GUIDANCE FOR ASSESSING CHEMICAL CONTAMINANT DATA
FOR USE IN FISH ADVISORIES
VOLUME 1: FISH SAMPLING AND ANALYSIS
SECOND EDITION
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC
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TABLE OF CONTENTS
TABLE OF CONTENTS
Section
Page
List of Figures vii
List of Tables ix
Acknowledgments xiii
List of Acronyms xvii
Executive Summary ES-1
1 Introduction 1-1
1.1 Historical Perspective 1-1
1.2 Purpose 1-3
1.3 Objectives 1-4
1.4 Relationship of Manual to Other Guidance Documents 1-6
1.5 Organization of this Manual 1-6
2 Monitoring Strategy 2-1
2.1 Screening Studies (Tier 1) 2-4
2.2 Intensive Studies (Tier 2) 2-14
3 Target Species 3-1
3.1 Purpose of Using Target Species 3-1
3.2 Criteria for Selecting Target Species 3-2
3.3 Freshwater Target Species 3-3
3.3.1 Target Finfish Species 3-5
3.3.2 Target Turtle Species 3-12
3.4 Estuarine/Marine Target Species 3-16
3.4.1 Target Shellfish Species 3-24
3.4.2 Target Finfish Species 3-2
4 Target Analytes 4-1
4.1 Recommended Target Analytes 4-1
4.2 Selection of Target Analytes 4-5
4.3 Target Analyte Profiles 4-5
4.3.1 Metals 4-5
4.3.2 Organochlorine Pesticides 4-13
4.3.3 Organophosphate Pesticides 4-21
4.3.4 Chlorophenoxy Herbicides 4-25
4.3.5 Polycyclic Aromatic Hydrocarbons (PAHs) 4-26
4.3.6 Polychlorinated Biphenyls (Total) 4-29
4.3.7 Dioxins and Dibenzofurans 4-35
iii
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TABLE OF CONTENTS
Section
Page
4.4 Target Analytes under Evaluation 4-36
4.4.1 Lead 4-38
5 Screening Values for Target Analytes 5-1
5.1 General Equations for Calculating Screening Values 5-2
5.1.1 Noncarcinogens 5-3
5.1.2 Carcinogens 5-3
5.1.3 Recommended Values for Variables in Screening
Value Equations 5-4
5.2 Recommended Screening Values for Target Analytes 5-5
5.3 Comparison of Target Analyte Concentrations with
Screening Values 5-14
5.3.1 Metals 5-14
5.3.2 Organics 5-16
6 Field Procedures 6-1
6.1 Sampling Design 6-1
6.1.1 Screening Studies (Tier 1) 6-2
6.1.2 Intensive Studies (Tier 2) 6-12
6.2 Sample Collection 6-22
6.2.1 Sampling Equipment and Use 6-22
6.2.2 Preservation of Sample Integrity 6-29
6.2.3 Field Recordkeeping 6-31
6.3 Sample Handling 6-39
6.3.1 Sample Selection 6-39
6.3.2 Sample Packaging 6-47
6.3.3 Sample Preservation 6-48
6.3.4 Sample Shipping 6-50
7 Laboratory Procedures I—Sample Handling 7-1
7.1 Sample Receipt and Chain-of-Custody 7-1
7.2 Sample Processing 7-3
7.2.1 General Considerations 7-3
7.2.2 Processing Fish Samples 7-7
7.2.3 Processing Turtle Samples 7-17
7.2.4 Processing Shellfish Samples 7-25
7.3 Sample Distribution 7-30
7.3.1 Preparing Sample Aliquots 7-30
7.3.2 Sample Transfer 7-33
8 Laboratory Procedures II—Sample Analyses 8-1
8.1 Recommended Analytes 8-1
8.1.1 Target Analytes 8-1
8.1.2 Lipid 8-1
iv
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TABLE OF CONTENTS
Section
Page
8.2 Analytical Methods 8-3
8.2.1 Lipid Method 8-3
8.2.2 Target Analyte Methods 8-6
8.3 Quality Assurance and Quality Control Considerations .... 8-10
8.3.1 QA Plans 8-14
8.3.2 Method Documentation 8-14
8.3.3 Minimum QA and QC Requirements for Sample
Analyses 8-15
8.4 Documentation and Reporting of Data 8-49
8.4.1 Analytical Data Reports 8-49
8.4.2 Summary Reports 8-51
9 Data Analysis and Reporting 9-1
9.1 Data Analysis 9-1
9.1.1 Screening Studies 9-1
9.1.2 Intensive Studies 9-2
9.2 Data Reporting 9-3
9.2.1 State Data Reports 9-3
9.2.2 Reports to the National Fish Tissue Data Repository . 9-3
10 Literature Cited 10-1
Appendix
A Use of Individual Samples in Fish Contaminant Monitoring
Programs A-1
B Fish and Shellfish Species for which State Consumption
Advisories Have Been Issued B-1
C Target Analytes Analyzed in National or Regional Monitoring
Programs C-1
D Pesticides and Herbicides Recommended as Target Analytes ... D-1
E Target Analyte Dose-Response Variables and Associated
Information E-1
F Quality Assurance and Quality Control Guidance F-1
G Recommended Procedures for Preparing Whole Fish
Composite Homogenate Samples G-1
H General Procedures for Removing Edible Tissues from
Freshwater Turtles H-1
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TABLE OF CONTENTS
Appendix
Pag©
I General Procedures for Removing Edible Tissues from Shellfish .. 1-1
J Comparison of Target Analyte Screening Values (SVs) with
Detection and Quantitation Limits of Current Analytical Methods .. J-1
K A Recommended Method for Inorganic Arsenic Analysis K-1
L Sources of Recommended Reference Materials and Standards ... L-1
M Statistical Methods for Comparing Samples: Spatial and
Temporal Considerations M-1
VI
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LIST OF FIGURES
LIST OF FIGURES
Number
Page
2-1 Recommended strategy for State fish and shellfish contaminant
monitoring programs 2-2
3-1 Geographic range of the common snapping turtule (Chelydra
serpentina) 3-13
3-2 Geographic distributions of three bivalve species used
extensively in national contaminant monitoring programs 3-29
4-1 States issuing fish and shellfish advisories for mercury ....... 4-11
4-2 States issuing fish and shellfish advisories for chlordane ...... 4-15
4-3 States issuing fish and shellfish advisories for PCBs 4-31
4-4 States issuing fish and shellfish advisories for dioxin/furans .... 4-37
6-1 Example of a sample request form 6-3
6-2 Example of a field record for fish contaminant monitoring
program—screening study 6-32
6-3 Example of a field record for shellfish contaminant monitoring
program—screening study 6-33
6-4 Example of a field record for fish contaminant monitoring
program—intensive study 6-34
6-5 Example of a field record for shellfish contaminant monitoring
program—intensive study 6-36
6-6 Example of a sample identification label 6-38
6-7 Example of a chain-of-custody tag or label 6-38
6-8 Example of a chain-of-custody record form 6-40
6-9 Recommended measurements of body length and size for
fish, shellfish, and turtles 6-44
7-1 Preparation of fish fillet composite homogenate samples 7-8
7-2 Example of a sample processing record for fish contaminant
monitoring program—fish fillet composites 7-10
7-3 Illustration of basic fish filleting procedure 7-13
7-4 Preparation of individual turtle homogenate samples 7-18
7-5 Example of a sample processing record for a contaminant
monitoring program—individual turtle samples 7-19
7-6 Illustration of basic turtle resection procedure 7-22
7-7 Preparation of shellfish edible tissue composite homogenate
samples 7-26
VII
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LIST OF FIGURES
Number
Page
7-8 Example of a sample processing record for shellfish
contaminant monitoring program—edible tissue composites. ... 7-28
7-9 Example of a fish and shellfish monitoring program sample
aliquot record 7-32
7-10 Example of a fish and shellfish monitoring program sample
transfer record 7-34
8-1 Recommended contents of analytical standard operating
procedures (SOPs) 8-15
9-1 Recommended data reporting requirements for screening and
intensive studies 9-4
viii
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LIST OF TABLES
LIST OF TABLES
Number
Page
2-1 Recommended Strategy for State Fish and Shellfish
Contaminant Monitoring Programs 2-5
3-1 Recommended Target Species for Inland Fresh Waters 3-4
3-2 Recommended Target Species for Great Lakes Waters 3-4
3-3 Comparison of Freshwater Finfish Species Used in Several
National Fish Contaminant Monitoring Programs 3-6
3-4 Freshwater Turtles Recommended for Use as Target Species ... 3-7
3-5 Average Fish Tissue Concentrations of Xenobiotics for Major
Finfish Species Sampled in the National Study of Chemical
Residues in Fish 3-8
3-6 Average Fish Tissue Concentrations of Dioxins and Furans
for Major Finfish Species Sampled in the National Study of
Chemical Residues in Fish 3-9
3-7 Principal Freshwater Fish Species Cited in State Fish
Consumption Advisories 3-10
3-8 Principal Freshwater Turtle Species Cited in State
Consumption Advisories 3-14
3-9 Summary of Recent Studies Using Freshwater Turtles as
Biomonitors of Environmental Contamination 3-15
3-10 Recommended Target Species for Northeast Atlantic
Estuaries and Marine Waters (Maine through Connecticut) .... 3-17
3-11 Recommended Target Species for Mid-Atlantic Estuaries and
Marine Waters (New York through Virginia) .. 3-18
3-12 Recommended Target Species for Southeast Atlantic
Estuaries and Marine Waters (North Carolina through Florida) .. 3-19
3-13 Recommended Target Species for Gulf of Mexico Estuaries
and Marine Waters (West Coast of Florida through Texas) .... 3-20
3-14 Recommended Target Species for Pacific Northwest
Estuaries and Marine Waters (Alaska through Oregon) 3-21
3-15 Recommended Target Species for Northern California
Estuaries and Marine Waters (Klamath River through Morro
Bay) 3-22
3-16 Recommended Target Species for Southern California
Estuaries and Marine Waters (Santa Monica Bay to Tijuana
Estuary) 3-23
3-17 Sources of Information on Commercial and Sportfishing
Species in Various Coastal Areas of the United States 3-25
IX
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LIST OF TABLES
Number
Page
3-18 Estuarine/Marine Species Used in Several National Fish and
Shellfish Contaminant Monitoring Programs 3-26
3-19 Principal Estuarine/Marine Fish and Shellfish Species Cited in
State Consumption Advisories 3-30
4-1 Recommended Target Analytes 4-2
4-2 Contaminants Resulting in Fish and Shellfish Advisories 4-4
4-3 Polychlorinated Biphenyl (PCB) Congeners Recommended
for Quantitation as Potential Target Analytes 4-33
4-4 Dibenzo-p-Dioxins and Dibenzofurans Recommended as
Target Analytes 4-36
5-1 Recommended Values for Mean Body Weights (BWs) and
Fish Consumption Rates (CRs) for Selected Subpopulations .... 5-6
5-2 Dose-Response Variables and Recommended Screening
Values (SVs) for Target Analytes 5-8
5-3 Example Screening Values (SVs) for Various Subpopulations
and Risk Levels (RLs) 5-13
5-4 Estimated Order of Potential Potencies of Selected PAHs 5-17
5-5 Toxicity Equivalency Factors (TEFs) for Tetra- through Octa-
Chlorinated Dibenzo-p-Oioxins and Dibenzofurans 5-20
6-1 Values of [2/n2m2(n-1)]1/2 for Various Combinations of
n and m 6-19
6-2 Estimates of Statistical Power of Hypothesis of Interest Under
Specified Assumptions j 6-21
6-3 Summary of Fish Sampling Equipment 6-23
6-4 Summary of Shellfish Sampling Equipment 6-25
6-5 Checklist of Field Sampling Equipment and Supplies for Fish
.and Shellfish Contaminant Monitoring Programs 6-27
6-6 Safety Considerations for Field Sampling Using a Boat 6-28
6-7 Recommendations for Preservation of Fish, Shellfish, and
Turtle Samples from Time of Collection to Delivery at the
Processing Laboratory 6-49
7-1 Recommendations for Container Materials, Preservation, and
Holding Times for Fish, Shellfish, and Turtle Tissues from
Receipt at Sample Processing Laboratory to Analysis 7-4
7-2 Weights (g) of Individual Homogenates Required for
Screening Study Composite Homogenate Sample 7-16
7-3 Recommended Sample Aliquot Weights and Containers for
Various Analyses 7-31
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LIST OF TABLES
Number
Page
8-1
8-2
8-3
8-4
8-5
8-6
8-7
8-8
9-1
Contract Laboratories Conducting Dioxin/Furan Analyses in
Fish and Shellfish Tissues 8-2
Current References for Analytical Methods for Contaminants
in Fish and Shellfish Tissues 8-4
Recommended Analytical Techniques for Target Analytes 8-8
Range of Detection and Quantitation Limits of Current
Analytical Methods for Recommended Target Analytes 8-11
Approximate Range of Costs per Sample for Analysis of
Recommended Target Analytes 8-13
Recommended Quality Assurance and Quality Control
Samples 8-18
Minimum Recommended QA and QC Samples for Routine
Analysis of Target Analytes 8-27
Fish and Shellfish Tissue Reference Materials 8-30
Hypothetical Cadmium Concentrations (ppm) in Target
Species A at Three River Locations
9-6
XI
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ACKNOWLEDGMENTS
ACKNOWLEDGMENTS
This report was prepared by the U.S. Environmental Protection Agency, Office
of Water, Fish Contamination Section. The EPA Project Manager for this docu-
ment was Jeffrey Bigler who provided overall project coordination as well as
technical direction. EPA was supported in the development of this document by
the Research Triangle Institute (RTI) and Tetra Tech, Inc. (EPA Contract Num-
ber 68-C3-0374). Pat Cunningham of RTI was the contractor's Project Manager.
Preparation of the First and Second Editions of this guidance was facilitated by
the substantial efforts of the numerous Workgroup members and reviewers listed
below. These individuals representing EPA Headquarters, EPA Regions, State
and Federal agencies, Native American groups and others provided technical
information, reviews, and recommendations throughout the preparation of this
document. Participation in the review process does not imply concurrence by
these individuals with all concepts and methods described in this document.
FISH CONTAMINANT WORKGROUP
EPA Headquarters Staff
Charles Abernathy EPA/Office
Thomas Armitage EPA/Office
Jeffrey Bigler EPA/Office
Carin Bisland EPA/Office
Dennis Borum EPA/Office
Robert Cantilli EPA/Office
Julie Du EPA/Office
Richard Hoffman EPA/Office
Clyde Houseknecht EPA/Office
Henry Kahn EPA/Office
Amal Mahfouz EPA/Office
Michael Kravitz EPA/Office
Elizabeth Southerland EPA/Office
Margaret Stasikowski EPA/Office
Irene Suzukida-Horner EPA/Office
Elizabeth Tarn EPA/Office
William Telliard EPA/Office
Charles White EPA/Office
Jennifer Orme Zavala EPA/Office
Tina Levine EPA/Office
Michael Metzger EPA/Office
Richard Whiting EPA/Office
Jacqueline Moya EPA/Office
of Water
of Water
of Water (Workgroup Chairman)
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Water
of Pesticide Programs
of Pesticide Programs
of Pesticide Programs
of Health and Environmental Assessment
xiii
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ACKNOWLEDGMENTS
Other EPA Office Staff
David DeVault
Brian Melzian
John Paul
Dennis McMulien
Laurence Burkhard
Michael Dourson
Donald Klemm
EPA Regional Staff
Charles Kanetsky
Jerry Stober
Peter Redmon
Diane Evans
Philip Crocker
Bruce Herbold
Other Federal Agency Staff
Michael Bolger
Leon Sawyer
Lee Barclay
Frank De Luise
Donald Steffeck
Jerry Schulte
Adriana Cantillo
Maxwell Eldridge
Betty Hackley
Alicia Jarboe
Bruce Morehead
Don Dycus
J. Kent Crawford
EPA/Great Lakes National Program Office
EPA/Office of Reserach and Development-
Narragansett, Rl
EPA/Office of Research and Development-
Narragansett, Rl
EPA/Environmental Monitoring and
Systems Laboratory-Cincinnati, OH
EPA/Office of Research and Development-
Duluth, MN
EPA/Office of Health and Environmental Assessment-
Cincinnati, OH
EPA/Office of Health and Environmental Assessment-
Cincinnati, OH
Region 3
Region 4
Region 5
Region 6
Region 7
Region 9
FDA
FDA
FWS
FWS
FWS
ORSANCO
NOAA
NOAA
NOAA
NOAA
NOAA
TVA
USGS
xiv
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ACKNOWLEDGMENTS
State Agency Staff
Robert Cooner
Brian Hughes
William Keith
Thomas McChesney
Randall Mathis
Gerald Pollock
Robert McConnell
Richard Green
Eldert Hartwig
Randall Manning
Robert Flentge
C. Lee Bridges
Emelise Cormier
Albert Hindrichs
Elaine Sorbet
Deirdre Murphy
Jack Schwartz
John Hesse
Richard Powers
Lisa Williams
Pamela Shubat
Alan Buchanan
David Tunink
Donald Normandeau
Paul Hauge
Lawrence Skinner
Ken Eagleson
Jay Sauber
Luanne Williams
Michael Ell
Martin Schock
Abul Anisuzzaman
Gene Foster
Barbara Britton
Peter Sherertz
Ram Tripathi
Jim Amrhein
Bruce Baker
Alabama
Alabama
Arkansas
Arkansas
Arkansas
California
Colorado
Delaware
Florida
Georgia
Illinois
Indiana
Louisiana
Louisiana
Louisiana
Maryland
Massachusetts
Michigan
Michigan
Michigan
Minnesota
Missouri
Nebraska
New Hampshire
New Jersey
New York
North Carolina
North Carolina
North Carolina
North Dakota
North Dakota
Ohio
Oregon
Texas
Virginia
Virginia
Wisconsin
Wisconsin
Other Organizations
James Wiener American Fisheries Society
Deborah Schwackhamer University of Minnesota
Alvin Braswell North Carolina State Museum of Natural Science
J. Whitfield Gibbons University of Georgia Savannah River
Ecology Laboratory
xv
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LIST OF ACRONYMS
LIST OF ACRONYMS
AFS
ANOVA
ATSDR
BCF
BW
CERCLA
COC
CR
CRADAs
CSOs
DOT
EPA
FDA
FWS
y-BHC
GC/ECD
GC/MS
GPS
HRGC/MRMS
IRIS
MDL
MQL
NAS
NCBP
American Fisheries Society
Analysis of Variance
Agency for Toxic Substances and Disease Registry
bioconcentration factor
body weight
Comprehensive Environmental Response, Compensation, and
Liability Act
chain-of-custody
consumption rate
Cooperative Research and Development Agreements
combined sewer overflows
U.S. Department of Transportation
U.S. Environmental Protection Agency
U.S. Food and Drug Administration
U.S. Fish and Wildlife Service
benzene hexachloride
hexachlorocyclohexane
gas chromatography/electron capture detection
gas chromatography/mass spectrometry
Global Positioning System
high-resolution gas chromatography/high-resolution mass
spectrometry
Integrated Risk Information System
method detection limit
method quantitation limit
National Academy of Sciences
National Contaminant Biomonitoring Program
xvii
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LIST OF ACRONYMS
NCR
NFTDR
NIST
NOAA
OAPCA
OAQPS
ODES
ODW
OHEA
OPPs
ORSANCO
PAHs
PCBs
PCDDs
PCDFs
PEC
PNAs
PTFE
QA
QC
RCRA
RfD
RPs
SF
SOPS
SVs
2,4,5-T
2,3,7,8-TCDD
2,3,7,8-TCDF
2,4,5-TCP
TECS
TVA
no-carbon-required
National Fish Tissue Data Repository
National Institute of Standards and Technology
National Oceanic and Atmospheric Administration
Organotin Antifouling Paint Control Act
Office of Air Quality Planning and Standards
Ocean Discharge Evaluation System
Office of Drinking Water
Office of Health and Environmental Assessment
Office of Pesticide Programs
Ohio River Valley Water Sanitation Commission
polycyclic aromatic hydrocarbons
polychlorinated biphenyls
polychlorinated dibenzo-p-dioxins
polychlorinated dibenzofurans
potency equivalency concentration
polynuclear aromatic hydrocarbons
polytetrafluoroethylene
quality assurance
quality control
Resource Conservation and Recovery Act
reference dose
relative potencies
slope factor
standard operating procedures
screening values
2,4,5-trichlorophenoxyacetic acid
2,3,7,8-tetrachlorodibenzo-p-dioxin
2,3,7,8-tetrachlorodibenzofuran
2,4,5-trichlorophenol
toxicity equivalent concentrations
Tennessee Valley Authority
XVIII
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LIST OF ACRONYMS
USDA
USGS
WHO
U.S. Department of Agriculture
United States Geological Survey
World Health Organization
XIX
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EXECUTIVE SUMMARY
EXECUTIVE SUMMARY
State, local, and Federal agencies currently use various methods to sample and
analyze chemical contaminants in fish and shellfish in order to develop fish
consumption advisories. A 1988 survey, funded by the U.S. Environmental
Protection Agency (EPA) and conducted by the American Fisheries Society,
identified the need for standardizing the approaches to evaluating risks and
developing fish consumption advisories that are comparable across different
jurisdictions (Cunningham et al., 1990; Cunningham et al., 1994). Four major
components were identified as critical to the development of a consistent risk-
based approach: standardized practices for sampling and analyzing fish,.
