation of in-field versus laboratory conducted measurements
(where appropriate). These evaluations will also include looking at the performance of the technology for
the samples which were analyzed both as extracts and as environmental soil/sediment samples.
Differences in results provided for the soil/sediment samples and their associated extract samples may
indicate a matrix or homogeneity issue if the comparison of the developer and HRMS results for the
extract samples are in better agreement than for the analysis of the associated soil/sediment sample. If the
soil/sediment sample HRMS and developer results are highly correlated, but the comparative results for
the extracts are not, it may indicate that the toluene extract was not compatible with the developer's
procedure if toluene is not their extraction solvent.
6.6.7 Primary Objective P7: Technology Costs
Since conventional laboratory-based analytical methods for measuring dioxins are relatively costly, the
cost of each field technology is an important evaluation factor. With input from each technology
developer, Battelle will document the full cost of each technology and compare those costs to typical and
actual costs for D/F and PCB analytical methods. At a minimum, cost inputs will include equipment,
consumable materials, mobilization and demobilization, and labor. Battelle will document what
equipment was provided by the developer and what was provided by Battelle.
6.6.8 Secondary Objective SI: Skills and Training Required to Properly Operate the Technology
As described in Section 6.5, Battelle observers will be assigned to each of the technologies. These notes
and observations will determine the number and skill-level of the operators. The observers will also
determine the type of background and training required to properly operate the technology. The
evaluation of this secondary objective will also include how user-friendly the technologies are. The
developers will have the opportunity to review and comment on the observers notes before the
observations are incorporated into the report to ensure accuracy.
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6.6.9 Secondary Objective S2: Document Health and Safety Aspects Associated with the Technology
Because dioxins are a class of toxic compounds, it is important to understand the health and safety aspects
associated with each technology. This will include health and safety issues when operating the
technology as well as the amount and type of hazardous and non-hazardous waste generated by the
technology. The outcomes from this evaluation will be based on the observer notes.
6.6.10 Secondary Objective S3: Document the Portability of the Technology
This evaluation will document if the technology can be readily transported to the field and how easy the
technology was to operate in the field. The Battelle observers will be responsible for the collection and
reporting of this information.
6.6.11 Secondary Objective S4: Evaluate Sample Throughput
Because HRMS methods are time consuming, it will be important to note the sample throughput (i.e.,
number of samples that can be processed in a typical work day). This secondary objective will be
evaluated based on the observer notes, which will make particular note of the time limiting steps of the
procedures, as well as the documentation of sample custodianship that will be recorded on the
COC/results forms (Appendix D). The number of operators involved in the sample analyses will also be
discussed.
6.7 Schedule of Events
Table 6-8 is a summary of events since the award of the contract to perform this demonstration. The
reports from the data generated in this demonstration will be submitted to EPA for publication in
December 2004.
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Table 6-8. Schedule of Demonstration Events
Event
Contract award
Prepare and distribute developer survey
First Conference Call
Distribute summary notes from the conference call
Develop preliminary strategy for sample
homogenization
Prepare one-page demonstration flyer for Dioxin
2003 Conference
Obtain dioxin-contaminated soil from one site and
test homogenization procedure
Draft homogenization procedure
Second Conference Call
Identify, obtain, and homogenize samples from
additional sites
Third Conference Call
First draft demonstration plan to EPA, developers,
peer reviewers, and 1 or 2 technical advisors
Final receipt of environmental samples
PE samples sent to dioxin laboratories and audits
scheduled
Comments due to Battelle on first draft
demonstration plan
Fourth Conference Call
Reference laboratory selected
Pre-demonstration samples distributed
Developer and reference laboratory pre-
demonstration results due to Battelle
Distribute second draft demonstration plan to
EPA, developers, and entire Dioxin SITE
Demonstration Panel (includes peer reviewers,
technical advisors, and observers) for final review
Pre-demonstration results distributed to developers
Fifth Conference Call
Comments due to Battelle on third draft
demonstration plan
Demonstration plan finalized
Field demonstration (Saginaw, Michigan)
Audit of reference laboratory
First draft report to EPA
Remaining five draft reports to EPA
Reports to developers for review
Reports to peer review
Final reports submitted to EPA
Contract ends
Schedule for Completion
n/a
July 18,2003
July 28, 2003
August 5, 2003
August 12, 2003
August 22, 2003
September 30, 2003
October 3, 2003
October 8, 2003
November 28, 2003
December 4, 2003
December 12, 2003
December 19, 2003
January 9, 2004
January 15,2004
February 5, 2004
February 3, 2004
February 10, 2004
March 3 1,2004
March 3 1,2004
April 9, 2004
April 8, 2004
Apnl 12, 2004
Apnl 16, 2004
April 26 through May 5,
2004 Visitor's Day on April
28
May 24, 2004
August 2, 2004
September 6, 2004
October 1, 2004
November 1,2004
December 17, 2004
December 30, 2004
Actual Completion Date
June 18, 2003
July 18,2003
July 29, 2003
July 31,2003
August 12, 2003
August 22, 2003
October 7, 2003
October 1,2003
October 8, 2003
November 13,2003
December 4, 2003
December 12, 2003
December 24, 2003
January 12, 2004
January 15,2004
February 5, 2004
February 20, 2004
Phase 1: February 12,2004
Phase 2: March 16, 2004
Apnl 16, 2004
April 2, 2004
Apnl 16, 2004
April 8, 2004
Apnl 12, 2004
Apnl 20, 2004
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Chapter 7
Confirmatory Process
This chapter describes the process performed by Battelle for the selection of the reference method and
laboratory. The reference laboratory has provided the method performance information that is presented
in this chapter.
7.1 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 both high resolution mass spectrometric methods. Method 1613B includes more
labeled internal standards which, therefore, 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
dioxin/furan reference method.
The Dioxin SITE Demonstration Panel (Appendix A) proposed that an equal amount of reference data be
generated to determine the PCB contribution to the TEQ, since risk assessment is often times based on
TEQ values that are not class specific. As such, the complimentary HRMS method for PCB TEQ
determinations, Method 1668A, was selected as the reference method for PCBs.
7.2 Reference Laboratory Selection
Battelle generated a list often prospective laboratories, based on conversations with EPA Regional staff
who routinely use dioxin laboratories, personal experiences, and recommendations from the Dioxin SITE
Demonstration Panel. The ten laboratories were contacted by Battelle and were sent a questionnaire to
complete (see Appendix F). The 14 questions were geared towards understanding the capabilities of the
laboratories, their experience with analyzing dioxin samples for EPA, and their ability to meet the needs
of this demonstration. The responses from the questionnaires were studied and two laboratories were
selected for the next phase of the selection process. The two laboratories were sent three blind audit
samples and were audited by two auditors from Battelle. The day-long audit at each laboratory 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 follow-up questions to each laboratory to
address gaps in the observations. This was done to ensure consistency in the process.
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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, so AXYS was selected as the
reference laboratory for this demonstration. The statement of work that was provided to AXYS to perform
the reference methods is provided in Appendix G.
7.3 Reference Laboratory Sample Preparation and Analytical Methods
The purpose of this section is to describe the reference methods that will be used in the demonstration
sample analyses. Both Method 1613B(6) and Method 1668A(7) allow for method modifications, as long as
the modifications are documented and the requirements for method performance (i.e., performance
specifications for quality control samples) can be met. The following sections briefly describe the
methods and the modifications to the methods that AXYS performs. For brevity, the entire methods are
not reproduced in this document, but the applicable sections where AXYS will perform modifications
have been cited for convenient reference.
7.3.1 Dioxin/Furan An alysis
All procedures are carried out according to protocols as described in AXYS Summary Method Doc
MSU-018 Rev 2 18-Mar-2004 (AXYS detailed SOP MLA-017 Rev 9 May-2004), which is based on EPA
Method 1613B, with the modifications summarized below. Briefly, samples are spiked with a suite of
isotopically labeled surrogate standards prior to analysis, solvent extracted, and cleaned up through a
series of chromatographic columns that may include gel permeation, silica, Florisil, carbon/Celite, and
alumina columns. The extract is concentrated and spiked with an isotopically labeled (internal) standard.
Analysis is performed using a high-resolution mass spectrometer coupled to a high-resolution gas
chromatograph equipped with a DB-5 capillary chromatography column (60 m, 0.25 mm i.d., 0.1 |im film
thickness). A second column, DB-225 (30 m, 0.25 mm i.d., 0.15 |im film thickness), is used for
confirmation of 2,3,7,8-TCDF identification.
The following is a summary of AXYS's 1613B method modifications. The italicized text is the portion of
the method that is being modified, followed by the AXYS modification.
Section 2.1.2 The labeled compounds are spiked into a sample containing 10 g (dry weight) of solids.
Samples containing multiple phases are pressure filtered and any aqueous liquid is discarded. Coarse
solids are ground or homogenized. Any non-aqueous liquid from multi-phase samples is combined with
the solids and extracted in an SDS extractor. The extract is concentrated for cleanup.
Modification: Non-aqueous liquid from the multiphase sample is combined with the solid phase and
extracted by Dean Stark soxhlet.
Section 7.2.1: Sodium sulfate, reagent grade, granular, anhydrous (Baker 3375, or equivalent), rinsed
with methylene chloride (20 mL/g), baked at 400 "Cfor one hour minimum, cooled in a dessicator, and
stored in a pre-cleaned glass bottle with screw-cap that prevents moisture from entering. If, after heating,
the sodium sulfate develops a noticeable grayish cast (due to the presence of carbon in the crystal
matrix), that batch of reagent is not suitable for use and should be discarded. Extraction with methylene
chloride (as opposed to simple rinsing) and baking at a lower temperature may produce sodium sulfate
that is suitable for use.
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Modification: Anhydrous sodium sulphate (NajSO^ is purchased in powder form (not granular) and is
baked overnight prior to use. There is no solvent rinse with dichloromethane.
Section 7.10: From stock solutions, or from pur chased mixtures, prepare this solution to contain the
labeled compounds in nonane at the concentrations shown in Table 3.
Note: See Appendix H for Table 3. All tables in Appendix H are reproduced verbatim from the method.
Modification: The concentration of the labeled compound spiking solution is 100 ng/mL (except for
OCDD which is 200 ng/mL), the concentration of the labeled clean-up standard solution is 10 ng/mL and
the concentration of the labeled injection internal standard solution is 100 ng/mL. The spiking volume for
all these labeled standard solutions is 20 |iL. The resulting concentrations in the final extracts are as
specified in the method.
Section 7.11: Prepare Cl -2,3,7,8-TCDD in nonane at the concentration shown in Table 3. The cleanup
standard is added to all extracts prior to cleanup to measure the efficiency of the cleanup process.
Modification: The concentration of the clean-up standard spiking solution is 10 ng/mL and the sample
spiking volume is 20 |iL. The resulting concentrations in the final extracts are as specified in the method.
Sections 7.13 Combine the solutions in Sections 7.9 through 7.12 to produce the five calibration solutions
shown in Table 4 in nonane. 14.0 HRGC/HRMS Analysis 15.0 System and Laboratory Performance.
Note: See Appendix H for Table 4.
Modification: An additional lower level calibration solution, 0.2 times the concentration of CS1, is
prepared and included in the initial calibration series. Initial calibration is based on a six-point series.
Section 7.14 Precision and Recovery (PAR) Standard-Used for determination of initial (Section 9.2) and
ongoing (Section 15.5) precision and recovery. Dilute 10 pL of the precision and recovery standard
(Section 7.9.1 or 7.9.2) to 2.0 mL with acetone for each sample matrix for each sample batch. One mL
each are required for the blank and OPR with each matrix in each batch.
Modification: The concentrations of the PAR spiking solutions are 0.2/1.0/2.0 ng/mL for tetra/penta,
hexa, and hepta/octa, respectively, and the spiking volume is 1 mL. The resulting final concentration in
the extracts are as specified in the method.
Section 11.5 Preparation of Samples Containing Greater Than 1% Solids.
Modification: Aqueous samples containing > 1% visible solids are prepared and extracted using the same
procedure as samples containing < 1% visible solids. This involves extracting the solids by soxhlet and the
filtrate by separatory funnel extraction and combining the extract from the two phases.
Section 12 Extraction procedures include separatory funnel (Section 12.1) and solid phase (Section 12.2)
for aqueous liquids; Soxhlet/Dean-Stark (Section 12.3) for solids, filters, and SPE disks; and Soxhlet
extraction (Section 12.4.1) and HCl digestion (Section 12.4.2) for tissues. Acid/base back-extraction
(Section 12.5) is used for initial cleanup of extracts. Macro-concentration procedures include rotary
evaporation (Section 12.6.1), heating mantle (Section 12.6.2), and Kuderna-Danish (K-D) evaporation
(Section 12.6.3). Microconcentration uses nitrogen blowdown (Section 12.7).
Modification: Samples with sufficiently low moisture content may be mixed with Na2SO4 and extracted
using the regular soxhlet apparatus in 80:20 toluene:acetone.
Section 12.4 Add 30-40 g of powdered anhydrous sodium sulfate to each of the beakers (Section 11.8.4)
and mix thoroughly. Cover the beakers with aluminum foil and allow to equilibrate for 12-24 hours.
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Modification: The equilibration time for the sodium sulphate drying step is that required to produce a dry,
free flowing powder (minimum thirty minutes). This may be less than the 12-hour minimum specified.
Section 12.5.1 Spike 1.0 mL of the cleanup standard (Section 7.11) into the separatory funnels
containing the sample and QC extracts from Section 12.1.4.1, 12.3.9.1.3, or 12.3.9.2.
Modification: Samples are spiked with cleanup standard right after extraction and before reduction; not
spiked into the separatory funnels containing the extracts prior to the acid/base wash.
Section 12.6.1.1 Assemble the rotary evaporator according to manufacturer's instructions, and warm the
water bath to 45 °C. On a daily basis, preclean the rotary evaporator by concentrating 100 mL of clean
extraction solvent through the system. Archive both the concentrated solvent and the solvent in the catch
flask for a contamination check if necessary. Between samples, three 2-3 mL aliquots of solvent should be
rinsed down the feed tube into a waste beaker.
Modification: Rotary evaporator baths are maintained at 35°C. Mimic proofs are collected instead of
collecting proofs each day and archiving.
Section 13.0 Extract Cleanup.
Modification: Extracts may be cleaned up on silica, alumina, and carbon chromatographic columns using
a Fluid Management System automated cleanup system.
Sections 14.0 HRGC/HRMS Analysis 15.0 System and Laboratory Performance. ,16.0 Qualitative
Determination, Table 8, Table 9:
Note: See Appendix H for Tables 8 and 9.
Modification: M/Z channels 354/356 and 366/368 are used to confirm and quantify the native and
surrogate penta-substituted dioxins, respectively; this change from the method's specification is made in
the instrument method in order to avoid a persistent interference in the 356/358 and 368/370 M/Z
channels. The theoretical ratio for the P5CDD M/M+2 ions is 0.61; therefore, the acceptance range is 0.52
-0.70.
Section 17.0 Quantitative Determination
Modification: Conct - the concentrations of target analytes, and the labeled compound concentrations and
recoveries, are calculated using the equations below. These procedures are equivalent to those described
in the method, but are more direct.
Cone, = —-X ^— x-
ASI RRFita Mx
where Ai = summed areas of the primary and secondary m/z channels for the analyte peak
of interest (compound/)
Asi = summed areas of the primary and secondary m/z channels for the labeled
surrogate peak used to quantify /')
Mx = mass of sample taken for analysis
Msi = mass of labeled surrogate (compound si) added to sample as calculated by the
concentration of standard spiked (pg/mL) multiplied by the volume spiked
(mL)
RRFisi = mean relative response factor of/' to si from the six-point calibration range and
defined individually as:
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^L M™
= 4* Msl
Concentrations of surrogate standards are calculated using the following equation:and, the percent
Cone = —— >
recoveries of the surrogate standards are calculated using the following equation:
%Recovery =—^x M" x—!— xlOO
A.
where Ars and Asi are the summed peak areas (from the primary and secondary m/z channels) of
recovery standard and labeled surrogate added to the sample
Mrs and M^ are the masses of recovery standard and labeled surrogate added to the sample
RRFsirs is the mean relative response factor of the labeled surrogate to the recovery standard as
determined by the six-point calibration range and defined individually as:
A. M
Section 17.5 If the selected ion current profile area at either quantitation m/z for any compound exceeds
the calibration range of the system, a smaller sample aliquot is extracted.
Modification: Extracts may be diluted with solvent and reanalyzed by GC/MS isotope-dilution to bring
the instrumental response to within the linear range of the instrument. For very high-level samples where
a smaller sample aliquot may not be representative, extracts may be diluted and respiked with labeled
quantification standards and reanalyzed by GC/MS to bring the instrumental response analytes within
range. Final results may be recovery corrected using the mean recovery of labeled quantification
standards.
7.3.1.1 Effects of these Modifications on Method Performance
It should be noted that the performance of this method including these modifications will be verified
during the in-process audit of the reference laboratory and by the laboratory's performance on QC
samples. These checks will confirm that the above-listed modifications will not negatively affect the
accuracy or precision of the reference laboratory results.
7.3.2 PCB Analysis
The method is 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 166 8A, with changes through August 20, 2003, incorporating the AXYS modifications described
below. Details of all procedures are documented in AXYS method MLA-010, Analytical Method for
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Determination of 209 PCB Congeners by EPA Method 1668A. Briefly, samples are spiked with
isotopically labeled surrogate standards, solvent extracted and cleaned up on a series of chromatographic
columns which may include silica, Florisil, alumina, carbon/Celite and gel permeation columns. The final
extract is spiked with isotopically labeled recovery (internal) standards prior to instrumental analysis.
Analysis of the extract is performed on an HRMS coupled to a HRGC equipped with a SPB-Octyl
chromatography column (30 m, 0.25 mm i.d., 0.25 |im film thickness). Resolution of the PCB 156/157
coelution may be achieved by high resolution GC/MS using a DB-1 chromatography column (30 m,
0.25 mm id, 0.25 |im film thickness). Where required to eliminate interferences, extracts maybe cleaned
up by carbon columning to isolate the toxic PCBs of interest; analysis may then be conducted using the
DB-1 column as the primary GC column rather than the SPB Octyl column.
The following is a summary of AXYS's 1668A method modifications. The italicized text is the portion of
the method that is being modified, followed by the AXYS modification.
Section 4.2.1 Glassware should be rinsed with solvent and washed with a detergent solution as soon after
use as is practical. Sonication of glassware containing a detergent solution for approximately 30 seconds
may aid in cleaning. Glassware with removable parts, particularly separatory funnels withfluoropolymer
stopcocks, must be disassembled prior to detergent washing. 4.2.2 After deter gent washing, glassware
should be rinsed immediately; first with methanol, then with hot tap water. The tap water rinse is
followed by another methanol rinse, then acetone, and then methylene chloride.
Modification: The protocol for washing reusable glassware includes a detergent wash, water rinse and
baking at 325°C for 8 hours. Immediately prior to use, glassware is solvent rinsed with toluene and
hexane.
Section 4.7 Cleanup oftissue-The natural lipid content of tissue can interfere in the analysis of tissue
samples for the CBs. The lipid contents of different species and portions of tissue can vary widely. Lipids
are soluble to varying degrees in various organic solvents and may be present in sufficient quantity to
overwhelm the column chromatographic cleanup procedures used for cleanup of sample extracts. Lipids
must be removed by the anthropogenic isolation column procedure in Section 13.6, followed by the gel
permeation chromatography procedure in Section 13.2. Florisil (Section 13.7) is recommended as an
additional cleanup step.
Modification: The first cleanup column for tissue extracts is a gravity gel permeation column (SX-3
Biobeads). An anthropogenic isolation column 7.5.3 is not used.
Section 6.5.1 Pyrex glass wool-Solvent-extracted using a Soxhlet or Soxhlet/Dean-Stark extractor for 3
hours minimum
Modification: Glass wool is cleaned by rinsing twice with toluene and twice with hexane.
Section 7.12 Labeled Toxics/LOC/window-defining standard spiking solution-This solution is
spiked into each sample (Section 9.3) and into the IPR (Section 9.2.1), OPR (Section 15.5), and blank
(Section 9.5) to measure recovery. Dilute the Labeled Toxics/LOC/window-defining stock solution
(Section 7.9.1) with acetone to produce a concentration of the labeled compounds at 2 ng/mL, as shown
in Table 3. When 1 mL of this solution is spiked into an IPR, OPR, blank, or sample and concentrated to
a final extract volume of 20 FL, the concentration in the final extract volume will be 100 ng/mL (100
pg/FL). Prepare only the amount necessary for each reference matrix with each sample batch. 7.13
Labeled cleanup standard spiking solution-This solution is spiked into each extract prior to cleanup to
measure the efficiency of the cleanup process. Dilute the Labeled cleanup standard stock solution
(Section 7.9.2) in methylene chloride to produce a concentration of the cleanup standards at 2 ng/mL, as
shown in Table 3. When 1 mL of this solution is spiked into a sample extract and concentrated to a final
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volume of 20 juL,the concentration in the final volume will be 100 ng/mL (100pg/juL). 9.0 Quality
assurance/quality control 11.0 Sample preparation
Note: See Appendix H for Table 3.All tables in Appendix H are reproduced verbatim from the method.
Modification: The concentration of the labeled toxics/LOC and the cleanup standard spiking solutions is
100 ng/mL and the sample spiking volume is 20 |iL. The resulting final concentrations in the extracts are
as specified in the method.
Section 7.14 Labeled injection internal standard spiking solution-This solution is added to each
concentrated extract prior to injection into the HRGC/HRMS. Dilute the Labeled injection internal
standard stock solution (Section 7.9.3) in nonane to produce a concentration of the injection internal
standards at 1000 ng/mL, as shown in Table 3. When 2 //Z of this solution is spiked into a 20 //Z extract,
the concentration of each injection internal standard will be nominally 100 ng/mL (100 pg/pL). Note: The
addition of 2 pL of the Labeled injection internal standard spiking solution to a 20 pL final extract has
the effect of diluting the concentration of the components in the extract by 10%. Provided all calibration
solutions and all extracts undergo this dilution as a result of adding the Labeled injection internal
standard spiking solution, the effect of the 10% solution is compensated, and correction for this dilution
should not be made.
Modification: Concentration of the labeled injection internal standard spiking solution (recovery
standard) is modified so that a volume of 5 |iL is added. The resulting amount of standard added to the
final extract is the same as specified in the method. The solution is spiked into a 15-|iL extract volume for
a final extract volume of 20 |iL.
Section 7.2.1 Solution drying-Sodium sulfate, reagent grade, granular, anhydrous (Baker 33 75, or
equivalent), rinsed with methylene chloride (20 mL/g), baked at 400 °Cfor 1 hour minimum, cooled in a
desiccator, and stored in a pre-cleaned glass bottle with screw-cap that prevents moisture from entering.
If, after heating, the sodium sulfate develops a noticeable grayish cast (due to the presence of carbon).
Modification: Powdered, not granular, sodium sulphate is baked at 325°C for 8 hours rather than at
600°Cfor24hrs.
Section 7.5.1 Activated silica gel-100-200 mesh, Supelco 1-3651 (or equivalent), rinsedwith methylene
chloride, baked at 180 °Cfor a minimum ofl hour, cooled in a desiccator, and stored in aprecleaned
glass bottle with screw-cap that prevents moisture from entering.
Modification: Silica is activated by baking at 450°C in a muffle oven for at least 8 hours.
Section 7.5.4.1.1 Fill a clean 1- to 2-L bottle 1/2 to 2/3 full with Florisil and place in an oven at 130-150
°C for a minimum of three days to activate the Florisil.
Modification: Florisil is baked at 450°C in a muffle oven for at least 8 hours, then deactivated with water
to 2.1% deactivation.
Section 11.5.6 Decant excess water. If necessary to remove water, filter the sample through a glass-fiber
filter and discard the aqueous liquid.
Modification: Unless requested by the client, the aqueous portion after filtration of aqueous samples with
> 1% solids is not discarded but is extracted.
Section 11.5 Preparation of samples containing greater than one percent solids 11.5.2 Spike 1.0 mL of
the Labeled Toxics/LOC/window-defining standard spiking solution (Section 7.12) into the sample.
12.3 Soxhlet/Dean-Stark extraction of samples containing particles.
Modification: Solid samples are dried by mixing with anhydrous sodium sulphate. The dried solid is
extracted using a soxhlet extraction apparatus. The surrogate spike is incorporated after the drying step.
The extracting solvent for solids is dichloromethane.
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Section 11.8 Fish and other tissues-Prior to processing tissue samples, the laboratory must determine
the exact tissue to be analyzed. Common requests for analysis offish tissue include
whole fish-skin on, whole fish-skin removed, edible fish fillets (filleted in the field or by
the laboratory), specific organs, and other portions. Once the appropriate tissue has been
determined, the sample must be homogenized. 12.4 Soxhlet extraction of tissue
Modification: The surrogate spike is incorporated into the sample after the drying step to eliminate the
possibility of disproportional loss of volatile labeled and target compounds.
Section 12.4.2 Assemble and pre-extract the Soxhlet apparatus per Sections 12.3.1-12.3.4, exceptuse the
methylene chloride :hexane (1:1) mixture for the pre-extraction and rinsing and omit the quartz sand.
Modification: The pre-cleaning of the soxhlet apparatus is carried out using toluene instead of
dichloromethane.
Section 12.4.9 Percent lipid determination-The lipid content is determined by extraction of tissue with the
same solvent system (methylene chloride:hexane) that was used in EPA's National Dioxin Study
(Reference 16) so that lipid contents are consistent with that study.
Modification: Lipid analysis is carried out by sub-sampling two 2-g portions of the extract from a total
30-g extract weight. The cleanup standard is spiked into the extract after soxhlet extraction and before any
lipid analysis or rotary evaporation is done.
Section 12.6.1.1: Assemble the rotary evaporator according to manufacturer's instructions, and warm the
water bath to 45 °C. On a daily basis, pre-clean the rotary evaporator by concentrating 100 mL of clean
extraction solvent through the system. Archive both the concentrated solvent and the solvent in the catch
flask for a contamination check if necessary. Between samples, three 2- to 3-mL aliquots of solvent should
be rinsed down the feed tube into a waste beaker.
Modification: Rotary evaporation is done at 30 deg. C. Daily cleaning of the rotary evaporators include
dismantling the vapor tube, sonicating the vapor tube for 20 minutes in toluene, and soaking the vapor
tube sheath and locking caps in soapy water overnight. The vapor tube is rinsed with hexane and the
vapor tube sheath and locking cap are rinsed with water, methanol, and hexane prior to air drying and
assembly. Mimic proofs are run periodically but are not archived daily.
Section 12.7.4 When the volume of the liquid is approximately 100 fjL, add 2 to 3 mL of the desired
solvent (methylene chloride for GPC andHPLC, or hexane for the other cleanups) and continue
concentration to approximately 100 FL. Repeat the addition of solvent and concentrate once more.
Modification; Before Florisil or alumina cleanup procedures, a solvent exchange is done by reducing
under nitrogen to 300 uL and bulking up to 1 mL in hexane. If toluene is present the extract is reduced to
50 uL under nitrogen and bulked up to 1 mL.
Section 12.7.7 If the extract is to be concentrated for injection into the GC/MS (Section 14), qantitatively
transfer the extract to a 0.3-mL conical vial for final concentration, rinsing the larger vial with hexane
and adding the rinse to the conical vial. Reduce the volume to approximately 100 pL. Add 20 pL of
nonane to the vial, and evaporate the solvent to the level of the nonane. Seal the vial and label with the
sample number. Store in the dark at room temperature until ready for GC/MS analysis. If GC/MS
analysis will not be performed on the same day, store the vial at < -10 °C.
Modification: Toluene (1 mL) is added to the eluate from the final column prior to rotary evaporation
and nitrogen blow down concentration steps.