standardized risk assessment methods, standardized procedures for making risk
management decisions, and standardized approaches for communicating risk to
the general public (Cunningham et al., 1990).
To address concerns raised by the survey respondents, EPA is developing a
series of four documents designed to provide guidance to State, local, regional,
and tribal environmental health officials responsible for designing contaminant
monitoring programs and issuing fish and shellfish consumption advisories. It is
essential that all four documents be used together, since no single volume
addresses all of the topics involved in the development of risk-based fish
consumption advisories. The documents are meant to provide guidance only
and do not constitute a regulatory requirement. This document series includes:
Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories
Volume I: Fish Sampling and Analysis
Volume II: Risk Assessment and Fish Consumption Limits
Volume III: Risk Management
Volume IV: Risk Communication.
Volume I was first released in September 1993 and this current revision to the
Volume I guidance provides the latest information on sampling and analysis
procedures based on new information provided by the Environmental Protection
Agency. The major objective of Volume I is to provide information on sampling
strategies for a contaminant monitoring program. In addition, information is
provided on selection of target species; selection of chemicals as target analytes;
development of human health screening values; sample collection procedures
including sample processing, sample preservation, and shipping; sample
analysis; and data reporting and analysis.
ES-1
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EXECUTIVE SUMMARY
Volume II was released in June 1994 and provides guidance on the development
of risk-based meal consumption limits for the high-priority chemical fish
contaminants (target analytes). In addition to the presentation of consumption
limits, Volume II contains a discussion of risk assessment methods used to
derive the consumption limits as well as a discussion of methods to modify these
limits to reflect local conditions.
Volume III will be released in FY 1996 and provides guidance on risk
management procedures. This volume provides information regarding the
selection and implementation of various options for reducing health risks
associated with the consumption of chemically contaminated fish and shellfish.
Using a human health risk-based approach, States can determine the level of the
advisory and the most appropriate type of advisory to issue. Methods to
evaluate population risks for specific groups, waterbodies, and geographic areas
are also presented.
Volume IV was released in March 1995 and provides guidance on risk commu-
nication as a process for sharing information with the public on the health risks
of consuming chemically contaminated fish and shellfish. This volume provides
guidance on problem analysis and program objectives,' audience identification
and needs assessments, communication strategy design, implementation and
evaluation, and responding to public inquiries.
The EPA welcomes your suggestions and comments. A major goal of this
guidance document series is to provide a clear and usable summary of critical
information necessary to make informed decisions regarding the development
of fish consumption advisories. We encourage comments, and hope this
document will be a useful adjunct to the resources used by the States, local
governments, and Tribal bodies in making decisions regarding the development
of fish advisories within their various jurisdictions.
ES-2
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1. INTRODUCTION
SECTION 1
INTRODUCTION
1.1 HISTORICAL PERSPECTIVE
Contamination of aquatic resources, including freshwater, estuarine, and marine
fish and shellfish, has been documented in the scientific literature for many
regions of the United States (NAS, 1991). Environmental concentrations of
some pollutants have decreased over the past 20 years as a result of better
water quality management practices. However, environmental concentrations of
other heavy metals, pesticides, and toxic organic compounds have increased
due to intensifying urbanization, industrial development, and use of new
agricultural chemicals. Our Nation's waterbodies are among the ultimate
repositories of pollutants released from these activities. Pollutants come from
permitted point source discharges (e.g., industrial and municipal facilities),
accidental spill events, and nonpoint sources (e.g., agricultural practices,
resource extraction, urban runoff, in-place sediment contamination, ground water
recharge, and atmospheric deposition).
Once these toxic contaminants reach surface waters, they may concentrate
through aquatic food chains and bioaccumulate in fish and shellfish tissues.
Aquatic organisms may bioaccumulate environmental contaminants to more than
1,000,000 times the concentrations detected in the water column (U.S. EPA,
1992c, 1992d). Thus, fish and shellfish tissue monitoring serves as an important
indicator of contaminated sediments and water quality problems, and many
States routinely conduct chemical contaminant analyses of fish and shellfish
tissues as part of their comprehensive water quality monitoring programs
(Cunningham and Whitaker, 1989). Tissue contaminant monitoring also enables
State agencies to detect levels of contamination in fish and shellfish tissue that
may be harmful to human consumers. If States conclude that consumption of
chemically contaminated fish and shellfish poses an unacceptable human health
risk, they may issue local fish consumption advisories or bans for specific
waterbodies and specific fish and shellfish species for specific populations.
In 1989, the American Fisheries Society (AFS), at the request of the U.S.
Environmental Protection Agency (EPA), conducted a survey of State fish and
shellfish consumption advisory practices. Questionnaires were sent to health
departments, fisheries agencies, and water quality/environmental management
departments in all 50 States and the District of Columbia. Officials in all 50
States and the District responded.
1-1
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1. INTRODUCTION
Respondents were asked to provide information on several issues including
Agency responsibilities
Sampling strategies
Sample collection procedures
Chemical residue analysis procedures
Risk assessment methodologies
Data interpretation and advisory development
State concerns
Recommendations for Federal assistance.
Cunningham et al. (-1990) summarized the survey responses and reported that
monitoring and risk assessment procedures used by States in their fish and
shellfish advisory programs varied widely. States responded to the question
concerning assistance from the Federal government by requesting that Federal
agencies
Provide a consistent approach for State agencies to use in assessing health
risks from consumption of chemically contaminated fish and shellfish
Develop guidance on sample collection procedures
Develop and/or endorse uniform, cost-effective analytical methods for
quantitation of contaminants
• Establish a quality assurance (QA) program that includes use of certified
reference materials for chemical analyses.
In March 1991, the National Academy of Sciences (NAS) published a report
entitled Seafood Safety (NAS, 1991) that reviewed the nature and extent of
public health risks associated with seafood consumption and examined the
scope and adequacy of current seafood safety programs. After reviewing ovef
150 reports and publications on seafood contamination, the NAS Institute of
Medicine concluded that high concentrations of chemical contaminants exist Hi
various fish species in a number of locations in the country. The report noted
that the fish monitoring data available in national and regional studies had two
major shortcomings that affected their usefulness in assessing human health
risks:
• In some of the more extensive studies, analyses were performed on
nonedible portions of finfish (e.g., liver tissue) or on whole fish, whict)
precludes accurate determination of human exposures.
Studies did not use consistent methods of data reporting (e.g., bofri
geometric and arithmetic means were reported in different studies) or failed
to report crucial information on sample size, percent lipid, mean values of
contaminant concentrations, or fish size, thus precluding direct comparison
1-2
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1. INTRODUCTION
of the data from different studies and complicating further statistical analysis
and risk assessment.
As a result of these MAS concerns and State concerns expressed in the AFS
survey, the EPA Office of Water established a Fish Contaminant Workgroup. It
is composed of representatives from EPA and the following State and Federal
agencies:
U.S. Food and Drug Administration (FDA)
• U.S. Fish and Wildlife Service (FWS)
• Ohio River Valley Water Sanitation Commission (ORSANCO)
• National Oceanic and Atmospheric Administration (NOAA)
Tennessee Valley Authority (TVA)
• United States Geological Survey (USGS)
and representatives from 26 States: Alabama, Arkansas, California, Colorado,
Delaware, Florida, Georgia, Illinois, Indiana, Louisiana, Maryland, Massachu-
setts, Michigan, Minnesota, Missouri, Nebraska, New Hampshire, New Jersey,
New York, North Carolina, North Dakota, Ohio, Oregon, Texas, Virginia, and
Wisconsin.
The objective of the EPA Fish Contaminant Workgroup was to formulate
guidance for States on how to sample and analyze chemical contaminants in fish
and shellfish where the primary end uses of the data included development of
fish consumption advisories. The Workgroup compiled documents describing
protocols currently used by various Federal agencies, EPA Regional offices, and
States that have extensive experience in fish contaminant monitoring. Using
these documents, they selected methods considered most cost-effective and
scientifically sound for sampling and analyzing fish and shellfish tissues. These
methods are recommended as standard procedures for use by the States and
are described in this manual.
1.2 PURPOSE
The purpose of this manual is to provide overall guidance to States on methods
for sampling and analyzing contaminants in fish and shellfish tissue that will
promote consistency in the data States use to determine the need for fish
consumption advisories. This manual provides guidance only and does not
constitute a regulatory requirement for the States. It is intended to describe
what the EPA Office of Water believes to be scientifically sound methods for
sample collection, chemical analyses, and statistical analyses of fish and
shellfish tissue contaminant data for use in fish contaminant monitoring programs
that have as their objective the protection of public health. This nonregulatory,
technical guidance manual is intended for use as a handbook by State and local
agencies that are responsible for sampling and analyzing fish and shellfish
tissue. Adherence to this guidance will enhance the comparability of fish and
shellfish contaminant data, especially in interstate waters, and thus provide more
standardized information on fish contamination problems.
1-3
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1. INTRODUCTION
In order to enhance the use of this guidance as a working document, the EPA
will issue additional information and updates to users as appropriate. It is
anticipated that updates will include minor revisions such as the addition or
deletion of chemicals from the recommended list of target analytes, new
screening values as new toxicologic data become available, and new chemical
analysis procedures for some target analytes as they are developed. A new
edition of the guidance will be issued to include the addition of major new areas
of guidance such as using frogs and waterfowl as target species for assessment
of human health risks or when major changes are made to the Agency's risk
assessment procedures.
The EPA Office of Water realizes that adoption of these recommended methods
requires adequate funding. In practice, funding varies among States and
resource limitations will cause States to tailor their fish and shellfish contaminant
monitoring programs to meet their own needs. States must consider tradeoffs
among the various parameters when developing their fish contaminant
monitoring programs. These parameters include
Total number of stations sampled
Intensity of sampling at each site
Number of chemical analyses and their cost
Resources expended on data storage and analysis, QA and quality control
(QC), and sample archiving.
These tradeoffs will limit the number of sites sampled, number of target analytes
analyzed at each site, number of target species collected, and number of
replicate samples of each target species collected at each site (Crawford and
Luoma, 1993).
1.3 OBJECTIVES
The specific objectives of the manual are to
1. Recommend a tiered monitoring strategy designed to
Screen waterbodies (Tier 1) to identify those harvested sites where
chemical contaminant concentrations in the edible portions of fish and
shellfish exceed human consumption levels of potential concern
(screening values [SVs]). SVs for contaminants with carcinogenic
effects are calculated based on selection of an acceptable cancer risk
level. SVs for contaminants with noncarcinogenic effects are
concentrations determined to be without appreciable noncancer health
risk. For a contaminant with both carcinogenic and noncarcinogenic
effects, the lower (more conservative) of the two calculated SVs is used.
1-4
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1. INTRODUCTION
• Conduct intensive followup sampling (Tier 2, Phase I) to determine the
magnitude of the contamination in edible portions of fish and shellfish
species commonly consumed by humans in waterbodies identified in the
screening process.
• Conduct intensive sampling at additional sites (Tier 2, Phase II) in a
waterbody where screening values were exceeded to determine the
geographic extent of contamination in various size classes of fish and
shellfish.
2. Recommend target species and criteria for selecting additional species if the
recommended target species are not present at a site.
3. Recommend target analytes to be analyzed in fish and shellfish tissue and
criteria for selecting additional analytes.
4. Recommend risk-based procedures for calculating target analyte screening
values.
5. Recommend standard field procedures including
• Site selection
• Sampling time
Sample type and number of replicates
Sample collection procedures including sampling equipment
Field recordkeeping and chain of custody
Sample processing, preservation, and shipping.
6. Recommend cost-effective, technically sound analytical methods and
associated QA and QC procedures, including identification of
• Analytical methods for target analytes with detection limits capable of
measuring tissue concentrations at or below SVs
Sources of recommended certified reference materials
Federal agencies currently conducting QA interlaboratory comparison
programs.
7. Recommend procedures for data analysis and reporting of fish and shellfish
contaminant data.
8. Recommend QA and QC procedures for all phases of the monitoring
program and provide guidance for documenting QA and QC requirements
in a QA plan or in a combined work/QA project plan.
1-5
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1. INTRODUCTION
1A RELATIONSHIP OF MANUAL TO OTHER GUIDANCE DOCUMENTS
This manual is the first in a series of four documents to be prepared by the EPA
Office of Water as part of a Federal Assistance Plan to help States standardize
fish consumption advisories. This series of four documents-—Guidance for
Assessing Chemical Contaminant Data for Use in Fish Advisories includes
• Volume I: Fish Sampling and Analysis (EPA 823-R-93-00), published
August 1993
• Volume II: Risk Assessment and Fish Consumption Limits (EPA 823-B-94-
004), published June 1994
• Volume III: Risk Management, to be published in FY 1996
• Volume IV: Risk Communication (EPA 823-R-95-001), published March
1995.
This sampling and analysis manual is not intended to be an exhaustive guide to
all aspects of sampling, statistical design, development of risk-based screening
values, laboratory analyses, and QA and QC considerations for fish and shellfish
contaminant monitoring programs. Key references are provided that detail
various aspects of these topics.
In addition, interested individuals may obtain a software program (on five 3.5-
inch diskettes) of all fish consumption advisories for the 50 States and U.S.
Territory waters entitled The National Listing of Fish Consumption Advisories
(EPA-823-C-95-001) by contacting:
U.S. Environmental Protection Agency
National Center for Environmental Publications and Information
11029 Kenwood Road
Cincinnati, OH 45242
(513) 489-8190
In October 1995, EPA also will make this database available for downloading
from the Internet. Point your World Wide Web browser to the following URL:
http://www.epa.gov/water
1.5 ORGANIZATION OF THIS MANUAL
This manual provides specific guidance on sampling, chemical analysis, and
data reporting and analysis procedures for State fish and shellfish contaminant
monitoring programs. Appropriate QA and QC considerations are integral parts
of each of the recommended procedures.
1-6
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1. INTRODUCTION
Monitoring Strategy: Section 2 outlines the recommended strategy for State
fish and shellfish contaminant monitoring programs. This strategy is designed
to (1) routinely screen waterbodies to identify those locations where chemical
contaminants in edible portions of fish and shellfish exceed human health
screening values and (2) sample more intensively those waterbodies where
exceedances of these SVs have been found in order to assess the magnitude
and the geographic extent of the contamination.
Target Species: Section 3 discusses the purpose of using target species and
criteria for selection of target species for both screening and intensive studies.
Lists of recommended target species are provided for inland fresh waters, Great
Lakes waters, and seven distinct estuarine and coastal marine regions of the
United States.
Target Analytes: Section 4 presents a list of recommended target analytes to
be considered for inclusion in screening studies and discusses criteria used in
selecting these analytes.
Screening Values: Section 5 describes the EPA risk-based procedure for
calculating screening values for target analytes.
Field Procedures: Section 6 recommends field procedures to be followed from
the time fish or shellfish samples are collected until they are delivered to the
laboratory for processing and analysis. Guidance is provided on site selection
and sample collection procedures; the guidance addresses material and
equipment requirements, time of sampling, size of animals to be collected,
sample type, and number of samples. Sample identification, handling,
preservation, shipping, and storage procedures are also described.
Laboratory Procedures: Section 7 describes recommended laboratory
procedures for sample handling including: sample measurements, sample
processing procedures, and sample preservation and storage procedures.
Section 8 presents recommended laboratory procedures for sample analyses,
including cost-effective analytical methods and associated QC procedures, and
information on sources of certified reference materials and Federal agencies
currently conducting Intel-laboratory comparison programs.
Data Analysis and Reporting: Section 9 includes procedures for data analysis
to determine the need for additional monitoring and risk assessment and for data
reporting. This section also describes the National Fish Tissue Data Repository
(NFTDR), a national database of fish and shellfish contaminant monitoring data.
Supporting documentation for this guidance is provided in Section 10, Literature
Cited, and in Appendixes A through M.
1-7
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2. MONITORING STRATEGY
SECTION 2
MONITORING STRATEGY
The objective of this section is to describe the strategy recommended by the
EPA Office of Water for use by States in their fish and shellfish contaminant
monitoring programs. A two-tiered strategy is recommended as the most cost-
effective approach for State contaminant monitoring programs to obtain data
necessary to evaluate the need to issue fish or shellfish consumption advisories.
This monitoring strategy is shown schematically in Figure 2-1 and consists of
• Tier 1—Screening studies of a large number of sites for chemical
contamination where sport, subsistence, and/or commercial fishing is
conducted. This screening will help States identify those sites where
concentrations of chemical contaminants in edible portions of commonly
consumed fish and shellfish indicate the potential for significant health risks
to human consumers.
Tier 2—Two-phase intensive studies of problem areas identified in
screening studies to determine the magnitude of contamination in edible
portions of commonly consumed fish and shellfish species (Phase I), to
determine size-specific levels of contamination, and to assess the
geographic extent of the contamination (Phase II).
This basic approach of using relatively low-cost, nonintensive screening studies
to identify areas for more intensive followup sampling is used in a variety of
water quality programs involving public health protection (California
Environmental Protection Agency, 1991; Oregon Department of Environmental
Quality, 1990; TVA, 1991; U.S. EPA, 1989d).
One key objective in the recommendation of this approach is to improve the data
used by States for issuing fish and shellfish consumption advisories. Other
specific aims of the recommended strategy are
To ensure that resources for fish contaminant monitoring programs are
allocated in the most cost-effective way. By limiting the number of sites
targeted for intensive studies, as well as the number of target analytes at
each intensive sampling site, screening studies help to reduce overall
program costs while still allowing public health protection objectives to be
met.
2-1
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2. MONITORING STRATEGY
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2. MONITORING STRATEGY
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2. MONITORING STRATEGY
To ensure that sampling data are appropriate for developing risk-based
consumption advisories.
To ensure that sampling data are appropriate for determining contaminant
concentrations in various size (age) classes of each target species so that
States can give size-specific advice on contaminant concentrations (as
appropriate).
To ensure that sampling designs are appropriate to allow statistical
hypothesis testing. Such sampling designs permit the use of statistical tests
to detect a difference between the average tissue contaminant concentration
at a site and the human health screening value for any analyte.
The following elements must be considered when planning either screening
studies or more intensive followup sampling studies:
• Study objective
Target species (and size classes)
Target analytes
Target analyte screening values
Sampling locations
Sampling times
Sample type
Sample replicates
Sample analysis
Data analysis and reporting.
Detailed guidance for each of these elements, for screening studies (Tier 1) and
for both Phase I and Phase II of intensive studies (Tier 2), is provided in this
document. The key elements of the monitoring strategy are summarized in
Table 2-1, with reference to the section number of this document where each
element is discussed.
2.1 SCREENING STUDIES (TIER 1)
The primary aim of screening studies is to identify frequently fished sites where
concentrations of chemical contaminants in edible fish and shellfish composite
samples exceed specified human health screening values and thus require more
intensive followup sampling. Ideally,-screening studies should include all
waterbodies where commercial, recreational, or subsistence fishing is practiced;
specific sampling sites should include areas where various types of fishing are
conducted routinely (e.g., from a pier, from shore, or from private and
commercial boats), thereby exposing a significant number of individuals to
potentially adverse health effects. Composites of skin-on fillets (except for
catfish and other scaleless species, which are usually prepared as skin-off fillets)
and edible portions of shellfish are recommended for contaminant analyses in
screening studies to provide conservative estimates of typical exposures for the
general population. Note: If consumers remove the skin and fatty areas from
2-4
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2. MONITORING STRATEGY
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a fish before preparing it for eating, exposures to some contaminants can be
reduced (Armbruster et al., 1987,1989; Cichy, Zabik, and Weaver, 1979; Foran,
Cox, and Croxton, 1989; Gall and Voiland, 1990; Reinert, Stewart, and Seagram,
1972; Sanders and Haynes, 1988; Skea et al., 1979; Smith, Funk, and Zabik,
1973; Voiland et al., 1991; Wanderstock et al., 1971; Zabik, Hoojjat, and Weaver,
1979).
Because the sampling sites in screening studies are focused primarily on the
most likely problem areas and the numbers of commonly consumed target
species and samples collected are limited, relatively little detailed information is
obtained on the magnitude and geographic extent of contamination in a wide
variety of harvestable fish and shellfish species of concern to consumers. More
information is obtained through additional intensive followup studies (Tier 2,
Phases I and II) conducted at potentially contaminated sites identified in
screening studies.
Although the EPA Office of Water recommends that screening study results not
be used as the sole basis for conducting a risk assessment, the Agency
recognizes that this practice may be unavoidable if monitoring resources are
limited or if the State must issue an advisory based on detection of elevated
concentrations in one composite sample. States have several options for
collecting samples during the Tier 1 screening study (see Figure 2-1), which can
provide additional information on contamination without necessitating additional
field monitoring expenditures as part of the Tier 2 intensive studies.
The following assumptions are made in this guidance document for sampling fish
and shellfish and for calculating human health SVs:
Use of commonly consumed target species that are dominant in the catch
and have high bioaccumulation potential
• Use of fish fillets (with skin on and belly flap tissue included) for scaled
finfish species, use of skinless fillets for scaleless finfish species, and use
of edible portions of shellfish
• Use of fish and shellfish above legal size to maximum size in the target
species
• Use of a 10"5 risk level, a human body weight of 70 kg (average adult), a
consumption rate of 6.5 g/d for the general population, and a 70-yr lifetime
exposure period to calculate SVs for carcinogens. Note: The EPA is
currently reviewing the 6.5-g/d consumption rate for the general population.
• Use of a human body weight of 70 kg (average adult) and a consumption
rate of 6.5 g/d for the general population to calculate SVs for noncar-
cinogens.
2-12
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2. MONITORING STRATEGY
Use of no contaminant loss during preparation and cooking or from
incomplete absorption in the intestines.
For certain site-specific situations, States may wish to use one or more of the
following exposure assumptions to protect the health of subpopulations at
potentially greater risk:
Use of commonly consumed target species that are dominant in the catch
and have the highest bioaccumulation potential
Use of whole fish or whole body of shellfish (excluding shell of bivalves),
which may provide a better estimate of contaminant exposures in sub-
populations that consume whole fish or shellfish
Use of the largest (oldest) individuals in the target species to represent the
highest likely exposure levels
• Use of a 10"6 or 10'7 risk level, body weights less than 70 kg for women and
children, site-specific consumption rates (i.e., 30 g/d for sport fisherman or
140 g/d for subsistence fishermen or other consumption rates based on
dietary studies of local fish-consuming populations), and a 70-yr exposure
period to calculate SVs for carcinogens. Note: The EPA is currently
reviewing the consumption rate for sport and subsistence fishermen.
Use of body weights less than 70 kg for women and children and site-
specific consumption rates (i.e., 30 g/d for sport fishermen or 140 g/d for
subsistence fishermen or other consumption rates based on dietary studies
of local fish-consuming populations) to calculate SVs for noncarcinogens.
There are additional aspects of the screening study design that States should
review because they affect the statistical analysis and interpretation of the data.
These include
Use of composite samples, which results in loss of information on the
distribution of contaminant concentrations in the individual sampled fish and
shellfish. Maximum contaminant concentrations in individual sampled fish,
which can be used as an indicator of potentially harmful levels of
contamination (U.S. EPA, 1989d), are not available when composite
sampling is used.
• Use of a single sample per screening site for each target species, which
precludes estimating the variability of the contamination level at that site and,
consequently, of conducting valid statistical comparisons to the target
analyte SVs.
2-13
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2. MONITORING STRATEGY
• Uncertainty factors affecting the numerical calculation of quantitative health
risk information (i.e., references doses and cancer slope factors) as well as
human health SVs.
The use of composite samples is often the most cost-effective method for esti-
mating average tissue concentrations of analytes in target species populations
to assess chronic human health risks. However, there are some situations in
which individual sampling can be more appropriate from both ecological and risk
assessment perspectives. Individual sampling provides a direct measure of the
range and variability of contaminant levels in target fish populations. Information
on maximum contaminant concentrations in individual fish is useful in evaluating
acute human health risks. Estimates of the variability of contaminant levels
among individual fish can be used to ensure that studies meet desired statistical
objectives. For example, the population variance of a contaminant can be used
to estimate the sample size needed to detect statistically significant differences
in contaminant screening values compared to the mean contaminant concentra-
tion. Finally, the analysis of individual samples may be desirable, or necessary,
when the objective is to minimize the impacts of sampling on certain vulnerable
target populations, such as predators in headwater streams and aquatic turtles,
and in cases where the cost of collecting enough individuals for a composite
sample is excessive. For States that wish to consider use of individual sampling
during either the screening or intensive studies, additional information on
collecting and analyzing individual samples is provided in Appendix A.
States should consider the potential effects of these study design features when
evaluating screening study results.
2.2 INTENSIVE STUDIES (TIER 2)
The primary aims of intensive studies are to assess '..e magnitude of tissue
contamination at screening sites, to determine the size class or classes of fish
within a target species whose contaminant concentrations exceed the SVs, and
to assess the geographic extent of the contamination for the target species in the
waterbody under investigation. With respect to the design of intensive studies,
EPA recommends a sampling strategy that may not be feasible for some site-
specific environments. Specifically, EPA recognizes that some waterbodies
cannot sustain the same intensity of sampling (i.e., number of replicate
composite samples per site and number of individuals per composite sample)
that others (i.e., those used for commercial harvesting) can sustain. In such
cases, State fisheries personnel may consider modifying the sampling strategy
(e.g., analyzing individual fish) for intensive studies to protect the fishery
resource. Although one strategy cannot cover all situations, these sampling
guidelines are reasonable for the majority of environmental conditions, are
scientifically defensible, and provide information that can be used to assess the
risk to public health. Regardless of the final study design and protocol chosen
for a fish contaminant monitoring program, State fisheries, environmental, and
2-14
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2. MONITORING STRATEGY
health personnel should always evaluate and document the procedures used to
ensure that results obtained meet State objectives for protecting human health.
The allocation of limited funds to screening studies or to intensive studies should
always be guided by the goal of conducting adequate sampling of State fish and
shellfish resources to ensure the protection of the public's health. The amount
of sampling that can be performed by a State will be determined by available
economic resources. Ideally, State agencies will allocate funds for screening as
many sites as is deemed necessary while reserving adequate resources to
conduct subsequent intensive studies at sites where excessive fish tissue
contamination is detected. State environmental and health personnel should use
all information collected in both screening and intensive studies to (1) conduct
a risk assessment to determine whether the issuance of an advisory is
warranted, (2) use risk management to determine the nature and extent of the
advisory, and then (3) effectively communicate this risk to the public. Additional
information on risk assessment, risk management, and risk communication
procedures will be provided in subsequent volumes in this series.
2-15
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3. TARGET SPECIES
SECTION 3
TARGET SPECIES
The primary objectives of this section are to: (1) discuss the purpose of using
target species, (2) describe the criteria used to select target species, and (3)
provide lists of recommended target species. Target species recommended for
freshwater and estuarine/marine ecosystems are discussed in Sections 3.3 and
3.4, respectively.
3.1 PURPOSE OF USING TARGET SPECIES
The use of target species allows comparison of fish, shellfish, and turtle tissue
contaminant monitoring data among sites over a wide geographic area.
Differences in habitat, food preferences, and rate of contaminant uptake among
various fish, shellfish, and turtle species make comparison of contaminant
monitoring results within a State or among States difficult unless the contaminant
data are from the same species. It is virtually impossible to sample the same
species at every site, within a State or region or nationally, due to the varying
geographic distributions and environmental requirements of each species.
However, a limited number of species can be identified that are distributed widely
enough to allow for collection and comparison of contaminant data from many
sites.
Three aims are achieved by using target species in screening studies. First,
States can cost-effectively compare contaminant concentrations in their State
waters and then prioritize sites where tissue contaminants exceed human health
screening values. In this way, limited monitoring resources can be used to
conduct intensive studies at sites exhibiting the highest degree of tissue
contamination in screening studies. By resampling target species used in the
screening study in Phase I intensive studies and sampling additional size classes
and additional target species in Phase II intensive studies as resources allow,
States can assess the magnitude and geographic extent of contamination in
species of commercial, recreational, or subsistence value. Second, the use of
common target species among States allows for more reliable comparison of
sampling information. Such information allows States to design and evaluate
their own contaminant monitoring programs more efficiently, which should further
minimize overall monitoring costs. For example, monitoring by one State of fish
tissue contamination levels in the upper reaches of a particular river can provide
useful information to an adjacent State on tissue contamination levels that might
be anticipated in the same target species at sampling sites downstream. Third,
the use of a select group of target fish, shellfish, and freshwater turtle species
3-1
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3. TARGET SPECIES
will allow for the development of a national database for tracking the magnitude
and geographic extent of pollutant contamination in these target species
nationwide and will permit analyses of trends in fish, shellfish, and turtle
contamination over time.
3.2 CRITERIA FOR SELECTING TARGET SPECIES
The appropriate choice of target species is a key element of any chemical
contaminant monitoring program. Criteria for selecting target species used in the
following national fish and shellfish contaminant monitoring programs were
reviewed by the EPA Fish Contaminant Workgroup to assess their applicability
for use in selecting target species for State fish contaminant monitoring
programs:
• National Study of Chemical Residues in Fish (U.S. EPA)
• National Dioxin Study (U.S. EPA)
• 301 (h) Monitoring Program (U.S. EPA)
• National Pesticide Monitoring Program (U.S. FWS)
• National Contaminant Biomonitoring Program (U.S. FWS)
• National Status and Trends Program (NOAA).
• National Water-Quality Assessment Program (USGS).
The criteria used to select target species in many of these programs are similar
although the priority given each criterion may vary depending on program aims.
The EPA Fish Contaminant Workgroup believes the most important criterion for
selecting target fish, shellfish, and turtle species for State contaminant monitoring
programs assessing human consumption concerns is that the species are
commonly consumed in the study area and are of commercial, recreational, or
subsistence fishing value. Two other criteria of major importance are that the
species have the potential to bioaccumulate high concentrations of chemical
contaminants and have a wide geographic distribution. EPA recommends thai:
States use the same criteria to select species for both screening and intensive
site-specific studies.
In addition to the three primary criteria for target species selection, it is also
important that the target species be easy to identify taxonomically because there
are significant species-specific differences in bioaccumulation potential. Because
many closely related species can be similar in appearance, reliable taxonomic
identification is essential to prevent mixing of closely related species with the
target species. Note: Under no circumstance should individuals of more than
one species be mixed to create a composite sample (U.S. EPA, 1991e). It is
also both practical and cost-effective to sample target species that are abundant,
easy to capture, and large enough to provide adequate tissue samples for
chemical analyses.
3-2
-------
3. TARGET SPECIES
It cannot be overemphasized that final selection of target species will require the
expertise of State fisheries biologists with knowledge of local species that best
meet the selection criteria and knowledge of local human consumption patterns.
Although, ideally, all fish, shellfish, or turtle species consumed from a given
waterbody by the local population should be monitored, resource constraints may
dictate that only a few of the most frequently consumed species be sampled.
In the next two sections, lists of recommended target species are provided for
freshwater ecosystems (inland fresh waters and the Great Lakes) and
estuarine/marine ecosystems (Atlantic, Gulf, and Pacific waters), and the
methods used to develop each list are discussed.
3.3 FRESHWATER TARGET SPECIES
As part of the two-tiered sampling strategy proposed for State fish contaminant
monitoring programs, EPA recommends that States collect one bottom-feeding.
fish species and one predator fish species at each freshwater screening study
site. Some suggested target species for use in State fish contaminant
monitoring programs are shown in Table 3-1 for inland fresh waters and in Table
3-2 for Great Lakes waters.
The lists of target species recommended by the EPA Fish Contaminant
Workgroup for freshwater ecosystems were developed based on a review of
species used in the following national monitoring programs:
• National Study of Chemical Residues in Fish (U.S. EPA)
• National Dioxin Study (U.S. EPA)
National Pesticide Monitoring Program (U.S. FWS)
• National Contaminant Biomonitoring Program (U.S. FWS)
• National Water-Quality Assessment Program (USGS)
and on a review of fish species cited in State fish consumption advisories or
bans (RTI, 1993). Separate target species lists were developed for inland fresh
waters (Table 3-1) and Great Lakes waters (Table 3-2) because of the distinct
ecological characteristics of these waters and their fisheries. Each target
species list has been reviewed by regional and State fisheries experts.