Section 13.1.1 Gel permeation chromatography (Section 13.2) removes high molecular weight
interferences that cause GC column performance to degrade. It should be used for all soil and sediment
71
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extracts. It may be used for water extracts that are expected to contain high molecular weight organic
compounds (e.g., polymeric materials, humic acids). It should also be used for tissue extracts after initial
cleanup on the anthropogenic isolation column (Section 13.6).
Modification: GPC chromatography, by a gravity column, is routinely used only for tissue extracts. The
GPC cleanup is optional for all other matrices.
Section 13.3.1 Place a glass-wool plug in a 15-mm ID chromatography column (Section 6.7.4.2). Pack
the column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel (Section 7.5.1.3), 1 g
silica gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g granular anhydrous sodium sulfate
(Section 7.2.1). Tap the column to settle the adsorbents.
Modification: Routine layered silica column is as follows: 0.5 g neutral silica, 2 g 28% basic silica, 0.5 g
neutral silica, 4 g 44% acidic silica, 4 g 22% acidic silica, 1 g neutral silica.
Section 13.3.4 Rinse the receiver twice with 1-mL portions ofhexane, and apply separately to the
column. Elute the CBs with 25 mL ofhexane and collect the eluate.
Modification: The sample is loaded onto the column followed by 2-3 rinses of a least 1 mL, and eluted
with 100 mL ofhexane.
Section 14.2 Add 2 //Z of the Labeled injection internal standard spiking solution (Section 7.14) to the
20 jiL sample extract immediately prior to injection to minimize the possibility of loss by evaporation,
adsorption, or reaction. If an extract is to be reanalyzed and evaporation has occurred, do not add more
Labeled injection internal standard spiking solution. Rather, bring the extract back to its previous volume
(e.g., 19 jiL) with pure nonane (18 pL if 2 pL injections are used).
Modification: The volume of labeled injection internal standard (recovery standard) added to the extract
is 5 |iL, for a final extract volume of 20 |iL. Hexane rather than nonane is used as the solvent to bring
extract back to volume for reanalysis or to dilute extracts.
Section 17.5 If the selected ion current profile area at either quantitation m/zfor any congener exceeds
the calibration range of the system, dilute the sample extract by the factor necessary to bring the
concentration within the calibration range, adjust the concentration of the Labeled injection internal
standard to 100 pg/pL in the extract, and analyze an aliquot of this diluted extract. If the CBs cannot be
measured reliably by isotope dilution, dilute and analyze an aqueous sample or analyze a smaller portion
of a soil, tissue, or mixed-phase sample. Adjust the CB congener concentrations, detection limits, and
minimum levels to account for the dilution.
Modification: Extracts are diluted with hexane. The concentration of the labeled injection internal
(recovery) standard is not readjusted to 100 pg/uL when dilutions are performed.
Table 6, page 92:
Note: See Appendix H for Table 6.
Modification: The acceptance ranges for the labeled compounds dichlorobiphenyls (PCBs 4L and 15L)
in OPRs, IPRs and samples have been lowered to 15% in recognition of the higher volatility of these
compounds. This is a project-specific change to Sections 4.2 and 10.0 of SOP MLA-010.
Section 17.0 Quantitative Determination
Modification: Cone,-the concentrations of target analytes, and the labeled compound concentrations and
recoveries, are calculated using the equations below. These procedures are equivalent to those described
in the method but are more direct.
72
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A,. RRF
si,rs
where A; = summed areas of the primary and secondary m/z's for the analyte
peak of interest (compound /)
Asi = summed areas of the primary and secondary m/z's for the labeled
surrogate peak used to quantify /')
Mx = mass of sample taken for analysis
Msi = mass of labeled surrogate (compound si) added to sample as
calculated by the concentration of standard spiked (pg/mL)
multiplied by the volume spiked (mL)
RRFisi = mean relative response factor of/' to si from the five-point
calibration range and defined individually as:
A. M
~
Concentrations of surrogate standards are calculated using the following equation:
Cone = —- x — x •
A* RRF^
and, the percent recoveries of the surrogate standards are calculated using the following equation:
%Recovery =x " x-— xlOO
Ars RRFsirs MS1
where Ars and Asi are the summed peak areas (from the primary and secondary m/z channels) of
recovery standard and labeled surrogate added to the sample;
Mrs ar\dMsi are the masses of recovery standard and labeled surrogate added to the
sample, and;
RRFsirs is the mean relative response factor of the labeled surrogate to the recovery
standard as determined by the five-point calibration range and defined individually as:
' Ars Msl
7.3.2.1 Effects of these Modifications on Method Performance
It should be noted that the performance of this method including these modifications will be verified
during the in-process audit of the reference laboratory and by the laboratory's performance on QC
73
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samples. These checks will confirm that the above-listed modifications will not negatively affect the
accuracy or precision of the reference laboratory results.
7.3.3 Options for High-Level Samples
For extremely high level samples (> 1,000,000 pg/g), AXYS may dilute with solvent to the limit for
reliable isotope dilution quantification (labeled response > low calibration native response and S:N >
10:1 for both ions). For those samples that do not meet this criterion, a diluted portion of the extract will
be respiked with an additional aliquot of surrogate standard, reinjected, and final concentrations for
dilution/respike factors and surrogate recoveries using the mean surrogate recovery (excluding any
surrogate responses compromised by high-level native presence) will be quantified.
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Chapter 8
Data Management
To ensure that the demonstration data are scientifically valid, defensible, and comparable, appropriate
procedures will be used to perform data management. This chapter describes (1) data reduction, (2) data
review, (3) data reporting, and (4) data storage procedures for the demonstration.
8.1 Data Reduction
Each analytical method selected for the demonstration and each developer technology's instruction
manual contain detailed instructions and equations for generation of results. Each developer will be
responsible for reducing their own data and providing final results to Battelle in the form described in
Table 6-7. The reference laboratory will generate concentration data for the dioxin/furan congeners using
EPA Method 1613B and for the WHO PCBs using modified Method 1668A. The reference laboratory
concentration data will be converted to TEQ using the WHO 1998 guidance as described in Chapters 1
and 6. The reference laboratory will generate final concentration and TEQ data, and Battelle will review
those results through data validation (see Section 8.2.3). Comparisons between the developer and
reference laboratory data will be dependent on how the developer is reporting their data (see Sections 1.3
and 6.6.3 for further discussion on this topic).
8.2 Data Review
A review of technology and laboratory analytical data will be conducted by each developer and the
reference laboratory, respectively. Battelle will also conduct a review of all field and laboratory data. The
review processes that will be used for developer and laboratory analytical data are described below.
8.2.1 Data Review by Developers
Each developer will review all results generated by its measurement technology. The developer will
review all demonstration sample data as well as QC results (such as positive controls/spikes and method
blanks) for their technology. The developer will report results to Battelle on the COC/results form
(Appendix D) and in the units described in Table 6-7.
Battelle will generate a Microsoft™ Excel spreadsheet of each developer's results, including only sample
number and developer result, and send the spreadsheet to each developer for review. This will allow the
developer to review the results that have been entered by Battelle in electronic form to minimize
transcription or interpretation errors. Battelle will not perform any data analysis with the developer data
until the spreadsheet has been reviewed by the developer.
75
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8.2.2 Data Review by Reference Laboratory
The procedures for data review by the reference laboratory are provided in Appendix I.
8.2.3 Data Review by Battelle
In addition to the review process that will be used by the reference laboratory, the Battelle project
manager or designee (e.g., such as a Battelle dioxin expert) will review all laboratory and developer
results, based on demonstration objectives. The Battelle project manager or designee (such as the QA
manager) will also conduct a complete data validation for 100 percent of the data as an independent check
of the reference laboratory results. If this validation reveals no oversights or problems, Battelle will
consider all data to be acceptable. If oversights or problems are identified, the reference laboratory project
manager will be consulted. The reference laboratory data will be compared to the data generated by
Battelle during the characterization analyses (see Section 4.3). This will be a key comparison which will
confirm the overall quality of the data set. Battelle's assessment of the data and QC results will be
summarized for discussion with the EPA program manager and incorporated into the DER. The checklists
for performing the data validations are presented in Appendix J.
During its data review, Battelle will identify project outlier data using statistical testing and will report
these data to the EPA program manager. Project outlier data are defined as sample data outside specified
acceptance limits established about the central tendency estimator (the arithmetic mean) of the data set for
a given area or for all areas taken together. For data known or assumed to be normally distributed, the
specified acceptance limits will be the 95 percent confidence limits defined by the Student's two-tailed
t-test. Consistent procedures will be used to identify outliers for both reference laboratory and developer
data. No data will be rejected simply because they are statistical outliers, but data may be reported with
and without the statistical outliers as appropriate. Battelle will conduct a thorough check to identify the
reasons for the outliers and will provide the EPA program manager with an explanation of why some data
appear to be outliers.
8.3 Data Reporting
Each developer and the reference laboratory will prepare and submit data packages reporting the results
of developer and laboratory results, respectively. The reference laboratory will also prepare and submit
electronic data deliverables (EDD). Battelle will use these data to prepare the ITVR for each developer
technology and the DER for the entire demonstration. Described below are the data reporting require-
ments for (1) developer data packages, (2) reference laboratory data packages, (3) ITVRs, and (4) the
DER.
8.3.1 Developer Data Packages
The developers will compile their results on standard forms provided by Battelle (see Appendix D). The
forms will contain sample identification numbers and spaces for a developer to enter their results as
appropriate (i.e., each form will be unique to each developer). Electronic reporting of results will not be
required. The developers will only be required to report their sample results. Developer-supplied QC
sample results will be requested for the DER. Raw data, copies of logbook pages, standards preparation
logs, etc. that are included in a typical laboratory data package will not be required from the developers.
76
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8.3.2 Reference Laboratory Data Packages
The procedure for data package preparation for the reference laboratory is presented in Appendix K.
8.3.3 Innovative Technology Verification Reports
In accordance with the demonstration plan, Battelle will evaluate the performance and cost data collected
for each dioxin measurement technology demonstrated and will prepare an ITVR for the technology.
Each ITVR will be a focused report of about 100 pages and will include the following:
• An introduction
• A description of the measurement technology
• Site descriptions and the demonstration design
• Deviations from the demonstration plan
• A description of the reference method and its performance
• A description of the technology's performance
• An economic analysis
• A summary of demonstration results
• A verification statement.
Battelle will prepare individual ITVRs in accordance with the format specified in the "Handbook for
Preparing Office of Research and Development Reports"00' and project-specific guidance from the EPA
program manager. The reports will be written in such a way that a reader with a basic science background
can understand their contents and make an informed decision regarding the performance of the
technologies. The ITVRs will undergo a rigorous review process that will include reviews by the EPA
program manager, the developers, and external peer reviewers provided in accordance with the detail and
content of previous reports.
8.4 Data Evaluation Report
Battelle will prepare a DER containing tabular summaries of investigative and QA/QC data from the
demonstration as well as results of technical system and performance audits. The DER will primarily
discuss the following:
Pre-demonstration activities
• Demonstration activities
Post-demonstration activities
• All developer and reference laboratory demonstration sample data
• QA/QC data, including completed audit, observer, and data validation checklists
Audit reports.
8.5 Data Storage
The reference laboratory analysts responsible for performing measurements will enter raw data into
logbooks or on datasheets. In accordance with standard document control procedures, the laboratory will
maintain on file the original logbooks or data sheets, which will be signed and dated by the laboratory
analysts responsible for them. Similar procedures will be used for all data entered directly into the
laboratory information management system. Separate instrument logs will also be maintained by the
laboratory to allow reconstruction of the run sequences for individual instruments. The reference
laboratory will maintain all raw data, including raw instrument output on tape or diskette, on file for
5 years after the submission of the data packages to Battelle. Data documents will be kept in secure
77
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archive file cabinets accessible only to designated laboratory personnel. The data will be disposed of upon
receipt of EPA instructions to do so or after 5 years, whichever is sooner. A central project file for the
demonstration will be established in the Records Management Office at Battelle's Columbus head-
quarters. This file will be a repository for all relevant field and laboratory project documentation. Battelle
will offer the central file to the EPA at the end of the demonstration project but will maintain in the
central file until the end of the SITE contract if requested to do so.
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Chapter 9
QA/QC Procedures
This chapter describes the QA/QC procedures that will be implemented in this demonstration to ensure
that the data generated are of high quality.
9.1 QA/QC Objectives
The overall QA objective for the demonstration is to produce well-documented data of known quality.
Data quality will be measured in terms of the data's precision, accuracy, representativeness, complete-
ness, and comparability. Table 9-1 contains the objectives for the data quality indicators which applies to
both the developer and reference laboratory data. If analytical data from the reference laboratory fail to
meet the QA objectives described in this section (except for comparability, which does not apply), the
source of the errors will be investigated and corrective actions will be taken if necessary and possible.
(Corrective actions associated with the reference method are discussed in detail in Section 9.2.) If
analytical data from the field technologies did not meet the QA objectives, the discrepancies will be
described in the ITVRs.
Table 9-1. Data Quality Indicator Objectives for Reference Laboratory and Developer Data
Data Quality Indicator
Precision
Accuracy
Representativeness
Comparability to reference method*
Completeness
Calculation
RSD of replicate samples
Percent recovery of certified or
spiked PE values
Valid samples from each soil and
sediment type
average absolute median RPD
Percent of total samples analyzed
and valid results provided
Objective
Average of all RSDs < 20 percent
75 percent to 125 percent
At least one valid sample result
generated from each soil and
sediment sampling location
< ± 25 percent
98 percent
a Applies only to developer data
9.2 Internal QC Checks
9.2.1 Reference Method QC Checks
Tables 9-2, 9-3, 9-4, and 9-5 summarize the QC checks that will be performed by the reference laboratory
as described in AXYS Summary Method Doc MSU-018 Rev. 2 18-Mar-2004 (AXYS detailed SOP
MLA-017 Rev 9 May-2004) and AXYS Summary Method Doc MSU-020 Rev 03 24-Mar-2004 (AXYS
detailed SOP MLA-010 Rev 5 Sep-2003).
79
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Table 9-2. QC Acceptance Criteria for EPA Method 1613B a
Native Compound
2,3,7,8-TCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HXCDD
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDD
OCDF
Surrogate Standards
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
13C12-l,2,3J,8-PeCDD
13C12-l,2,3J,8-PeCDF
13C12-2,3,4,7,8-PeCDF
13C12-l,2,3,4,7,8-HxCDD
13C12-l,2,3,6,7,8-HxCDD
13C12-l,2,3,4,7,8-HxCDF
13C12-l,2,3,6,7,8-HxCDF
13C12-l,2,3,7,8,9-HxCDF
1JC12-2,3,4,6,7,8-HxCDF
13C12-l,2,3,4,6,7,8-HpCDD
13C12-l,2,3,4,6,7,8-HpCDF
13C12-l,2,3,4,7,8,9-HpCDF
13C,,-OCDD
Cleanup Standard
37CL-2,3J,8-TCDD
Test Cone
ng/mL
10
10
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
10
IP
RSD
(%)
28
20
15
15
17
19
15
22
17
13
13
15
15
13
16
19
27
37
35
39
34
38
41
38
43
35
40
37
35
41
40
48
36
Rb
X(%)
83-129
87-137
76-132
86-124
72-150
78-152
84-124
74-142
82-108
92-120
84-122
74-158
76-130
90-112
86-126
86-126
74-146
28-134
31-113
27-184
27-156
16-279
29-147
34-122
27-152
30-122
24-157
29-136
34-129
32-110
28-141
20-138
39-154
OPRC
(%)
70-130
75-130
70-130
80-130
70-130
70-130
76-130
70-130
72-130
84-130
78-130
70-130
70-130
82-122
78-130
78-130
70-130
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
25-120
40-120
I-CAL
%
20
20
20
20
20
20
20
35
20
20
20
20
20
20
20
20
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
CAL/VER"
(%)
78-125
84-120
78-130
82-120
82-122
78-125
78-125
82-122
90-112
88-114
90-112
88-114
86-116
90-110
86-116
79-125
75-125
82-121
71-130
70-130
76-130
77-130
85-117
85-118
76-130
70-130
74-130
73-130
72-130
78-129
77-129
70-130
79-127
Labeled
Cmpd
%Rec. in
Sample
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
40-120
25-120
40-120
QC acceptance criteria for IPR, OPR, and samples based on a 20-|iL extract final volume.
b IPR: Initial precision and recovery demonstration.
c OPR: Ongoing precision and recovery test run with every batch of samples.
d CAL/VER: Calibration verification test run at least every 12 hours.
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Table 9-3. Method 1613B Specifications for QC Samples, Instrumental Analysis, and Analyte Quantification
QC Parameter
Analysis Duplicate
Procedural Blank
Detection Limit
Analyte/Surrogate Ratios
Ion Ratios
Sensitivity
Specification
Must agree to within ±20% of the mean (applicable to concentrations 1 0 times the
DL)a
TCDD/F < 0.5 pg/sample, PeCDD/F, HxCDD/F, HpCDD/F <
OCDD/F < 5 pg/sample
1.0 pg/sample,
SDL requirements
1 pg/sample
Response must be within the calibrated range of the instrument
data from more than one chromatogram to get the responses in
Coders may use
the calibrated range.
Must be within ±15% of theoretical
S:N>10: 1 for all compounds for 0. 1 pg/|_iL (CS-0.2), plus
For bloods: S:N>3:1 for 0.025 pg/|_iL 2,3,7,8-T4CDD
a Duplicate criterion is a guideline; final assessment depends upon sample characteristics, overall batch QC and on-going lab
performance.
Table 9-4. Method 1668A QC Acceptance Criteria for Chlorinated Biphenyls in CAL/VER, IPR, OPR, and
Samples a
Congener
2-MoCB
4-MoCB
2,2'-DiCB
4,4'-DiCB
2,2'6-TrCB
3,4,4'-TrCB
2,2'6,6'TeCB
3,3',4,4'-TeCB
3,4,4',5-TeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB
2,3,4,4',5-PeCB
2,3',4,4',5-PeCB
2',3,4,4',5-PeCB
3,3',4,4',5-PeCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB
2,3,3',4,4',5'-HxCBf
2,3',4,4',5,5'-HxCB
3,3',4,4',5,5'-HxCB
2,2',3,4',5,6,6'-HpCB
2,3,3',4,4',5,5'-HpCB
2,2',3,3',5,5',6,6'-OcCB
2,3,3',4,4',5,5',6-OcCB
2,2',3,3',4,4',5,5',6-NoCB
Congener
number1"
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
Test
cone
(ng/mL)
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
CAL/VERC
(%)
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
75-125
IPR"
RSD
(%)
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
X
(%)
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
OPRe
(%)
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
Labeled compound
recovery in samples
(%)
% (no
carbon
column)
%
(carbon
column)
81
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Congener
2,2',3,3;4,5,5',6,6'-NoCB
DeCB
Labeled Compounds
13C12-2-MoCB
13C12-4-MoCB
13C12-2,2'-DiCB
13C12-4,4'-DiCB
13C12-2,2',6-TrCB
13C12-3,4,4'-TrCB
13C12-2,2',6,6'-TeCB
13C12-3,3',4,4'-TCB
13C12-3,4,4',5-TeCB
13C12-2,2',4,6,6'-PeCB
13C12-2,3,3',4,4'-PeCB
13C12-2,3,4,4',5-PeCB
13C12-2,3',4,4',5-PeCB
13C12-2',3,4,4',5-PeCB
13C12-3,3',4,4',5-PeCB
13C12-2,2'54,4',6,6'-HxCB
13C12-2,3,3',4,4',5-HxCBf
13C12-2,3,3',4,4'55'-HxCBf
13C12-2,3',4,4',5,5'-HxCB
13C12-3,3',4,4',5,5'-HxCB
13C12-2,2',3,4',5,6,6'-HpCB
13C12-2',3,3',4,4',5,5'-HpCB
13C12-2,2',3,3',5,5',6,6'-OcCB
13C12-2,3,3',4,4',5,5',6-OcCB
13C12-2,2',3,3',4,4',5,5',6-NoCB
13C12-2,2',3,3',4,5,5',6,6'-NoCB
13C12-2,2'53,3',454'55,5',656'-
DeCB
Congener
number1"
208
209
1L
3L
4L
15L
19L
37L
54L
77L
81L
104L
105L
114L
118L
123L
126L
155L
156L
157L
167L
169L
188L
189L
202L
205L
206L
208L
209L
Test
cone
(ng/mL)
50
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
CAL/VERC
(%)
75-125
75-125
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
65-135
IPR"
RSD
(%)
40
40
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
X
(%)
60-140
60-140
20-135
20-135
20-135
20-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
OPRe
(%)
70-130
70-130
15-140
15-140
20-140
20-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
Labeled compound
recovery in samples
(%)
% (no
carbon
column)
15-130
15-130
20-130
20-130
30-130
30-130
30-130
30-130
30-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
40-130
Cleanup Standard
13C12-2,4,4'-TnCB
13C12-2,3,3',5,5'-PeCB
13C19-2,2',3,3',5,5',6-HpCB
28L
111L
178L
100
100
100
60-130
60-130
60-130
45
45
45
45-120
45-120
45-120
40-125
40-125
40-125
40-130
40-130
40-130
%
(carbon
column)
-
-
-
-
-
-
-
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
25-130
-
-
-
-
-
-
-
-
a QC acceptance criteria for IPR, OPR, and samples based on a 20-|iL extract final volume.
b Suffix "L" indicates labeled compound.
c CAL/VER: Calibration verification test run at least every 12 hours.
d IPR: Initial precision and recovery demonstration.
e OPR: Ongoing precision and recovery test run with every batch of samples.
f PCBs 156 and 157 are tested as the sum of two concentrations.
82
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Table 9-5. Method 1668A Specifications for QC Samples, Instrumental Analysis, and Analyte Quantification
QC Parameter
Analysis Duplicate
Procedural Blank
Detection Limit
Initial Calibration
Continuing CAL/VER
Analyte/Surrogate Ratios
Ion Ratios
Sensitivity
Specification
Must agree to within ±20% of the mean (applicable to concentrations >10 times
the DL)a
Analyte concentrations in blank samples for PCB congeners 77, 81, 1 14, 123,
126 and 169 must be less than 2 pg/congener, and concentrations of PCB
congeners 156, 157, 167 and 189 must be less than 10 pg/congener.
Concentrations of all other individual PCB congeners or coelutions must be less
than 50 pg/congener in blank samples. The sum of all 209 congeners must be
less than 200 pg.
Typical detection limits for individual congeners range from 0.5 to 2.0 pg.
For 5-point calibration, a relative standard deviation of the RRF's <20% for all
compounds. Ion ratios for all congeners must be within ±15% of theoretical for
CS-0.2. Minimum S:N ratio 10:1 forCS-0.2
Concentrations for all labeled surrogate PCB congeners must fall within ±35%
of expected concentrations. Concentrations for all native PCB congeners must
fall within ±25% of expected concentrations. Concentrations for labeled cleanup
PCB Congeners must fall within 60% - 1 30% of expected concentrations.
Response must be within the calibrated range of the instrument. Coders may
use data from more than one chromatogram to get the responses in the
Ion ratios must fall within ±15% of the theoretical values for positive
identification of all targets in the calibration standards and samples.
Minimum S:N ratio 10:1 for CS-0.2
Duplicate criterion is a guideline; final assessment depends upon sample characteristics, overall batch QC, and on-going lab
performance.
9.2.2 Developer Technology QC Checks
Quality control checks to be performed by the developers will be at each developer's discretion, although it is
highly recommended that quality controls such as blanks, spikes, and duplicates, be systematically analyzed
throughout the demonstration. Developer QC data will be reported to Battelle for inclusion in the DER. The
developers process for QC sample analysis will be evaluated and recorded by the Battelle observers.
9.3 Audits, Corrective Actions, and QA Reports
The assessment stage involves procedures to verify that project efforts are in compliance with the quality system
as the project is being implemented, and that upon conclusion of the data gathering stage of the project, the
collected data meet the performance and acceptance criteria (e.g., data quality objectives) specified in the
planning stage of the project. The QA manager or designee conducts audits at planned, scheduled intervals;
implements provisions for timely responses and implementation of corrective actions if needed; and completes
the evaluation process with written reports to technical and management staff. The Battelle project manager will
ensure that this individual has sufficient authority, access to project staff, access to documents and records, and
organizational freedom to conduct the assessment.
9.3.1 Technical Systems Audits
Battelle will conduct a TSA during the time when the reference laboratory is analyzing the demonstration
samples. This in-process audit will include a technical systems audit and a quality systems audit and will last
one full-day. The work plan for this audit is included in Appendix L. This audit is very similar in focus and
scope as the audit that was performed to select AXYS as the reference laboratory. The audit will consist of a
short opening conference, a full day of observation of laboratory procedures, records, and interviews with
laboratory staff, and a brief closing meeting. Auditors will inspect whether the observations/questions/issues
83
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identified during the first TSA (conducted prior to selection of the reference laboratory) are adequately
addressed/implemented by the reference laboratory. The TSA will focus on topics specifically related to this
project such as: sample and standards tracking, sample and standard preparation, instrument calibration, sample
analysis, sample integration and data reduction, and acceptability of quality control data. Records to be
reviewed are:
Sample receipt records and holding location/conditions
Standard (spiking and calibration solution) receipt records and certificates of analysis
Standard preparation records
• Support equipment records (balances, thermometers)
Sample preparation and spiking records
Preparation of instrument standards
• Preparation of spiking solutions
Instrument calibration and maintenance records
• Instrument sample run logs
• Instrument set-up conditions, temperature programs, calibration and acquisition methods
Sample chromatograms
• Instrument reports
• Quantification techniques
• QC sample results
• Pre-demonstration sample results
Data management procedures
• Quality assurance procedures
• Data validation and verification procedures
Auditors will also review corrective actions from the pre-selection audit. Auditors may submit follow-up
questions to the reference laboratory after the audit to address gaps in the observations.
A separate TSA will be performed by Battelle at the demonstration site to ensure that the demonstration plan is
being implemented properly by Battelle. This will be a self evaluation. The Battelle QA manager will review
notebooks, logsheets, chain-of-custody documentation, and observer checklists. The Battelle project manager
will be debriefed by the Battelle QA manager at the conclusion of the on-site TSA.
The EPA Quality Manager has the authority to conduct an independent technical systems audit at any time
during this demonstration.
9.3.2 Corrective Action Procedures
Corrective action procedures from TSA findings for the in-process reference laboratory audit or the field
demonstration TSA will be documented by the responder and approved by the auditor. Acknowledgment of the
response will be provided by the Battelle project manager. The Battelle auditor will then establish a time line for
monitoring that the required parties will properly act upon the corrective action(s) within the required time
frame. With the Battelle project manager, the Battelle auditor will set measurable milestones for determining
that progress is being made on the corrective action and that the corrective action is effective.
9.3.3 QA Reports
The outcome of each assessment will be fully documented. The Battelle project manager will archive all audit
documentation collected during the project and include it in the DER.
The Battelle QA manager or his designee will report the findings of each audit to the Battelle or AXYS Project
Manager, as appropriate, who will then address the audit findings and provide an appropriate response. QA
84
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reports require a written response by the person performing the inspected activity, and acknowledgment of the
audit by the Battelle project manager.
Authority to report all TSA results is designated to the Battelle QA manager or his designee. These reports
should
• Identify and document problems that affect quality and the achievement of objectives required by the
demonstration and quality assurance project plan and any associated SOPs,
• Identify and cite noteworthy practices that may be shared with others to improve the quality of their
operations and products,
• Propose recommendations (if requested) for resolving problems that affect quality,
• Independently confirm implementation and effectiveness of solutions, and
• Provide documented assurance (if requested) to line management that, when problems are identified, further
work performed is monitored carefully until the problems are suitably resolved.
Responses to adverse findings are addressed immediately during a debriefing after the assessment is completed,
and preferably at the site of the assessment.
Responses to each adverse finding will be documented in a letter or memo to the Battelle project manager. The
letter or memo will indicate for each adverse finding the corrective action(s) taken or planned. The letter or
memo will be signed by the appropriate developer or reference laboratory representative.