Use of two distinct ecological groups of finfish (i.e., bottom-feeders and
predators) as target species in freshwater systems is recommended. This
permits monitoring of a wide variety of habitats, feeding strategies, and
physiological factors that might result in differences in bioaccumulation of
contaminants. Bottom-feeding species may accumulate high contaminant
concentrations from direct physical contact with contaminated sediment and/or
by consuming benthic invertebrates and epibenthic organisms that live in
contaminated sediment. Predator species are also good indicators of persistent
pollutants (e.g., mercury or DDT and its metabolites) that may be biomagnified
through several trophic levels of the food web. Species used in several Federal
3-3
-------
3. TARGET SPECIES
Table 3-1. Recommended Target Species for Inland Fresh Waters
Family name
Perclchthyidae
Centrarchidae
Percidae
Cyprinidae
Catostomidae
Ictaluridae
Esocidae
Salmonidae
Common name
White bass
Largemouth bass
Smallmouth bass
Black crappie
White crappie
Walleye
Yellow perch
Common carp
White sucker
Channel catfish
Flathead catfish
Northern pike
Lake trout
Brown trout
Rainbow trout
Scientific name
Morone chrysops
Micropterus salmoides
Micropterus dolomieui
Pomoxis nigromaculatus
Pomoxis annularis
Stizostedion vitreum
Perca flavescens
Cyprinus carpio
Catostomus commersoni
Ictalurus punctatus
Pylodictis olivaris
Esox lucius
Salvelinus namaycush
Salmo trutta
Oncorhynchus myldss*
aFormeriy Salmo gairdneri.
Table 3-2. Recommended Target Species for Great Lakes Waters
Family name
Percichthyidae
Centrarchidae
Percidae
Cyprinidae
Catostomidae
Ictaluridae
Esocidae
Salmonidae
Common name
White bass
Smallmouth bass
Walleye
Common carp
White sucker
Channel catfish
Muskellunge
Chinook salmon
Lake trout
Brown trout
Rainbow trout
Scientific name
Morone chrysops
Micropterus dolomieui
Stizostedion vitreum
Cyprinus carpio
Catostomus commersoni
Ictalurus punctatus
Esox masquinongy
Oncorhynchus tschawytscha
Salvelinus namaycush
Salmo trutta
Oncorhynchus mykissa
"Formerly Salmo gairdneri.
3-4
-------
3. TARGET SPECIES
programs to assess the extent of freshwater fish tissue contamination nationwide
are compared in Table 3-3.
In addition to finfish species, States should consider monitoring the tissues of
freshwater turtles for environmental contaminants in areas where turtles are
consumed by recreational, subsistence, or ethnic populations. Interest has been
increasing in the potential transfer of environmental contaminants from the
aquatic food chain to humans via consumption of freshwater turtles. Turtles may
bioaccumulate environmental contaminants in their tissues from exposure to
contaminated sediments or via consumption of contaminated prey. Because
some turtle species are long-lived and occupy a medium to high trophic level of
the food chain, they have the potential to accumulate high concentrations of
chemical contaminants from their diets (Hebert et al., 1993). Some suggested
target turtle species for use in State contaminant monitoring programs are listed
in Table 3-4.
The list of target turtle species recommended by the EPA Fish Contaminant
Workgroup for freshwater ecosystems was developed based on a review of turtle
species cited in State consumption advisories or bans (RTI, 1993) and a review
of the recent scientific literature. The recommended target species list has been
reviewed by regional and State experts.
3.3.1 Target Finfish Species
3.3.1.1 Bottom-Feeding Species
EPA recommends that, whenever practical, States use common carp (Cyprinus
carpio), channel catfish (Ictalurus punctatus), and white sucker (Catostomus
commersoni) in that order as bottom-feeding target species in both inland fresh
waters (Table 3-1) and in Great Lakes waters (Table 3-2). These bottom-feeders
have been used consistently for monitoring a wide variety of contaminants
including dioxins/furans (Crawford and Luoma, 1993; U.S. EPA, 1992c, 1992d;
Versar Inc., 1984), organochlorine pesticides (Crawford and Luoma, 1993;
Schmitt et al., 1983, 1985, 1990; U.S. EPA, 1992c, 1992d), and heavy metals
(Crawford and Luoma, 1993; Lowe et al., 1985; May and McKinney, 1981;
Schmitt and Brumbaugh, 1990; U.S. EPA, 1992c, 1992d). These three species
are commonly consumed in the areas in which they occur and have also
demonstrated an ability to accumulate high concentrations of environmental
contaminants in their tissues as shown in Tables 3-5 and 3-6. Note: The
average contaminant concentrations shown in Tables 3-5 and 3-6 for fish
collected for the National Study of Chemical Residues in Fish (U.S. EPA, 1992c,
1992d) were derived from concentrations in fish from undisturbed areas and from
areas expected to have elevated tissue contaminant concentrations. The mean
contaminant concentrations shown, therefore, may be higher or lower than those
found in the ambient environment because of site selection criteria used in this
study.
3-5
-------
3. TARGET SPECIES
Table 3-3. Comparison of Freshwater Finfish Species Used in Several
National Fish Contaminant Monitoring Programs
U.S. EPA
National
Dioxin Study
U.S. FWS
NPMP*and
NCBPb
U.S. EPA
NSCRF6
USGS
NAWQA"
BOTTOM FEEDERS
Family Cypmidao
Carp (Cyprinus carpio)
Family Ictaluridas
Channel catfish (Ictaluius punctatus)
Family Catostomidae
White sucker (Catastomus commersoni)
Longnoae sucker (C. catostomus)
Largescate sucker (C. macrocheilus)
Spotted sucker (Minytoema melanops)
Redhoree sucker (Moxosloma sp.)
included variety of species:
Silver redhorae (M. arisunim)
Grey redhorae (M. congestion)
Black redhorse (M. duquesnei)
Golden redhorse (M. oiythrurum)
Shorthead redhorse (M. macrolepidotum)
Blacktail redhoree (M. poedlumm)
Or other ictalurid
Or other catostomid
PREDATORS
Family Sahnonidae
Rainbow trout (Oncoihynchus myfcfss,1
[formerty Salmo gairdne^l
Brown trout (Salmo tn.
Brook ttout (Salvelinus fontinalis)
Lake trout (Salmo namaycush)
FamKy Perridae
WaUaye (Stizostedion vitneum)
Sauger (Stizostedion canadensa)
Yellow perch (Psrca flavescens)
Family Percichthyidae
White bass (Morone chtysops)
Family Centrarchidae
Largemouth bass(Microptetvssalmoides)
Smallmouth bass (Microptewsdolomieui)
Black crappie (Pomoxisrigromaculatus)
White crappie (Pomoxis annularis)
Bluegill sunfish (Lepomis maciochlrvs)
Family Esocidae
Northern pike (Esoxlucius)
Family Ictaluridao
Flathead catfish (Pylodictis olhraris)
Or other percid
O
O
Or other centrarchid
O
O
O
Or other percid
O
O
Or other centrarchid
O
O
O
• Recommended target species
O Alternate target species
Sources: Versar, Inc., 1964; Schmiltetal.. 1990; Sohmitt
Crawford and Luoma, 1993.
"National Pesticide Monitoring Program
''National Contaminant Biomonitoring Program
''National Study of Chemical Residues in Fish
"'National Water Quality Assessment Program
etal., 1983; May and McKinney, 1981; U.S. EPA, 1992c, 1992d;
3-6
-------
3. TARGET SPECIES
Table 3-4. Freshwater Turtles Recommended for Use as Target Species
Family name
Common name
Scientific name
Chelydridae
Emydidae
Trionychidae
Snapping turtle
Yellow-bellied turtle
Red-eared turtle
River cooter
Suwanee cooter
Slider
Texas slider
Florida cooter
Peninsula cooter
Smooth Softshell
Eastern Spiny Softshell
Western Spiny Softshell
Gulf Coast Spiny Softshell
Florida Softshell
Chelydra serpentina
Trachemys scripta scripta
Trachemys scripta elegans
Pseudemys concinna concinna
Pseudemys concinna suwanniensis
Pseudemys concinna hieroglyphica
Pseudemys concinna texana
Pseudemys floridana floridana
Pseudemys floridana penisularis
Apalone muticus
Apalone spinifera spinifera
Apalone spinifera hartwegi
Apalone spinifera aspera
Apalone ferox
In addition, these three species are relatively widely distributed throughout the
continental United States, and numerous States are already sampling these
species in their contaminant monitoring programs. A review of the database
National Listing of State Fish and Shellfish Consumption Advisories and Bans
(RTI, 1993) indicated that the largest number of States issuing advisories for
specific bottom-feeding species did so for carp (21 States) and channel catfish
(22 States), with eight States issuing advisories for white suckers (see Table
3-7). Appendix B lists the freshwater fish species cited in consumption
advisories for each State.
3.3.1.2 Predator Species
EPA recommends that, whenever practical, States use predator target species
listed in Tables 3-1 and 3-2 for inland fresh waters and Great Lakes waters,
respectively. Predator species, because of their more definitive habitat and
water temperature preferences, generally have a more limited geographic
distribution. Thus, a greater number of predator species than bottom feeders
have been used in national contaminant monitoring programs (Table 3-3) and
these are recommended for use as target species in freshwater ecosystems.
Predator fish that prefer relatively cold freshwater habitats include many
members of the following families: Salmonidae (trout and salmon), Percidae
3-7
-------
3. TARGET SPECIES
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3-8
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3. TARGET SPECIES
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3-9
-------
3. TARGET SPECIES
Table 3-7. Principal Freshwater Fish Species Cited in State Fish
Consumption Advisories"
Family name
Common name
Scientific name
Number of Statas
with advisories"
Perdchthyidae .
Centrarchidae
Perddae
Cyprinldae
Aclpenserldae
Catostomidae
Ictaluridaa
Sciaenidae
Esocidae
Salmonidae
White bass
Striped bass
White perch
Largemouth bass
Smallmouth bass
Black crappie
White crappie
Bluegill
Rock bass
Yellow perch
Sauger
Walleye
Common carp
Shovelnose sturgeon
Lake sturgeon
Smallmouth buffalo
Bigmoulh buffalo
Shorthead redhorse
White sucker
Quillback carpsucker
White catfish
Channel catfish
Flathead catfish
Black bullhead
Brown bullhead
Yellow bullhead
Freshwater drum
Northern pike
Muskellunge
Coho salmon
Chinook salmon
Brown trout
Lake trout
Rainbow trout
Brook trout
Lake whitefish
Morone chrysops
Morone saxatilis
Morone americana
Microptorus salmoidas
Micropterus dolomioui
Pomoxis nigromaculatus
Pomoxis annularis
Lepomis macrochirus
Ambloplites rupestris
Perca flavescens
Stizostedion canadense
Stizostadion vitroum
Cyprinus carpio
Scaphirhynchus platorynchus
Adpenser fulvescens
Ictiobus bubalus
Ictiobus cyprinellus
Moxostoma macrolepidotum
Catostomus commersoni
Carpiodes cyprinus
Ictalurus catus
Ictalurus punctatus
Pylodictis olivaris
Ictalurus melas
Ictalurus nebulosus
Ictalurus natalis
Aplodinotus grunniens
Esox lucius
Esox masquinongy
Oncorhynchus kisutch
Oncorhynchus tschawytscha
Salmo trutta
Salvelinus namaycush
Oncorhynchus mykissF
Salvelinus fontinalis
Coregonus clupea formis
10
6
4
15
9
5
2
5
3
8
4
9
21
1
2
4
4
2
8
2
5
22
4
2
7
2
3
7
4
6
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10
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3
2
Anouillidae
American eel
Anguilla rostrata
6
"Species in boldface are EPA-recommended target species for inland fresh waters (see Table 3-1) and the
Great Lakes waters (Table 3-2).
bMany States did not identify individual species of finfish in their advisories.
cFormerry Salmo gairdneri.
Source: RTI, 1993.
3-10
-------
3. TARGET SPECIES
(walleye and yellow perch), and Esocidae (northern pike and muskellunge).
Members of the Centrarchidae (large- and smallmouth bass, crappie, and
sunfish), Percichthyidae (white bass), and Ictaluridae (flathead catfish) families
prefer relatively warm water habitats. Only two predator species (brown trout
and largemouth bass) have been used in all four of the national monitoring
programs reviewed (Table 3-3). However, most of the other predator species
recommended as target species have been used in at least one national
monitoring program.
To identify those predator species with a known ability to bioaccumulate
contaminants in their tissues, the EPA Workgroup reviewed average tissue
concentrations of xenobiotic contaminants for major predator fish species
sampled in the National Study of Chemical Residues in Fish. Unlike the bottom-
feeders (common carp, channel catfish, and white suckers), no single predator
species or group of predator species consistently exhibited the highest tissue
concentrations for the contaminants analyzed (Tables 3-5 and 3-6). However,
average fish tissue concentrations for some contaminants (i.e., mercury, mirex,
chlorpyrifos, DDE, 1,2,3-trichlorobenzene [123-TCB], and trifluralin) were higher
for some predator species than for the bottom-feeders despite the fact that only
the fillet portion rather than the whole body was analyzed for predator species.
This finding emphasizes the need for using two types of fish (i.e., bottom-feeders
and predators) with different habitat and feeding strategies as target species.
The current fish consumption advisories for these predator target species are
further justification for their recommended use. As was shown for the bottom-
feeder target species, States are already sampling the recommended predator
target species listed in Table 3-7. The largest number of States issuing
advisories for specific predator species did so for largemouth bass (15), lake
trout (10), white bass (10), smallmouth bass (9), brown trout (9), walleye (9),
rainbow trout (8), yellow perch (8), Chinook salmon (7), northern pike (7), black
crappie (5), flathead catfish (4), and muskellunge (4) (RTI, 1993).
Because some freshwater finfish species (e.g., several Great Lake salmonids)
are highly migratory, harvesting of these species may be restricted to certain
seasons because sexually mature adult fish (i.e., the recommended size for
sampling) may make spawning runs from the Great Lakes into tributary streams.
EPA recommends that spawning populations not be sampled in fish contaminant
monitoring programs. Sampling of target finfish species during their spawning
period should be avoided because contaminant tissue concentrations may
decrease during this time (Phillips, 1980) and because the spawning period is
generally outside the legal harvest period. Note: Target finfish may be sampled
during their spawning period, however, if the species can be legally harvested
at this time.
State personnel, with their knowledge of site-specific fisheries and human
consumption patterns, must be the ultimate judge of the species selected for use
in freshwater fish contaminant monitoring programs within their jurisdiction.
3-11
-------
3. TARGET SPECIES
3.3.2 Target Turtle Species
EPA recommends that, in States where freshwater turtles are consumed by
recreational, subsistence, or ethnic populations, States consider monitoring
turtles to assess the level of environmental contamination and whether it poses
a human health risk. In all cases, the primary criterion for selecting the target
turtle species is whether it is commonly consumed. To identify those turtle
species with a known ability to bioaccumulate contaminants in their tissues, the
EPA Workgroup reviewed turtle species cited in State consumption advisories
and those species identified in the scientific literature as having accumulated
high concentrations of environmental contaminants.
Based on information in State advisories and a number of environmental studies
using turtles as biological indicators of pollution, one species stands out as an
obvious choice for a target species, the common snapping turtle (Chelydra
serpentine). This turtle has been recommended by several researchers as an
important bioindicator species (Olafsson et al., 1983; Stone et al., 1980) and has
the widest geographic distribution of any of the North American aquatic turtles
(see Figure 3-1). In addition, this species is highly edible, easily identified, easily
collected, long-lived (>20 years), grows to a large size, and has been extensively
studied with respect to a variety of environmental contaminants. Other species
that should be considered for use as target species are also listed in Table 3-4.
Four States (Arizona, Massachusetts, Minnesota, and New York) currently have
consumption advisories in force for various turtle species (New York State
Department of Health, 1994; RTI, 1993). The species cited in the State
advisories and the pollutants identified in turtle tissues as exceeding acceptable
levels of contamination with respect to human health are listed in Table 3-8.
New York State has a statewide advisory directed specifically at women of
childbearing age and children under 15 and advises these groups to avoid eating
snapping turtles altogether. The advisory also recommends that members of the
general population who wish to consume turtle meat should trim away all fat and
discard the liver tissue and eggs of the turtles prior to cooking the meat or
preparing other dishes. These three tissues have been shown to accumulate
extremely high concentrations of a variety of environmental contaminants in
comparison to muscle tissue (Bryan et al., 1987; Hebert et al., 1993; Olafsson
et al 1983; 1987; Ryan et ai., 1986; Stone et al., 1980). The Minnesota advisory
also recommends that consumers remove all fat from turtle meat prior to cooking
as a risk-reducing strategy (Minnesota Department of Health, 1994). States
should consider monitoring pollutant concentrations in all three tissues (fat, liver,
and eggs) in addition to muscle tissue if resources allow. If residue analysis
reveals the presence of high concentrations of any environmental contaminant
of concern, the State should consider making the general recommendation to
consumers to discard these three highly lipophilic tissues (fat, liver, and eggs)
to reduce the risk of exposure particularly to many organic chemical
contaminants.
3-12
-------
3. TARGET SPECIES
Source: Conant and Collins, 1991.
Figure 3-1. Geographic range of the common snapping turtle (Chelydra serpentine).
3-13
-------
3. TARGET SPECIES
Table 3-8. Principal Freshwater Turtle Species Cited
In State Consumption Advisories
Family name
Chetydridae
Trionychidae
Common name
Snapping turtle8
Snapping turtle8
(and other unspecified
turtle species)
Snapping turtleb
Western Spiny Softshell3
Scientific name
Chelydra serpentina
Chelydra serpentina
Chelydra serpentina
Apalone spiniferus
Pollutant
Mercury
PCBs
PCBs
DDT,
mercury
State
MN
MA
NY
AZ
PCB - Polychlorinated biphenyls.