The Battelle QA manager or his designee will review the responses to each adverse finding and will follow up
with the Battelle, developer, or reference laboratory representative on any findings that were not adequately
addressed. Once all corrective actions associated with the QA report have been verified, the Battelle QA
manager or designee will approve the QA report. The QA report and responses to adverse findings will be sent
to the Battelle project manager for review and approval. The QA report and responses will be maintained in the
QA project files and will be included in the DER
85
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Chapter 10
Health and Safety Plan
This chapter contains the site health and safety plan for field activities for the dioxin monitoring and
measurement demonstration. This plan will be reviewed and signed by all demonstration participants before
work begins at the site. The Battelle Project Manager will conduct daily safety briefings before the start of
analysis each day to advise and remind the developers of health and safety issues.
86
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SECTION 1: GENERAL INFORMATION AND DISCLAIMER
CLIENT NAME: PROJECT NAME: SITE DEMONSTRATION
U. S. Environmental Protection Agency OF DIOXIN MONITORING AND
Superfund Innovative Technology Evaluation (SITE) Program MEASUREMENT TECHNOLOGIES FOR
SOIL AND SEDIMENT
EPA PROJECT MANAGER: Steve Billets
BATTELLE Project Manager: Amy Dindal
BATTELLE Site Safety Officer: Amy Dmdal
BATTELLE ES&H Representative: Gary Carlin DATE: April 26, 2004
The purpose of this demonstration is to gather technologies capable of measuring dioxin and dioxin-like compounds
in soil and sediment in one field location. The demonstration will be conducted near (but not on) a dioxin-
contaminated site. The demonstration site will be in the parking lot of the Green Point Environmental Learning
Center in Saginaw, MI. Developers of measurement technologies will operate their own technologies inside a trailer
or mobile laboratory designated for their use. Battelle will distribute the samples for analyses and observe the
operation of the technologies. Battelle will have its own trailer to support overseeing the demonstration, and it will be
known as the Battelle Headquarters.
This Site Specific Health and Safely Plan (HASP) has been prepared for use by participants in this SITE MMT
Demonstration. The plan is written for the specific LEVEL D site conditions, purposes, tasks, dates and personnel
specified. If these conditions change, this plan must be amended and reviewed by those named in Section 18. This
HASP satisfies the requirements of 29 Code of Federal Regulations (CFR) 1910.
Although it does not specifically apply, all site activities shall be performed in accordance with 1910.120(b)(l)(iv)
and (v). Battelle will inform the demonstration participants of the site emergency response procedures and any
potential fire, explosion, health, safely or other hazards by making this HASP and site information obtained by others
available on-site. All participants are responsible for: (1) attending the health and safely briefing given by the Battelle
Site Safely Officer (SSO) covering the requirements of this HASP; (2) providing their own personal protective
equipment (PPE); (3) providing documentation that their employees have been health and safely trained in
accordance with applicable federal, state and local laws and regulations; (4) providing evidence of medical
surveillance and medical approvals for their employees (as applicable); and (5) complying with the direction and
guidance provided by the SSO.
Prior to any demonstration samples being analyzed on site, a pre-work health and safely briefing shall be conducted
by the SSO. This briefing shall discuss the chemical and physical hazards associated with this demonstration.
Visitors will be briefed on the site-specific health and safely procedures by the SSO. Visitors will be escorted by
Battelle staff or by the developers trained on this HASP.
Under this plan, the SSO and the Battelle project manager, or their designees, are authorized and obligated to stop any
site work activity that place or may place any staff member, subcontractor, visitor, or participant at risk of being
injured or that violate specific requirements set forth within the HASP.
The requirements described within this HASP apply to all site activities and all Battelle staff, EPA, participants, or
visitors working on the site. Violators of this HASP will be addressed on a case-by-case basis. Actions taken will
range, at a minimum, from a verbal warning up to removal from the site depending on severity of the infraction.
Initial enforcement shall be implemented by the SSO, the Battelle project manager, or their designee. All
enforcement activities must be documented and maintained by the SSO. If the SSO is not on-site, an alternate will be
designated.
87
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SECTION 2: PROJECT INFORMATION
(1) SITE INFORMATION
Site Name: Green Point Environmental
Learning Center
Address
3010 Maple St.
Saginaw, MI 48602
Site Contact:
Phone Number:
Site Safety Officer
(SSO) Contact:
Site Phone Number
Becky Goche
989-759-1669
Amy Dindal
614-893-2260 (Battelle
headquarters cellular)
(2) SITE CLASSIFICATION (check all that apply)
(3) ENTRY OBJECTIVES (check all that apply)
Hazardous (RCRA/CERCLA/State)
Site Inspection (General)
Construction
Well Drilling Observation
Landfill (Non-Hazardous)
Sampling, Air
Q UST/LUST
Sampling, Water
Manufacturing
Sampling, Soil
Active
Other:
Inactive
Demonstration of dioxin monitoring and
measurement technologies. All work to be
conducted within temporary containment
facilities (i.e., trailers and mobile
laboratories).
Other:
All Battelle related activities will be conducted
outside of HAZWOPER site.
DATE(S) OF FIELD VISIT(S):
April 26 - May 6
2004
(4) BATTELLE TASKS
B1. Coordinate site activities associated
with the dioxin monitoring and
measurement technologies project.
B2. Assist developers, visitors, client as
necessary.
TASKS PERFORMED BY OTHERS
1 Developers will conduct analysis of
soil, sediment, and extraction samples
utilizing their respective equipment.
(5) PROJECT ORGANIZATION AND COORDINATION - The following personnel are designated to carry out
the stated project job functions on site. (Note: One person may carry out more than one job function.)
PROJECT MANAGER
SITE SAFETY OFFICER (SSO)
ALTERNATIVE SITE SAFETY OFFICER(S)
PUBLIC INFORMATION OFFICER
SITE RECORD KEEPER
SAMPLE CUSTODIAN
ALTERNATE SAMPLE CUSTODIAN(S)
SITE PERSONNEL WITH CPR/FA*
* If this person is not on-site, medical facilities that are
~ 3 miles away will be relied upon for medical care.
Amy Dindal
Amy Dindal
Gary Carlin; Rachel Sell
Steve Billets, U.S. EPA
Amy Dindal
Robyn Kroeger
Amy Dindal; Rachel Sell
Mark Misita
-------
SECTION 2: PROJECT INFORMATION
(6) ON SITE CONTROL
• Entry to the demonstration site will be controlled. Authorized personnel (developers, Battelle staff, EPA,
Michigan Department of Environmental Quality, U.S. Fish and Wildlife Service) will be allowed free access to
demonstration site. Unauthorized personal, such as visitors, will only be allowed to enter the trailers and mobile
labs on Visitor's Day or with approval from Battelle.
• An on-site Battelle Headquarters trailer will be established and clearly marked and will be the central
communication location.
• Facility gates will be closed when Green Point is not open (typical hours of operation are 7:30 am to 4:00 pm).
Security guards will patrol the grounds when participants are not there.
• Battelle staff will be present whenever sample analyses are on-going.
• All temporary containment facilities (e.g., trailers and mobile labs) will be locked when not occupied.
• On Visitor's Day, parking areas will be clearly marked and visitors will be given specific instructions concerning
entry into the demonstration site.
SECTIONS: PHYSICAL HAZARDS
(1)
IDENTIFY POTENTIAL PHYSICAL HAZARDS TO PARTICIPANTS (check all that apply)
Q
Q
Q
Q
Q
Q
Q
Confined Space
Heavy Equipment
Moving Parts
Heavy Lifting
Electrical
Overhead Hazards
Fall (>6; Vertical)
Q
Q
y
Q
Q
y
Q
Steep/Uneven Terrain
Heat Stress
Extreme Cold
Cold weather conditions may
exist during the time of this
project.
Ionizing Radiation
Traffic
Biological Hazards
Various wildlife may be
encountered on the site
Surface Water (Immersion)
Q
Q
Q
Q
Drums Handling
Noise
Non-Ionizing Radiation
Other:
Site hazards will be mitigated by:
1 . Briefing site personnel as to identified physical hazards.
2. Traffic will be directed by site staff to well -marked traffic areas on visitor days to minimize the potential
for injury to vehicular traffic.
3. Antiseptic ointment or solution will be included in the first aid kit.
4. All temporary containment facilities will be equipped with heating systems which can be utilized by the
occupants as needed.
(2) SAFETY EQUIPMENT REQUIRED (check all that apply)
Q
Q
y
y
Q
y
y
Q
Explosimeter
Fall Protection Equipment
Barrier Tape
Traffic Cones
Stretcher
First Aid Kit
A-B-C- Fire Extinguisher
Snake Bite Kit
y
Q
y
Q
y
Q
Q
Q
Eye Wash
Emergency Shower
Emergency Air Horn
Lights
Lights - emergency
(Minimum 2 Flashlights)
Ladder
Tick Repellant
Flotation Device (USCG Type
HI)
Q
y
y
Q
y
y
y
Confined Space Warning
Signs
Communications - On Site
Communications - Off Site
Hard Hats
Weather Radio
Air Horn
Portable Fume Hoods with
confirmed face velocity of
100 fpm +/- 20%. Vented to
the outside.
89
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SECTIONS: PHYSICAL HAZARDS
Emergency equipment will be located at the Battelle Headquarters. See Sections 10 and 12 for communication
procedures. A phone will be available on site and a cellular phone will carried by the Site Safely Officer. Due to
size of the demonstration area (approximately 120 feet by 140 feet) and the close proximity of all the containment
units, primary communication on site communication will be verbal. An air hom alarm system will be used for fire
or other potentially dangerous situations that need site personal to immediately respond to the site muster point
(Battelle Headquarters).
SECTION 4: CHEMICAL HAZARDS INFORMATION
(1) IDENTIFIED CONTAMINANTS
Known or suspected hazardous/toxic material
Media
SL/SD/EX
SL/SD/EX
SL/SD/EX
SL/SD/EX
Media types
Characterization
Substances
Involved
Dioxin/Furans
Characteristics
Estimated
Concentrations
<5o 13,000 pg/g
TLV
See Attachment 1
OT - Low
concentrations in
soil, sediment or
extract.
PCBs OT - Low < 300 ppm
concentrations in
soil, sediment, or
extract
PAHs OT - Low < 100 ppm
concentrations in
soil, sediment, or
extract
PCP OT-Low < 100 ppm
concentrations in
soil, sediment, or
extract
GW (ground water), SW (surface water), WW (wastewater), ALR (air), SL (soil), SD (sediments),
WL (waste, liquid), WS (waste, solid), WD (waste, sludge), WG (waste, gas), EX (extracts in
toluene), OT (other).
CA (corrosive, acid) CC, (corrosive, caustic), IG (ignitable), RA (radioactive), VO (volatile), TO
(toxic), RE (reactive), BIO (infectious), UN (unknown), OT (other, describe)
See Attachment 1
See Attachment 1
See Attachment 1
Material Safely Data Sheets (MSDSs) for the Health and Safely contaminants of concern will be available on-site in
the Battelle Headquarters. The data sheets include information on the chemical/toxicological properties of the site
contaminants and signs and symptoms of over exposure.
90
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(2) DESCRIBE POTENTIAL FOR CONTACT WITH EACH MEDIA TYPE FOR EACH OF THE BATTELLE
TASKS LISTED IN SEC 2.4:
BATTELLE
TASK*
Bl
B2
ROUTE OF
EXPOSURE
Inhal/Contact
Inhal/Contact
POTENTIAL FOR
CONTACT
Low
Low
METHOD OF CONTROL
• Latex/Nitrile Gloves,
Safety Glasses,
disposable lab coat
• Portable Fume Hoods
• Latex Nitrile Gloves,
Safely Glasses,
disposable lab coat, shoe
covers
• Portable Fume Hoods
The BATTELLE SSO will brief the participants on interpretation of the attached MSDSs and particularly on
symptoms and signs of over exposure to chemical hazards.
(3) DESCRIBE POTENTIAL FOR CONTACT WITH EACH MEDIA TYPE FOR EACH OF THE OTHER
TASKS LISTED IN SEC 2.4:
OTHER TASK #
01
ROUTE OF
EXPOSURE
Inhal/Contact
POTENTIAL FOR
CONTACT
Low
METHOD OF CONTROL
• Latex/Nitrile Gloves,
Safely Glasses,
disposable lab coat,
shoe covers
• Portable Fume Hoods
The BATTELLE SSO will brief the participants on interpretation of the MSDSs and particularly on symptoms and
signs of over exposure to chemical hazards.
SECTION 5: HAZARD COMMUNICATION PROGRAM
If chemicals are introduced to the site by Battelle (e.g., decontamination liquids, preservatives, etc.), bring a copy of
the MSDSs to the site. The Battelle SSO will review this information with all field personnel prior to the start of
the field activities. The current list of chemicals for this site is:
Hexane
Methanol
Toluene
Dimethyl sulfoxide (DMSO)
Nitrogen
Carbon dioxide
Sulfuric acid
91
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SECTION 6: ENVIRONMENTAL MONITORING
(1)
Q
Q
Q
Q
Q
Q
Q
Q
Q
The following environmental monitoring instruments shall be used on site at the specified intervals for
breathing zone monitoring:
EQUIPMENT
Combustible Gas Indicator
O2 Meter
PID (Lamp 10.6 eV)
FID
Radiation Meter (Gamma)
Respirable Dust Meter
Other
MONITORING PERIOD
daily/hourly/continuous/other
daily/hourly/continuous/other
daily/hourly/continuous/other
daily/hourly/continuous/other
daily/hourly/continuous/other
daily/hourly/continuous/other
daily/hourly/continuous/other
daily/hourly/continuous/other
daily/hourly/continuous/other
ACTION LEVEL
NO MONITORING WILL BE CONDUCTED AS ALL HANDLING OF SAMPLES AND CHEMICALS
WILL BE PERFORMED IN A FUME HOOD.
(2) Monitoring equipment is to be calibrated according to the manufacturers' instructions. Workers will record
calibration data and air concentration in the Health and Safety on-site logbook. (NOT APPLICABLE)
(3) Action Levels are for work stoppage and on-site assessment. These are average values. Consideration should
be given to the potential for release of highly toxic compounds from the waste or from reaction by-products.
Levels are for persistence (> 10 min). If action levels are exceeded, work will stop until levels drop to below
the action level. Personnel with respirator training may reenter the work zone with proper respirator equipment.
Battelle personnel without respirator training will not reenter the work zone until levels drop below the action
level (NOT APPLICABLE)
ACTION LEVEL
Uncharacterized Airborne Vapors or Gases >5 ppm Not Applicable
Characterized Airborne Gases, Vapor, Particulates >50% PEL, REL, TLV Not Applicable
Oxygen <19.5;>23.5 Not Applicable
Flammability >10%EL Not Applicable
(4) Military and/or civilian personnel in charge of buildings adjacent to invasive monitoring activities will be
notified via a health and safely kickoff meeting of site activities. A copy of this HASP will be provided. If any
action levels are reached at the work area as described above or if discernible odors are released as a result of
field activities, the personnel in charge or their designated representative will be notified immediately. (NOT
APPLICABLE)
SECTION 7: HEALTH AND SAFETY TRAINING/MEDICAL MONITORING PROGRAM
The Battelle project staff is included in the Battelle Health and Safety Training and Medical Monitoring Programs in
conformance with 29 CFR 1910.120(f) as applicable. As deemed necessary by the Battelle SSO, training records will
be kept on file for all on-site Battelle personnel.
NAME
Mark Misita
Robyn Kroeger
Zachary Willenberg
MEDICAL
(Date)
Not Applicable. Not a
HAZWOPER site.
Not Applicable. Not a
HAZWOPER site.
Not applicable. Not a
HAZWOPER site.
INITIAL
(Hrs/Date)
Not Applicable. Not a
HAZWOPER site.
Not Applicable. Not a
HAZWOPER site.
Not applicable. Not a
HAZWOPER site.
HAZWOPER TRAINING
REFRESHER
(Date)
CPR/FA/BBP
(Dates)
Not Applicable. Not a CPR (4/04)
HAZWOPER site. FA (4/04)
BBP (4/04)
Not Applicable. Not a
HAZWOPER site.
BBP (4/04)
Not applicable. Not a
HAZWOPER site.
BBP (8/03)
92
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SECTIONS: PERSONAL MONITORING
No personal exposure monitoring or heat/cold stress monitoring will take place on site as test articles and laboratory
chemicals will be handled within a portable lab hood. If the need for such monitoring is anticipated, this HASP will be
modified as accordingly. Temporary containment facilities will be climate controlled.
SECTION 9: CONFINED SPACE ENTRY
No confined space and/or trench entries will take place on site. If the possibility of such entries taking place exists,
this HASP will be modified accordingly.
SECTION 10: COMMUNICATION PROCEDURES
Verbal communication will be used to communicate among staff in the mobile laboratories/trailers for routine
information exchange.
For emergencies requiring immediate muster of all site personnel, an air horn will be used.
A land line will be available at the Green Point Environmental Learning Center. The Battelle Project Manager
and the SSO will have cell phones.
Signs will be posted to announce critical safely procedures (e.g., "Safety Glasses Must Be Worn Upon Entry To
The Mobile Lab/Trailer")
Important Phone Numbers
Battelle Headquarters
Amy Dindal
Battelle Project Manager
Battelle Site Safety Officer
(Cell) 614-893-2260
(Cell) 561-721-5826
Gary Carlin, CIH
Battelle ES&H Representative
(Office) 614-424-4929
(Cell) 614-348-5785
(Home) 614-853-2024
Green Point Environmental Learning Center
(Office) 989-759-1669
Michigan Department of Environmental Quality Contact
Michael Jury
(Office) 989-686-8025, X 8311
(Cell) 989-860-6646
(Pager) 989-253-3942
93
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SECTION 11: DECONTAMINATION PROCEDURES
Temporary Containment Facility Clearance Sampling
Each temporary containment facility (i.e., mobile laboratory or trailer) used to house the developer technologies
when analyzing samples will be wipe tested after initial set-up but before sample analyses begin. The trailers and
mobile laboratories will be also be cleaned and then wipe tested at the conclusion of the demonstration. Wipe
test results must demonstrate that the total TEQ (dioxin/furan and PCB) are <20 pg TEQ/lOOcm2
(http://www.epa. gov/wtc/wipe_samples/censuspdfs/wipe_benchmarks.htm) before a containment facility can be
certified as "clean." Samples will be collected in accordance with EPA Methods 1613B and 1668A
respectively. Analyses of these samples will be performed by Battelle. A letter describing the cleaning
procedure and the analytical results will be provided to the suppliers of the mobile labs and trailers as soon as
results are available.
Wipe Clearance Test Sampling Overview
Samples taken before analyses being will be taken on the floor near the exterior door. Samples taken after
analysis by the developers within each temporary containment facility in the following places: 1 sample-fume
hood, 1 sample-floor directly in front of fume hood, 1 sample-door knob, 1 blank. The wipe samples will be
analyzed for dioxins/furans and the 12 World Health Organization PCBs using EPA Methods 1613B and
Method 1668A, respectively.
Sampling Procedure
Each wipe test sample will be collected in the following fashion:
Step 1: A new pair of disposable nitrile gloves will be used to collect each sample.
Step 2: Each sample jar will be labeled with the sample ID.
Step 3: The sampling solvent will be poured into the j ar containing the wipe test paper.
Step 3: The wipe test paper will be removed from the jar after it has been saturated with the sample solvent.
Step 4: The wipe test paper will be used to collect a surface sample (one sample per jar).
Step 5: The sample collection area will be set as a 10 cm x 10 cm area. This will determined by using a pre-
cut 10 cm x 10 cm square as a reference. If the area is of irregular size or shape, the sample area will
be estimated so total surface area on the wipe will equal approximately 100 cm2.
Step 6: The wipe will be placed back into its respective jar and the lid snuggly screwed back on.
Step 7: The sampling time, sampling date, sampler's initials, location of the wipe sample, location of the wipe
sample, trailer/mobile lab the sample was taken from, and any comments will be documented.
Step 8: Steps 1-7 will be repeated for all samples.
The following decontamination/spill response equipment is required (check all that apply)
y
y
Q
y
y
Shoe covers
Trash Cans/Bags
Buckets
Dust Pan
Safety Glasses w/Side
shields
y
Q
y
y
y
Dry Brushes
Wet Brushes
Water
Waste Tags
Nitrile gloves
y
Q
4
y
y
Detergent Soap
Other Decontamination
Solution
Small hand shovel
Barricade tape
Disposable lab coat
94
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SECTION 12: EMERGENCY PROCEDURES
All on-site personnel will use the following standard emergency procedures. The Battelle SSO shall be notified
of all on-site emergencies and be responsible for ensuring that the procedures are followed. The site SSO has
ultimate authority during any emergency until relieved by a ranking responder (i.e., local fire department, police
department) or until the emergency is brought under control.
Personal Injury in the demonstration Designated Emergency Signal: Verbal communication as needed.
test area
Upon notification of an injury, the Battelle SSO or alternate should evaluate the nature of the injury, and the
affected person should be decontaminated to the extent possible prior to movement off site. The on-site
CPR/FA personnel shall initiate the appropriate first aid, and contact should be made for an ambulance (and
other emergency services as needed) and with the designated medical facility (if required).
Fire/Explosion Muster Designated Emergency Signal: 2 short horn blasts
Upon notification of a fire or explosion on site, all site personnel will assemble at the Battelle Headquarters.
The fire department shall be alerted and all personnel moved to a safe distance from the involved area.
Severe Inclement Weather Designated Emergency Signal: Verbal communication as needed.
In severe inclement weather (i.e., thunderstorm, tornado, hail, etc.), participants will discontinue operations and go
to the Green Point ELC for shelter. If the severe inclement weather occurs when Green Point ELC is closed,
participants will discontinue operations and leave the site.
Equipment Failure Designated Emergency Signal: Verbal communication as needed.
If any other equipment (i.e., portable fume hood) on-site fails to operate properly, the Battelle SSO shall be
notified and then determine the effect of this failure on continuing operations on site. If the failure affects the
safely of personnel or prevents completion of the demonstration plan tasks, all personnel shall leave the affected
temporary containment facility until the situation is evaluated and appropriate actions taken.
In all situations, when an on-site emergency results in evacuation of an area, personnel shall not reenter the area
until:
• The conditions resulting in the emergency have been corrected.
• The hazards have been reassessed by the Battelle SSO.
• The Site Safely Plan has been reviewed by the Battelle SSO and the Battelle project manager.
SECTION 13: SPILL CONTROL PROCEDURES
The following procedures will be followed in the event of a spill involving the demonstration samples or the
other analytical chemicals brought on site. Spill clean-up materials will be assessed on a case-by-case basis.
Spills of Demonstration samples inside of a Temporary Containment Facility
Concentrations of contaminants in the test media will be extremely low. Primary concern is migration of trace
contaminates. In case of a spill
• Barricade the spill area and contact the Battelle SSO.
• Don safety glasses and nitrile or latex gloves and obtain a spill cleanup kit from the SSO.
• Clean up the spill by first removing any bulk materials using a brush and dust pan or scraper and dust pan
(Do not pick up any glass shards by hand). Next, thoroughly wipe down the spill area using a lab towel
dampened with acetone. Finally, wash the spill area with a soap and water mixture.
• All waste generated from the spill will be placed into a plastic bag (or box if glass is involved) and a yellow
waste tag filled out and attached to the bag. The bag will be returned to the Battelle Columbus site for proper
disposal.
• The Battelle SSO shall determine if wipe test sampling of the area is required. The area should have a piece
of plastic taped over it until it is determined if wipe test sampling is required.
95
-------
SECTION 13: SPILL CONTROL PROCEDURES
Spills of Demonstration samples on Soil or Parking Lot
Concentrations of contaminants in the test media will be extremely low. Primary concern is migration of trace
contaminates. In case of a spill
• Barricade the spill area and contact the Site Safely Officer.
• Don safety glasses and nitrile or latex gloves and obtain a spill cleanup kit.
• Clean up the spill by first removing any bulk materials using a brush and dust pan or scraper and dust
pan (do not pick up any glass shards by hand). Glass should be placed into a puncture-resistant
container. Next, remove any soil or debris that has come into direct contact with the spilled material.
• All waste generated from the spill will be placed into a plastic bag and a yellow waste tag filled out and
attached to the bag. The bag will be returned to the Battelle Columbus site for proper disposal.
• The SSO will determine if wipe test sampling of the area is required. The area should have a piece of
plastic taped over it until it is determined if wipe test sampling is required.
Spills of Lab chemicals
Spills of lab chemicals will be evaluated on a case by case basis. Small spills (involving < 50 ml) of a laboratory
chemical in the fume hood can be cleaned up by staff with lab towels. For larger spills, barricade the spill area
and contact the SSO.
All waste generated from a spill must be segregated and labeled. The SSO will address the disposal of the
materials. Spill areas may need to be certified free of dioxin/PCB contamination depending on the type and
nature of the spill. The SSO will determine if the certification analysis is necessary.
Gas Cylinder leaks
In the event of gas leak from any cylinder, the effected trailer will be evacuated until the SSO deemed it safe to
reentry.
96
-------
SECTION 14: EMERGENCY INFORMATION
(2)
LOCAL RESOURCES
Ambulance (name):
Hospital (name):
Police (local or state):
Fire (name):
On-Site CPR/FA(s):
Mobile Medical Response Phone:
Covenant Health Care Phone:
St Mary's Medical Center
Saginaw Police Phone:
Saginaw Fire Dept Phone:
Mark Misita Phone:
989-758-2900
989-583-2000
989-776-8000
989-759-1288
989-759-1376
614-893-2260
For life-threatening emergencies or emergency trauma care dial 911.
The above hospital is approximately 3 miles from the furthest work area and the ambulance response time is
approximately 5 minutes.
DIRECTIONS TO NEAREST HOSPITALS - See written directions (Attachment 2)
BATTELLE RESOURCES
Project Manager
Amy Dindal
ES&H Representative
Gary Carlin
Hazardous Waste Coordinator
Jim Walters
Important Phone Numbers
Battelle Main Number
Legal Department
Medical Services (Emergency)
Amy Dindal
Gary Carlin
Jim Walters
(614) 424-6424
(614) 424-6585
(614) 424-4622
(561) 422-0113 (office)
(561)721-5826 (cell)
(614) 424-4929 (office)
(614) 348-5785 (cell)
(614) 424-4746 (office)
NOTE: All accidents/injuries/spills must be immediately reported to the SSO. The SSO will ensure any
additional actions are taken (as appropriate).
97
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SECTION 15: PERSONAL PROTECTIVE EQUIPMENT FOR ENTRY INTO TEMPORARY
CONTAINMENT FACILITIES INVOLVED IN SAMPLE ANALYSIS
(In effect AFTER temporary containment facility setup is completed)
1. Respiratory protection is not anticipated to be needed on this site.
2. Any contaminated PPE will be immediately removed, cleaned or replaced (as appropriate). (Contaminated
items being discarded will be placed in tagged laboratory debris containers.)
3. Shoe covers will be worn in only one trailer/mobile lab and not transferred for use in other trailers/mobile labs.
4. Safety glass must be worn upon entry into a trailer/mobile. Safety glasses will be worn at all times that work is
on-going.
5. Developers will be required to wear a minimum of safety glasses when receiving/returning samples. The
sample custodian will don safety glasses, gloves, and lab coat. Anyone working in the Battelle HQ trailer while
samples are being distributed must wear safety glasses.
V
CLOTHING |
Disposable Lab
Coat
Must be removed
prior to exiting
temporary
containment facility
V
GLOVES |
Lab gloves (either Latex
or Nitrile)
V
BOOTS |
Shoe Covers
(Tyvek® or
equivalent)
Must be removed
prior to exiting
temporary
containment facility
V
OTHER
Safety
Glasses
w/Side
shields
SECTION 16: SAFE WORK PRACTICES
THE FOLLOWING PRACTICES MUST BE FOLLOWED BY PERSONNEL ON SITE
1. Smoking, eating, chewing gum or tobacco, or drinking are forbidden except in clean or designated areas.
2. Ignition of flammable liquids within or through improvised heating devices (e.g., barrels) is forbidden.
3. Contact with samples, excavated materials, or other contaminated materials must be minimized.
4. All electrical equipment used in outside locations, wet areas, or near water must be plugged into ground
fault circuit interrupter (GFCI) protected outlets.
5. Good housekeeping practices are to be maintained.
6. Flammables needing refrigeration must be stored in a refrigerator rated for flammable use.
7. Where the eyes or body may be exposed to corrosive materials, water suitable for quick drenching or
flushing shall be available for immediate use.
SECTION 17: WASTE DISPOSAL
Waste/Effluent/Residual Sample Handling will be handled according to the plan outlined in Attachment 4.
98
-------
- 29CFRI9JO
* U.S. EPA Standard Operating Safety Guidelines fpr H
Waste Operations. June 1992
PLAN REVIEWED BY;
Officer
Battelfc BS&H
Representative
Battelie Pityect Manager:
DATE
92
-------
SFTE HEALTH AND SAFETY PLAN FOR FIELD ACTIVITIES FOR THE SITE
DEMONSTRATION OF DIOX1N MONITORING AND MEASUREMENT TECHNOLOGIES
FOR SOIL AND SEDIMENT
Review Sign-Off
I acknowledge that I have read the information in this HASP form and the MSDSs. I understand the site
hazards as described and agree to comply with the contents of the plan.
Date
Employee Name (Print)
INDICATE:
Battelle Staff =
Developer
EPA
Visitor
Other (specify)
"B"
"D"
= "E"
= "V"
"O"
Ml
p.
14-0/1
D
6
-------
SITE HEALTH AND SAFETY PLAN FOR FIELD ACTIVITIES FOR THE SITE
DEMONSTRATION OF DIOXIN MONITORING AND MEASUREMENT TECHNOLOGIES
FOR SOIL AND SEDIMENT
Review Sign-Off
I acknowledge that I have read the information in this HASP form and the MSDSs. I understand the site
hazards as described and agree to comply with the contents of the plan.
INDICATE:
Battelle Staff
Developer
EPA
Visitor
Other (specify)
Employee Name (Print)
Employee Name (Signature)
93
-------
Attachment 1
Identified or Suspected Contaminants
101
-------
IDENTIFIED OR SUSPECTED CONTAMINANTS
Table 1. Known or Suspected Contaminants that may be Encountered during Analytical Activities. No employee
exposures are expected as all handling of samples and laboratory chemicals will be completed in a portable fume hood.
Media
Soil, Sediment,
Toluene Extract
Soil, Sediment,
Toluene Extract
Soil, Sediment,
Toluene Extract
Soil, Sediment,
Toluene Extract
Lab Chemical
Lab Chemical
Lab Chemical
Lab Chemical
Substances
Involved
Dioxins
(Various)
PCBs
PAHs
PCP
Hexane
Methanol
Toluene
DMSO
Characteristics
Low Concentrations in
Soil, Sediment, or Extract.
Low Concentrations in Soil
or Sediment.
Low Concentrations in Soil
or Sediment.
Low Concentrations in Soil
or Sediment.
Ignitable, Volatile Organic,
Toxic.
Ignitable, Volatile Organic,
Toxic.
Ignitable, Volatile Organic.
Irritant.
Estimated
Concentrations
<5to 13,000
PR/R
< 300 ppm
< 100 ppm
< 100 ppm
Laboratory Grade
(Essentially pure)
Laboratory Grade
(Essentially pure)
Laboratory Grade
(Essentially pure)
Laboratory Grade
(Essentially pure)
American Conference of
Governmental
Industrial Hygienists
Limits
None Established.
42% Chlorine: 1 mg/m3
(8 hr TWA)
54% Chlonne: 0.5 mg/m3
(8 hr TWA)
0.2 mg/m3 (8 hr TWA)
(as coal tar pitch volatiles
- benzene soluble)
0.5 mg/m3 (8 hr TWA)
50 ppm (8 hr TWA)
200 ppm (8 hr TWA)
250 ppm (STEL)
50 ppm (8 hr TWA)
None
ppm= parts per million
hr = hour
mg/m3 = milligrams per cubic meter
TWA= time-weighted average
102
-------
Attachment 2
Written Directions to the Nearest Hospitals
103
-------
EMERGENCY INFORMATION
(POST ON SITE)
EMERGENCY CONTACTS AND ROUTE TO CONVENANT HEALTHCARE
GREEN POINT ENVIRONMENTAL LEARNING CENTER
Emergency Contact
Telephone No.
Amy Dindal
614-893-2260
Medical Emergency
Hospital Name: Covenant Healthcare
Hospital Address: 1447 N. Harrison St.
Saginaw, MI 48602
Hospital Telephone No.:
Ambulance Telephone No.:
Emergency - 911
General (989) 583-0000
Mobile Medical Response
989-758-2900
Route to Hospital: (Approximate Distance: 3 miles)
From the site, start out going north on Maple Street toward Beacon Drive and turn right on S. Michigan Ave. for
about 1.6 miles. S. Michigan Ave. will turn into N. Michigan Ave. Continue on N. Michigan Ave. and then turn left
onto Houghton Street. Another option would be to take N. Michigan Ave all the way to Cooper St. and take a left.
There are emergency entrances at both Houghton St. and Cooper St. Signs to the hospital are well-marked throughout
the area.
104
-------
EMERGENCY INFORMATION
(POST ON SITE)
EMERGENCY CONTACTS AND ROUTE TO ST. MARY'S
GREEN POINT ENVIRONMENTAL LEARNING CENTER
Emergency Contact Telephone No.
AmyDmdal 614-893-2260
Medical Emergency
Hospital Name: St. Mary's Medical Center (http://www.saintmarys-saginaw.org/)
Hospital Address: 800 S. Washington Ave.
Saginaw MI 48601
Hospital Telephone No.: Emergency - 911
General (989) 776-8000
Ambulance Telephone No.: Mobile Medical Response
989-758-2900
Route to Hospital: (Approximate Distance: 3.7 miles)
From the site, start out going north on Maple Street toward Beacon Drive. Turn right onto S. Michigan Ave. for about
a mile, and then take another right onto Stephens St./MI-46 E. Continue to follow MI-46 E. There is a set of one-way
pairs that merge into what is known as Rust St. just before the Saginaw River. Stay on Rust, (going east) to S.
Washington Ave./M-13. Turn left (going north) onto S. Washington Ave/MI-13. St. Mary's Medical Center is
located on S. Washington Ave.
105
-------
Attachment 3
Waste Disposal Plan
106
-------
Waste/Effluent/Residual Sample Handling
Effluents (e.g., solvent/aqueous collected from analytical processes, and sample by-products), used samples,
and unused sample will be collected for quantification and be reported as part of the verification of each
technology. These items are necessary to aid in the complete characterization of each developer's analytical
process and will be transported back to the Battelle Columbus facility for analysis and quantity
confirmation/verification (see Table A-l, below). These items will be retained by Battelle until such time that
they are no longer needed. At that time, they will be considered waste, characterized, and properly disposed.
Table A-l: Effluent Handling
Effluent Stream
Solvent effluent from analytical
processes
Used samples
Unused sample
Cell culture (non-human; non-
hazardous; non-biohazard)
Container Type
5-gallon steel can with screw cap
5-gallon steel can with screw cap
Original container
Double ziplock bag; 30-gallon
plastic drum
Quantity on Hand
6 - 5-gallon steel cans with screw
cap
6 - 5-gallon steel cans
N/A
1 - 30-gallon plastic drum
The miscellaneous non-RCRA hazardous wastes generated on the test site (such as booties, gloves, lab
towels, and broken glass) will be taken from the site and disposed of at the Battelle - Columbus location (see
Table A-2 below). The only exception to this is general refuse (e.g., coffee cups, food waste, and office
waste). General refuse will be disposed of in regular public refuse containers.
Table A-2. Waste Handling
Waste Stream
Miscellaneous non-hazardous waste
(e.g., booties, gloves, lab towels,
etc.)
Non-hazardous broken glass
Container Type
55-gallon drum (plastic)
Broken glass box
Quantity on Hand
2 - 55-gallon containers (A small
collection container will be placed in
each developer's area; the amount of
times this is emptied into the central
55-gallon container will be
recorded.)
2 - glass boxes in central location
107
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Chapter 11
References
1. EPA. 1996. "A Guidance Manual for the Preparation of Site Characterization and Monitoring
Technology Demonstration Plans." NERL. October.
2. EPA. 1998. "Quality Assurance Project Plan Requirements for Applied Research Projects."
Unpublished. National Risk Management Laboratory.
3. Kutz, F. W., Barnes, D. G., Bottimore, D. P., Greim, H., and Bretthauer, E. W. 1990. The international
toxicity weighting factor (I-TEF) method of risk assessment for complex mixtures of dioxins
and related compounds. Chemosphere 20: 751-758.
4. 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.
5. EPA. 2001. Database of Sources of Environmental Release of Dioxin-like Compounds in the United
States, EPA/600/C-01/012, March.
6. 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.
7. EPA Method 1668A. 1999. Chlorinated biphenyl congeners by HRGC/HRMS, EPA/82l/R-00-02,
December.
8. 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.
9. De Rosa, Christopher T., et al. 1997a. Dioxin and dioxin-like compounds in soil, Part 1: ATSDR Interim
Policy Guideline. Toxicology and Industrial Health, Vol. 13, No. 6, 1997. pp. 759-768.
10. EPA. 1995. (updated March 16, 1998) "Handbook for Preparing Office of Research and Development
Reports." ORD. Washington, DC, EPA/600/K-95-002. August.
108
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ADDENDUM
Shortly before making a final commitment to participate in the field demonstration, Paracelsian decided that
it did not have the resources to fulfill all of the requirements described in this document. Paracelsian
committed to participate in the Visitor's Day activities, but did not plan to analyze any of the samples in the
field. Subsequently, Paracelsian determined that it could only commit to analyzing about 25% of the
demonstration sample set in their laboratories. It was decided that this was insufficient to draw meaningful
conclusions regarding the performance of this technology. As a result, Paracelsian withdrew from further
consideration as a full demonstration participant, but agreed to analyze the 15 pre-demonstration samples.
The Paracelsian pre-demonstration results will be included in the Data Evaluation Report (DER, see Section
8.4), which may be obtained from the EPA. The DER also will include a limited review of the Paracelsian
technology by the Battelle observers during the Visitor's Day. The DER will not be published, and no other
published report regarding the performance of the Paracelsian technology will be prepared.
-------
APPENDIX A
LIST OF DEMONSTRATION PANEL MEMBERS
-------
Dioxin SITE Demonstration Panel
Participant
Fernando Rubio
Bob Harrison
Randy Allen
Noriyoshi Inoue
John Gordon, Jeff Sturkey, George Clark
Masako Hayakawa/Emmy Leung/
Hiroyuki Hayashi
Steve Billets
Andy Beliveau
Christopher Stitt
William Sy
Stevie Wilding
Nardina Turner
Greg Rudloff
Allen Debus
Barry Evans
Craig Smith
David Williams
Vance Fong
Bill Coakley
Dwain Winters
David E. Cooper
Jon Josephs
Bob Mouringhan
Terry Smith
Joe Ferrario
Deana Crumbling
Mike Jury, Sue Kaelber-Matlock, Al Taylor
Becky Goche, Doug Spencer
Laurie Phillips, Coreen Hamilton, Georgina
Brooks, Dale Hoover
Rosanna Buhl, Gary Carlin, Amy Dindal,
Tim Pivetz, Mary Schrock, Rachel Sell,
Zack Willenberg
Organization
Abraxis LLC
CAPE Technologies
Hybrizyme Corporation
Paracelsian Inc.
Xenobiotic Detection Systems
Wako Pure Chemical
Industries, Ltd.
ORD/ESD-LV
Region 1
Region 2
Region 2
Region 3
Region 4
Region 5
Region 5
Region 7
Region 7
Region 7
Region 9
OSRTI/ERTC-NJ
OPPTS/NPCD-DC
OSWER/OERR-DC
ORD/HSTL-R2
ORD/HSTL-R7
OSRTI/DC
OPPTS/MS
OSRTI/DC
Michigan Department of
Environmental Quality
U.S. Fish and Wildlife Service
AXYS Analytical Services
Battelle
Role
Developer
Developer
Developer
Developer
Developer
Developer
Program Manager
Technical Advisor
Observer
Observer
Technical Advisor
Technical Advisor
Technical Advisor
Technical Advisor
Observer
Technical Advisor
Technical Advisor
Observer
Technical Advisor
Technical Advisor
Observer
Technical Advisor
Technical Advisor
Technical Advisor
Technical Advisor
Observer
Technical Advisors
Demonstration Site
Representatives
Reference Laboratory
Demonstration Coordinator
-------
APPENDIX B
REQUEST FOR AND OBTAINING OF SPECIAL USE PERMIT
-------
February 3, 2004
Douglas G. Spencer
Refuge Manager
Shiawassee National Wildlife Refuge
6975 Mower Road
Saginaw, Michigan 48601
Dear Mr. Spencer:
The purpose of this letter is to request the use of the Green Point Environmental Learning Center
("Green Point") located in the Shiawassee National Wildlife Refuge managed by the U.S. Fish
and Wildlife Service to serve as the host site for an upcoming demonstration of dioxin monitoring
and measurement technologies.
Performance verification of innovative environmental sampling, monitoring, and measurement
technologies is an integral part of the regulatory and research mission of the U.S. Environmental
Protection Agency (EPA). To address this need, the Superfund Innovative Technology
Evaluation (SITE) Program was established by EPA. Additional information regarding the
history of this program may be found at our website: http://www.epa.gov/ORD/SITE. One
component of SITE is the Monitoring and Measurement Technology (MMT) Program. This
Program evaluates the performance of innovative technologies that sample, detect, monitor, or
measure hazardous and toxic substances in soil, water, and sediment samples. The SITE MMT
Program is currently planning a demonstration of technologies that can be deployed in the field or
in a mobile laboratory for the rapid measurement of dioxins in soil and sediment. The EPA has
awarded a technical support contract to the Battelle Memorial Institute to assist with this
demonstration.
We have been working closely with the Michigan Department of Environmental Quality (MDEQ)
in planning this demonstration, and dioxin-contaminated samples from the Saginaw area were
supplied by MDEQ to be included in this study. As a result of our discussions with the MDEQ
and our site selection survey, the EPA requests the use of Green Point Center's parking lot as the
demonstration site. We request access for a two-week timeframe, from approximately from April
22, 2004 to May 6, 2004.
Use of Green Point's parking lot for this demonstration will entail the following:
• Access to site approximately seven trailers in a remote area of the parking lot at Green
Point so as not to disturb or impede normal daily activities at Green Point;
• Contain all of the analysis to within the trailers;
• Conduct a half-day Visitors Day during the demonstration period (approximately April
28), including the use of Green Point's auditorium. For the Visitors Day, the
demonstration site would be open to the public so that those interested in using these
technologies can learn more about them.
• Use of the Green Point building itself would be limited to bathrooms, and possibly phone
use should cellular service be an issue.
• Not to disturb the grounds or parking area of Green Point in any way. We are committed
to leave the area as we found it before conducting the demonstration.
-------
This demonstration is fully funded by the SITE Program. Any infrastructure requirements that we
have, such as supplying power for the trailers, will be coordinated and paid for through this
contract by Battelle. All logistics for this demonstration will be coordinated by Becky Goche.
For more information about the dioxin demonstration and this request for use of Green Point's
parking lot, please do not hesitate to contact me at 702-798-2232 or by e-mail at
billets. stephen@epa.gov.
We look forward to working with you and would appreciate your support of this demonstration.
Sincerely,
Stephen Billets
Program Manager
U.S. EPA MMT Program
cc:
G. Rudloff, U.S. EPA, Region 5
A. Dindal and R. Sell, Battelle Memorial Institute
S. Kaelber-Matlock, Michigan Department of Environmental Quality
A. Taylor, Michigan Department of Environmental Quality
-------
UNITED STATES DEPARTMENT OF THE INTERIOR
FISH AND WILDLIFE SERVICE
Shiawassee NWR
6975 Mower Rd.
Saginaw, Mi. 48601
SPECIAL USE PERMIT
Station No. to be Credited Permit No,
31520 - 04006
Date
March 4,2004
Period of Use (inclusive)
From April 19,2004
TO May 6,2004
Permitte Name
Stephen Billets
Permittee Address
USEPA
P.O. Box 93478
Las Vegas, NV 89193
Purpose (specify in detail privilege requested, or units of products involved)
Utilized Green Point Environmental Learning Center faciirtes as the host site for an upcoming demonstation of dioxin monitoring and
measurement technologies.
Description (specify unit numbers: metes and bounds, or other recongizable designations)
Superfund Innovative Technology Evaluation (SITE) Program and Monitoring and Measurement Technology (MMT) Program is
currently planning a demonstration of technologies that can be deployed in the field or in a mobile laboratory for the rapid
measurement of dloxins in soil and sediment The USEPA has awarded a techical support contract to the Battefle Memorial Institute
to assist with this demonstration to held at Green Point ELC, Michigan Department of Environmental Quality is assisting with the
demonstration by supplying dtoxin-contaminated samples from ttte local area.
Amount of fee
D Payment Exempt
D Full Payment
D Partial Payment
$0 if not a feted payment, specify rate and unit of charge:
- Jusfficatton:
- Balance of payments to be made as follow*:
Record of Payments
Special Conditions
1. Access to site for approximately seven trailers in a remote area of the parking lot at Green Point ELC so as not to disturb or
impede normal daily access to the Center.
2, Contain all of the analysis to within the trailers.
3, Conduct a half-day "Visitors Day' during the demonstraflon period (approamatety April 28), including the use of Green Points
auditorium. For the Visitor's Day, the demonstration sits would be open to the public so that those interested in using these
technologies can learn more about them,
4, Use of the Green Point building teelf would be totted to bathrooms, and possibly phone use should cellular service be an issue.
The area of use will be restored back to the way it was found before conducting the demonstration.
This permit is issued by the U.S. Fish and Wildlife Service and accepted by the undersigned, subject to the terms, covenants,
oblicatloos, and reservations, expressed or implied herein, and to the conditions and requir«nerto^jpearinfl on the reverse tide.
Permittee Signature
t=H
Issuing. Office
Form 3-1383 mev fi/851
ff
-------
APPENDIX C
EXAMPLE COVER LETTER FOR SAMPLE COLLECTION
-------
Batreiie
The Business of Innovation
October 13, 2003
Al Taylor
Waste and Hazardous Materials Division
Michigan Department of Environmental Quality
Constitution Hall, Atrium North
525 West Allegan Street
Lansing, MI 48933
SAMPLING AND SHIPPING INSTRUCTIONS FOR SEDIMENT SAMPLES
DearAl:
Thank you so much for agreeing to provide Battelle with Saginaw River sediment samples for use in U.S.
Environmental Protection Agency's Superfund Innovative Technology Evaluation (SITE) Demonstration
of Monitoring and Measurement Technologies for Dioxin in Soil and Sediment. Twelve pre-cleaned one-
gallon sample containers are enclosed for you to fill with sediment (6 one-gallon samples in duplicate).
Please label two containers containing the same sample as "1 of 2" and "2 of 2". Please attach a
description or label each container with a description of the sample including the location where the
sample was collected and the estimated concentrations of dioxin and PCBs.
The collected samples are to be shipped back to Battelle via the provided coolers. Federal Express labels
that include an account number and the shipping address are enclosed. Please notify me by phone or
email once the samples have been shipped so we can expect delivery in our laboratories. We would like to
have the samples returned by December 2, 2003 if that is at all possible.
Any questions on sample shipment or receipt should be directed to Kim Andrews,
andrewsk(g),battelle.org. 614-424-5254. Questions about the EPA project should be directed to Amy
Dindal. dindala@,battelle.org. 561-422-0113.
Sincerely,
Amy B. Dindal
Project Manager
Atmospheric Science and Applied Technology
ABDllg
cc: Steve Billets, U.S. Environmental Protection Agency (letter only)
-------
APPENDIX D
EXAMPLE CHAIN OF CUSTODY/RESULTS FORM
-------
CHAIN-OF-CUSTODY/RESULTS FORM
SITE Demonstration for Monitoring and Measurement of Dioxin in Soil and Sediment
To: Developer
From: Battelle
COC #; Developer 01
Sample Number/
Analysis Order
Developer 1
Developer 2
Developer 3
Developer 4
Developer 5
Developer 6
Developer 7
Developer 8
Developer 9
Developer 10
Received by
Developer
(•)
Result
Measured
Value
Unit
Analyzed in
Lab (L) or
Field (F)
REMINDERS: Report results consistently (i.e., to the same number of decimal places); Non-detect results should be reported as "< reporting limits" with the
reporting limits of the technology specified.
Relinquished by (signature)
Relinquished by (signature)
Relinquished by (signature)
Relinquished by (signature)
Date
Date
Date
Date
Time
Time
Time
Time
Received by (signature)
Received by (signature)
Received by (signature)
Received by (signature)
Date
Date
Date
Date
Time
Time
Time
Time
COMMENTS:
-------
APPENDIX E
OBSERVER CHECKLISTS
-------
Procedural Observations and Questionnaire
Of
ABRAXIS LLC Coplanar PCB ELISA Kit
Procedure witnessed by:
Date witnessed:
Time/Date procedure started:
Name of Kit Used:
Lot Number of Kit:
Expiration Date of Kit:
Time/Date procedure ended:
Individuals Witnessed:
Answer the following questions:
Question
Could this kit be performed in the
field without a mobile lab/trailer?
Would it take long to set up in the
field before first samples could be
processed?
How long?
How many samples could be
prepared and analyzed in one day in
the field once set up is complete?
By an experienced kit user?
By the novice kit user?
Would sample throughput be faster
in the lab than in the field?
If so by how much?
Are the instructions supplied with
the kit the same as the operating
procedure listed in the demo plan?
If not, why ?
If not, use the kit instructions for
evaluation.
Y
N
NA
Comments
-------
Question
Was testing carried out at kit
recommended temperature of 20° C
to 25° C?
How was temperature measured?
Was measuring device calibrated?
Are the following equipment and
reagents supplied with the kit?
(Note if item not used at all, also
note grade and supplier of solvents)
thermometer
soil collector bottle (containing
dispersion device)
digital balance
30-mL high-density polyethylene
(HOPE) bottle
steel mixing ball
anhydrous sodium sulfate
acetone
hexane
shaker/rotater
filter
centrifuge
extraction tube
concentrated sulfuric acid
nitrogen evaporator
methanol
water
1 : 10 in 50% methanol/water
anti-coplanar PCB antibody solution
controls
standards
parafilm
strip holder
pippetor
enzyme conjugate solution
waste container
IX wash solution
paper towels
color solution
stop solution
microplate reader
graph paper
commercial ELISA program
Y
N
NA
Comments
Actual Temp
-------
Question
Were any supplies or equipment
used that were not listed in the
instructions?
If so, please list.
What are recommended hold times
and storage conditions for:
Samples?
Extracts?
Reagents?
Standards?
Would you know based on the
instructions provided (if not how
did you decide):
How much sample to extract?
How many sample to extract in a
"batch"?
How much sodium sulfate to mix
with sample?
Which solvent and how much to
extract with?
How long to extract?
How many controls and standards
to prepare with "batch"?
How long to agitate during
oxidation cleanup (acid wash)
before letting phases separate and
removing top layer?
Maximum number of oxidation
(acid wash steps) that can be
complete before results are
affected?
Y
N
NA
Comments
-------
Question
After acid wash, is sample
evaporated to complete dryness
during nitrogen evaporation step?
Is additional cleanup ever
necessary?
How do you know and what
additional cleanup options are
there?
Are all samples diluted? If not how
do you know which ones to dilute?
How long do you mix the wells by
moving in a circular motion? ( If
measured what did you measure
with?)
How long to incubate? (If measured
what did you measure with?)
What temperature to incubate? (If
measured, what did you measure
with? Is it calibrated?) How critical
is this temperature?
How long do you mix the wells
with the enzyme conjugate
solution?
How long to incubate? (If
measured, what did you measure
with?)
What temperature to incubate?(If
measured, what did you measure
with? Is it calibrated?) How critical
is this temperature?
How dry do the wells have to be
after the IX wash step?
Y
N
NA
Comments
-------
Question
Do you have to mix in the color
solution? How long does color
solution incubate. Is its incubation
temp critical- what temp is
recommended?
How critical is it that the plate be
read within 1 5 minutes of adding
the stop solution?
How to use/measure with the
microplate reader? Is it calibrated,
if so, how?
How much sample solution needs
to be used with the microplate
reader?
How do you calculate PCDD/PCDF
amounts from the data generated?
Is it clear how to account for
dilutions?
For the cross-reactivity factor?
Additional Comments
Y
N
NA
Comments
-------
Question
Must all procedures be completed
in the same day?
If not, when can procedure be
stopped and how must samples be
stored?
Is that in the instructions?
Were any procedural steps
performed differently than you
interpreted from the instructions?
Were any of the instructions
confusing?
If so please comment:
What QC samples are required with
this approach and at what
frequency?
What are recommended QC
acceptance criteria?
Did QC samples meet acceptance
criteria?
If not, is it clear what corrective
action to take?
What QC samples would vendor
recommend, but not require and at
what frequency?
Do you recommend that some of
the data be verified by conventional
methods?
What method?
What frequency?
How accurate do weights and
volumes used with this technique
have to be?
Were all balances, pipettes, and
thermometers calibrated?
Y
N
NA
Comments
-------
Following the procedure you just
observed, including QC
requirements, how many samples
do you, the observer, think you
could process in a day?
In a week?
Does the vendor provide training in
kit use? Is this extra charge?
Video?
Classes?
Phone support?
What education/experience would
vendor recommend kit users have?
What do you think would be
required education/experience for
successful operation of this
technology?
Additional Comments:
-------
Abraxis Operating Procedure
To ensure accurate and reliable results, every effort should be made to perform the coplanar PCB ELISA kit at
temperatures between 20°C and 25°C and to allow the reagents to be at the same temperature. The following
sample preparation was designed for a quick screen at 625 pg/g. Other sample preparation options are available
for lower concentration.
3.4.2.1 Preparation of Sample Extracts From Soil
1. Label soil collection bottles and extract collection vials.
2. Remove the screw cap from the soil collector bottle (containing dispersion device) and collect soil by
weight using a digital balance. Place the bottle in an upright position on the balance and tare weight. Weigh
2 ± 0.02 g of soil into the tube. Record the soil weight.
3. Weigh 2 g of soil in a 30-mL high-density polyethylene (HDPE) bottle.
4. Add 6 g of anhydrous sodium sulfate and mix until sample is free flowing.
5. Add 1 steel mixing ball.
6. Add 10 mL of 20% acetone in hexane. Rotate for one hour.
7. Remove the organic extract from the soil particulates by filtration, sedimentation, or centrifugation.
8. Transfer extract to a 40 mL screw cap extraction tube and oxidize using concentrated sulfuric acid (4 mL).
Mix by agitation for one minute and allow phases to separate.
9. Remove the organic phase (top layer), transfer to a fresh extraction tube and add 4 mL of concentrated
sulfuric acid. Mix by agitation for one minute and allow phases to separate.
10. Repeat step 9 until the acid phase is colorless.
11. Evaporate 1 mL of the organic phase using a nitrogen stream.
12. Redissolve in 0.25 mL of methanol.
13. Add 0.25 mL of water. If cloudy, centrifuge.
NOTE: This extraction procedure dilutes the sample by a factor of 2.5. Therefore, assay results need to be
multiplied by 2.5 to obtain the final coplanar PCB concentration in the sample.
3.4.2.2 Dilution of Sample Extracts
Dilute sample (1:10) by adding 50 jiL of extract to 950 jiL of 50% methanol/water.
-------
3.4.2.3 Assay Procedure
1. Add 50 jiL of anti-coplanar PCB antibody solution successively to each well.
2. Add 50 jiL of the appropriate standard, control, or sample. Using duplicates or triplicates is recommended.
Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a circular motion
on the benchtop. Be careful not to spill the contents. Incubate at room temperature for 30 minutes.