DDT- 1,1,1-trichloro-2,2 bis(p-chlorophenyl)ethane.
"Source: RTI, 1993.
bSource: New York State Department of Health, 1994.
To identify those freshwater turtle species with a known ability to bioaccumulate
chemical contaminants in their tissues, the EPA Workgroup reviewed several
studies that identified freshwater turtle species as useful biomonitors of PCBs
(Bryan et al.F 1987; Hebert et al., 1993; Helwig and Hora, 1983; Olafsson et al.,
1983; 1987; Safe, 1987; and Stone et al., 1980), dioxins and dibenzofurans
(Rappe et al., 1981; Ryan et al., 1986), organochlorine pesticides (Hebert et al.,
1993; Stone et al., 1980), heavy metals (Helwig and Hora, 1983; Stone et al.,
1980), and radioactive nuclides (cesium-137 and strontium-90) (Lamb et al.,
1991; Scott et al., 1986). The turtle species used in these studies, the pollutants
monitored, and the reference sources are summarized in Table 3-9.
State personnel, with their knowledge of site-specific fisheries and human
consumption patterns, must be the ultimate judge of the turtle species selected
for use in contaminant monitoring programs within their jurisdictions. Because
several turtle species are becoming less common as a result of habitat loss or
degradation or overharvesting, biologists need to ensure that the target species
selected for the State toxics monitoring program is not of special concern within
their jurisdiction or designated as a threatened or endangered species. For
example, two highly edible turtle species, the Alligator snapping turtle
(Macroclemys temmincki) and the Northern diamondback terrapin (Malaclemys
terrapin terrapin) are protected in some States or designated as species of
concern within portions of their geographic range and are also potential
candidates for Federal protection (Sloan and Lovich, 1995). Although protected
to varying degrees by several States, George (1987) and Pritchard (1989)
concluded that the Alligator snapping turtle should receive range-wide protection
from the Federal government as a threatened species under the Endangered
Species Act. Unfortunately, basic ecological and life history information
3-14
-------
3. TARGET SPECIES
Table 3-9. Summary of Recent Studies Using Freshwater Turtles as
Blomonltors of Environmental Contamination
Species
Pollutant Monitored
Source
Snapping turtle
(Chelydra serpentina)
Snapping turtle
(Chelydra serpentina)
Snapping turtle
(Chelydra serpentina)
Snapping turtle
(Chelydra serpentina)
Snapping turtle
(Chelydra serpentina)
Snapping turtle
(Chelydra serpentina)
Snapping turtle
(Chelydra serpentina)
Snapping turtle
(Chelydra serpentina)
Yellow-bellied turtle
(Trachemys scripts)
Yellow-bellied turtle
(Trachemys scn'pta)
PCBs
Total DDT
Mirex
PCBs
PCBs
PCBs
Dioxins and furans
PCBs
Mercury
Cadmium
PCDFs
Organochlorine pesticides
DDE
Dieldrin
Hexachlorobenzene
Heptachlor epoxide
Mirex
PCBs
Cadmium
Mercury
Cesium-137
Strontium-90
Cesium-137
Strontium-90
Hebert et al., 1993
Olafsson et al., 1987
Olafsson et al., 1983
Safe, 1987
Bryan et al., 1987
Ryan et al., 1986
Helwig and Hora, 1983
Rappe et al., 1981
Stone et al., 1980
Lamb et al., 1991
Scott et al., 1986
PCBs = Polychlorinated biphenyls.
DDT = 1,1,1 -Trichloro-2,2 bis(p-chlorophenyl)ethane.
PCDFs = Polychlorinated dibenzofurans.
DDE - 1,1-Dichloro-2,2-bis(p-chlorophenyl)-ethylene.
3-15
-------
3. TARGET SPECIES
necessary to make environmental management decisions (i.e., Federal listing as
endangered or threatened species) is often not available for turtles and other
reptiles (Gibbons, 1988).
Several species of freshwater turtles already have been designated as
endangered or threatened species in the United States including the Plymouth
red-bellied turtle (Pseudemys rubriventris bangs!), Alabama red-bellied turtle
(Pseudemys alabamensis), Flattened musk turtle (Stemotherus depressus),
Ringed map (=sawback) turtle (Graptemys oculifera), and the Yellow-blotched
map (=sawback) turtle (Graptemys flavimaculata) (U.S. EPA, 1994; U.S. Fish
and Wildlife Service, 1994). In addition, all species of marine sea turtles
including the Green sea turtle (Chelonia mydas), Hawksbill sea turtle
(Eretmochelys imbricata), Kemp's ridley sea turtle (Lepidochelys kempii), Olive
ridley sea turtle (Lepidochelys olivacea), Loggerhead sea turtle (Caretta caretta),
and the Leatherback sea turtle (Dermochelys coriacea) have been designated
as endangered (U.S. EPA, 1994; U.S. Fish and Wildlife Service, 1994).
3.4 ESTUARINE/MARINE TARGET SPECIES
EPA recommends that States collect either one shellfish species (preferably a
bivalve mollusc) and one finfish species or two finfish species at each
estuarine/marine screening site. In all cases, the primary criterion for selecting
the target species is that it is commonly consumed. Ideally, one shellfish
species and one finfish species should be sampled; however, if no shellfish
species from the recommended target species list meets the primary criterion,
EPA recommends that States use two finfish species selected from the
appropriate regional estuarine/marine target species lists. If two finfish are
selected as the target species, one should be a bottom-feeding species.
EPA recommends that, whenever practical, States use target species selected
from fish and shellfish species identified in Tables 3-10 through 3-16 for the
following specific estuarine/marine coastal areas:
Northeast Atlantic region (Maine through Connecticut)—Table 3-10
Mid-Atlantic region (New York through Virginia)—Table 3-11
Southeast Atlantic region (North Carolina through Florida)—Table 3-12
Gulf Coast region (west coast of Florida through Texas)—Table 3-13
Pacific Northwest region (Alaska through Oregon)—Table 3-14
Northern California waters (Klamath River through Morro Bay)—Table 3-15
Southern California waters (Santa Monica Bay to Tijuana Estuary)—Table
3-16.
The seven separate regional lists of target species recommended by the EPA
Workgroup for estuarine/marine ecosystems were developed because of differ-
ences in species' geographic distribution and abundance and the nature of the
3-16
-------
3. TARGET SPECIES
Table 3-10. Recommended Target Species for Northeast Atlantic
Estuaries and Marine Waters (Maine through Connecticut)
Family name
^i$ii$ JlpttfaT " > ""
Anguillidae
Percichthyidae
Pomatomidae
Sparidae
Sciaenidae
Bothidae
Common name
" -' ;,
American eel
Striped bass
Bluefish
Scup
Weakfish
Summer flounder
Scientific name
•. " .. ;,;, ;
Anguilla rostrata
Morone saxatilis
Pomatomus saltatrix
Stenotomus chrysops
Cynoscion regalis
Paralichthvs dentatus
Pleuronectidae
Species
Bivalves
Crustaceans
Four-spotted flounder
Winter flounder
Yellowtail flounder
American dab
Soft-shell clam
Hard clam
Ocean quahog
Surf clam
Blue mussel
American lobster
Eastern rock crab
Paralichthys oblongus
Pseudopleuronectes
americanus
Limanda ferruginea
Hippoglossoides
platessoides
Mya arenaria
Mercenaria mercenaria
Arctica islandica
Spisula solidissima
MytHus edulis
Homarus americanus
Cancer irroratus
3-17
-------
3. TARGET SPECIES
Table 3-11. Recommended Target Species for Mid-Atlantic
Estuaries and Marine Waters (New York through Virginia)
Family name
Anguillidae
Icialun'dae
Percichthyidae
Pomatomidae
Sparidae
Sciaenidae
Bothidae
Pleuronectidae
Common name
>>,,,, % v -'
\ ..>.,''' f
American eel
Channel catfish
White catfish
White perch
Striped bass
Bluefish
Scup
Weakfish
Spot
Atlantic croaker
Red drum
Summer flounder
Winter flounder
Scientific name
Anguilla rostrata
Ictalurus punctatus
Ictalurus catus
Morone americana
Morone saxatilis
Pomatomus saltatrix
Stenotomus chrysops
Cynoscion regalis
Leistomus xanthurus
Micropogonias undulatus
Sciaenops ocellatus
Paralichthys dentatus
Pseudopleuronectes
americanus
Bivalves
Crustaceans
Hard clam
Soft-shell clam
Ocean quahog
Surf clam
Blue mussel
American oyster
Blue crab
American lobster
Eastern rock crab
Mercenaria mercenaria
Mya arenaria
Arctica islandica
Spisula solidissima
Mytilus edulis
Crassostrea virginica
Callinectes sapidus
Homarus americanus
Cancer irroratus
3-18
-------
3. TARGET SPECIES
Table 3-12. Recommended Target Species for Southeast Atlantic
Estuaries and Marine Waters (North Carolina through Florida)
Family name
Anguillidae
Ictaluridae
Percichthyidae
Sciaenidae
Bothidae
Bivalves
Crustaceans
Common name
American eel
Channel catfish
White catfish
White perch
Striped bass
Spot
Atlantic croaker
Red drum
Southern flounder
Summer flounder
Hard clam
American oyster
West Indies spiny lobster
Blue crab
Scientific name
Anguilla rostrata
Ictalurus punctatus
Ictalurus catus
Morone americana
Morone saxatilis
Leistomus xanthurus
Micropogonias undulatus
Sciaenops ocellatus
Paralichthys lethostigma
Paralichthys dentatus
Mercenaria mercenaria
Crassostrea virginica
Panulirus argus
Callinectes sapidus
3-19
-------
3. TARGET SPECIES
Table 3-13. Recommended Target Species for Gulf of Mexico
Estuaries and Marine Waters (West Coast of Florida through Texas)
Family name
Common name
Scientific name
Ictaluridae
Arildae
Sciaenidae
Bothidae
Blue catfish
Channel catfish
Hardhead catfish
Spotted seatrout
Spot
Atlantic croaker
Red drum
Gulf flounder
Southern flounder
American oyster
Hard clam
White shrimp
Blue crab
Gulf stone crab
West Indies spiny lobster
Ictalurus furcatus
Ictalurus punctatus
Arius felis
Cynoscion nebulosus
Leistomus xanthurus
Micropogonias undulatus
Sciaenops ocellatus
Paralichthys albigutta
Paralichthys lethostigma
Crassostrea virginica
Mercenaria mercenaria
Penaeus setiferus
Callinectes sapidus
Menippe adina
Panulirus argus
3-20
-------
3. TARGET SPECIES
Table 3-14. Recommended Target Species for Pacific Northwest
Estuaries and Marine Waters (Alaska through Oregon)
Family name
•f * VS S % %
Embiotocidae
Scorpaenidae
Bothidae
Pleuronectidae
Salmonidae
Sheimsh Species
Bivalves
Crustaceans
Common name
Redtail Surf perch
Copper rockfish
Black rockfish
Speckled sanddab
Pacific sanddab
Starry flounder
English sole
Coho salmon
Chinook salmon
Blue mussel
California mussel
Pacific oyster
Horseneck clam
Pacific littleneck clam
Soft-shell clam
Manila clam
Dungeness crab
Red crab
Scientific name
Amphistichus rhodoterus
Sebastes caurinus
Sebastes melanops
Citharichthys stigmaeus
Citharichthys sordidus
Platichthys stellatus
Parophrys vetulus
Onchorhynchus kisutch
Onchorhynchus tshawytscha
Mytilus edulis
Mytilus californianus
Crassostrea gigas
Tresus capax
Protothaca staminea
Mya arenaria
Venerupis japonica
Cancer magister
Cancer productus
3-21
-------
3. TARGET SPECIES
Table 3-15. Recommended Target Species for Northern California
Estuaries and Marine Waters (Klarnath River through Morro Bay)
Family name
'
f$rafpMli#**>'
§K«£s^w£«^i*iwK*R-. > ^ s.vv.'-v.'- s
Triakidae
Sciaenidae
Embiotocidae
Scorpaenidae
Bothidae
Pleuronectidae
Salmonidae
Common name
Leopard shark
White croaker
Redtailed surfperch
Striped seaperch
Black rockfish
Yellowtail rockfish
Bocaccio
Pacific sanddab
Speckled sanddab
Starry flounder
English sole
Coho salmon
Chinook salmon
Scientific name
Triakis semifasciata
Genyonemus lineatus
Amphistichus rhodoterus
Embiotoca lateralis
Sebastes melanops
Sebastes flavidus
Sebastes paucispinis
Citharichthys sordidus
Citharichthys stigmaeus
Platichthys stellatus
Parophrys vetulus
Onchorhynchus kisutch
Onchorhynchus tshawytscha
„ —.-
BOS'**'
Bivalves
Crustaceans
Blue mussel
California mussel
Pacific littleneck clam
Soft-shell clam
Dungeness crab
Red crab
Pacific rock crab
Mytilus edulis
Mytilus californianus
Protothaca staminea
Mya arenaria
Cancer magister
Cancer productus
Cancer antennarius
3-22
-------
3. TARGET SPECIES
Table 3-16. Recommended Target Species for Southern California
Estuaries and Marine Waters (Santa Monica Bay to Tijuana Estuary)
Family name
Common name
Serranidae
Sciaenidae
Embiotocidae
Scorpaenidae
Pleuronectidae
Bivalves
Crustaceans
Kelp bass
Barred sand bass
White croaker
Corbina
Black perch
Walleye surf perch
Barred surfperch
California scorpionfish
Widow rockfish
Blue rockfish
Bocaccio
Diamond turbot
Dover sole
Blue mussel
California mussel
Pacific littleneck clam
Pacific rock crab
Red crab
California rock lobster
Scientific name
Paralabrax clathratus
Paralabrax nebulifer
Genyonemus lineatus
Menticirrhus undulatus
Embiotoca jacksoni
Hyperprosopan argenteum
Amphistichus argenteus
Scorpaena guttata
Sebastes entomelas
Sebastes mystinus
Sebastes paucispinis
Hypsopetta guttulata
Microstomus pacificus
Mytilus edulis
Mytilus californianus
Protothaca staminea
Cancer antennarius
Cancer productus
Panulirus interruptus
3-23
-------
3. TARGET SPECIES
regional fisheries and were developed based on a review of species used in the
following national monitoring programs:
- National Dioxin Study (U.S. EPA)
Section 301 (h) Monitoring Program (U.S. EPA)
National Status and Trends Program (NOAA)
National Study of Chemical Residues in Fish (U.S. EPA).
Because some of these programs identified some fish and shellfish species that
are not of commercial, sportfishing, or subsistence value, several recent
literature sources identifying commercial and sportfishing species were also
reviewed (Table 3-17). Some sources included information on seasonal
distribution and abundance of various life stages (i.e., adults, spawning adults,
juveniles) of fish and shellfish species. This information was useful in delineating
seven regional estuarine/marine areas nationwide. The EPA Workgroup also
reviewed fish and shellfish species cited in State consumption advisories for
estuarine/marine waters (Appendix B). Each of the final regional lists of target
species has been reviewed by State, regional, and national fisheries experts.
Use of two distinct ecological groups of organisms (shellfish and finfish) as target
species in estuarine/marine systems is recommended. This permits monitoring
of a wide variety of habitats, feeding strategies, and physiological factors that
might result in differences in bioaccumulation of contaminants. Estuarine/marine
species used in several national contaminant monitoring programs are compared
in Table 3-18.
3.4.1 Target Shellfish Species
Selection of shellfish species (particularly bivalve molluscs) as target species
received primary consideration by the EPA Workgroup because of the
commercial, recreational, and subsistence value of shellfish in many coastal
areas of the United States. Bivalve molluscs (e.g., oysters, mussels, and clams)
are filter feeders that accumulate contaminants directly from the water column
or via ingestion of contaminants adsorbed to phytoplankton, detritus, and
sediment particles. Bivalves are good bioaccumulators of heavy metals
(Cunningham, 1979) and polycyclic aromatic hydrocarbons (PAHs) and other
organic compounds (Phillips, 1980; NOAA, 1987) and, because they are sessile,
they may reflect local contaminant concentrations more accurately than more
mobile crustacean or finfish species.
Three bivalve species—the blue mussel (Mytilus edulis), the California mussel
(Mytilus californianus), and the American oyster (Crassostrea virginica)—were
recommended and/or used in three of the national monitoring programs. Two
other bivalve species—the soft-shell clam (Mya arenaria) and the Pacific oyster
(Crassostrea gigas)—were also recommended and/or used in two national pro-
grams. Although no bivalve species was identified by name in State fish and
shellfish consumption advisories (Appendix B), seven coastal States issued
advisories for unspecified bivalves or shellfish species that may have included
3-24
-------
3. TARGET SPECIES
Table 3-17. Sources of Information on Commercial and Sportflshlng
Species In Various Coastal Areas of the United States
Goographic
•raa
Source
Atlantic Coast National Marine Fisheries Service. 1987. Marine Recreational Fishery Statistics Survey, Atlantic and Gulf
Coasts, 1986. Current Fishery Statistics Number 8392. National Oceanic and Atmospheric Administration,
U.S. Department of Commerce, Rockville, MD.
Leonard, O.L., M.A. Broutman, and K.E. Harkness. 1989. The Quality of Shellfish Growing Waters on the
East Coast of the United States. Strategic Assessment Branch, National Oceanic and Atmospheric
Administration, U.S. Department of Commerce, Rockville, MD.
Nelson, D.M., M.E. Monaco, E.A. Irlandi, L.R. Settle, and L Coston-Ctements. 1991. Distribution and
Abundance of Fishes and Invertebrates in Southeast Estuaries. ELMR Report No. 9. Strategic
Assessment Division. National Oceanic and Atmospheric Administration, U.S. Department of Commerce,
Rockville, MD.