3. After the incubation, remove the covering and add 50 jil of enzyme conjugate solution to the individual
wells. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a circular
motion on the benchtop. Be careful not to spill the contents. Incubate at room temperature for 90 minutes.
4. After the incubation, remove the covering and vigorously shake the contents of the wells into a waste
container. Wash the strips 3 times using the IX wash solution with a volume of at least 250 uL per each
wash step. Any remaining buffer in the wells should be removed by patting the plate on a dry stack of paper
towels.
5. Add 150 uL of color solution successively to each well. Incubate for 20 to 30 minutes.
6. Add 50 uL of stopping solution to each well in the same sequence as for the other reagents.
7. Read absorbance using a microplate reader at 450 nm within 15 minutes after adding the stopping solution.
8. Construct a standard curve by plotting the %B/Bo for each standard on a vertical logit (y axis) versus the
corresponding PCB 126 standard concentration on the horizontal logarithmic axis (x) on a graph paper.
Alternatively commercial ELISA programs can be used.
9. To obtain the total coplanar PCB TEQ in a sample, multiply sample assay results by the cross-reactivity
factor 0.01.
-------
Procedural Observations and Questionnaire
Of
CAPE Technologies Dioxin/Furan Immunoassay Kit
Procedure witnessed by:
Date witnessed:
Time/Date procedure started:
Name of Kit Used:
Lot Number of Kit:
Expiration Date of Kit:
Time/Date procedure ended:
Individuals Witnessed:
Answer the following questions:
Question
Could this kit be performed in the field
without a mobile lab/trailer?
Would it take to set up in the field before
first samples could be processed?
How long?
How many samples could be prepared and
analyzed in one day in the field once set
up is complete?
By an experienced kit user?
By the novice kit user?
Would sample throughput be faster in the
lab than in the field?
If so by how much?
Are the instructions supplied with the kit
the same as the operating procedure listed
in the demo plan?
If not, why?
Use the kit instructions for evaluation.
Y
N
NA
Comments
-------
Question
Was testing carried out at kit
recommended temperature of 20° C to 25°
C?
How was temperature measured?
Was measuring device calibrated?
Are the following equipment and reagents
supplied with the kit? (Note if item not
used at all, also note grade and supplier of
solvents)
Thermometer
Shaker
DMF
Sodium sulfate
Hexane
Fuming sulfuric acid
Water-miscible organic solvent solution
Carbon adsorption cleanup
Toluene
Triton X- 100
TEG
Methanol
Negative control
Standards
Aqueous sample diluent
HRP competitor conjugate
Repeater pippetor
Chromogenic HRP substrate/hydrogen
peroxide
Stop solution
Tube reader or spectrophotometer
Were any supplies or equipment used that
were not listed in the instructions? If so,
please list.
What are recommended hold times and
storage conditions for:
Samples?
Extracts?
Reagents?
Standards?
Y
N
NA
Comments
Actual Temp
-------
Question
Would you know based on the instructions
provided (if not how did you decide):
How much sample to extract?
How many samples to extract in a "batch"?
How much sodium sulfate to mix with sample?
Which solvent /how much to extract with?
How long to extract?
How many negative controls and standards to
prepare with "batch"?
How to do the chemical oxidation cleanup?
If additional cleanup was necessary?
How to do additional cleanup?
How long to incubate? (If measured what did
you measure with?)
What temperature to incubate?(If measured,
what did you measure with?). How critical is
incubation temperature?
What to wash tubes with after incubation?
How to remove unbound conjugate?
How much chromogenic HRP substrate and
hydrogen peroxide to add?
How long do you let color develop before adding
stop solution? (If time was measured, what did
you use to measure with?)
How quickly must you read the results after
addition of the stop solution?
How to use/measure with the tube
reader/spectrophotometer? Is it calibrated?
How?
How much sample solution needs to be used
with the tube reader/spectrophotometer?
How do you calculate PCDD/PCDF amounts
from the data generated?
Y
N
NA
Comments
-------
Must all procedures be completed in the
same day?
If not, when can procedure be stopped and
how must samples be stored?
Is that in the instructions?
Did samples need, or would they ever
need dilution for successful analysis?
Would you know when and how to do
this?
Were any procedural steps performed
differently than you interpreted from the
instructions? Were any of the instructions
confusing? If so please comment:
What QC samples are required with this
approach and at what frequency?
What are recommended QC acceptance
criteria?
Did QC samples meet acceptance criteria?
If not, is it clear what corrective action to
take?
What QC samples would vendor
recommend, but not require and at what
frequency?
Do you recommend that some of the data
be verified by conventional methods?
What method?
What frequency?
How accurate d weights and volumes used
with this technique have to be?
Were all balances, pipettes, and
thermometers calibrated?
-------
Following the procedure you just
observed, including QC requirements, how
many samples do you think you could
process in a day?
In a week?
Does the vendor provide training in kit
use? Is this extra charge?
Video?
Classes?
Phone support?
What education/experience would vendor
recommend kit users have?
What do you think would be required
education/experience for successful
operation of this technology?
Additional Comments:
-------
Cape Technologies Operating Procedure
PCDD/Fs are typically extracted with organic solvents that are incompatible with EIA; therefore, a solvent
exchange is required. PCDD/Fs have very low volatility and are retained during this solvent exchange in a small
volume of a keeper solution (Triton X-100 detergent in TEG) after evaporation of the original solvent.
Methanol is added to dilute this solution, and the methanol-TEG-Triton mixture is added directly to the EIA
tubes. The solubility of PCDD/Fs in methanol is augmented significantly by adding TEG and Triton X-100.
During the first EIA incubation, PCDD/Fs are specifically bound by the anti-dioxin antibodies, which have been
immobilized on the EIA tube surface. After washing away the unbound material, the bound PCDD/Fs remain,
and a competitor-URP conjugate is added. Bound PCDD/Fs occupy the dioxin binding sites of the antibodies in
proportion to the PCDD/F content of the sample and prevent binding of the competitor-URP conjugate. After a
short incubation, unbound conjugate is removed, and the test tubes are washed thoroughly. The amount of
conjugate bound by the anti-dioxin antibody is inversely related to the amount of PCDD/Fs originally present in
the sample. Finally, a solution of chromogenic HRP substrate and hydrogen peroxide is added to the test tubes.
Color development is directly proportional to enzyme concentration and inversely related to the PCDD/F
concentration in the original sample. The test tubes are analyzed using a tube reader or spectrophotometer to
measure the OD. The OD values of unknown samples are compared to the OD values of standards to determine
the level of PCDD/Fs in the samples.
To ensure accurate and reliable results, every effort should be made to perform the dioxin/furan immunoassay at
temperatures between 20* C (68* F) and 25* C (77°F). The following sample preparation procedure was
designed for same-day analysis at 500 pg/g, using a one-step cleanup. Other sample preparation options are
available for lower concentrations. All sample extraction and extract cleanup components are in a kit form and
are disposable.
1. Sodium sulfate is added to a soil sample and mixed. Dimethylformamide (DMF) is added to the soil sample,
and the soil is extracted by shaking for two hours. The supernatant DMF extract is removed. DMF extracts
are stable for weeks to months at room temperature.
2. Interferences are removed by chemical oxidation. Hexane is added to an aliquot of the DMF extract, then
treated with 15% SO3 in concentrated H2SO4 (fuming sulfuric acid). The supernatant hexane is removed and
exchanged to a water-miscible organic solvent solution. This hexane-based fuming sulfuric acid cleanup is
sufficient for most samples; but, in certain circumstances, an additional cleanup step may be required. This
is the case for samples that contain large amounts of non-volatile aliphatic oils. When the DMF extracts of
such soils are cleaned using fuming sulfuric acid, the oil is not oxidized; and it remains after evaporation of
the hexane, causing a biphasic system when introduced to the EIA first incubation. Such EIA samples
appear opalescent or milky, and their results will be invalid because the biphasic system prevents capture of
analyte by the antibody. For these samples, a new aliquot of DMF extract is cleaned by carbon adsorption.
In this case, the final solvent in the cleanup procedure is toluene rather than hexane.
3. The cleaned sample in hexane or toluene is exchanged to a water-miscible organic solvent solution for EIA
analysis. PCDD/Fs have very low volatility and are retained during this solvent exchange in Triton X-100
detergent in TEG after evaporation of the original solvent. Methanol is added to dilute this solution, and the
methanol-TEG-Triton mixture is added directly to the EIA tubes.
-------
4. An accurately measured volume of negative control, standard, or prepared sample is mixed with an aqueous
sample diluent in test tubes with anti-dioxin antibody immobilized on the surface, and the mixture is
incubated.
5. After incubation, antibody tubes are washed, and 0.5 mL of HRP competitor conjugate is added to each tube
using a repeater pipettor. Bound PCDD/Fs occupy the dioxin binding sites of the antibodies in proportion to
the PCDD/F content of the sample and prevent binding of the competitor-HRP conjugate. After a short
incubation, unbound conjugate is removed, and the test tubes are washed thoroughly.
6. A solution of chromogenic HRP substrate and hydrogen peroxide is added to the test tubes. Color
development is directly proportional to enzyme concentration and inversely related to the PCDD/F
concentration in the original sample. Stop solution is added to each tube using a repeater pipettor to fix the
amount of color development.
7. The test tubes are analyzed using a tube reader or spectrophotometer to measure the OD at 450 nanometers.
The test is interpreted by measuring the signal produced by a sample and determining the concentration
from a dose-response curve constructed from standards tested at the same time.
-------
Procedural Observations and Questionnaire
Of
Hybrizyme Corporation AhRC PCR™ Kit
Procedure witnessed by:
Date witnessed:
Time/Date procedure started:
Name of Kit Used:
Lot Number of Kit:
Expiration Date of Kit:
Time/Date procedure ended:
Individuals Witnessed:
Answer the following questions:
Question
Could this kit be performed in the field
without a mobile lab/trailer?
Would it take long to set up in the field
before first samples could be processed?
How long?
How many samples could be prepared and
analyzed in one day in the field once set
up is complete?
By an experienced kit user?
By the novice kit user?
Would sample throughput be faster in the
lab than in the field?
If so by how much?
Are the instructions supplied with the kit
the same as the operating procedure listed
in the demo plan?
If not, why?
Use the kit instructions for evaluation.
Y
N
NA
Comments
-------
Question
Is there a recommended temperature for
processing the test kits? If so, what?
How was temperature measured?
Was measuring device calibrated?
Are the following equipment and reagents
supplied with the kit? (Note if item not
used at all, also note grade and supplier of
solvents)
Thermometer
Methanol
Activation solution
Shaker
Capture strips
Assay buffer
Capture reagent
Primer/probe
PCR wash concentrate
Glass vials (rack with 96 vials)
Distilled or DI water
Automated plate washer
Standards
Multichannel pippetter
2x Universal Master Mix
adhesive tape
compression pads
thermocycler
Were any supplies or equipment used that
were not listed in the instructions? If so,
please list.
What are recommended hold times and
storage conditions for:
Samples?
Extracts?
Reagents?
Standards?
Y
N
NA
Comments
Actual Temp
-------
Question
Would you know based on the
instructions provided (if not how did you
decide):
How much sample to extract?
How many samples to extract in a
"batch"?
How to extract and with what solvent?
How long to extract?
How many controls and standards to
prepare with "batch"?
Is extract cleanup necessary? If so what
cleanups are a must and what are
optional?
How do you know when to use optional
cleanups?
Is the automated plate washer difficult to
use? How much does an automated plate
washer cost?
Can the plates be washed manually?
Would that be difficult?
At what temperature does step 14 need to
be completed?
How long were samples shaken for step
14? (If measured what did you measure
with?)
At what temperature does step 17 need to
be completed?
Y
N
NA
Comments
-------
Question
How long were samples shaken for step
17? (If measured what did you measure
with?)
At what temperature does step 1 8 need to
be completed?
How long were samples shaken for step
18? (If measured what did you measure
with?)
How long did the PCR wash step take?
Did strips need to incubate before
analyzing on thermocycler? If so how
long and at what temperature?
How quickly must you read the results
after addition of the 1 X Master Mix?
How to use/measure with the
thermocycler? Is it calibrated? How?
How much sample solution needs to be
used with the thermocycler?
How do you calculate PCDD/PCDF
amounts from the data generated?
Must all procedures be completed in the
same day?
If not, when can procedure be stopped and
how must samples be stored?
Is that in the instructions?
Y
N
NA
Comments
-------
Question
Did samples need, or would they ever
need dilution for successful analysis?
Would you know when and how to do
this?
Were any procedural steps performed
differently than you interpreted from the
instructions? Were any of the instructions
confusing? If so please comment:
What QC samples are required with this
approach and at what frequency?
What are recommended QC acceptance
criteria?
Did QC samples meet acceptance criteria?
If not, is it clear what corrective action to
take?
What QC samples would vendor
recommend, but not require and at what
frequency?
Do you recommend that some of the data
be verified by conventional methods?
What method?
What frequency?
How accurate d weights and volumes used
with this technique have to be?
Were all balances, pipettes, and
thermometers calibrated?
Following the procedure you just
observed, including QC requirements,
how many samples do you think you
could process in a day?
In a week?
Y
N
NA
Comments
-------
Question
Does the vendor provide training in kit
use? Is this extra charge?
Video?
Classes?
Phone support?
What education/experience would vendor
recommend kit users have?
What do you think would be required
education/experience for successful
operation of this technology?
Additional Comments:
Y
N
NA
Comments
-------
Hybrizyme Corporation Operating Procedure
The general steps for analyzing sample extracts are as follows:
1. Add 5 jiL of sample extract in methanol to the activation solution, mix, and shake for 1 hour at
room temperature.
2. Transfer to a capture strip Amicrowell@ and shake for 30 minutes at room temperature.
3. Wash the capture strip.
4. Add PCR mix.
5. PCR amplify.
Real-time PCR is analyzed by examining "primary growth curves" generated by fluorescent probes within
newly synthesized DNA. For quantitative and comparative purposes, a threshold cycle (Ct) is defined for each
sample. The threshold may be an arbitrary signal above background, or a certain number of standard deviations
above background.
The approximate detection limit of the AhRC is less than 0.2 pg TCDD in the assay, according to Hybrizyme.
The AhRC PCR™ Kit includes
Activation solution (12 vials, 0.5 mL each): Source of Ah receptor and DNA probe (store at -80oC).
• Capture strips (1 plate, 8x12 wells): Keep unused strips sealed and in the pouch (store at room
temperature). Strip rack included.
• Assay buffer (1 bottle, 20 mL): Ready to use (store at room temperature for up to 2 weeks or at 4oC for
extended periods of time).
Capture reagent (red cap) (1 vial, 0.60 mL): A 16-fold concentrated solution (store at 4oC). Prepare for
use by adding 40 L to 600 L of assay buffer.
• Primer/probe (white cap) (1 vial, 600 jiL): Forward and reverse primers containing Taqman MGB FAM
probe (aliquot, store at 20oC when not in use).
• PCR wash concentrate (25X) (2 bottles, 40 mL each): A 25-fold concentrated solution (store at 4oC).
Prepare for use by mixing entire contents of one bottle with 960 milliliter (mL) of deionized water.
• Glass vials: Rack with 96 vials (flat-bottomed 0.5-mL glass vials with plate).
3.2.2 Operating Procedure
12. Reconstitute PCR wash solution to 1 X with distilled or deionized water for use with an automated plate
washer. Prime the plate washer with PCR wash solution.
-------
13. Prepare standards and unknowns in methanol.
14. Prepare the 1 X capture reagent by diluting 40 jiL of stock reagent into 600 jiL of assay buffer for each
strip used. Place desired number of strips in the strip frame and re-seal the remainder in the foil pouch. Wash
the strips using the "3XWASH" program of the plate washer. Using the multichannel pipetter, dispense 50 jiL
to each well in the strip. Shake 60 to 90 minutes.
15. Thaw the activation solution, mixing gently during the process. Do not allow the activation solution to
remain at room temperature for more than 20 minutes prior to use. For best results, mix the activation solution
vials together prior to dispensing when performing multiple strips.
16. Dispense 50 jiL of the assay buffer into each glass vial using a multichannel pipette. Assay buffer
should be at room temperature prior to use. Add 5 jiL of standard or sample. The use of a pipetter (i.e., P-10
Pipetman) with a filter tip is preferred. After adding sample to the entire row, tap gently to mix. Repeat the
process for each row of glass vials.
17. Add 50 jiL of activation solution to each glass vial using a multichannel pipette. Shake for 1 hour at
room temperature.
18. Wash the strips using the "3XWASH" program of the plate washer. Using a multichannel pipetter,
transfer 30 jiL of each reaction to each well. Shake 30 minutes.
19. 8. Wash the strips using the "PCRWash" program of the plate washer. This series of soaks and
washes takes about 15 minutes. Thaw the primer/probe solution at this time.
20. Add 100 jiL of primer/probe solution, 400 jiL of water, and 500 jiL of 2 X Universal Master Mix to
make each 1,000 jiL of 1 X Master Mix. Dispense 40 jiL into each well.
21. Seal the wells with adhesive tape and cover with two compression pads. Insert the strips into
thermocycler and run the "PCR" template.
22. Analyze the data.
-------
Procedural Observations and Questionnaire
Of
Paracelsian, Inc., Ah-IMMUNOASSAY®
Procedure witnessed by:
Date witnessed:
Time/Date procedure started:
Name of Kit Used:
Lot Number of Kit:
Expiration Date of Kit:
Time/Date procedure ended:
Individuals Witnessed:
Answer the following questions:
Question
Could this kit be performed in the field
without a mobile lab/trailer?
Would it take long to set up in the field
before first samples could be processed?
How long?
How many samples could be prepared and
analyzed in one day in the field once set
up is complete?
By an experienced kit user?
By the novice kit user?
Would sample throughput be faster in the
lab than in the field?
If so by how much?
Are the instructions supplied with the kit
the same as the operating procedure listed
in the demo plan?
If not, why?
Use the kit instructions for evaluation.
Y
N
NA
Comments
-------
Question
Is there a recommended temperature for
using the kit? If so, what is it?
How was temperature measured?
Was measuring device calibrated?
Are the following equipment and reagents
supplied with the kit? (Note if item not
used at all, also note grade and supplier of
solvents)
Refrigerator/Freezer
Thermometer
Cytosol
ORE Oligo
ARNT extract
Activator
Beakers
Icebath
50-mL mixing tube
ice
DMSO
Tubes
ELISA plates
TCDD reference standard
NAP standard
Multichannel pippettor
20X wash buffer
reagent grade water
AB1
AB diluent
AB2
Detection tablets
Detection buffer
Paper towels
Automated plate washer
Plate reader
Graph paper
Curve fitting statistical software
Y
N
NA
Comments
Actual Temp
-------
Question
Were any supplies or equipment used that
were not listed in the instructions? If so,
please list.
What are recommended hold times and
storage conditions for:
Samples?
Extracts?
Reagents?
Standards?
Would you know based on the
instructions provided (if not how did you
decide):
Which kit components are to be stored
refrigerated and which are frozen?
How many of the cytosol vials need to be
thawed at once?
Which mixtures are intermediates and
need to be kept on an ice bath?
How to decide how much DRE Oligo is
needed for the amount of cytsol thawed?
How long the does tube need to be rocked
in step 5 for sufficient mixing? Is this
done by hand or automated shaking?
How to decide how much ARNT to add in
step 6?
Y
N
NA
Comments
-------
Question
How long the does tube need to be rocked
in step 6 for sufficient mixing? Is this
done by hand or automated shaking?
How to decide how much activator is
needed for step 8?
How long the does tube need to be rocked
in step 9 for sufficient mixing? Is this
done by hand or automated shaking?
How are samples extracted?
How much sample is used to extract?
How many samples to extract in a
"batch"?
Which solvent and how much to extract
with?
How long to extract?
Does the extract require any cleanup? If
so, what?
Does extract have to be solvent exchanged
to DMSO?
How many negative controls and
standards to prepare with "batch"?
When should the TCDD reference
standard be used and when should the
NAP standard be used?
Y
N
NA
Comments
-------
Question
Steps 1 1 through 14 says you can use four
dilutions of 10 samples. What dilution
levels would you use and why?
How long were samples incubated for step
16? (If measured what did you measure
with?)
What temperature to incubate?(If
measured, what did you measure with?).
How critical is incubation temperature?
How was temp controlled?
How to prepare the IX wash buffer?
How to wash tubes after incubation?
Was wash done by hand or with an
automated washer? Which is easier to
use? Faster?
How to prepare the primary antibody
stock?
How long was plate incubated after
adding primary antibody stock? What
temperature was used?
How to prepare the secondary antibody
stock?
How long was plate incubated after
adding secondary antibody stock? What
temperature was used?
Y
N
NA
Comments
-------
Question
How to prepare the detection reagent
stock? How do you know how many
tablets to dissolve and volume of
detection buffer to use? How was this
protected from light? Does it have to be
stored protected from light?
How long was plate incubated after
adding detection reagent stock? What
temperature was used? How was this
protected from light?
How critical is it that readings be taken at
15, 30, 45, and 60 minutes? Must these
times be exact? Is the plate kept
incubating between each reading?
How to use/measure with the plate reader?
Is it calibrated? How?
How much sample solution needs to be
used with the plate reader?
How do you calculate PCDD/PCDF
amounts from the data generated?
Must all procedures be completed in the
same day?
If not, when can procedure be stopped and
how must samples be stored?
Is that in the instructions?
Y
N
NA
Comments
-------
Question
Did samples need, or would they ever
need dilution for successful analysis?
Would you know when and how to do
this?
Were any procedural steps performed
differently than you interpreted from the
instructions? Were any of the instructions
confusing? If so please comment:
What QC samples are required with this
approach and at what frequency?
What are recommended QC acceptance
criteria?
Did QC samples meet acceptance criteria?
If not, is it clear what corrective action to
take?
What QC samples would vendor
recommend, but not require and at what
frequency?
Do you recommend that some of the data
be verified by conventional methods?
What method?
What frequency?
How accurate do weights and volumes
used with this technique have to be?
Were all balances, pipettes, and
thermometers calibrated?
Following the procedure you just
observed, including QC requirements,
how many samples do you think you
could process in a day?
In a week?
Y
N
NA
Comments
-------
Question
Does the vendor provide training in kit
use? Is this extra charge?
Video?
Classes?
Phone support?
What education/experience would vendor
recommend kit users have?
What do you think would be required
education/experience for successful
operation of this technology?
Additional Comments:
Y
N
NA
Comments
-------
Paracelsian Operating Procedure
3.3.2 Operating Procedure
The following protocol is for an assay that uses all 96 wells in the 96-well version of the kit. Steps 18 through
21, describing the procedure for the three replicate washes following each incubation, can be automated by a
commercial plate washer. Likewise, any residual fluid remaining in the plate wells can be completely removed
by a properly adjusted plate washer, thereby eliminating the need for the manual blotting in step 31.
1. Remove the refrigerated components of the kit from cold storage and warm to room
temperature.
2. Remove the cytosol components (cytosol, DRE Oligo, ARNT extract, activator) from frozen storage.
Thaw up to four 7.5-mL vials of cytosol in a beaker of tepid water. Steps 3 through 9 will generate the activated
cytosol. Keep all intermediate mixtures in an ice bath as much as possible.
3. Pool the thawed tubes of cytosol into the 50-mL mixing tube.
4. Add the required volume of DRE Oligo to the pooled cytosol. Return unused DRE Oligo to the freezer.
5. Mix by rocking the tube gently.
6. Add the required volume of ARNT to the cytosol, DRE Oligo mixture. Return unused ARNT to the
freezer.
7. Mix by rocking the tube gently.
8. Add the required volume of activator to the cytosol, DRE Oligo, ARNT mixture. Return unused
activator to the freezer.
9. Mix immediately (to avoid high local concentrations of salt) by rocking the tube gently. This mixture is
the activated cytosol and must be kept on ice.
10. Add 10 L each of one reference standard, one negative control, and up to 10 samples in DMSO (the
negative control) to 1.0-mL aliquots of the activated cytosol and mix gently. Tubes for these preparations are
supplied by the user. These mixtures compose the treated stock for each type of determination.
11. The 96-well ELISA plate layout will accommodate one negative control, seven dilutions of the TCDD
reference standard (or a single dilution of the NAP Standard), and four dilutions of 10 samples, all in duplicate.
If less than 96 wells are used, return the unused ELISA strips to 4 C storage in the foil bag with desiccant.
12. Remove the ELISA plate from the desiccated plastic bag and add 400 L of each treated stock to the top
row of the wells to give duplicate runs of each of the 10 samples and the single reference standard.
13. With a multichannel pipettor, add 200 L of activated cytosol to all the remaining wells. One of the kit
reservoirs will facilitate loading the pipettor.
-------
14. Beginning with the first sets of duplicate wells, make serial dilutions (seven dilutions for the TCDD
reference or the single dilution of the NAP, four dilutions for each sample) down the respective plate columns.
Individual dilutions are mixed by a minimum of six repetitive aspiration and redispensing cycles of the well
contents. The residual waste following the final dilution must be collected for proper disposal.
15. Attach the lid to the plate.
16. Incubate the plate for two hours at 30 C. Incubation at room temperature (i.e., 20 C) is satisfactory, but
will result in a slightly lower response. Incubation at a higher temperature (i.e., 37 C) will also result in a lower
response.
17. Prepare the 1 X wash buffer by diluting the contents of the 20 X wash buffer bottle to 500 mL with
reagent-grade water (250 mL for the 48-well kit). Store unused 1 X wash buffer at 4 C.
18. After the two-hour incubation, remove the well contents by aspiration into a waste receptacle for proper
disposal.
19. Add 400 L 1 X wash buffer to all wells, loading the multichannel pipette from a kit reservoir.
20. Wait two minutes. Remove the well contents by aspiration into the waste receptacle for proper disposal.
21. Repeat Steps 19 and 20 twice for a total of three separate washes.
22. Prepare the primary antibody stock by aliquoting the required amount of AB 1 into the appropriate
volume of AB diluent. Mix gently. Store unused AB 1 and AB diluent at 4 C.
23. Deliver 200 L AB 1 stock to all wells of the plate, loading the multichannel pipette from a kit reservoir.
24. Incubate the plate for one hour at 30 C.
25. After the one hour incubation, repeat steps 18 through 21.
26. Prepare the secondary antibody stock by aliquoting the required amount of AB 2 into the appropriate
volume of AB diluent. Mix gently. Store unused AB 2 and AB diluent at 4 C.
27. Deliver 200 L AB 2 stock to all wells of the plate, loading the multichannel pipette from a kit reservoir.
28. Incubate the plate for one hour at 30 C.
29. After the one-hour incubation, repeat Steps 18 through 21.
30. Prepare the detection reagent stock by dissolving the required number of detection tablets in the
appropriate volume of detection buffer with gentle mixing while protected from light. Allow a minimum of 15
minutes for the tablets to completely dissolve. Unused detection buffer and tablets are kept at 4 C.
-------
31. Following the incubation of the final wash, strike the inverted plate against paper toweling to remove
residual fluid before proceeding to the next step.
32. Deliver 200 L of the detection reagent stock from Step 30 to all wells of the plate, loading the
multichannel pipette from a kit reservoir.
33. Incubate the plate at 30 C while protected from light. Residual moisture from condensation and/or from
contact with incubator water must be carefully blotted from the plate before reading. The plate is read at 405
nanometers (nm) after 15, 30, 45, and 60 minutes. Variations in incubation temperatures and plate reader
characteristics may dictate the best incubation times for individual conditions.
A standard curve is used to determine the amount of TCDD equivalents present in a sample. The standard curve
is generated by plotting the average absorbance (OD) measured at 405 nm for each of the TCDD reference
standards on the vertical (Y) axis versus the corresponding quantity (pg) of TCDD on the horizontal (X) axis.
Results are calculated manually using graph paper or a curve-fitting statistical software package. The TCDD
equivalents for the samples are determined from the standard curve by interpolating from the absorbance value
(Y axis) to the quantity (pg) of TCDD (X axis). The initial TCDD concentration in the sample is found by
correcting for the sample dilution. The supplied NAP solution, at 2 L per test well, may optionally be used as a
single-point positive control that is equivalent to 32 pg TCDD.
-------
Procedural Observations and Questionnaire
Of
WAKO Dioxin ELISA Kit
Procedure witnessed by:
Date witnessed:
Time/Date procedure started:
Name of Kit Used:
Lot Number of Kit:
Expiration Date of Kit:
Time/Date procedure ended:
Individuals Witnessed:
Answer the following questions:
Question
Could this kit be performed in the
field without a mobile lab/trailer?
How long would it take to set up in the
field before first samples could be
processed?
How many samples could be prepared
and analyzed in one day in the field
once set up is complete?
By an experienced kit user?
By the novice kit user?
Would sample throughput be faster in
the lab than in the field?
If so by how much?
Are the instructions supplied with the
kit the same as the operating
procedure listed in the demo plan?
If not, why?
Use the kit instructions for evaluation.
Y
N
NA
Comments
-------
Question
Was testing carried out at kit
recommended temperature of 20° C to
25° C?
How was temperature measured?
Was measuring device calibrated?
Instructions say kit is to be stored at 2
to 10°C. How long can it be kept at
room temperature before the kit is
compromised?
Are the following equipment and
reagents supplied with the kit? How
many of each are needed? What grade
and supplier of solvents are used?
Thermometer
PC tube
Methanol
Stirring rod
Sample solubilizer
Tubes
Tube Rack
Acetone
Buffer B
Vortex mixer
Centrifuge
Primary antibody tube
Purified water
Paper towels
Buffer A
POD conjugate solution tube
Scotch tape
Refrigerator
Parafilm
Reaction mixture
Wash solution concentrate
Color developing solution
Citrate buffer
Aluminum foil
Stop solution
Pipettes
Mi cr opiate reader
Y
N
NA
Comments
Actual Temp
-------
Question
Were any supplies or equipment used
that were not listed in the instructions?
If so, please list.
What are recommended hold times
and storage conditions for:
Samples?
Extracts?
Reagents?
Standards?
Would you know based on the
instructions provided (if not how did
you decide):
How much sample to extract?
How to extract/what sample
pretreatment to use?
Is any extract cleanup was necessary?
If so, what?
How many samples to include in a
"batch"?
How many controls and standards to
prepare with "batch"?
How to prepare the PC solutions?
Y
N
NA
Comments
-------
Question
How to prepare the primary antibody
working solution?
How to prepare the POD-conjugate
solution?
How long to leave tubes in ice water
in step 15?
What is the temperature of the
refrigerator used to store the
microplates in step 17?
How long were the microplates
actually refrigerated in step 17?
How to prepare wash solution?
How to prepare color developing
solution?
How long do you let color develop
before adding stop solution? (If time
was measured, what did you use to
measure with?)
How critical is it that reading be taken
within 1 5 minutes of adding stop
solution?
How to use/measure with the tube
reader/spectrophotometer?
How much sample solution needs to
be used with the tube
reader/spectrophotometer?
How do you calculate PCDD/PCDF
amounts from the data generated?
Y
N
NA
Comments
-------
Question
Must all procedures be completed in
the same day? If not, when can
procedure be stopped and how must
samples be stored? Is that in the
instructions?
Did samples need, or would they ever
need dilution for successful analysis?
Would you know when and how to do
this?
Were any procedural steps performed
differently than you interpreted from
the instructions? Were any of the
instructions confusing? If so please
comment:
What QC samples are required with
this approach and at what frequency?
What are recommended QC
acceptance criteria?
Did QC samples meet acceptance
criteria?
If not, is it clear what corrective action
to take?
What QC samples would vendor
recommend, but not require and at
what frequency?
How accurate do weights and volumes
used with this technique have to be?
Were all balances, pipettes, and
thermometers calibrated?
Y
N
NA
Comments
-------
Question
Following the procedure you just
observed, including QC requirements,
how many samples do you think you
could process in a day?
In a week?
Do you recommend that some of the
data be verified by conventional
methods?
What method?
What frequency?
Does the vendor provide training in kit
use? Is this extra charge?
Video?
Classes?
Phone support?
What education/experience would
vendor recommend kit users have?
What do you think would be required
education/experience for successful
operation of this technology?
Additional Comments:
Y
N
NA
Comments
-------
WAKODioxin ELISA Kit Operating Procedure
The Dioxin ELISA Kit should be used at room temperature (20« «to 25« C) and stored at 2« «to 10« C.
1. To prepare the PC solution (5,000 pg/mL), add 2.0 mL of methanol to the tube containing the PC. Then, gently stir the tube four
or five times, and leave it at room temperature for 10 minutes. Gentle stirring is repeated once more before use. Do not vigorously
shake or vortex the solution to avoid PC material adhering to the tube wall. The solution can be used within four weeks at 2« «to
10« C after reconstitution.
2. Prepare the dilution solution by dispensing 2 mL of sample solubilizer in a tube and adding 2 mL of methanol.
3. Prepare 1/10-fold diluted PC solution (500 nanograms/mL) by adding 900 uL of dilution solution to a disposable culture tube
rinsed with acetone and dried. Then add 100 uL of concentrated PC solution and gently stir the tube two or three times. Leave the
tube at room temperature. The solution can be used after pipetting it up and down two or three times.
4. Prepare PC dilution solutions for the standard curve using the 1/10-fold diluted PC solution described above as follows:
Tube Number:
Concentration
Dilution solution
Concentrated PC
solution
1/1 0-fold diluted
PC solution
Total volume
1
0 pg/mL
500 uL
-
0 uL
500 uL
2
40 pg/mL
460 uL
-
40 uL
500 uL
3
100 pg/mL
400 uL
-
lOOuL
500 uL
4
250 pg/mL
250 uL
-
250uL
500 uL
5
500 pg/mL
450 uL
50 uL
-
500 uL
6
l,OOOpg/mL
400 uL
100 uL
-
500 uL
7
2,500 pg/mL
250 uL
250 uL
-
500 uL
5. Dry the contents of each tube.
6. To prepare a dilution series of PC solutions, add 500 \\L of Buffer B to each tube, lightly stir it with a vortex mixer, and centrifuge it at 2,000
revolutions per minute (rpm) at room temperature to collect the solution at the bottom.
7. To prepare the primary antibody stock solution, add 200 uL of purified water to the tube and gently stir it, then leave the tube for 10 minutes at
room temperature. The solution can be used within four weeks.
8. Rinse a beaker or a 5- to 10-mL vial with acetone and air dry on clean paper towels.
9. Prepare the primary antibody working solution using a 100-fold dilution of the primary antibody stock solution described above with Buffer A
according to assay sample numbers and volumes. Stir the diluted solution gently several times, and leave it at room temperature for 10 minutes.
Stir the solution several times just before use. The solution cannot be stored prior to use.
10. Prepare the POD-conjugate solution by adding 4 mL of Buffer A to the tube and gently stir several times. Leave the tube at room temperature
for 10 minutes. Gently stir the tube just before use. The solution can be used within two weeks when stored at 2- 4o 10- C.
11. Fix the necessary number of microplate wells (coated with the secondary antibody) to a microplate holder with Scotch tape. Place the holder in a
closed box, and store it in a refrigerator for at least 30 minutes before use.
12. Add 100 uL of Buffer B to the tube containing the material obtained by sample pretreatment, and gently stir the solution along the inner surface
of the tube several times with vortex mixer. Briefly centrifuge the tube at 2,000 rpm for five minutes to collect the solution at the bottom.
13. Line up the tubes containing the dilution series of PC solutions and the samples resolved in Buffer B in a tube rack. Dispense an equal volume
of the primary antibody working solution to each tube.
14. Immediately, agitate the solutions several times, then seal the tubes with Parafilm and leave them at room temperature for 30 minutes.
15. During the incubation, prepare chilled water containing ice in a water bath and sink the tubes under the water in the bath. After the 30
minute-incubation at room temperature, add 250 uL and 50 uL of POD-conjugate solution to the tubes containing the diluted PC solutions and
those containing sample material, respectively.
16. Gently agitate the tubes several times, and leave them for 10 minutes. Avoid vigorous shaking with the vortex because dioxins may adsorb to
the tube wall.
-------
17. Take the microplate holder from the box stored in a refrigerator. Dispense 100 uL of the reaction mixture to the wells. Briefly agitate the
solutions in the wells. Seal the wells with Parafilm, return the holder to the box, and store the box in a refrigerator at 2- -to 10- C for 18 to 20
hours.
18. On the second day, prepare the wash solution by diluting the wash solution concentrate (x 6) with five volumes of purified water. The solution
volume prepared can be adjusted depending upon the number of wells used. Usually, one well needs 1.2 mL of the wash solution. The solution
can be used within two weeks after dilution when stored at 2' -to 10' C.
19. Prepare the necessary volume of color-developing solution by mixing the substrate and the citrate buffer in a ratio of 1:50 just before use. (The
tube used in this preparation should be cleaned with purified water.) Add the citrate buffer to the tube, then the substrate. Avoid vigorous
agitation of the mixed solution because it may cause crystalization.
20. Take the well holder from the box stored in a refrigerator, and remove the seal from the wells. Remove the reaction mixture from the wells,
being careful not to spread the solution to the holder. Lightly tap the holder upside down on sheets of paper to remove the residual mixture in the
wells. Dispense approximately 0.35 mL of wash solution to each well, and then drain the solution, being careful not to spread the solution to the
holder. Remove the solution from the well walls by tapping the holder upside down on the papers. Repeat washing twice. At the last washing,
remove the solution by tapping as much as possible.
21. For the enzyme reaction, dispense 100 uL of the color-developing solution to all the wells. Seal with Parafilm, cover the holder with aluminum
foil to interrupt light, and leave the holder at room temperature for 30 minutes. Stop the reaction by adding 100 uL of stop solution in the same
order as the color-developing solution was added. Tap the side of the holder to mix the solution. Read the signal with a microplate reader at 450
nm or at 450/650 nm within 15 minutes after stopping the reaction. Construct the standard curve obtained with the diluted PC solutions and
estimate the dioxin concentrations of samples as 3,7,8-TCCD. The primary antibody used in this kit reacts with 2,3,7,8-TCCD and 3,7,8-TCCD
with equal intensity.
22. In estimating the dioxin concentration, it is useful to plot the obtained values with a regression of polynomial quadratic equation or
four-parameter logic after log-logit conversion.
-------
Procedural Observations and Questionnaire
Of
Xenobiotics Detection Systems, Inc. CALUX®
Procedure witnessed by:
Date witnessed:
Time/Date procedure started:
Name of Kit Used:
Lot Number of Kit:
Expiration Date of Kit:
Time/Date procedure ended:
Individuals Witnessed:
Answer the following questions:
Question
Is this product intended for use by anyone
other than XDS?
Could this kit be performed in the field
without a mobile lab/trailer?
Would it take long to set up in the field
before first samples could be processed?
How long?
How many samples could be prepared and
analyzed in one day in the field once set
up is complete?
By an experienced kit user?
By the novice kit user?
Would sample throughput be faster in the
lab than in the field?
If so by how much?
Y
N
NA
Comments
-------
Question
Are the instructions supplied with the kit
the same as the operating procedure listed
in the demo plan?
If not, why?
Use the kit instructions for evaluation.
Is there a recommended temperature for
carrying out these analyses? If so, what?
How was temperature measured?
Was measuring device calibrated?
Are the following equipment and reagents
supplied with the kit? (Note if item not
used at all, also note grade and supplier of
solvents)
Thermometer
Glass vials with PTFE-lined caps
Methanol
Toluene
Ultrasonic water bath
Filter
Vacuum centrifuge
Hexane
DMSO
Cell culture medium
96-well culture plates
2,3,7,8-TCDD standard curve
humidified CO2 incubator
microscope
Promega luciferase assay kit
Y
N
NA
Comments
Actual Temp
-------
Question
Were any supplies or equipment used that
were not listed in the instructions?
If so, please list.
What are recommended hold times and
storage conditions for:
Samples?
Extracts?
Reagents?
Standards?
Would you know based on the
instructions provided (if not how did you
decide):
How much sample to extract?
How many samples to extract in a
"batch"?
How to dry the sample?
Which solvent and how much to extract
with?
How long to extract?
How many controls and standards to
prepare with "batch"?
Y
N
NA
Comments
-------
During extraction is there a recommended
temperature for the ultrasonic water bath?
How are extracts filtered?
How much are they concentrated by vacuum
centrifugation?
If cleanup is proprietary, how does kit user
know what to do?
After cleanup, how is extract concentrated into
DMSO?
How do you suspend the DMSO/extract
concentrate in the cell culture medium?
How do you prepare the 96-well culture plates?
How long do the plates incubate in the
humidified C02 incubator? Is this step
temperature sensitive? What humidity range is
acceptable?
How do you tell if the cells are microscopically
viable?
How do you use the Promega luciferase assay
kit to quanitify? Is it clear how this converts to
dioxin and PCB TEQ?
Do cells have to be read within a certain amount
of time from incubation? How critical is this?
How much sample solution needs to be
used with the detection technique?
-------
Must all procedures be completed in the
same day?
If not, when can procedure be stopped and
how must samples be stored?
Is that in the instructions?
Did samples need, or would they ever
need dilution for successful analysis?
Would you know when and how to do
this?
Were any procedural steps performed
differently than you interpreted from the
instructions? Were any of the instructions
confusing? If so please comment:
What QC samples are required with this
approach and at what frequency?
What are recommended QC acceptance
criteria?
Did QC samples meet acceptance criteria?
If not, is it clear what corrective action to
take?
What QC samples would vendor
recommend, but not require and at what
frequency?
Do you recommend that some of the data
be verified by conventional methods?
What method?
What frequency?
How accurate do weights and volumes
used with this technique have to be?
-------
Were all balances, pipettes, and
thermometers calibrated?
Following the procedure you just
observed, including QC requirements,
how many samples do you think you
could process in a day?
In a week?
Does the vendor provide training in kit
use? Is this extra charge?
Video?
Classes?
Phone support?
What education/experience would vendor
recommend kit users have?
What do you think would be required
education/experience for successful
operation of this technology?
Additional Comments:
-------
Xenobiotics Detection Systems, Inc. CALVX ® Operating Procedure
3.6.2 Operating Procedure
Xenobiotic Detection Systems, Inc. has a patented genetically engineered cell line (mouse hepatoma H1L1) that
contains the gene for firefly luciferase under transactivational control of the AhR. This cell line can be used for
the detection and relative quantification of a sample's total dioxin I-TEQ. Using a patent pending sample
processing procedure, it is also possible to use the CALUX® by Xenobiotic Detection Systems assay to
estimate the I-TEQ contributions of PCDDs/Fs or the I-TEQ contributions of the coplanar PCBs. The assay that
uses this cell line is called the Chemical-Activated Luciferase Expression or CALUX® by Xenobiotic
Detection Systems assay.
The samples are extracted using a modification of the EPA 8290 extraction method. Briefly, the dried samples
are ground and 1-g aliquots are placed in solvent-cleaned glass vials with polytetrafluoroethylene-lined caps.
The sample is extracted with a 20% solution of methanol in toluene then twice with toluene. During each
extraction step, the samples are incubated in an ultrasonic water bath. The three extracts from each sample are
filtered, pooled, and concentrated by vacuum centrifugation. The sample extract is suspended in hexane and
prepared for the bioassay by a proprietary cleanup method. The eluate from the cleanup method is concentrated
under vacuum into DMSO. The DMSO solution is used to dose the genetically engineered cells in the
CALUX® by Xenobiotic Detection Systems assay.
Prior to dosing the cells, the sample extracts in DMSO are suspended in cell culture medium. This medium is
then used to expose monolayers of the H1L1 cell line grown in 96-well culture plates. In addition to the
samples, a standard curve of 2,3,7,8-TCDD is assayed (161, 80.5, 40.2, 20.1, 10.1, 5.0, 2.5, 1.2, and 0.6 ppt
TCDD). The plates are incubated for a time to produce optimal expression of the luciferase activity in a
humidified CO2 incubator. Following incubation, the medium is removed and the cells are examined
microscopically for viability. The induction of luciferase activity is quantified using the luciferase assay kit
from Promega.
-------
APPENDIX F
AXYS ANALYTICAL SERVICES STATEMENT OF WORK
-------
Statement of Work
AXYS Analytical Services Ltd.
SITE Demonstration for Dioxin Monitoring and Measurement Technologies
AXYS Project Manager: Georgina Brooks Battelle Project Manager: Amy Dindal
Phone: 250-655-5800 Phone: 561-422-0113
Email: gbrooks@axys.com Email: dindala@battelle.org
Battelle Memorial Institute is conducting a field demonstration of dioxin monitoring and
measurement technologies under the U.S. Environmental Protection Agency's (EPA)
Superfund Innovative Technology Evaluation (SITE) Program. In April 2004, developers
of innovative dioxin measurement technologies will gather at a field demonstration site in
Saginaw, MI to analyze dioxin-contaminated soil, sediment, and extract samples. A
reference laboratory will be concurrently analyzing replicate splits of the samples by high
resolution mass spectrometry (HRMS). The reference laboratory, AXYS Analytical
Services, Ltd. (Sidney, British Columbia, Canada), will analyze samples for 17
dioxin/furans and the 12 World Health Organization dioxin-like poly chlorinated biphenyl
(WHO PCB) congeners in 13 soil/sediment samples and 2 extracts for the pre-
demonstration phase of the project and 186 soil/sediment samples and 23 extracts in the
demonstration phase of the project. AXYS will also be responsible for the preparation of
the extract samples.
The purpose of this statement of work is to describe in detail the analytical services to be
procured from AXYS for this project. AXYS will provide reference analytical
measurements for dioxin/furans (D/F) using EPA Method 1613B and for the 12 WHO
PCBs using EPA Method 1668A. The congeners to be reported and their associated toxic
equivalency factors (TEFs) are provided in Table 1. The following is a list of the major
tasks that will be completed by AXYS.
Task 1: D/F and PCB Analysis. Approximately 200 soil/sediment samples will be
provided by Battelle to AXYS for analysis by both 1613B and 1668A. AXYS will
receive -13 samples as soon as this purchase order is in place (pre-demonstration phase),
and the remaining 186 samples in mid-April (demonstration phase). AXYS will also
generate and analyze 25 extracts in Tasks 2 and 3 below for 1613B and 1668A analysis.
Two extracts will be prepared and analyzed immediately after this purchase order is in
place (pre-demonstration phase). Twenty-three extracts will be prepared and analyzed in
late April (demonstration phase).
Task 2: Soil Extract Preparation. Battelle will provide AXYS with approximately
ninelOO g soil samples. AXYS will extract each 100 g sample according to Dean Stark
extraction procedure for 1613B, with the exception of surrogate spiking which will be
spiked (after aliquot splitting step) into the AXYS aliquot only. AXYS will split the
extract into 11 equal aliquots. Each aliquot will be concentrated to 10 mL and put in a
heat-sealed ampoule. Each ampoule will be labeled per Battelle's instructions. Six of the
11 ampoules will be shipped by Federal Express according to Battelle's directions. One
-------
Table 1. Congeners to be Reported and Toxicity Equivalency Factors (TEF) Values
TEF Value
Compound*3' J TEF WHO
PCDDs
2,3,7,8-TCDD 1 1
1,2,3,7,8-PeCDD 0.5 1
1,2,3,4,7,8-HxCDD 0.1 0.1
1,2,3,6,7,8-HxCDD 0.1 0.1
1,2,3,7,8,9-HxCDD 0.1 0.1
1,2,3,4,6,7,8-HpCDD 0.01 0.01
OCDD 0.001 0.0001
Dioxins-like PCBs
co-planar
3,3',4,4'-TCB (PCB 77) 0.0005 0.0001
3,4,4',5-TCB (PCB 81) - 0.0001
3,3',4,4',5-PeCB (PCB 126) 0.1 0.1
3,3',4,4',5,5'-HxCB (PCB 169) 0.01 0.01
Compound
PCDFs
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,7,8,9-HxCDF
1,2,3,6,7,8-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDF
mono-ortho
2,3,3',4,4'-PeCB (PCB 105)
2,3,4,4',5-PeCB(PCB114)
2,3,'4,4',5-PeCB(PCB118)
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)
TEF
I-TEF
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.001
0.0001
0.0005
0.0001
0.0001
0.0005
0.0005
0.00001
0.0001
Value
WHO
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
(a) T = Tetra, Pe = Penta, Hx = Hexa, Hp = Hepta, O = Octa, CDD = chlorinated dibenzo-/i-dioxin, CDF = chlorinated
dibenzofuran, CB = chlorinated biphenyl
ampoule each will be analyzed by AXYS by 1613B and 1668A. Three ampoules will be
archived. AXYS will repeat this procedure for all nine soil samples. One soil will be
extracted for the pre-demonstration phase and eight soils will be extracted for the
demonstration phase.
Task 3: Spiked Extract Preparation. AXYS will provide spiked extracts prepared in
the same solvent (toluene) as the soil extracts in Task 2. AXYS will prepare four sets of
spiked extracts. The details for these samples are described in Table 2. Spike #0 will be
-------
prepared for the pre-demonstration phase (by -March 23), while Spike #1, 2, and 3 will
be prepared for the demonstration phase (by April 21).
Table 2. Summary of spiked extracts
Spike #
0
1
2
3
No. of
ampoules
11
60
35
35
Sample
Description
100 pg/mL
TCDD
0.5 pg/mL
TCDD
100 pg/mL
TCDD and
1,000 pg/mL
all 12 WHO
PCBs
10,000 pg/mL
all 12 WHO
PCBs
No. to be
analyzed by
AXYS using
1613B and
1668A
1,1
7
4
4
No. to be
shipped by
AXYS
according to
Battelle-
provided
address(es)
6
42
24
24
No. to be
archived
by
AXYS
3
11
7
7
Task 4: Technical Support/Project Management. AXYS will provide written text
describing the extraction, clean-up, analysis, quality control checks, data reporting, data
review, and data packaging procedures for the project demonstration plan. AXYS will
also participate in project conference calls as requested. AXYS will also host an on-site
audit while the demonstration samples are being analyzed. Battelle anticipates that this
audit will occur in mid-May, around the time when the first batch of data are being
finalized and reported.
Project Requirements
The following are technical requirements for this project that AXYS is expected to
follow. Any deviation from these requirements should be discussed in advance with
Battelle's project manager.
• AXYS's standard electronic data deliverable (EDD) will be delivered to Battelle's
project manager by email, at no additional charge, by the due date committed by
AXYS in Table 3. A complete, auditable data package is also required for each
sample. The paginated data package will be delivered to the Battelle project
manager by hard copy and CD within 5 days of the EDD being delivered. The
narrative for the data package will include the sample extraction weight for each
sample.
-------
• Final reporting deliverable turn around time for the pre-demonstration samples
will be 21 days. Data packages for the demonstration samples will be delivered to
Battelle according to the schedule in Table 3 (at 1-week intervals starting with
Week 4 of the project). Final reporting deliverable turn around time for the
demonstration samples will be no more than 90 days from receipt of samples.
Table 3. AXYS Anticipated Reporting Schedule
Date3
Mar-22
Apr-7
Apr- 19
Apr-21
Apr-26
May-03
May- 10
May- 17
May-24
May-31
Jun-07
Jim- 14
Jun-21
Jun-28
Jul-05
Jul-12
Jul-19
Start
D/F
Start
WHO PCB
Report
D/F
Report
WHO
PCB
One soil extract and one spiked extract prepared and distributed to
participants
Pre-demo results reported for D/F and PCBs for 13 soils, one soil
extract, one spiked extract
Group 1 20 samples
group 1 20 samples
Soil and spiked extracts distributed to location specified by Battelle
Group2 20 samples
Group3 20 samples
Group4 20 samples
GroupS 20 samples
Group6 20 samples
Group7 20 samples
GroupS 20 samples
Group9 20 samples
group 10 20 samples
group2 20 samples
group3 20 samples
group4 20 samples
groupS 20 samples
group6 20 samples
group7 20 samples
groupS 20 samples
group9 20 samples
group 10 20 samples
group 1
group2
group3
group4
groupS
group6
group7
groupS
group9
group 10
group 1
group2
group3
group4
groupS
group6
group7
groupS
group9
group 10
Date may shift, depending on when samples are received at AXYS.
• Dean Stark extraction will be used for the dioxin/furan analysis. Regular soxhlet
extraction will be used for the PCB analysis.
• Percent moisture measurements do not need to be performed.
• A six-point initial calibration, including a low level calibration point (at 0.2
ng/mL), will be performed for 1613B. The standard five-point calibration will be
performed for 1668A.
• All sample processing, clean-up, analysis, and reporting will follow EPA Method
1613B and 1668 A except for the method modifications defined in the AXYS's
standard operating procedures (SOPs).
• The data will be reported as both concentration (for each target analyte), total
concentration, TEQ (for each target analyte), and total TEQ. TEQ will be
calculated and reported two ways: using both the WHO TEFs (listed in Table 1)
and using both a value of zero and l/2 the detection limit when the analyte is not
detected. The extract samples are to be reported in pg/mL TEQ.
• The static mass resolution check will be demonstrated every 12 hours.
-------
The following will be standard with each analytical batch. Results for quality
control (QC) samples must meet method/SOP requirements.
o No more than 20 samples will be included in each batch.
o One procedural blank will be included in each batch.
o One demonstration sample, of AXYS's choosing, will be analyzed in
duplicate with each batch.
o Surrogates will be spiked in each sample, except for the soil and spiked
extracts that are prepared for the participants.
o A spiked matrix (OPR or SRM) will be analyzed and reported with each
batch.
o Daily calibration check will occur every 12 hours.
o A column carryover solvent blank will be run after each calibration
standard to demonstrate instrumental carry over rate. Sample
responses will be evaluated using the demonstrated instrumental
carryover information to ensure that results are not significantly affected
by carryover from a previously injected sample of higher concentration.
Where the data indicates this may have occurred the sample in question
will be re-injected.
The pre-demonstration samples will be analyzed completely blind by AXYS. For
the demonstration samples, Battelle will identify which sites the samples are
from, and AXYS can refer to the pre-demonstration results so that congener
patterns and approximate dilutions can be estimated. Battelle will also identify
which samples are anticipated to contain high levels (> 10,000 pg/g) so that
AXYS can take pre-cautionary measures to deal with these samples.
AXYS will report sample specific detection limits, as specified in their response
to the SITE demonstration questionnaire. AXYS will flag any low level detections
that are lower than the lowest calibration standard.
A carbon column cleanup step to isolate toxic PCBs will be performed and the
DB-1 column will be used as the primary column.
Secondary column confirmation for 2,3,7,8-TCDF will be performed for every
sample.
Analytical data generated by AXYS for this project will be backed-up on a
weekly basis. The data shall remain on the instruments (or, at a minimum, readily
accessible) until the reports for this project are finalized in December 2004.
All project files and supporting data will be archived for a minimum of 5 years.
-------
APPENDIX G
QUESTIONNAIRE FOR REFERENCE LABORATORY SELECTION
-------
Inquiry Regarding Participation in EPA SITE Demonstration Program
Statement of Work
The purpose of this inquiry is to identify laboratories that are interested in providing dioxin analyses in
support of the US Environmental Protection Agency (EPA) Superfund Innovative Technology Evaluation
(SITE) Program. In April 2004, developers of innovative dioxin monitoring and measurement
technologies will gather at a field demonstration site in Saginaw, MI to analyze approximately 200
dioxin-contaminated soil and sediment samples. A reference laboratory will be concurrently analyzing a
replicate split of the samples by Method 1613B for the 17 dioxin/furan congeners and by EPA Method
1668A for the 12 World Health Organization PCBs, with concentrations ranging from < 1 pg/g to >
10,000 pg/g. The data generated during the field demonstration will be used to prepare Innovative
Technology Evaluation Reports for each of the participating technologies. Each report will be a
comprehensive evaluation of the technology's ability to measure dioxin in contaminated soil and
sediment, including a comparison to the HRMS method results, so the selection of a laboratory to perform
these analyses is a critical step in the process.