Stone, S.L., T.A. Lowery, J.D. Field, C.D. Williams, D.M. Nelson, S.H. Jury, M.E. Monaco, and L.
Andreasen. 1994. Distribution and Abundance of Fishes and Invertebrates in Mid-Altantic Estuaries.
ELMR Rep. No. 12. NOAA/NOS Strategic Environmental Assessments Division, Sliver Spring, MD.
Gulf Coast National Marine Fisheries Service. 1987. Marine Recreational Fishery Statistics Survey, Atlantic and Gulf
Coasts, 1986. Current Fishery Statistics Number 8392. National Oceanic and Atmospheric Administration,
U.S. Department of Commerce, Rockville, MD.
Broutman, MA, and D.L Leonard. 1988. The Quality of Shellfish Growing Waters in the Gulf of Mexico.
Strategic Assessment Branch, National Oceanic and Atmospheric Administration, Rockville, MD.
Monaco, M.E., D.M. Nelson, T.C. Czapla, and M.E. Patillo. 1989. Distribution and Abundance of Fishes
and Invertebrates in Texas Estuaries. ELMR Report No. 3. Strategic Assessment Branch, National
Oceanic and Atmospheric Administration, U.S. Department of Commerce, Rockville, MD.
Williams, C.D., D.M. Nelson, M.E. Monaco, S.L Stone, C. lancu, L. Coston-Clements, LR. Settle, and E.A.
Irlandi. 1990. Distribution and Abundance of Fishes and Invertebrates in Eastern Gulf of Mexico
Estuaries. ELMR Report No. 6. Strategic Assessment Branch, National Oceanic and Atmospheric
Administration, U.S. Department of Commerce, Rockville, MD.
Czapla, T.C., M.E. Patillo, D.M. Nelson, and M.E. Monaco. 1991. Distribution and Abundance of Fishes
and Invertebrates in Central Gulf of Mexico Estuaries. ELMR Report No. 7. Strategic Assessment Branch,
National Oceanic and Atmospheric Administration, U.S. Department of Commerce, Rockville, MD.
Nelson, D.M. (editor). 1992. Distribution and Abundance of Fishes and Invertebrates in Gulf of Mexico
Estuaries, Volume I: Data Summaries. ELMR Rep. No. 10. NOAA/NOS Strategic Environmental
Assessments Division, Rockville, MD.
West Coast National Marine Fisheries Service. 1987. Marine Recreational Fishery Statistics Survey, Pacific Coast,
1986. Current Fishery Statistics Number 8393. National Oceanic and Atmospheric Administration, U.S.
Department of Commerce, Rockville, MD.
Leonard, D.L., and E.A. Slaughter. 1990. The Quality of Shellfish Growing Waters on the West Coast of
the United States. Strategic Assessment Branch, National Oceanic and Atmospheric Administration, U.S.
Department of Commerce, RockviHe, MD.
Monaco, M.E., D.M. Nelson, R.L. Emmett, and S.A. Hinton. 1990. Distribution and Abundance of Fishes
and Invertebrates in West Coast Estuaries. Volume I: Data Summaries. ELMR Report No. 4. Strategic
Assessment Branch, National Oceanic and Atmospheric Administration, Rockville, MD.
Emmett, R.L, S.A. Hinton, S.L. Stone, and M.E. Monaco. 1991. Distribution and Abundance of Fishes
and Invertebrates in West Coast Estuaries. Volume II: Life History Summaries. ELMR Report No. 8.
Strategic Environmental Assessment Division, Rockville, MD.
Jury, S.H., J.D. Field, S.L Stone, D.M. Nelson, and M.E. Monaco. 1994. Distribution and Abundance of
Fishes and Invertebrates in North Atlantic Estuaries. ELMR Rep. No. 13. NOAA/NOS Strategic
Environmental Assessments Division, Sliver Spring, MD.
3-25
-------
3. TARGET SPECIES
Table 3-18. Estuarine/Marle Species Used In Several National Fish and Shellfish
Contaminant [Monitoring Programs
RNF1SH ' V > V •^'-\-S^;:v^^:il-^;S^:^-:--:*!^J-^^
Family Acipenseridas
White sturgeon (Acipensertransmontanus)
Family Ariidae
Hardhead catfish (Arius falls)
Family Perchhthyidae
White perch (Morone americana)
Family Pomatomidae
Bluefish (Pomatomus saftatrix)
Family Lutjanldae
Red snapper (Lutjanus campechanus)
Family Sparidae
Sheepshead (Archosargus probatocephalus)
Family Sciaenidae
Spotted seatrout (Cynoscion nebulosus)
Weakfish (Cynoscion regalis)
Spot (Leiostomus xanthurus)
White croaker (Genyonemus lineatus)
Atlantic croaker (Micropogonias undulatus)
Black drum (Pogonias cromis)
Red drum (Sclaenpps ocellatus)
Family Serranidae
Barred sand bass (Paralabrax nebulifer)
Family Mugilidae
Striped mullet (Mugil cephalus)
Family Bothldas
Southern flounder (Paralichthys lethostigma)
Windowpane flounder (Scophthalmus aquosus)
Family Pleuronectidae
Pacific sanddab (Citharichthys sordidus)
Flathead sole (Hlppoglossoides elassodon)
Diamond turbot (Hypsopsetta guttulata)
Starry flounder (Platichthys stellatus)
Homyhead turbot (Pleuronichthys verticalis)
Winter flounder (Pseudopleuronectes americanus)
English sole (Parophrys vetulus)
Dover sole (Microstomus pacificus)
U.S. EPA
National
Dloxin
Study*
• .'•;','"• ••::• .•-- • .-: '
'-',''. '.'-'•', '•'•'• ' • ' -'••- '
NOAA
Status
and
Trends
•
•
•
•
•
U.S. EPA
301 (h)
Program
', V>-:':t ;•%;/• ir
•
•
•
«
•
U.S. EPA
NSCRFb(C
•
•
•
•
•
•
•
•
•
•
•
See notes at end of table.
(continued)
3-26
-------
3. TARGET SPECfES
Table 3-18 (continued)
SHELLFISH
Bivalves
Hard clam (Marcenaria mercenaria)
Soft-shell clam (Mya arenaria)
Ocean quahog (Arctica islandia)
Surf clam (Spisula solidissima)
Blue mussel (Mytilus edulis)
California mussel (Mytilus califomianus)
American oyster (Crassostrea virginica)
Hawaiian oyster (Ostrea sandwichensis)
Pacific oyster (Crassostrea gigas)
Bent-nosed macoma (Macoma nasuta)
Baltic macoma (Macoma baltica)
White sand macoma (Macoma secta)
Crustaceans
American lobster (Homarus americanus)
West Indies spiny lobster (Panulirus argus)
California rock lobster (Panulirus interrupts)
Hawaiian spiny lobster (Panulirus penicillatus)
Eastern rock crab (Cancer irroratus)
Dungeness crab (Cancer magister)
Pacific rock crab (Cancer antennarius)
Yellow crab (Cancer anthonyi)
Red crab (Cancer productus)
U.S. EPA
National •
Dioxln
Study"
j
•
•
•
NOAA
Status
and
Trends
•
•
•
•
U.S. EPA
301 (h)
Program
U.S. EPA
NSCRF"-0
•
•
•
"Only freshwater finfish were identified as target species; bivalves were identified as estuarin e/marine target species.
bSpecies listed were those collected at more than one site nationally; Salmonidae were not listed because they were
included on freshwater lists.
°National Study of Chemical Residues in Rsh.
3-27
-------
3. TARGET SPECIES
these and other bivalve species. All three species are known to bioaccumulate
a variety of environmental contaminants (Phillips, 1988). The wide distribution
of these three species makes them useful for comparisons within a State or
between States sharing coastal waters (Figure 3-2). Because these three
species meet all of the selection criteria, they are recommended as target
species for use in geographic areas in which they occur.
In addition, several species of edible clams were added to the various
estuarine/marine target species lists based on recommendations received from
specific State and regional fisheries experts.
Crustaceans are also recommended as target species for estuarine/marine
sampling sites. Many crustaceans are bottom-dwelling and bottom-feeding
predator and/or scavenger species that are good indicators of contaminants that
may be biomagnified through several trophic levels of the food web. Several
species of lobsters and crabs have been recommended in one national
monitoring program, and the Dungeness crab has been recommended in two
national monitoring programs (Table 3-18). These crustaceans, although of
fishery value in many areas, are not as widely distributed nationally as the three
bivalve species (Figure 3-2). However, they should be considered for selection
as target species in States where they are commonly consumed.
Only two crustaceans—the American lobster (Homarusamericanus) and the blue
crab (Callinectes sapidus)—were specifically identified in State advisories (RTI,
1993). However, seven coastal States reported advisories in estuarine/marine
waters for unspecified shellfish species that may have included these and other
crustacean species (Table 3-19). All of the shellfish species cited in State
advisories are included as EPA-recommended target species on the appropriate
estuarine/marine regional lists.
3.4.2 Target Finfish Species
Two problems are encountered in the selection of target finfish species for
monitoring fish tissue contamination at estuarine/marine sites regionally and
nationally. First is the lack of finfish species common to both Atlantic and Gulf
Coast waters as well as Pacific Coast waters. Species used in several Federal
fish contaminant monitoring programs are compared in Table 3-18. Members
of the families Sciaenidae (seven species), Bothidae (two species), and
Pleuronectidae (eight species) were used extensively in these programs.
Bottom-dwelling finfish species (e.g., flounders in the families Bothidae and
Pleuronectidae) may accumulate high concentrations of contaminants from direct
physical contact with contaminated bottom sediments. In addition, these finfish
feed on sedentary infaunal or epifaunal organisms and are at additional risk of
accumulating contaminants via ingestion of these contaminated prey species
(U.S. EPA, 1987a). For finfish species, two Atlantic coast species, spot
(Leiostomus xanthurus) and winter flounder (Pseudopleuronectes americanus),
are recommended and/or used in three of the national monitoring programs, and
the Atlantic croaker (Micropogonias undulatus) is recommended and/or used in
3-28
-------
3. TARGET SPECIES
CD
O
o
1
o
3-29
-------
3. TARGET SPECIES
Table 3-19. Principal Estuarlne/Marlne Fish and Shellfish Species Cited In State
Consumption Advisories8'*
Species
group name
Percichthyldae
Ictaluridae
Anguillidae
Pomatomidae
Belonidae
Serranidae
Sciaenidae
Crustacean^
Common name
^S™ \^s '•• s •> ••
Striped bass
White perch
White catfish
Channel catfish
American eel
Bluefish
Atlantic needlefish
Kelp bass
Black croaker
White croaker
Queenfish
Corbina
7^7 ^ ^
American lobster
Blue crab
Scientific name
- f
Morone saxatilis
Morone americana
Ictalurus catus
Ictalurus punctatus
Anguilla rostrata
Pomatomus saltatrix
Strongylura marina
Paralabrax clathratus
Cheilotrema saturnum
Genyonemus lineatus
Seriphus politus
Menticirrhus undulatus
", ' , ,,,
Homarus americanus
Callinectes sapidus
Number of States
with advisories
'-' ',-''-.
5
3
4
5
6
4
1
1
1
1
1
1
1
3
a Species in boldface are EPA-recommended target species for regional estuarine/marine waters (see
Tables 3-10 through 3-16).
b Many coastal States issued advisories for fish and shellfish species and thus did not identify specific
finfish and shellfish species in their advisories.
6 Seven coastal States (American Samoa, California, Louisiana, Massachusetts, New Jersey, South
Carolina, and Texas) report advisories for unspecified shellfish or bivalve species.
Source: RTI, 1993.
3-30
-------
3. TARGET SPECIES
two national monitoring programs. Three Pacific coast species, Starry flounder
(Platichthys stellatus), English sole (Parophrys vetulus), and Dover sole
(Microstomus pacificus), are recommended or used in two of the national
monitoring programs.
Second, because some estuarine/marine finfish species are highly migratory,
harvesting of these species may be restricted to certain seasons because
sexually mature adult fish (i.e., the recommended size for sampling) may enter
the estuaries only to spawn. ' EPA recommends that neither spawning
populations nor undersized juvenile stages be sampled in fish contaminant
monitoring programs. Sampling of target finfish species during their spawning
period should be avoided as contaminant tissue concentrations may decrease
during this time (Phillips, 1980) and because the spawning period is generally
outside the legal harvest period. Note: Target finfish species may be sampled
during their spawning period if the species can be legally harvested at this time.
Sampling of undersized juveniles of species that use estuaries as nursery areas
is precluded by EPA's recommended monitoring strategy because juveniles may
not have had sufficient time to bioaccumulate contaminants or attain harvestable
size.
Because of these problems, the EPA Workgroup consulted with regional and
State fisheries experts and reviewed the list of current State fish consumption
advisories and bans to determine which estuarine/marine finfish species should
be recommended as target species. As shown in Table 3-19, the largest number
of States issuing advisories for specific estuarine and marine waters did so for
the American eel (6), channel catfish (5), striped; bass (5), bluefish (4), white
catfish (4), and white perch (3). Several other estuarine/marine species were
cited in advisories for one State each (Table 3-19). Many coastal States did not
identify individual finfish species by name in their advisories (see Appendix B);
however, almost all of the species that have been cited in State advisories are
recommended as target species by EPA (see Tables 3-10 through 3-16).
These seven regional lists of recommended estuarine/marine target species are
provided to give guidance to States on species commonly consumed by the
general population. State personnel, with their knowledge of site-specific
fisheries and human consumption patterns, must be the ultimate judge of the
species selected for use in estuarine/marine fish contaminant monitoring
programs within their jurisdiction.
3-31
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4. TARGET ANALYTES
SECTION 4
TARGET ANALYTES
The selection of appropriate target analytes in fish and shellfish contaminant
monitoring programs is essential to the adequate protection of the health of fish
and shellfish consumers. The procedures used for selecting target analytes for
screening studies and a list of recommended target analytes are presented in
this section.
4.1 RECOMMENDED TARGET ANALYTES
Recommended target analytes for screening studies in fish and shellfish
contaminant monitoring programs are listed in Table 4-1. This list was
developed by the EPA Fish Contaminant Workgroup from a review of the
following information:
1. Pollutants analyzed In several national or regional fish contaminant
monitoring programs—The monitoring programs reviewed included
• National Study of Chemical Residues in Fish (U.S. EPA)
• National Dioxin Study (U.S. EPA)
• 301 (h) Monitoring Program (U.S. EPA)
• National Pollutant Discharge Elimination System (U.S. EPA)
National Pesticide Monitoring Program (U.S. FWS)
National Contaminant Biomonitoring Program (U.S. FWS)
• National Status and Trends Program (NOAA)
Great Lakes Sportfish Consumption Advisory Program
FDA recommendations
National Water-Quality Assessment Program (USGS).
Criteria for selection of the target analytes in these programs varied widely
depending on specific program objectives. The target analytes used in these
major fish contaminant monitoring programs are compared in Appendix C.
Over 200 potential contaminants are listed, including metals, pesticides,
base/neutral organic compounds, dioxins, dibenzofurans, acidic organic
compounds, and volatile organic compounds.
2. Pesticides with active registrations—The EPA Office of Pesticide
Programs (OPPs) Fate One Liners Database (U.S. EPA, 1993a) containing
information for more than 900 registered pesticides was reviewed to identify
pesticides and herbicides with active registrations that met four criteria. The
screening criteria used were
4-1
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4. TARGET ANALYTES
Table 4-1. Recommended Target Analytes*
Itotals
Arsenic (inorganic)
Cadmium
Mercury
Selenium
Tributyltin
Ofqanochlofine Pesticides
Chlordane, total (cis- and trans-chlordane,
els- and trans-nonachlor, oxychlordane)
DDT, total (2,4'-DDD, 4,4'-DDD, 2,4'-DDE,
4,4'-DDE, 2,4'-DDT, 4,4'-DDT)
Dicofol
Dieldrin
Endosulfan (I and II)
Endrin
Heptachlor epoxide
Hexachlorobenzene
LJndane (y-hexachlorocydohexane; y-HCH)c
Mirex0
Toxaphene
Orqanophosphate Pesticides"
Chlorpyrifos
Diazinon
Disulfoton
Ethion
Terbufos
Chlorophenoxv Herbicides
Oxyfluorfen
PAHS'
PCBs
Total Aroclors0
Dioxins/furansh|1
PAHs - PotycycKc aromatic hydrocarbons.
PCBs = Polychlorinated biphenyls.
• -States should include all recommended target analytos in screening studies, if resources allow, unless historic tissue or
sediment data indicate that an analyte is not present at a level of concern for human health. Additional target analytes
should be included in screening studies if States have site-specific information (e.g., historic tissue or sediment data,
discharge monitoring reports from municipal and industrial sources, pesticide use application information) that these
chemicals may be present at levels of concern for human health.
b Heptachlor epoxide is not a pesticide but is a metabolite of the pesticide heptachlor.
0 Also known as Y-benzene hexachloride (y-BHC).
d Mirex should be regarded primarily as a regional target analyte in the southeast and Great Lakes States, unless historic
tissue, sediment, or discharge data indicate the likelihood of its presence in other areas.
• The reader should note that carbophenothton was included on the original list of target analytes. Because the registrant did
not support reragistration of this chemical, it will no longer be used. For this reason and because of its use profile,
carbophenothion was removed from the recommended list of target analytes.
1 It is recommended that, in both screening and intensive studies, tissue samples be analyzed for benzo[a]pyrene, benzfa]-
anthracene, benzo[b]fluoranthene, benzo[A]fluoranthene, chrysene, dibenz[a,/?]anthracene, and indeno/?,2,3-co]pyrene. and
that the order-of-magnitude relative potencies given for these PAHs in the EPA provisional guidance for quantitative risk
assessment of PAHs (U.S. EPA, 1993c) be used to calculate a potency equivalency concentration (PEC) for each sample
for comparison with the recommended SV for benzo[a]pyrene (see Section 5.3.2.3). At this time, EPA's recommendation for
risk assessment of PAHs (U.S. EPA, 1993c) is considered provisional because quantitative risk assessment data are not
avaDabte for all PAHs. This approach is under Agency review and over the next year will be evaluated as new health effects
benchmark values are developed. Therefore, the method provided in this guidance document is subject to change pending
results of the Agency's revaluation.
0 Analysis of total PCBs, as the sum of Arodor equivalents, is recommended in both screening and intensive studies because
of the lack of adequate toxicologic data to develop screening values (SVs) for individual PCB congeners (see Section 4.3.5).
However, because of the wide range of toxidties among different PCB congeners and the effects of metabolism and degra-
dation on Arodor composition in the environment, congener analysis is deemed to be a more scientifically sound and accu-
rate method for determining total PCB concentrations. Consequently, States that currently do congener-specific PCB
analyses should continue to do so. Other States are encouraged to develop the capability to conduct PCB congener
analysis.
h Note: The EPA Office of Research and Development is currently reassessing the human health effects of dioxins/furans.
' Dioxins/furans should be considered for analysis primarily at sites of pulp and paper mills using a chlorine bleaching process
and at industrial sites where the following organic compounds are formulated: herbicides (containing 2,4,5-trichlorophenoxy
acids and 2,4,5-trichtorophenol), hexachlorophene, pentachlorophenol, and PCBs (U.S. EPA, 1987d). It is recommended
that the 2.3,7,8-substituted tetra- through octa-chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) be
determined and a toxicity-weighted total concentration calculated for each sample (Barnes and Bellin, 1989; U.S. EPA,
1987d) (see Section 5.3.2.4). If resources are limited. 2,3,7.8-TCDD and 2,3,7,8-TCDF should be determined at a minimum.
4-2
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4. TARGET ANALYTES
• Oral toxicity, Class I or II
• Bioconcentration factor greater than 300
Half-life value of 30 days or more
Initial use application profile.
At the time of this review, complete environmental fate information was
available for only about half of the registered pesticides. As more data
become available, additional pesticides will be evaluated for possible
inclusion on the target analyte list.
Use of the OPP Database was necessary because many pesticides and
herbicides with active registrations have not been monitored extensively
either in national or State fish contaminant monitoring programs.
3. Contaminants that have triggered States to issue fish and shellfish
consumption advisories or bans—The database, National Listing of State
Fish and Shellfish Consumption Advisories and Bans (RTI, 1993), was
reviewed to identify specific chemical contaminants that have triggered
issuance of consumption advisories by the States. As shown in Table 4-2,
four contaminants have triggered advisories in the largest number of States:
polychlorinated biphenyls (PCBs), mercury, chlordane, and dioxins/furans.
4 Published literature on the chemistry and health effects of potential
contaminants—The physical, chemical, and toxicologic factors considered
to be of particular importance in developing the recommended target analyte
list were
Oral toxicity
Potential of the analyte to bioaccumulate
Prevalence and persistence of the analyte in the environment
• Biochemical fate of the analyte in fish and shellfish
• Human health risk of exposure to the analyte via consumption of
contaminated fish and shellfish
Analytical feasibility.
Final selection of contaminants for the recommended target analyte list (Table
4-1) was based on their frequency of inclusion in national monitoring programs,
on the number of States issuing consumption advisories for them, and on their
origins, chemistry, potential to bioaccumulate, estimated human health risk, and
feasibility of analysis. Primary consideration was given to the recommendations
of the Committee on Evaluation of the Safety of Fishery Products, published in
Seafood Safety (NAS, 1991), and to the recommendations of the EPA Fish
Contaminant Workgroup.
4-3
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4. TARGET ANALYTES
Table 4-2. Contaminants Resulting in Fish and Shellfish Advisories
Contaminant
Number of States
Issuing advisories
Metals
Arsenic (total)
Cadmium
Chromium
Copper
Lead
Mercury
Selenium
Zinc
Organometallics
Unidentified metals
Pesticides
Chlordane
DDT and metabolites
Dieldrin
Heptachlor epoxide
Hexachlorobenzene
Kepone
Mirex
Photomirex
Toxaphene
Unidentified pesticides
Polycycllc aromatic hydrocarbons (PAHs)
Polychlorlnated blphenyls (PCBs)
Dloxlns/furans
Other chlorinated organlcs
Dichlorobenzene
Hexachlorobutadiene
Pentachlorobenzene
Pentachlorophenol
Tetrachlorobenzene
Tetrachloroethane
Others
Creosote
Gasoline
Multiple pollutants
Phthalate esters
Polybrominated biphenyls (PBBs)
Unspecified pollutants
1
2
1
1
4
27
5
1
1
3
24
9
3
1
2
1
3
1
2
2
3
31
22
1
1
1
1
2
1
2
1
2
1
1
3
Source: RTI, 1993.
4-4
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4. TARGET ANALYTES
4.2 SELECTION OF TARGET ANALYTES
States should include all recommended target analytes (Table 4-1) in screening
studies, if resources allow, unless historic tissue or sediment or pollutant source
data indicate that an analyte is not present at a level of concern (see Section 5).
For the pesticides with active registrations, use and rate application information
maintained by the State's Department of Agriculture should be reviewed to
identify watersheds where these pesticides have been used historically or are
currently used and are likely to be present in aquatic systems as a result of
agricultural runoff or drift.
It is important to note that pesticide uses and labels may change over time. The
State agency responsible for designing the fish contaminant monitoring program
should be aware of all historic and current uses of each pesticide within its State,
including the locations, application rates, and acreage where the pesticide has
been or currently is applied to ensure that all potentially contaminated sites are
included in the sampling plan.
Additional target analytes should be included in screening programs if States
have site-specific chemical information (e.g., historic tissue or sediment data,
discharge monitoring reports from municipal and industrial sources, or pesticide
use data) that these contaminants may be present at levels of concern for
human health. Compounds that are currently under review by the EPA Office
of Water for inclusion as recommended target analytes are discussed in Section
4.4. Specific factors that were considered in the selection of currently
recommended target analytes are summarized in the following sections.
4.3 TARGET ANALYTE PROFILES
4.3.1 Metals
Five metals—arsenic, cadmium, mercury, selenium and tributyltin—are
recommended as target analytes in screening studies. Arsenic, cadmium, and
mercury have been included in six major fish contaminant monitoring programs
(see Appendix C). It should be noted, however, that with respect to arsenic, all
monitoring programs measured total arsenic rather than inorganic arsenic.
Selenium has been monitored in five national monitoring programs. Tributyltin
has been recommended for analysis in the FDA monitoring program.
Consumption advisories are currently in effect for arsenic, cadmium, mercury,
selenium, and tributyltin in one, two, twenty-seven, five, and one States
respectively (Table 4-2). Also, with the exception of tributyltin, these metals have
been identified as having the greatest potential toxicity resulting from ingestion
of contaminated fish and shellfish (NAS, 1991).
4-5
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4. TARGET ANALYTES
4.3.1.1 Arsenic
Arsenic is the twentieth most abundant element in the earth's crust and naturally
occurs as a sulfide in a variety of mineral ores containing copper, lead, iron,
nickel, cobalt, and other metals (Eisler, 1988; Merck Index, 1989; Woolson,
1975). Arsenic is released naturally to the atmosphere from volcanic eruptions
and forest fires (Walsh et al., 1979) and to water via natural weathering
processes (U.S. EPA, 1982b). Arsenic also has several major anthropogenic
sources including industrial emissions from coal-burning electric generating
facilities, releases, as a byproduct of nonferrous-metal (gold, silver, copper, lead,
uranium, and zinc) mining and smelting operations (Eisler, 1988; May and
McKinney, 1981; NAS, 1977), releases associated with its production and use
as a wood preservative (primarily as arsenic trioxide), and application as an
insecticide, herbicide, algicide, and growth stimulant for plants and animals
(Eisler, 1988). Releases are also associated with leaching at hazardous waste
disposal sites and discharges from sewage treatment facilities. Arsenic trioxide.
is the arsenic compound of chief commercial importance (U.S. EPA, 1982b) and
was produced in the United States until 1985 at the ASARCO smelter near
Tacoma, Washington. Arsenic is no longer produced commercially within the
United States in any significant quantities, but arsenic compounds are imported
into the United States primarily for use in various wood preservative and
pesticide formulations.
The toxicity of arsenicals is highly dependent upon the nature of the compounds,
and particularly upon the valency state of the arsenic atom (Frost, 1967;
Penrose, 1974; Vallee et al., 1960). Typically, compounds containing trivalent
(+3) arsenic are much more toxic that those containing pentavalent (+5) arsenic.
The valency of the arsenic atom is a more important factor in determining toxicity
than the organic or inorganic nature of the arsenic-containing compound
(Edmonds and Francesconi, 1993). With respect to inorganic arsenic
compounds, salts of arsenic acid (arsenates) with arsenic in the pentavalent
state are less toxic than arsenite compounds with arsenic in the trivalent state
(Penrose, 1974). Because some reduction of arsenate (pentavalent arsenic) to
arsenite (trivalent arsenic) might occur in the mammalian body (Vahter and
Envall, 1983), it would be unwise to disregard the possible toxicity of inorganic
arsenic ingested in either valency state (Edmonds and Francesconi, 1993).
Seafood is a major source of trace amounts of arsenic in the human diet.
However, arsenic in the edible parts of fish and shellfish is predominantly present
as the arsenic-containing organic compound arsenobetaine (Cullen and Reimer,
1989; Edmonds and Francesconi, 1987a; NAS, 1991). Arsenobetaine is a stable
compound containing a pentavalent arsenic atom, which has been shown to be
metabolically inert and nontoxic in a number of studies (Cannon et al., 1983;
Jongen et al., 1985; Kaise et al., 1985; Sabbioni et al., 1991; Vahter et al.,
1983), and is not generally considered a threat to human health (ATSDR, 1989).
Inorganic arsenic, although a minor component of the total arsenic content of fish
and shellfish when compared to arsenobetaine, presents potential toxicity
problems. To the degree that inorganic forms of arsenic are either present in
4-6
-------
4. TARGET ANAL YTES
seafood or, upon consumption, may be produced as metabolites of organic
arsenic compounds in seafood, some human health risk, although small, would
be expected (NAS, 1991).
Inorganic arsenic is very toxic to mammals and has been assigned to Toxicity
Class I based on oral toxicity tests (Farm Chemicals Handbook, 1989). Use of
several arsenical pesticides has been discontinued because of the health risks
to animals and man. Inorganic arsenic also has been classified as a human
carcinogen (A) (IRIS, 1995) and long-term effects include dermal hyperkeratosis,
dermal melanosis and carcinoma, hepatomegaly, and peripheral neuropathy
(NAS, 1991) (Appendix D).
Total arsenic (inclusive of both inorganic and organic forms) has been included
in six national monitoring programs (Appendix C); however, no national program
is currently monitoring total inorganic arsenic in fish or shellfish tissues. Arsenic
and arsenic-containing organic compounds have not been shown to
bioaccumulate to any great extent in aquatic organisms (NAS, 1977).
Experimental evidence indicates that inorganic forms of both pentavalent and
trivalent arsenic bioaccumulate minimally in several species of finfish including
rainbow trout, bluegill, and fathead minnows (ASTER, 1995). A BCF value of
350 was reported for the American oyster (Crassostrea virginica) exposed to
trivalent arsenic (Zaroogian and Hoffman, 1982). Only one State (Oregon)
currently has an advisory in effect for arsenic contamination (RTI, 1993).
Edmonds and Francesconi (1993) summarized existing data from studies
conducted outside the United States comparing concentrations of total arsenic,
organic arsenic, and inorganic arsenic in marine fish and shellfish. Inorganic
arsenic was found to represent from 0 to 44 percent of the total arsenic in
marine fish and shellfish species surveyed. Residue concentrations of inorganic
arsenic in the tissues typically ranged from 0 to 5.6 ppm (wet weight basis); but
were generally less than 0.5 ppm for most species. In a study of six species of
freshwater fish monitored as part of the Lower Columbia River study, inorganic
arsenic represented from 0.1 to 27 percent of the total arsenic, and tissue
residues of inorganic arsenic ranging from 0.001 to 0.047 ppm (wet weight) were
100 times lower than those reported for marine species (Tetra Tech, 1995).
Because it is the concentration of inorganic arsenic in fish and shellfish that
poses the greatest threat to human health, EPA recommends that total inorganic
arsenic (not total arsenic) be analyzed in contaminant monitoring programs.
Total inorganic arsenic should be considered for inclusion in State fish and
shellfish monitoring programs in areas where its use is or has been extensive.
States should contact their appropriate State agencies to obtain information on
the historic and current uses of arsenic particularly as a wood preservative and
in agricultural pesticides.
4-7
-------
4. TARGET ANALYTES
4.3.1.2 Cadmium—
Cadmium is commonly found in zinc, lead, and copper deposits (May and
McKinney, 1981). It is released into the environment from several anthropogenic
sources: smelting and refining of ores, electroplating, application of phosphate
fertilizers, surface mine drainage (U.S. EPA, 1978), and waste disposal
operations (municipal incineration and land application) (U.S. EPA, 1979a,
1987c). Cadmium is also used in the manufacture of paints, alloys, batteries,
and plastics and has been used in the control of moles and plant diseases in
lawns.
Cadmium is a cumulative human toxicant; it has been shown to cause renal
dysfunction and a degenerative bone disease, itai-itai, in Japanese populations
exposed via consumption of contaminated rice, fish, and water. Because
cadmium is retained in the kidney, older individuals (over 40-50 years of age)
typically have both the highest renal concentrations of cadmium and the highest
prevalence of renal dysfunction (U.S. EPA, 1979a). Cadmium is a known
carcinogen in animals, and there is limited evidence of the carcinogenicity of
cadmium or cadmium compounds in humans. It has been classified as a
probable human carcinogen by inhalation (B1) by EPA (IRIS, 1992).
Cadmium has been found to bioaccumulate in fish and shellfish tissues in fresh
water (Schmitt and Brumbaugh, 1990) and in estuarine/marine waters (NOAA,
1987, 1989a) nationwide. In the National Contaminant Biomonitoring Program
(NCBP), geometric mean concentrations of cadmium in freshwater fish were
found to have declined from 0.07 ppm in 1976 to 0.03 ppm in 1984 (Schmitt and
Brumbaugh, 1990). This trend contradicts the general trend of increasing
cadmium concentrations in surface waters, which Smith et al. (1987) attribute to
increasing U.S. coal combustion (Schmitt and Brumbaugh, 1990). Two States
(New York and Ohio) have issued advisories for cadmium contamination (RTI,
1993).
Cadmium should be considered for inclusion in all State fish and shellfish
contaminant monitoring programs.
4.3.1.3 Mercury-
The major source of atmospheric mercury is the natural degassing of the earth's
crust, amounting to 2,700 to 6,000 tons per year (WHO, 1990). Primary points
of entry of mercury into the environment from anthropogenic sources are
industrial discharges and wastes (e.g., the chlorine-alkali industry) and
atmospheric deposition resulting from combustion of coal and municipal refuse
incinerators (Glass et al., 1990). Primary industrial uses of mercury are in the
manufacture of batteries, vapor discharge lamps, rectifiers, fluorescent bulbs,
switches, thermometers, and industrial control instruments (May and McKinney,
1981), and these products ultimately end up in landfills or incinerators. Mercury
has also been used as a slimicide in the pulp and paper industry, as an
antifouling and mildew-proofing agent in paints, and as an antifungal seed
4-8
-------
4. TARGET ANALVTES
dressing and in chlor-alkali production facilities (Farm Chemicals Handbook,
1989; Friberg and Vostal, 1972).
Although mercury use and losses from industrial processes in the United States
have been reduced significantly since the 1970s, mercury contamination
associated with increased fossil fuel combustion is of concern in some areas and
may pose more widespread contamination problems in the future. An estimated
5,000 tons of mercury per year are released into the environment from fossil fuel
burning (Klaassen et al., 1986). There is also increasing evidence of elevated
mercury concentrations in areas where acid rain is believed to be a factor,
although the extent of this problem has not been documented with certainty
(Sheffy, 1987; Wiener, 1987). Volatilization from surfaces painted with mercury-
containing paints, both indoors and outdoors, may have been a significant
source in the past (Agocs et al., 1990; Sheffy, 1987). The United States
estimated that 480,000 pounds of mercuric fungicides were used in paints and
coatings in 1987 (NPCA, 1988). In July 1990, EPA announced an agreement
with the National Paint and Coatings Association to cancel all registrations for
use of mercury or mercury compounds in interior paints and coatings. In May
1991, the paint industry voluntarily canceled all remaining registrations for
mercury in exterior paints.
Cycling of mercury in the environment is facilitated by the volatile character of
its metallic form and by bacterial transformation of metallic and inorganic forms
to stable alkyl mercury compounds, particularly in bottom sediments, which leads
to bioaccumulation of mercury (Wood, 1974). Practically all mercury in fish
tissue is in the form of methylmercury, which is toxic to humans (NAS, 1991;
Tollefson, 1989).
The EPA has determined that the evidence of carcinogenicity of mercury in both
animals and humans is inadequate and has assigned this metal a D
carcinogenicity classification (IRIS, 1992). Both inorganic and organic forms of
mercury are neurotoxicants. Fetuses exposed to organic mercury have been
found to be born mentally retarded and with symptoms similar to those of
cerebral palsy (Marsh, 1987). Individuals exposed to mercury via long-term
ingestion of mercury-contaminated fish have been found to exhibit a wide range
of symptoms, including numbness of the extremities, tremors, spasms,
personality and behavior changes, difficulty in walking, deafness, blindness, and
death (U.S. EPA, 1981 a). Organomercury compounds were the causative
agents of Minamata Disease, a neurological disorder reported in Japan during
the 1950s among individuals consuming contaminated fish and shellfish (Kurland
et al., 1960), with infants exposed prenatally found to be at significantly higher
risk than adults. The EPA is especially concerned about evidence that the fetus
is at increased risk of adverse neurological effects from exposure to
methylmercury (e.g., Marsh et al., 1987; Piotrowski and Inskip, 1981; Skerfving,
1988; WHO, 1976, 1990).