If you are interested in being considered for selection, please answer the following questions and provide
your answers to Battelle Memorial Institute, who is the contractor that is conducting the demonstration for
EPA. Responses are to be submitted by email or fax no later than Tuesday, December 23, 2003 to Amy
Dindal, dindala@battelle.org, phone: 561-422-0113, fax: 561-258-0777.
1) List all dioxin and PCB methods that you routinely perform. State whether you are running
modifications of methods and note what the modifications are. List any certifications that you hold for
performing each analysis, as applicable. List the approximate number of analyses your lab performed by
each method in 2003. Also provide a quote on a per sample basis if you were to receive 15 soil samples
in one batch in February 2004 and 200 soil samples in one batch in March 2004 for analysis by those
methods.
EPA Method
Modifications
(Y/N - if Y explain on
separate sheet)
Certifications
(Y/N- if Y please
attach)
Number of
analyses in
2003
Quote for
215 sample
analyses
-------
2) List the make, model, age, and number of analytical instruments that would be available for use in this
study.
3) Describe how many samples will constitute a sample batch and the associated quality control samples
that will be analyzed with each batch.
4) Define the congeners and concentrations in the lowest calibration standard typically analyzed in your
lab.
5) Describe how you determine the method detection limit with and without background present. Be sure
to include the size of sample required and list the detection limits on a congener specific basis.
6) List the most recent EPA contracts you held for the analysis of dioxin in soil or sediment and how
many samples were analyzed for each contract.
-------
7) Will you accept high level samples (> 10,000 pg/g D/F; > 100 ppm PCB)? If so, does the quote
provided in the table include dilution/re-analysis or re-extraction with smaller amounts to accommodate
higher level samples, or will there be an additional fee?
8) Are you willing to have a representative of your laboratory serve on the project's advisory panel?
This would include participating in conference calls and reviewing documents (10-20 hours). Would there
be an additional charge for this service or would this be included as part of the project participation?
9) Describe your process for data review from the time the data is generated until it is reported to the
client (e.g., what percentage is reviewed by the analyst, QA officer, manager, etc.).
10) What would you need/want to know about a sample prior to analysis?
11) What is your current sample load for these methods? Could you complete the 15 samples received in
February within 21 days? Could you complete 200 samples received in March within 160 days of
receipt?
12) Are you willing to run three PE samples and host an onsite audit of your laboratory in January? Can
you provide the results and a data package for these samples within!4 days of receipt? Would you be
willing to do these analyses free of charge and, if not, what would be the cost per sample?
-------
13) Would you provide a representative data package for review prior to receiving the PE samples?
NAME OF PERSON COMPLETING THE FORM:
TITLE:
DATE:
-------
APPENDIX H
METHOD 1613B and METHOD 1668A TABLES
Note: All tables are reproduced verbatim from the methods and do not represent AXYS modifications.
-------
Method 1613
TABLE 3. CONCENTRATION OF STOCK AND SPIKING
SOLUTIONS
CONTAINING
CDDS/CDFS AND LABELED COMPOUNDS
CDD/CDF
2,3,7,8-TCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDD
OCDF
Labeled Labeled
Compound Stock Compound
Solution 1 Spiking Solution
(ng/mL) (ng/mL)
— —
— —
— —
— —
— —
— —
— —
— —
— —
— —
— —
— —
— —
PAR Stock
2 Solution 3
(ng/mL)
40
40
200
200
200
200
200
200
200
200
200
200
200
200
200
400
400
PAR Spiking
Solution 4
(ng/mL)
0.8
0.8
4
4
4
4
4
4
4
4
4
4
4
4
4
8
8
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
13C12-l,2,3,7,8-PeCDD
13C12-l,2,3,7,8-PeCDF
13C12-2,3,4,7,8-PeCDF
13C12-l,2,3,4,7,8-HxCDD
13C12-l,2,3,6,7,8-HxCDD
13C12-l,2,3,4,7,8-HxCDF
13C12-l,2,3,6,7,8-HxCDF
13C12-l,2,3,7,8,9-HxCDF
13C12-2,3,4,6,7,8-HxCDF
13C12-l,2,3,4,6,7,8-HpCDD
13C12-l,2,3,4,6,7,8-HpCDF
13C12-l,2,3,4,7,8,9-HpCDF
13C12-OCDD
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
October 1994
65
-------
Method 1613
TABLE 3. CONCENTRATION OF STOCK AND SPIKING SOLUTIONS CONTAINING
CDDS/CDFS AND LABELED COMPOUNDS
Labeled Labeled
Compound Stock Compound PAR Stock PAR Spiking
Solution 1 Spiking Solution 2 Solution 3 Solution 4
CDD/CDF (ng/mL) (ng/mL) (ng/mL) (ng/mL)
Concentration
(ng/mL)
Cleanup Standard5
37Cl4-2,3,7,8-TCDD 0.8
Internal Standards 6
13C12-1,2,3,4-TCDD 200
13C12-l,2,3,7,8,9-HxCDD 200
1 Section 7.10—prepared in nonane and diluted to prepare spiking solution.
2 Section 7.10.3—prepared in acetone from stock solution daily.
3 Section 7.9—prepared in nonane and diluted to prepare spiking solution.
4 Section 7.14—prepared in acetone from stock solution daily.
5 Section 7.11—prepared in nonane and added to extract prior to cleanup.
6 Section 7.12—prepared in nonane and added to the concentrated extract immediately prior
to injection into the GC (Section 14.2).
October 1994
-------
Method 1613
TABLE 4. CONCENTRATION OF CDDS/CDFS IN CALIBRATION
CALIBRATION VERIFICATION SOLUTIONS ' (section 15.3)
CDD/CDF
2,3,7,8-TCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDD
OCDF
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
13C12-l,2,3,7,8-PeCDD
13C12-PeCDF
13C12-2,3,4,7,8-PeCDF
13C12-l,2,3,4,7,8-HxCDD
13C12-l,2,3,6,7,8-HxCDD
13C12-l,2,3,4,7,8-HxCDF
13C12-l,2,3,6,7,8-HxCDF
13C12-l,2,3,7,8,9-HxCDF
13C12-l,2,3,4,6,7,8-HpCDD
13C12-l,2,3,4,6,7,8-HpCDF
13C12-l,2,3,4,7,8,9-HpCDF
13C12-OCDD
Cleanup Standard
37Cl4-2,3,7,8-TCDD
Internal Standards
13C12-1,2,3,4-TCDD
13C12-l,2,3,7,8,9-HxCDD
0.5
0.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
5.0
5.0
100
100
100
100
100
100
100
100
100
100
100
100
100
200
0.5
100
100
CS2
(ng/mL)
2
2
10
10
10
10
10
10
10
10
10
10
10
10
10
20
20
100
100
100
100
100
100
100
100
100
100
100
100
100
200
2
100
100
CSS
(ng/mL)
10
10
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
10
100
100
CS4
(ng/mL)
40
40
200
200
200
200
200
200
200
200
200
200
200
200
200
400
400
100
100
100
100
100
100
100
100
100
100
100
100
100
200
40
100
100
AND
CSS
(ng/mL)
200
200
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
2000
2000
100
100
100
100
100
100
100
100
100
100
100
100
100
200
200
100
100
October 1994
67
-------
Method 1613
TABLE 8. DESCRIPTORS, EXACT M/Z's, M/Z TYPES, AND ELEMENTAL
COMPOSITIONS OF THE CDDs AND CDFs
Descriptor Exact M/Z
M/Z Type Elemental Composition Substance'
392.9760
401.8559
403.8529
430.9729
445.7555
4 407.7818
409.7789
417.8253
419.8220
423.7766
425.7737
430.9729
435.8169
437.8140
479.7165
5 441.7428
442.9728
443.7399
457.7377
459.7348
469.7779
471.7750
513.6775
Lock
M+2
M+4
QC
M+4
M+2
M+4
M
M+2
M+2
M+4
Lock
M+2
M+4
M+4
M+2
Lock
M+4
M+2
M+4
M+2
M+4
M+4
r F
^9 M5
13c12H235c^37ciq
13cl2n235c\37c\ q
^9 ^17
C12 H2 35Q 37Q 0
C12 H 35C\ 37C1 O
C12 H 35Cl5 37C\ O
13C12H35CV 0
13C12H35CV7C10
c12 H 35c\ 37ci q
c12 H 35c\ 37c| q
^9 ^17
13c12H35cV7ciQ
13c12H35c^37c| q
C12 H 35C\ 37C\ O
C12 35C17 37C1 0
^10 ^17
C12 35C]s 37Q O
c12 35ci7 37ci q
c12 35c]e 37c^ q
13c1235ci737ciq
13C1235CL37C^ Q
C12 35C18 37C^ O
PFK
HxCDD 3
HxCDD 3
PFK
OCDPE
HpCDF
HpCDF
HpCDF3
HpCDF3
HpCDD
HpCDD
PFK
HpCDD 3
HpCDD 3
NCDPE
OCDF
PFK
OCDF
OCDD
OCDD
OCDD3
OCDD3
DCDPE
Nuclidic masses used:
H = 1.007825
O = 15.994915
C
35C1
= 12.00000
= 34.968853
13,
36,
2 TCDD = Tetrachlorodibenzo-p-dioxin
PeCDD = Pentachlorodibenzo-p-dioxin
HxCDD = Hexachlorodibenzo-p-dioxin
HpCDD = Heptachlorodibenzo-p-dioxin
OCDD = Octachlorodibenzo-p-dioxin
HxCDPE = Hexachlorodiphenyl ether
OCDPE = Octachlorodiphenyl ether
DCDPE = Decachlorodiphenyl ether
3 Labeled compound.
4There is only one m/z for37 CVZ.SJ.S.-TCDD (cleanup standard).
13C
37C1
TCDF
PeCDF =
HxCDF =
HpCDF =
OCDF =
HpCDPE =
NCDPE =
PFK
003355 F = 18.9984
965903
Tetrachlorodibenzofuran
Pentachlorodibenzofuran
Hexachlorodibenzofuran
Heptachlorodibenzofuran
Octachlorodibenzofuran
Heptachlorodiphenyl ether
Nonachlorodiphenyl ether
Perfluorokerosene
72
October 1994
-------
Method 1613
TABLE 9. THEORETICAL ION ABUNDANCE RATIOS AND QC LIMITS
Number of
Chlorine Atoms
42
5
6
63
7
74
8
M/Z's
Forming Ratio
M/(M+2)
(M+2)/(M+4)
(M+2)/(M+4)
M/(M+2)
(M+2)/(M+4)
M/(M+2)
(M+2)/(M+4)
Theoretical
Ratio
0.77
1.55
1.24
0.51
1.05
0.44
0.89
QC Limit l
Lower
0.65
1.32
1.05
0.43
0.88
0.37
0.76
Upper
0.89
1.78
1.43
0.59
1.20
0.51
1.02
1 QC limits represent ±15% windows around the theoretical ion abundance ratios.
2 Does not apply to37 014-2,3,7,8-1000 (cleanup standard).
3 Used for13 C12-HxCDF only.
4 Used for13 C12-HpCDF only.
October 1994 73
-------
Method 1668, Revision A
Table 3. Concentrations of native and labeled chlorinated biphenyls in stock solutions, spiking
solutions, and final extracts
CB congener
Solution concentrations
Stock
(Mg/mL)
Spiking
(ng/mL)
Extract
(ng/mL)
Native Toxics/LOC1
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
Native congener mix stock solutions2
MoCB thru TrCB
TeCB thru HpCB
OcCB thru DeCB
2.5
5.0
7.5
Labeled Toxics/LOC/window-defining3
1L
3L
4L
1.0
1.0
1.0
2.0
2.0
2.0
100
100
100
85
-------
Method 1668, Revision A
Table 6. QC acceptance crtiteria for chlorinated biphenyls in VER, IPR, OPR, and samples1
Congener
2-MoCB
4-MoCB
2,2'-DiCB
4,4'-DiCB
2,2'6-TrCB
3,4,4'-TrCB
2,2'6,6'TeCB
3,3',4,4'-TeCB
3,4,4',5-TeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB
2,3,4,4',5-PeCB
2,3',4,4',5-PeCB
2',3,4,4',5-PeCB
3,3',4,4',5-PeCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB5
2,3,3',4,4',5'-HxCB5
2,3',4,4',5,5'-HxCB
3,3',4,4',5,5'-HxCB
2,2',3,4',5,6,6'-HpCB
2,3,3',4,4',5,5'-HpCB
2,2',3,3',5,5',6,6'-OcCB
2,3,3',4,4',5,5',6-OcCB
2,2',3,3',4,4',5,5',6-NoCB
2,2',3,3,'4,5,5',6,6'-NoCB
DeCB
13C12-2-MoCB
13C12-4-MoCB
13C12-2,2'-DiCB
i3ci2-4,4'-DiCB
13C12-2,2',6-TrCB
13C12-3,4,4'-TrCB
13C12-2,2',6,6'-TeCB
13C12-3,3',4,4'-TCB
13C12-3,4,4',5-TeCB
IUPAC
number2
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
1L
3L
4L
15L
19L
37L
54L
77L
81L
Test cone
(ng/mL)3
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
100
100
100
100
100
100
100
VER4
(%)
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
IP
RSD (%)
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
50
50
50
50
50
50
50
50
50
R
X (%)
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
60-140
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
OPR
(%)
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
Labeled compound
recovery in samples
(%)
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
93
-------
Method 1668, Revision A
Congener
13C12-2,2',4,6,6'-PeCB
13C12-2,3,3',4,4'-PeCB
13C12-2,3,4,4',5-PeCB
13C12-2,3',4,4',5-PeCB
13C12-2',3,4,4',5-PeCB
13C12-3,3',4,4',5-PeCB
13C12-2,2',4,4',6,6'-HxCB
13C12-2,3,3',4,4',5 -HxCB5
13C12-2,3,3',4,4',5'-HxCB5
13C12-2,3',4,4',5,5'-HxCB
13C12-3,3',4,4',5,5'-HxCB
13C12-2,2',3,4',5,6,6'-HpCB
13C12-2',3,3',4,4',5,5'-HpCB
13C12-2,2',3,3',5,5',6,6'-OcCB
13C12-2,3,3',4,4',5,5',6-OcCB
13C12-2,2',3,3',4,4',5,5',6-NoCB
13C12-2,2',3,3',4,5,5',6,6'-NoCB
13C12-2,2',3,3',4,4',5,5',6,6'-DeCB
Cleanup standard
13C12-2,4,4'-TrCB
13C12-2,3,3',5,5'-PeCB
13C,,-2,2',3,3',5,5',6-HpCB
IUPAC
number2
104L
105L
114L
118L
123L
126L
155L
156L
157L
167L
169L
188L
189L
202L
205L
206L
208L
209L
28L
111L
178L
Test cone
(ng/mL)3
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
VER4
(%)
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
60-130
60-130
60-130
IP
RSD (%)
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
45
45
45
R
X (%)
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
35-135
45-120
45-120
45-120
OPR
(%)
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
30-140
40-125
40-125
40-125
Labeled compound
recovery in samples
(%)
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
25-150
30-135
30-135
30-135
1 . QC acceptance criteria for IPR, OPR, and samples based on a 20
2. Suffix "L" indicates labeled compound.
3. See Table 5.
4. Section 15.3.
5. PCBs 156 and 157 are tested as the sum of two concentrations
extract final volume
94
-------
Method 1668, Revision A
CB congener
15L
19L
37L
54L
77L
81L
104L
105L
114L
118L
123L
126L
155L
156L
157L
167L
169L
188L
189L
202L
205L
206L
208L
209L
Solution concentrations
Stock
(Mg/mL)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Spiking
(ng/mL)
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Extract
(ng/mL)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Labeled clean-up4
28L
111L
178L
1.0
1.0
1.0
2.0
2.0
2.0
100
100
100
Labeled injection internal5
9L
52L
101L
138L
194L
5.0
5.0
5.0
5.0
5.0
1000
1000
1000
1000
1000
100
100
100
100
100
86
-------
Method 1668, Revision A
Diluted combined 209 congener6
Standard
Native congeners
MoCB thru TrCB
TeCB thru HpCB
OcCB thru DeCB
Labeled Toxics/LOC/window-defming
Labeled Cleanup
Labeled Injection internal
Solution concentration (//g/mL)
Native
50
100
150
Labeled
100
100
100
1. Stock solution: Section 7.8.1; Spiking solution: Section 7.11
2. Section 7.8.1.2
3. Stock solution: Section 7.9.1; Spiking solution: Section 7.12
4. Stock solution: Section 7.9.2; Spiking solution: Section 7.13
5. Stock solution: Section 7.9.3; Spiking solution: Section 7.14
6. Section 7.10.2.2.2
87
-------
APPENDIX I
REFERENCE LABORATORY PROCEDURES FOR DATA REVIEW
-------
This document is the property of Axys Analytical Services Ltd and contains proprietary and confidential information.
It may not be reproduced or distributed without the written permission of the owner. Copyright 24 April 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Internal Quality Review of PCDD and PCDF
Data from USEPA Method 1613B
Area: QA/QC
SOP#:
Rev. No.:
Date:
Page:
SQA-018(QAQC-18)
3
28 May 2002
1 of 5.
Purpose
This document describes specific protocols for quality review of all PCDD/F results and data
packages from application of USEPA Method 1613B to environmental samples.
Scope
Axys SOP # QAQC-01 describes the general procedures for conducting a final quality review of
analytical data and should be consulted along with this document which contains complimentary
method specific details for review of USEPA 1613B results.
Requirement and Authority
This review must be completed for all data reports and data packages prior to release to clients.
The review is conducted by designated QA Chemists fully trained in application of Method 1613B
who report to the QA Manager. QA Chemists assess the acceptability of results and have the
authority to request corrective actions to bring any results not meeting specifications into
conformance. The QA Manager, or the designee, has final responsibility for ensuring that data
reviews are conducted properly and for final decisions on data acceptability.
Definitions of Terms Used
Terminology used in this document is consistent with that of Method 1613B. The following
additional terms are used:
PCDD - Polychlorinated dibenzo-p-dioxins
PCDF - Polychlorinated dibenzofurans
TCDD -Tetrachlorinated dibenzo-p-dioxin
TCDF - Tetrachlorinated dibenzofurans
Sample Specific Detection Limit (SDL) - detection limits calculated from an estimation of
instrumental noise converted to a final concentration in the same manner that target compound
peak responses are converted to final concentrations.
Procedures
Sample Receipt, Holding Times, Preservation
Sample Receipt forms must be completed fully including USEPA Sample Login Sheet DC-1
where required.
-------
This document is the property of Axys Analytical Services Ltd and contains proprietary and confidential information.
It may not be reproduced or distributed without the written permission of the owner. Copyright 24 April 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Internal Quality Review of PCDD and PCDF SOP#: SQA-018 (QAQC-18)
Data from USEPA Method 1613B Rev. No.: 3
Date: 28 May 2002
Area: QA/QC Page: 2 of 5.
Method 1613B requires that aqueous samples be stored at 0-4 °C and that solids/semi-solids/
oily/mixed phase/tissue samples be stored at <4 °C from time of collection until receipt at the
laboratory. Contract specific guidelines may also apply. USEPA contract guidelines require that
the client be informed if the shipping cooler temperature exceeds 10 °C upon receipt at
laboratory.
Method 1613B requires that aqueous samples be stored at 0-4 °C and solids/semi-solids/
oily/mixed phase/tissue samples be stored at <10 °C in the laboratory.
There are no demonstrated storage times for PCDD/F and Method 1613B defaults to a one-
year holding time.
Method 1613B specifies that if the PH of aqueous samples exceeds 9.0 they be adjusted to PH
7-9 with sulphuric acid and that any free chlorine be neutralized with 80 mg/l of sodium
thiosulfate. These procedures are to be done at the sampling stage. Some clients may choose
to waive this requirement or arrange for it to be done upon receipt at the lab. Note any
departures from the 1613B method protocol in the narrative.
Method Performance Documentation Inspection
- Annual Initial Performance and Recovery study results fall within limits in Method 1613B
Table 6.
- Annual Method Detection Limit study results fall below one-third the minimum levels in
Method 1613BTable2.
Laboratory Work-up Documentation Inspection
Consistency of client and laboratory sample identifiers on laboratory work-sheets with
those on instrumental run file listings, LIMS listings, sample receiving records, and client
supplied documentation, verified every sample.
Sample size, moisture and lipid content determinations - verified every sample.
- Amount, time, and analysts' initials documenting addition of labelled compounds, verified
every sample.
Correct extraction and clean-up procedures, verified every sample.
Instrumental Performance and Calibration Inspection
Mass resolution demonstration >10,000, verified at the beginning and end of 12 hour
periods
-------
This document is the property of Axys Analytical Services Ltd and contains proprietary and confidential information.
It may not be reproduced or distributed without the written permission of the owner. Copyright 24 April 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Internal Quality Review of PCDD and PCDF SOP#: SQA-018 (QAQC-18)
Data from USEPA Method 1613B Rev. No.: 3
Date: 28 May 2002
Area: QA/QC Page: 3 of 5.
Mass calibration software routine ensures <5 ppm mass drift, verified every 12 hours.
S:N > 10:1 for target PCDD/F in CS1 calibration standard, verified every initial calibration.
Retention time of 13C12-1234-TCDD > 25 minutes on DB-5 column and >15 minutes on
DB-225, verified every initial calibration.
First/last eluting target compounds as per Table 5 of Method 1613B within acquisition
window for window defining mixture run, verified with every initial calibration sequence and
at the beginning of every 12 hour period during which samples are analysed.
Isomer specificity test valley height <25 % as per Figure 6 and 7 of Method, every initial
calibration and at the beginning of every 12 hour period during which samples are
analysed.
Instrument carryover test <0.3 % for 2378-TCDD/F, verified every 12 hours.
Initial calibration RSD of response factors <20 % for all target compounds except 123789-
HxCDD, OCDF and <35 % for labelled standards, 123789-HxCDD, OCDF; all ion ratios
within the acceptance ranges in Table 9, verified every 30 days, as required to maintain
calibration verification specifications, or after instrumental changes (i.e. new GC column)
Calibration verification concentrations and ion ratios within acceptance limits in Method
1613B Table 6 and 9 respectively, S:N > 10:1 for all compounds, verified at the beginning
and end of 12 hour sample brackets.
Calibration verification SICP areas for labelled internal standards within 50-200 % of initial
calibration CSS standard run, verified every 12 hours.
Lock mass variation < ±20 % for all lock mass ions over the acquired RT range, verified
every injection.
Batch Quality Control Sample Inspection
Sample batch size maximum 20 samples, verified every batch.
Laboratory blank concentrations less than one-tenth the minimum levels in Method 1613B
Table 2, except for OCDD and OCDF which can be up to one-fifth the minimum levels in
Method 1613B Table 2; labelled compound recoveries within acceptance limits in Method
1613B Table 7, verified every blank.
OPR concentrations within acceptance ranges in Method 1613B Table 6, verified every
OPR.
QC samples and samples were processed identically in the same analysis batch- this to
ensure that any systematic errors in sample data do not go undetected
Sample Data Inspection
-------
This document is the property of Axys Analytical Services Ltd and contains proprietary and confidential information.
It may not be reproduced or distributed without the written permission of the owner. Copyright 24 April 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Internal Quality Review of PCDD and PCDF SOP#: SQA-018 (QAQC-18)
Data from USEPA Method 1613B Rev. No.: 3
Date: 28 May 2002
Area: QA/QC Page: 4 of 5.
Target compound responses within calibration range, verified every sample.
Labelled compound recoveries within Method 1613B limits in Table 7, verified every
sample.
Responses for target compounds detected meet all RT, ion ratio, and S:N identification
criteria specified in Method 1613B section 16.0, verified every sample.
Concentrations calculated in accordance with Method 1613B section 17.0 using correct
input data and scaling factors, verified very sample.
DB-5 column concentration of 2378-TCDF above the reporting limit is confirmed on a DB-
225 column, verified every sample.
Sample results are not affected by instrumental sample to sample carryover, verified every
sample.
- Absence of diphenylether interferences, verified every sample
General chromatography meets Axys quality standard, verified every sample.
General Documentation Inspection
Record keeping practices consistent with good laboratory protocols - (ink, revisions made
neatly and initialled and dated, no obscuring of original entries, no use of corrective fluid or
impermanent attachments).
- Sign-off on sample worksheets, instrument acquisition lists, instrument data reports, final
reports as per Axys Document SQA-019, 'General Documentation Policies'
Data package complete and assembled in accordance with Axys Document SAD-023, 'Data
Package Preparation'.
Electronic Data Submissions:
Electronic file submissions compiled in accordance with Axys Document SAD-023,
'Electronic Data Template Management'
hard copy printouts of electronic data file submissions initialled to document verification
against hard copy client reports
Remedial Actions
Quality Assurance Chemists must initiate remedial action to correct any deficiencies noted in
results during the final data review. These instructions are documented on the laboratory
worksheets used for the original sample analysis. They may include re-calculations, re-coding of
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It may not be reproduced or distributed without the written permission of the owner. Copyright 24 April 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Internal Quality Review of PCDD and PCDF SOP#: SQA-018 (QAQC-18)
Data from USEPA Method 1613B Rev. No.: 3
Date: 28 May 2002
Area: QA/QC Page: 5 of 5.
chromatograms, additional extract clean-up, instrumental re-injections or complete sample re-
analysis.
Reporting results which fall outside the specifications in Method 1613B and this document
requires the approval of the Quality Manager. Non-conformances to Method 1613B must be
documented in the data package narrative with an explanation of the cause and the impact on
final data interpretation.
References and Related Documents
1. USEPA Method 1613B
2. Axys Document SQA-001 Final Data Checking
3. Axys Document SCO-004 Determination of Carryover
4. Axys Document SCO-009 Hand Calculation of Relative Response Factors,
Concentrations, and Detection Limits of Target Analytes
5. Axys Document SCO-011 Code a Chromatogram
6. Axys Document SAD-025 'Electronic Data Template Management'
7. Axys Document SQA-019 'General Documentation Policies'
8. Axys Document SAD-023 Data Package Preparation
Approval:
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This document is the property of Axys Analytical Services Ltd and contains proprietary and confidential information.
It may not be reproduced or distributed without the written permission of the owner. Copyright 01 May 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title:
Area:
Final Data Checking
QA/QC
SOP#:
Rev. No.:
Date:
Page:
QAQC-1
4
01 May 2001
1 of 4
Purpose:
To review analytical data prior to release to ensure reported results satisfy all quality and contract
specific criteria
Scope:
Analytical raw data are converted to sample concentrations and final data reports by a team of
data interpretation and report preparation specialists in the Data Production Group. In addition to
the data quality checks performed during that process, a final data quality review is conducted by
the Quality Assurance Group prior to release of results. This SOP describes the general
procedures for conducting the final data review.
Requirement and Authority
All final data reports are subjected to a comprehensive review prior to release and the results of
the review are documented. The review is conducted by designated QA Chemists reporting to the
QA Manager. QA Chemists judge the acceptability of results and are responsible for ensuring the
accuracy and completeness of work. They have the authority to request confirmation or corrective
action such as additional documentation, re-injections, or analysis repeats. QA chemists provide
the Supervisors, Lab Manager, Chemists, and QA Manager with information and advice regarding
work quality, corrective action and special job requirements. Feedback is provided on the quality of
work-up, instrumental analysis, interpretation, coding, calculation, reporting, tracking, interlab
communication, and documentation. The QA Manager, or the designee, has final responsibility for
ensuring that data reviews are conducted properly and for the accuracy of final reports.
Definitions:
Surrogate standard - isotopically labeled compound added at the beginning of the analysis for
calculation of targets by isotope dilution
LIMS - Laboratory Information Management System
Instrument Carryover - elevation of instrument response for a target compound due to residual
contamination of the injection system from a proceeding sample
General Procedure and Corrective Actions:
1. Review raw data, including calculations and chromatograms, and final analytical reports to
ensure that all results are correctly interpreted and accurately coded, calculated, and
reported. The data review must cover the following as a minimum:
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It may not be reproduced or distributed without the written permission of the owner. Copyright 01 May 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Final Data Checking SOP #: QAQC-1
Area: QA/QC Rev. No.: 4
Date: 01 May 2001
Page: 2 of 4
a) header information on final reports must be confirmed, especially sample identification
which must match that on client custody forms, Axys receiving forms, LIMS entry,
sample work-up sheets, and instrument run file listings;
b) the demonstration of instrument carryover (instrument blank following calibration
standard) meets the required standard;
c) the demonstration of instrument sensitivity, linearity, and stability meets the required
standard;
d) sample to sample carryover has not affected results;
e) chromatography quality and GC resolution meet standard;
f) analyte responses are within calibration range;
g) surrogate recoveries meet method, contract and internal control specifications;
h) correct addition of quantification standards;
i) correct completion of worksheet;
j) absence of sample contamination by inspection of laboratory and instrumental blanks,
replicate analysis and injection data;
k) calibration data meet specifications;
I) correct quantification procedure used - manual check required;
m) accuracy of all coding, transcription, and typing;
n) blanks, duplicate tests and reference spikes/CRMs meet quality specifications;
o) detection limit criteria are met;
p) data pass reasonableness test;
q) documentation is complete, accurate and documentation practices are consistent with
good laboratory protocols (all records in permanent ink form, revisions made by a line
through original entry, new entry initialled; no use of correction fluid or 'sticky' labels);
r) data packages are complete and contain all contract required supporting quality
documentation such as method detection limit and initial performance/recovery studies;
s) inspection for systematic errors that could be undetected by QC sample results - verify
that samples and QC samples have been processed in an identical manner and within
the same analysis batch;
t) electronic data file submissions compiled in accordance with Axys SOP# ADMIN-24
'Electronic Data Template Management' and hardcopy printouts of electronic data file
submissions initialled to document verification against hard copy client reports;
u) any departures from sample acceptance criteria (containers, storage, preservation,
labelling, handling) have been authorized and are properly documented in the narrative
and/or data reports.
The frequency of checks, general quality criteria, and recommended corrective actions are
listed in Specification Chart/04 attached to this SOP.
Specific quality criteria for particular analyses are listed in the Method Specification Table
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It may not be reproduced or distributed without the written permission of the owner. Copyright 01 May 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Final Data Checking SOP #: QAQC-1
Area: QA/QC Rev. No.: 4
Date: 01 May 2001
Page: 3 of 4
attached to each method in the 'Axys Analytical Methods Manual' and in analyte specific
Quality Review SOPs.
Detailed procedures covering the complete analytical procedure from sample receipt to final
report are documented in the 'Axys Analytical Standard Operating Procedures Manual' and
should be consulted as necessary.
2. Document the acceptability of results by initialling the QA/QC sections of the Batch Summary
List. Clearly flag any problem data and alert the QA Manager. If it is necessary to report
results for which all QA/QC parameters have not been satisfied, clearly flag the data and
specify the parameters affected. Describe the probable impact on the results.
3. Initiate corrective action by completing the 'Additional Work' section of the Sample
Worksheet. Inform the Production Manager, who will up-date the sample tracking records,
and the supervisor of the appropriate group, who will assign the work to an analyst.
4. Review results from sub-contracting laboratories. Ensure that the sub-contracting lab is
authorized to perform the work as listed on the "List of Approved Contracting Laboratories".
Verify that all requested QC information has been supplied and meets accepted standards.
Inform the QA Manager of any concerns regarding quality of sub-contracted work.
5. Prepare Letters of Transmittal for final data reports to present results completely, clearly, and
unambiguously. Avoid the use of acronyms that may mislead or confuse the client. Explain
any shortfalls in data quality or completeness, and recommend further action where required.
6. Document any client questions or complaints regarding data quality on Client Complaint
Form (Client Services/03), according to standard operating procedure ADMIN-11. Provide a
copy to the QA Manager and the contract administration file.
7. Routine requests for additional information or for help in understanding results may be
handled directly by the QA Chemist who authorized release of the final data report.
References and Related Documents
1. Axys Analytical QA/QC Policies and Procedures Manual
2. Axys Analytical Standard Operating Procedures Manual
3. Axys Analytical Methods Manual
4. SOP CODE-1 Visual Inspection of Chromatogram
5. SOP CODE-4 Determination of Carryover
6. SOP CODE-9 Hand Calculation of Relative Response Factors, Concentrations, and
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It may not be reproduced or distributed without the written permission of the owner. Copyright 01 May 2001.
Axys Analytical Services Ltd, PO Box 2219, Sidney, BC, Canada, V8L 3S8. Tel. (250) 655-5800 FAX (250) 655-5811
Axys Analytical Services Ltd.
Standard Operating Procedure
Title: Final Data Checking SOP #: QAQC-1
Area: QA/QC Rev. No.: 4
Date: 01 May 2001
Page: 4 of 4
Detection Limits of Target Analytes
7. SOP CODE-11 Code a Chromatogram
8. SOP ADMIN-23 Data Package Preparation
Attachments
1. Specification Chart 704.
Approval:
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APPENDIX J
DATA VALIDATION CHECKLISTS FOR REFERENCE LABORATORY
DATA REVIEW
-------
Dioxin/Furan Data Review Checklist
Data Package:
Sample ID Nos:
Initials/Date
Activity
Were storage and holding times met?
• Held at <-1 OC after receipt
• Analyzed within 1 yr of receipt
Track chain-of-custody from sample receipt to analytical
injection.
Confirm correct concentration of standards spiked into
samples.
6-Point Initial Calibration meets criteria of <20% RSD for
items quantified by isotope dilution, <35% for items quantified
by internal standard.
Column performance checked at beginning of each 12 hr
period.
• <25% valley from 2,3,7,8-TCDD (2,3,7,8-TCDF for
confirms) and closest eluters.
• 1 ,2,3,4 TCDD13Ci2 elutes >25 minutes on DB5
and> 15 minutes on DB225
10K resolution documented every 12 hours.
Continuing calibration every 12 hours.
• Results within Demonstration Plan Table 9-2
CAL/VER criteria
• S:N of concal peaks at least 10:1
Method blanks with every 20 test samples
• less than 0.5 pg/sample fortetras, 1 pg/sample for
penta-hepta, 5 pg/sample for octas or 20x lower
than the sample concentration.
OPR with every 20 test samples
• Within Demonstration Plan Table 9-2 for OPR
Duplicate with every 20 test samples
• <20% difference between duplicates for total TEQ.
Note in comments if any individual congeners >1 0 x
DL are >20% difference.
All 2,3,7,8-isomers are accounted for: concentration reported,
flagged for reason not used, or obviously not present.
Ether masses were monitored and there are no ether
contributions to furans.
Lock masses were monitored and there were no variations
which could affect reported results.
Internal standard recoveries 40-120% except OCDDIJCi2at
25-120%?
Review 10% data transfer into final report spreadsheets.
If confirmations, check separation of column, calibration,
COC, and transfer of 10% of confirmation data to final
spreadsheet.
Review report narrative for accurately reflecting raw data and
resulting spreadsheets.
Were TEQs reported two ways?
• All ND and K values assigned zero
• ND = 1/2 the detect limit, K = EMPC concentration
Have data flags been applied appropriately?
• J for values between the detection limit and the low
calibration level
• K for estimated maximum possible concentration
• TEQs with 1 0% or greater contribution from J or K
flagged data also flagged J or K as appropriate
Y
N
NA
Comments
-------
PCB Data Review Checklist
Data Package:
Sample ID Nos:
Initials/Date
Activity
Were storage and holding times met?
• Held at <-1 OC after receipt
• Analyzed within 1 yr of receipt
Track chain-of-custody from sample receipt to analytical
injection.
Confirm correct concentration of standards spiked into
samples.
5-Point Initial Calibration meets criteria of <20% RSD for
all compounds and CS-1 has S:N of 10:1
Column performance checked at beginning of each 12 hr
period.
• For SPB Octyl: 34 resolved from 23, 187
resolved from 182, 156 and 157 coelute.
• For SPB Octyl: PCB 209 elutes >55 minutes
• ForDBI: PCB elutes > 55 minutes, PCB
156/157 resolution < 20% valley high.
10K resolution documented every 12 hours.
Continuing calibration every 12 hours.
• Results within Demonstration Plan Table 9-4
CAL/VER criteria
• S:N of concal peaks at least 10:1
Method blanks with every 20 test samples
• PCB 77, 81,114,123,126, and 169 < 2
pg/congener
• PCB 156, 157, 167, 189 < 10 pg/congener
• All other PCB < 50 pg/congener
• Total PCB < 200 pg or 20x below sample
concentration.
OPR with every 20 test samples
• Within Demonstration Plan Table 9-4 for OPR
Duplicate with every 20 test samples
• <20% difference between duplicates for total
TEQ. Note in comments if any each congener
>10 x DL are > 20% difference.
Each 12 WHO PCB accounted for: concentration
reported, flagged for reason not used, or obviously not
present.
Lock masses were monitored and there were no
variations which could affect reported results.
Internal standard recoveries within Demonstration Plan
Table 9-4 Labeled Compound Recovery criteria
Review 10% data transfer into final report spreadsheets.
Review report narrative for accurately reflecting raw data
and resulting spreadsheets.
Were TEQs reported two ways?
• All ND and K values assigned zero
• ND = 1/2 the detect limit, K = EMPC concentration
Have data flags been applied appropriately?
• J for values between the detection limit and the
low calibration level
• K for estimated maximum possible concentration
• TEQs with 1 0% or greater contribution from J or
K flagged data also flagged J or K as appropriate
Y
N
NA
Comments
-------
APPENDIX K
REFERENCE LABORATORY PROCEDURES FOR DATA PACKAGE
PREPARATION
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This document is the property of AXYS Analytical Services Ltd. and contains proprietary and confidential information. It may not
be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation SOP #: SAD-023
Area: Administration Rev. No.: 4
Date: 18-Mar-2004
Page: 1 of 8
Purpose:
To assemble and submit to a client a detailed data package that includes all final reports,
raw data and supporting documentation such that an independent data reviewer can validate all
final results through the analytical process.
Scope:
The following procedures apply to all samples for which a data package style of report has
been requested, unless the client has specified an alternate format. Data packages may be in a
paper format or in CD format. Data packages are assembled and prepared for shipment by a Data
Packager.
Abbreviations and Definitions:
Data package (DP) - A compilation of final data reports, raw data, and supporting documentation
for a pre-defined set of samples.
Instrument Run List- A sequential listing of instrumental run files including sample IDs, data
filenames and acquisition times.
Instrument Run Table - A subset of the instrumental run list, listing sample data file names
directly below the calibration data used to quantify them.
Analysis Worksheets - AXYS forms containing the information on sample extraction and
cleanup, one form per sample and QC sample. Other worksheets with data may be
included as specified by the Project Chemist.
Workgroup (WG) - A laboratory analysis batch including QC samples. The composition of a
workgroup is listed on a Batch List
PC - Project Chemist
Procedures:
Paper Data Packages
1. Refer to the Project Notes to determine which samples are to be included in the data package.
Depending upon the client request, this may be by AXYS log-in ("L" number), by workgroup or
by the client's Sample Delivery Group (SDG). Determine what type of data package is required
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This document is the property of AXYS Analytical Services Ltd. and contains proprietary and confidential information. It may not
be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation SOP #: SAD-023
Area: Administration Rev. No.: 4
Date: 18-Mar-2004
Page: 2 of 8
(hard copy or CD ROM).
2. To prepare a paper data package, assemble all the appropriate records for the data package.
Use the originals of the analysis worksheets. For all other records use photocopies or copies of
scanned originals. For information recorded in bound logbooks include page photocopies. In
some cases the client may request originals of all records or may specify which records are to
be included in the data package. Refer to the Project Notes for details of required records.
Each data package must be 'self-contained' and include all records relevant to the samples
included in the data package.
3. Ensure all photocopies are legible and are an accurate reproduction of the original copy. Poor
copies of the custody seals should be excluded from the data package.
4. Where documents relate to more than one data package and original records have been
included, place the originals in the data package with the lowest AXYS log-in number. Place a
photocopy in all other data packages .
6. To assemble the data package, compile the records listed below. Note that it may be
convenient to first compile the sample data, QC sample date, instrumental QC and raw data
prior to assembling the initial records, provided the records are ordered in the data package in
sequence below.
i. Hardcover title page
ii. Title page
iii. Narrative
iv. Method Summary (as available)
v. GC Column Temperature Program
vi. Cover page(s) with signature line
vii. AXYS Correlation Table (correlates AXYS ID with client ID)
viii. Individual Sample Receiving Forms (use copies unless Project Notes specify
otherwise)
• Client Chain of Custody
• FedEx waybill
• AXYS Sample Receiving Record
• Custody Seals
• Field Notes (documentation about the samples taken in the field-if available)
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be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation SOP #: SAD-023
Area: Administration Rev. No.: 4
Date: 18-Mar-2004
Page: 3 of 8
• AXYS Login Chain of Custody (do not include Sample Acknowledgement Form
• Selective Documentation Between AXYS and Client- only as requested. Documents
for inclusion will be stamped with 'Data Package'.
ix. Sample Preparation Records (if applicable, order numerically by AXYS ID number)
x. Sample Pretreatment Records (if applicable, order numerically by AXYS ID number)
xi. Analysis Workup Sheets (order numerically by workgroup and AXYS ID number)
xii. Sample Data (order numerically by AXYS ID number and then by analysis (ordered
by date for multiple analyses, including dilutions) - refer to the example at the end of
this document for guidance.
NOTE: in a paper data package, place coloured sheets between samples, so
complete reports for a given sample are visible at a glance
xiii. QC Sample Data including the following:
• Procedural Blank (order numerically by Workgroup Number)
• OPRs/SPMs (order numerically by WG number)
• MS/MSDs (order numerically by WG number)
• CRMs (order numerically by WG number)
xiv. Instrument QC (order chronologically by Analysis Run Date, using the date and time
of the starting Run List or Injection Log to order each set). Start with run list for each
"set" or bundle of related QC. Each set or bundle may include:
• Run List
• Mass resolutions, opening and closing (HRMS PCBs and PCDD/F only, opening
mass resolution only for pesticides)
• Run Table
• Linearity ( Form 3s, if present in run)
• Calibration Verification. Forms 4 and 6, opening only unless closing required.
• Isomer Specificity (Form 5 (Dx) or single page from PCB chromatogram)
• Full Calibration form 3A (PCB)
• Client Standard (if applicable)
xv. Raw Data
• Sample Raw Data (order in the same sequence as the Sample Data Reports above,
usually by AXYS sample ID). Bind the chromatograms with an elastic band, but do
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be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation SOP #: SAD-023
Area: Administration Rev. No.: 4
Date: 18-Mar-2004
Page: 4 of 8
not bind in the final paper copy.
• Laboratory blank chromatograms (order in the same sequence as Blank reports).
• OPR/SPM (or MS/MSD, CRM) chromatograms (order in same sequence as data
reports).
• Instrumental chromatograms (order chronologically by Analysis Run Date and Time-
to mirror the order of the Instrument QC hardcopy reports
-Run List
-Linearity, with OpusQuan RRF summary sheet
-Calibration Verification
-Window
-Client Standards (if applicable)
5. Prepare a title page, a cover page and a correlation table.
6. Submit the data package to the Project Chemist for review and for a narrative. Track the
progress of the data package on the Data package and Shipment Log (FSA-023).
7. When the completed, reviewed data package has been returned, make any necessary
corrections as directed.
8. Stamp each page with a sequential number.
9. If additional copies of the data package are required, make the appropriate number of
photocopies.
10. Bind the hardcopy data package(s) using AXYS cover pages. Unless otherwise instructed by
the client (refer to Project Notes).
11. Scan the data package as AXYS' backup copy.
CD Data Packages
1. To prepare a CD from a paper data package, reorder the paper data package so that the
chromatogram for each sample appears after the sample analysis report. Otherwise, the
sample data are ordered numerically by AXYS ID, as described for the paper data package.
Scan the Sample Data, QC Data, Instrument QC and Raw Data (items 6.xii to 6.xv above).
-------
This document is the property of AXYS Analytical Services Ltd. and contains proprietary and confidential information. It may not
be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation SOP #: SAD-023
Area: Administration Rev. No.: 4
Date: 18-Mar-2004
Page: 5 of 8
2. If a paper data package has not been prepared, assemble the required documents, (as
specified in 6.N to 6.xv above) either as hardcopy or as PDF files. Order the files such that the
chromatogram for each sample appears after the sample analysis report. Scan the documents
as appropriate and import the chromatograms. All scanned data is located AXYS' network at
G:/PDF_Data.
3. Create links to the Sample Data, Lab Blanks, QC Samples (a separate link for each type i.e.
OPR, MS/MSD) and Instrument QC. Within each section, bookmark each sample as it
appears in the data package. Notify the Project Chemist that the Data Package is ready for
the narrative and checking.
4. When the checked data package is returned with the narrative, scan the narrative and client
information (steps 6.N to 6.xi above) and create a bookmark to the file.
5. Arrange for another Data Packager who has not been involved in the preparation to check the
scanned data package.
6. Burn the final checked data package onto a non-rewriteable CD.
Quality Measures and Remedial Actions
1. Data Packages are prepared and stored in a secure area that is locked during non-working
hours.
2. A secure back-up copy of all data in electronic medium is stored as described in AXYS SOP
SAD-024 "Data Management and Handling".
3. Completeness and consistency of assembled data packages are verified by the Project
Chemist who writes and a narrative.
4. Procedural deficiencies detected in assembled data packages are returned to the Data
Packager for correction.
5. A re-submission of an entire data package must contain a revised narrative documenting the
reasons for re-submission.
6. Minor corrections to a data package may be resubmitted with a covering letter describing
-------
This document is the property of AXYS Analytical Services Ltd. and contains proprietary and confidential information. It may not
be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation SOP #: SAD-023
Area: Administration Rev. No.: 4
Date: 18-Mar-2004
Page: 6 of 8
the correction(s). If the original data package was a paper copy, the relevant revised pages
accompany the letter. These pages are reported with a revision date. If the data package
was originally issued as a CD, then the entire data package with revisions is sent on a CD
with the covering letter.
7. Client complaints and/or requests for further explanations are handled as per AXYS SOP#
SAD-011 "Settlement of Client Complaints".
Transmission Procedures:
1. Prepare four copies of a covering letter to the client indicating the contents of the package.
A Project Chemist must sign the original covering letter.
2. Package up the data packages(s) in an appropriate sized box or envelope.
3. Place the original letter in the box and tape the second copy on the outside of the box. Do a
check to verify that the two letters accompanying the data package are the same. Seal the
box for shipping.
4. Give the package to the Receptionist for shipping.
5. Complete the shipping information on the Data Package and Shipment Log (FSA-023).
6. Prior to shipment, the Receptionist verifies the shipping address on the letter with previous
shipping information for the client and reports any discrepancies to a Data Packager. If
necessary, the Data Packager prepares a new covering letter. The Receptionist places a
waybill on the box and checks that the name and address on the waybill are the same as on
the letter taped to the box. When the waybill is in place, the letter is removed from the
outside of the box.
7. The Receptionist tracks the package with the courier and maintains files of the shipping
information.
8. The Receptionist sends original copies of shipping records to the accounting department.
9. File the third copy of the covering letter in the Data Package binder and give the fourth copy
to the Accounts Receivable Technician.
-------
This document is the property of AXYS Analytical Services Ltd. and contains proprietary and confidential information. It may not
be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation
Area: Administration
SOP #: SAD-023
Rev. No.: 4
Date: 18-Mar-2004
Page: 7 of 8
References
SAD-024 Data Management and Handling
FSA-023 Data Package And Shipment Log
SAD-011 Settlement of Client Complaints
Approval:
Coreen Hamilton, Technical Director
Date
Dale Hoover, Quality Manager
Date
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This document is the property of AXYS Analytical Services Ltd. and contains proprietary and confidential information. It may not
be reproduced or distributed without the written permission of the owner. Copyright 18-Mar- 2004.
AXYS Analytical Services Ltd., PO Box 2219, Sidney, BC, Canada V8L 3S8 Tel (250) 655-5800 FAX (250) 655-5811
AXYS Analytical Services Ltd.
Standard Operating Procedure
Title: Data Package Preparation SOP #: SAD-023
Area: Administration Rev. No.: 4
Date: 18-Mar-2004
Page: 8 of 8
Example of Sequence for Sample Data Reports, Method EPA 1668A - ordered by
instrumental analysis date
• Initial Analysis Data (target reports, surrogate reports). Forms 1A,2
• Extra Work Analysis Data: i.e. dilution, recolumn, reinjection (target reports,
surrogate reports). Forms 1A, 2 (organize multiple dilutions by run date)
• Client Standards, if applicable. Form 1A
• Homologue Totals - if required or not on initial analysis report
• TEQ - if required or not on initial analysis report. Form 1A
• Aroclors - if required
• If there is Lipid weight basis analysis results, organize similarly (do not worry
about surrogate pages being with reports)- For a paper data package all wet wt
basis data is organized by L number, Samples -1 through -x and all lipid wt data
is in a separate section also organized by L number samples -1 through -x. In
the paper DP this is separated by a coloured cover page. ON A CD DP, the lipid
data comes immediately after the chromatogram for the wet wt data for a given L
number.
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APPENDIX L
WORK PLAN FOR REFERENCE LABORATORY IN-PROCESS AUDIT
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AUDIT PLAN
SITE Demonstration of Monitoring and Measurement Technologies
for Dioxin and Dioxin-like Compounds in Soil and Sediment
Subtask: In-Process Laboratory Audit
Client: EPA NERL, SITE Program (FEATS contract)
Project Manager: Amy Dindal (Battelle Columbus Laboratories)
Assessee Organizations: AXYS Analytical Services, LTD
Location: 2045 Mills Rd.
PO Box 2219
Sidney, BC
Canada V8L3S8
Senior Official/Title: Ms. Laurie Phillips
QA Manager: Mr. Dale Hoover
Authorizing Entity: US EPA
Review and Concurrence by Amy Dindal (signature on file)
Assessment Team: Ms. Rosanna Buhl and Mr. Mark Misita
Lead Assessor: Ms. Rosanna Buhl
Anticipated Dates of Assessment: May 26, 2004
Authority to Conduct Assessment:
The US EPA Task Order Manager (TOM) has authorized Battelle to conduct an independent
assessment of AXYS, the reference laboratory for the Technologies for Monitoring and
Measurement of Dioxin in Soil and Sediment demonstration.
Criteria for Assessment: Compliance with the following documents:
• MLA-017 Rev 09 (To be completed) (Analytical method for the determination ofdioxins and
furans by EPA Method 1613B)
• MLA-010 Rev 5 24-Sept-2003 (Analytical method for the determination of 209 PCB
congeners by EPA Method 1668A)
• Quality Assurance/Quality Control (QA/QC) Policies and Procedures Manual Rev 8 (17-Jul-
2003)
• Second Draft Demonstration Plan (April 2004)
Purpose and Scope of Assessment:
The purpose of this audit is to verify the compliance of AXYS Analytical Services, LTD vs. their
SOPs, QA/QC Manual, the Project Notes directives, and the project Demonstration/Quality
Assurance Plan. The scope will specifically include a review of dioxin and PCB congener sample
processing, analysis, and data reduction; sample receipt, handling, and tracking; supporting
Revision 1 May 24, 2004
Page 1 of4
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AUDIT PLAN
SITE Demonstration of Monitoring and Measurement Technologies
for Dioxin and Dioxin-like Compounds in Soil and Sediment
laboratory systems; and responses to the report generated as a result of the February 11, 2004
audit.
Issues Selected:
• Sample and standards tracking
• Sample and standards preparation
• Instrument calibration
• Sample analysis
• Sample integration and data reduction
• Acceptability of quality control data
Personnel to Be Interviewed:
• Laboratory Point of Contact
• Analyst
• Sample prep personnel
• Data management personnel
• Sample custodian
Documents to be Reviewed:
0 SOP MLA 010 (Analytical method for the determination of 209 PCB congeners by EPA Method
1668A)
El Quality Assurance/Quality Control (QA/QC) Policies and Procedures Manual Rev 8 (17-Jul-
2003)
bd Demonstration and Quality Assurance Project Plan (April 2004)
CH MLA-017 (Analytical method for the determination ofdioxins andfurans by EPA Method 1613B)
D Current Organization Chart (if revised after QDO-005 Rev 24 (23 January 2004)
CH Q-Pulse Project Notes
D Results of current sample analysis
Anticipated Date for Receipt of Records:
The auditors have three of the seven documents needed to prepare for the audit. The auditors
have requested the unchecked documents above be supplied by May 19th.
Records to be Reviewed:
• Data generated to date
• Sample receipt records and holding location/conditions
• Standard (spiking and calibration solution) receipt records and certificates of analysis
• Standard preparation records
• Support equipment records (Balances, refrigerators)
• Sample preparation and spiking records
• Preparation of instrument standards
• Preparation of spiking solutions
• Instrument calibration and maintenance records
• Instrument sample run logs
Revision 1 May 24, 2004
Page 2 of4
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AUDIT PLAN
SITE Demonstration of Monitoring and Measurement Technologies
for Dioxin and Dioxin-like Compounds in Soil and Sediment
• Instrument set-up conditions, temperature programs, calibration and acquisition methods
• Sample chromatograms
• Instrument reports
• Quantification techniques
• QC sample results
• Data management
• Data validation and verification
Anticipated Opening Meeting: May 26, 2004 (8:00 AM)
Opening Meeting Participants:
• Audit team
• Laboratory Point of Contact
• Analyst
• Laboratory QA Officer
• Others at the discretion of the laboratory
Anticipated Assessment Schedule:
May 26, 2004
(times are
approximate)
8AM
8:30 AM
9:00
9:30
10:00
11:00
Noon
1:00 pm
2:00 PM
3:OOPM
3:45 PM
4: 15PM
5PM
Quality Systems Audit
(Rosanna Buhl)
Technical System Audit
(Mark Misita)
Opening Meeting
Sample Receiving & Storage
Dioxin/Furan Sample Preparation
Support equipment calibration and
maintenance
Calibration standard tracking
Spiking solution tracking
Reagent and solvent tracking
Dioxin/Furan Sample Preparation
D/F Instrument calibration and operation
D/F Sample analysis, Integration, and
Chromatograms
D/F Data reduction and reporting
Lunch - The audit team would like to order a simple lunch to eat in.
Instrument maintenance
Data validation
Quality Control data and corrective
actions
PCB Sample Preparation
PCB Instrument calibration and operation
PCB Sample analysis, Integration, and
Chromatograms
Audit team prepares for the debriefing
Debriefing
Audit ends
Anticipated Closing Meeting: May 26, 2004 (4:15 PM)
Closing Meeting Participants:
Audit team
Laboratory Point of Contact
Analyst
Laboratory QA Officer
Others at the discretion of the laboratory
Revision 1 May 24, 2004
Page 3 of4
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AUDIT PLAN
SITE Demonstration of Monitoring and Measurement Technologies
for Dioxin and Dioxin-like Compounds in Soil and Sediment
Anticipated Reporting Schedule: See above
Report Routing Pathway:
The Lead Auditor will submit the audit reports to the Battelle Project Manager. Comments from
the Battelle Project Manager and the EPA TOM will be provided to the Lead Auditor by the
Battelle Project Manager. The audit is not closed until the laboratory has responded to each
finding.
Confidentiality of Findings Report:
Dissemination of the report to anyone besides the Battelle Project Manager, the EPA TOM, and
the laboratory QA Manager is at the discretion of the EPA TOM.
Dissemination of Findings Report: See Confidentiality of Findings Report.
Revision 1 May 24, 2004
Page 4 of4
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