Mercury has been found in both fish and shellfish from estuarine/marine (NOAA,
1987, 1989a) and fresh waters (Schmitt and Brumbaugh, 1990) at diverse
4-9
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4. TARGET ANALYTES
locations nationwide. In contrast to cadmium and selenium, concentrations of
mercury in freshwater fish tissue did not change between 1976 and 1984
(Schmitt and Brumbaugh, 1990). Mercury, the only metal analyzed in the
National Study of Chemical Residues in Fish, was detected at 92.2 percent of
374 sites surveyed. Maximum, arithmetic mean, and median concentrations in
fish tissue were 1.80,0.26, and 0.17 ppm, respectively (U.S. EPA, 1991 h, 1992c,
1992d). Fish consumption advisories have been issued in 27 States as a result
of mercury contamination (see Figure 4-1). In particular, mercury is responsible
for a large number of the fish advisories currently in effect for lakes in Wisconsin,
Michigan, and Minnesota and for rivers and lakes in Florida (RTI, 1993).
Mercury should be considered for inclusion in all State fish and shellfish
contaminant monitoring programs. Only two national programs (301 (h) and the
FDA) currently analyze specifically for methylmercury; however, six programs
analyze for total mercury (Appendix C). Because of the higher cost of methyl-
mercury analysis, EPA recommends that total mercury be determined in State
fish contaminant monitoring programs and the conservative assumption be made
that all mercury is present as methylmercury in order to be most protective of
human health.
It should be noted that Bache et al. (1971) analyzed methylmercury
concentrations in lake trout of known ages and found that methylmercury
concentration and the ratio of methylmercury to total mercury increased with age.
Relative proportions of methylmercury in fish varied between 30 and 100
percent, with methylmercury concentrations lower than 80 percent occurring in
fish 3 years of age or younger. Thus, when high concentrations of total mercury
are detected, and if resources are sufficient, States may wish to repeat sampling
and obtain more specific information on actual concentrations of methylmercury
in various age or size classes of fish.
4.3.1.4 Selenium—
Selenium is a natural component of many soils, particularly in the west and
southwest regions of the United States (NAS, 1991). It enters the environment
primarily via emissions from oil and coal combustion (May and McKinney, 1981;
Pillay et al., 1969). Selenium is an essential nutrient but is toxic to both humans
and animals at high concentrations and has been shown to act as a mutagen in
animals (NAS, 1991). Long-term adverse effects from ingestion by humans have
not been studied thoroughly. The EPA has determined that the evidence of
carcinogenicity of selenium in both humans and animals is inadequate and,
therefore, has assigned this metal a D carcinogenicity classification. However,
selenium sulfide has been classified as a probable human carcinogen (B2) (IRIS,
1992).
Selenium is frequently detected in ground and surface waters in most regions of
the United States and has been detected in marine fish and shellfish (NOAA,
1987,1989a) and in freshwater fish (Schmitt and Brumbaugh, 1990) from several
areas nationwide. Selenium has been monitored in five national fish contaminant
4-10
-------
4. TARGET ANALYTES
in
O
O
CO
1
0)
fi
.2
I
I
I
«=
O)
(0
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4-11
-------
4. TARGET ANALYTES
monitoring programs (Appendix C). Definitive information concerning the
chemical forms of selenium found in fish and shellfish is not available (NAS,
1976,1991). Five States (California, Colorado, North Carolina, Texas, and Utah)
have issued advisories for selenium contamination in fish (RTI, 1993).
Selenium should be considered for inclusion in all State fish and shellfish
monitoring programs.
4.3.1.5 Trlbutyltln
Tributyltin belongs to the organometallic family of tin compounds that have been
used as biocides, disinfectants, and antifoulants. Antifoulant paints containing
tributyltin compounds were first registered for use in the United States in the
early 1960s. Tributyltin compounds are used in paints applied to boat and ship
hulls as well as to crab pots, fishing nets, and buoys to retard the growth of
fouling organisms. These compounds are also registered for use as wood
preservatives, disinfectants, and biocides in cooling towers, pulp and paper mills,
breweries, leather processing facilities, and textile mills (U.S. EPA, 1988c).
Tributyltin compounds are acutely toxic to aquatic organisms at concentrations
below 1 ppb and are chronically toxic to aquatic organisms at concentrations as
low as 0.002 ppb (U.S. EPA, 1988c). The Agency initiated a Special Review of
tributyltin compounds used as antifoulants in January of 1986 based on concerns
over its adverse effects on nontarget aquatic species. Shortly thereafter the
Organotin Antifouling Paint Control Act (OAPCA) was enacted in June 1988,
which contained interim and permanent tributyltin use restrictions as well as
environmental monitoring, research, and reporting requirements. The Act
established interim release rate restrictions under which only tributyltin-containing
products that do not exceed an average daily release rate of 4 \ig organotin/
cm2/day can be sold or used. The OAPCA also contained a permanent provi-
sion to prohibit the application of tributyltin antifouling paints to non-aluminum
vessels under 25 meters (82 feet) long (U.S. EPA, 1988c).
Tributyltin compounds are highly toxic to mammals (i.e., LD50 values ranged from
0.04 to 60 mg/kg) based on animal testing (Eisler, 1989; IRIS, 1995). Immuno-
toxicity and neurotoxicity are the critical effects produced by tributyltin.
Insufficient data are available to evaluate the carcinogenicity of tributyltin
compounds (IRIS, 1995) (Appendix D).
Tributyltins have been found to bioaccumulate in fish, bivalve mollusks, and
crustaceans. Bioconcentration factors (BCF) ranging from 200 to 4,300 for
finfish, from 2,000 to 6,000 for bivalves, and of 4,400 have been reported for
crustaceans (U.S. EPA, 1988c). Tributyltin used to control marine fouling
organisms in an aquaculture rearing pen has been found to bioaccumulate in fish
tissue (Short and Thrower, 1987a and 1987b). Tsuda et al. (1988) reported a
BCF value of 501 for tributyltin in carp (Cyprinus carpio) muscle tissue. Martin
et al. (1989) reported a similar BCF value of 406 for tributyltin in rainbow trout
(Salmo gairdnerf) and Ward et al. (1981) reported a BCF value of 520 for the
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4. TARGET ANALYTES
sheepshead minnow (Cyprinodon variegatus). In an environmental monitoring
study conducted in England, a BCF value of 1,000 was reported for tributyltin in
seed oysters (Crassostrea gigas) (Ebdon et al.f 1989).
Tributyltin is recommended for monitoring by the FDA but has not been
monitored in any other national fish contaminant monitoring program (Appendix
C). Only one State, Oregon, currently has an advisory in effect for tributyltin
contamination in shellfish (RTI, 1993).
Tributyltin should be considered for inclusion in all State fish and shellfish
contaminant monitoring programs, particularly in States with coastal waters,
States bordering the Great Lakes, or States with large rivers where large ocean-
going vessels are used for commerce. Tributyltin concentrations are reported to
be highest in areas of heavy boating and shipping activities including shipyards
where tributyltin-containing antifouling paints are often removed and reapplied.
Before recpating, old paint containing tributyltin residues is scraped from the
vessel hull and these paint scrapings are sometimes washed into the water
adjacent to the boat or shipyard despite the tributyltin label prohibiting this
practice (U.S. EPA, 1988c). Tributyltin should be considered for inclusion in State
fish and shellfish monitoring programs in areas where its use is or has been
extensive. States should contact their appropriate agencies to obtain information
on the historic and current uses of tributyltin, particularly with respect to its uses
in antifouling paints and wood preservatives.
4.3.2 Organochlorlne Pesticides
The following organochlorine pesticides and metabolites are recommended as
target analytes in screening studies: total chlordane (sum of cis- and trans-
chlordane, cis- and trans-nonachlor, and oxychlordane), total DDT (sum of 2,4'-
and 4,4'-isomers of DDT, ODD, and DDE), dicofol, dieldrin, endosulfan I and II,
endrin, heptachlor epoxide, hexachlorobenzene, lindane (y-hexachlorocyclo-
hexane), mirex, and toxaphene (see Appendix D). Mirex is of particular concern
in the Great Lakes States and the southeast States (MAS, 1991). All of these
compounds are neurotoxins and most are known or suspected human carcino-
gens (IRIS, 1992; Sax, 1984).
With the exception of endosulfan I and II, dicofol and total DDT, each of the
pesticides on the recommended target analyte list (Table 4-1) has been included
in at least five major fish contaminant monitoring programs (Appendix C), and
seven of the compounds have triggered at least one State fish consumption
advisory (Table 4-2). Although use of some of these pesticides has been
terminated or suspended within the United States for as long as 20 years
(Appendix D), these compounds still require long-term monitoring. Many of the
organochlorine pesticides were used in large quantities for over a decade and
are present in sediments at high concentrations. Organochlorine pesticides are
not easily degraded or metabolized and, therefore, persist in the environment.
These compounds are either insoluble or have relatively low solubility in water
but are quite lipid soluble. Because these compounds are not readily
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4. TARGET ANALYTES
metabolized or excreted from the body and are readily stored in fatty tissues,
they can bioaccumulate to high concentrations through aquatic food chains to
secondary consumers (e.g., fish, piscivorous birds, and mammals including
humans).
Pesticides may enter aquatic ecosystems from point source industrial discharges
or from nonpoint sources such as aerial drift and/or runoff from agricultural use
areas, leaching from landfills, or accidental spills or releases. Agricultural runoff
from crop and grazing lands is considered to be the major source of pesticides
in water, with industrial waste (effluents) from pesticide manufacturing the next
most common source (Li, 1975). Significant atmospheric transport of pesticides
to aquatic ecosystems can also result from aerial drift of pesticides, volatilization
from applications in terrestrial environments, and wind erosion of treated soil (Li,
1975). Once in water, pesticide residues may become adsorbed to suspended
material, deposited in bottom sediment, or absorbed by organisms in which they
are detoxified and eliminated or accumulated (Nimmo, 1985).
The reader should note that two of the organochlorine pesticides have active
registrations: endosulfan and dicofol. States should contact their appropriate
State agencies to obtain information on both the historic and current uses of
these pesticides.
4.3.2.1 Chlordane (Total)—
Chlordane is a multipurpose insecticide that has been used extensively in home
and agricultural applications in the United States for the control of termites and
many other insects (Appendix D). This pesticide is similar in chemical structure
to dieldrin, although less toxic (Toxicity Class II), and has been classified as a
probable human carcinogen (B2) by EPA (IRIS, 1992; Worthing, 1991).
Although the last labeled use of Chlordane as a termiticide was phased out in the
United States beginning in 1975, it has been monitored in eight national fish
contaminant programs (Appendix C) and has been widely detected in freshwater
fish (Schmittet al., 1990) and in both estuarine/marine finfish (NOAA, 1987) and
marine bivalves (NOAA, 1989a) at concentrations of human health concern. The
cis- and trans-isomers of Chlordane and nonachlor, which are primary
constituents of technical-grade Chlordane, and oxychlordane, the major
metabolite of chlordane, were monitored as part of the National Study of
Chemical Residues in Fish. These compounds were detected in fish tissue at
the following percentage of the 362 sites surveyed: cis-chlordane (64 percent),
trans-chlordane (61 percent), cis-nonachlor (35 percent), trans-nonachlor (77
percent), and oxychlordane (27 percent) (U.S. EPA, 1992c, 1992d). Chlordane's
presence in fish tissue has resulted in consumption advisories in 24 States (see
Figure 4-2).
Total chlordane (i.e., sum of cis- and trans-chlordane, cis- and trans-nonachlor,
and oxychlordane) should be considered for inclusion in all State fish and
shellfish contaminant monitoring programs (NAS, 1991).
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4. TARGET ANALYTES
D D
I
o
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I
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CO
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4. TARGET ANALYTES
4.3.2.2 DDT (Total)—
Although the use of DDT was terminated in the United States in 1972, DDT and
its DDE and DDD metabolites persist in the environment and are known to
bioaccumulate (Ware, 1978). DDT, DDD, and DDE have all been classified by
EPA as probable human carcinogens (B2) (IRIS, 1992).
DDT or its metabolites have been included as target analytes in eight major fish
and shellfish monitoring programs (Appendix C) and contamination has been
found to be widespread (NOAA, 1987, 1989a; Schmitt et al., 1990). DDE, the
only DDT metabolite surveyed in fish tissue in the National Study of Chemical
Residues in Fish, was detected at more sites than any other single pollutant (99
percent of the 362 sites sampled) (U.S. EPA, 1992c, 1992d). Nine States
(Alabama, American Samoa, Arizona, California, Delaware, Massachusetts,
Nebraska, New York, and Texas) currently have fish consumption advisories in
effect for DDT or its metabolites (RTI, 1993).
Total DDT (i.e., sum of the 4,4'- and 2,4'- isomers of DDT and of its metabolites,
DDE and DDD) should be considered for inclusion in all State fish and shellfish
contaminant monitoring programs.
4.3.2.3 Dicofol—
This chlorinated hydrocarbon acaricide was first registered in 1957 and is
structurally similar to DDT (U.S. EPA, 1992c, 1992d). Technical-grade dicofo!
may contain impurities of the p,p' and o,p' isomers of DDT, DDE, DDD, and a
compound known as extra-chlorine DDT (CI-DDT) that are inherent as a result
of the manufacturing process (U.S. EPA, 1983b). Historically, dicofol has been
used to control mites on cotton and citrus (60 percent), on apples (10 percent),
on ornamental plants and turf (10 percent), and on a variety of other agricultural
products (20 percent) including pears, apricots, and cherries (Farm Chemical
Handbook, 1989), as a seed crop soil treatment, on vegetables (e.g., beans and
corn) and on shade trees (U.S. EPA, 1992c, 1992d).
Dicofol is moderately toxic to laboratory rats and has been assigned to Toxicity
Class III based on oral exposure studies (Appendix D). Technical-grade dicofol
induced hepatocellular (liver) carcinomas in male mice; however, results were
negative in female mice and in rats (NCI, 1978). EPA has classified dicofol as
a possible human carcinogen (C) (U.S. EPA, 1992a). Because of concern that
dicofol would have the same effect as DDT on thinning of egg shells, the FDA
required ail dicofol products to contain less than 0.1 percent DDT and related
contaminants after June 1, 1989 (51 FR 19508).
Dicofol was recommended for monitoring by the EPA Office of Water as part of
the Assessment and Control of Bioconcentratable Contaminants in Surface
Waters Program and has been included in two national monitoring programs
(see Appendix C). In the National Study of Chemical Residues in Fish, dicofol
was detected at 16 percent of the sites monitored (U.S. EPA, 1992c, 1992d).
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4. TARGET ANALYTES
Dicofol concentrations were greater than the quantification limit (2.5 ppb) in
samples from 7 percent of the sites. Most of the sites where dicofol was
detected were in agricultural areas where citrus and other fruits and vegetables
are grown (U.S. EPA, 1992c, 1992d). It should be noted that this national study
did not specifically target agricultural sites where this pesticide historically had
been or currently is used. Dicofol residues in fish could be much higher if
sampling were targeted for pesticide runoff. Experimental evidence indicates this
compound bioaccumulates in Bluegill sunfish (BCF from 6,600 to 17,000) (U.S.
EPA, 1993a); however, no consumption advisories are currently in effect for
dicofol (RTI, 1993).
Dicofol should be considered for inclusion in State fish and shellfish contaminant
monitoring programs, in areas where its use is or has been extensive. States
should contact their appropriate State agencies to obtain information on the
historic and current uses of this pesticide.
4.3.2.4 Dieldrin—
Dieldrin is a chlorinated cyclodiene that was widely used in the United States
from 1950 to 1974 as a broad spectrum pesticide, primarily on termites and other
soil-dwelling insects and on cotton, corn, and citrus crops. Because the toxicity
of this persistent pesticide posed an imminent danger to human health, EPA
banned the production and most major uses of dieldrin in 1974, and, in 1987, all
uses of dieldrin were voluntarily canceled by industry (see Appendix D).
Dieldrin has been classified by EPA as a probable human carcinogen (B2) (IRIS,
1992) and has been identified as a human neurotoxin (ATSDR, 1987a). Dieldrin
has been included in eight national minitoring programs (Appendix C) and is still
detected nationwide in freshwater finfish (Schmitt et al., 1990) and
estuarine/marine finfish and shellfish (NOAA, 1987, 1989a). Dieldrin was
detected in fish tissue at 60 percent of the 362 sites surveyed as part of the
National Survey of Chemical Residues in Fish (U.S. EPA, 1992c, 1992d).
Because it is a metabolite of aldrin, the environmental concentrations of dieldrin
are a cumulative result of the historic use of both aldrin and dieldrin (Schmitt et
al., 1990). Three States (Arizona, Illinois, and Nebraska) have issued advisories
for dieldrin contamination in fish (RTI, 1993).
Dieldrin should be considered for inclusion in all State fish and shellfish
contaminant monitoring programs.
4.3.2.5 Endosulfan—
Endosulfan is a chlorinated cyclodiene pesticide that is currently in wide use
primarily as a noncontact insecticide for seed and soil treatments (Appendix D).
Two stereoisomers (I and II) exist and exhibit approximately equal effectiveness
and toxicity (Worthing, 1991).
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4. TARGET ANALYTES
Endosulfan is highly toxic to humans and has been assigned to Toxicity Class
I. To date, no studies have been found concerning carcinogenicity in humans
after oral exposure to endosulfan (ATSDR, 1990). EPA has given endosulfan
the carcinogenicity classification E, indicating there is no evidence of
carcinogenicity for humans (U.S. EPA, 1992a).
Agricultural runoff is the primary source of this pesticide in aquatic ecosystems.
Endosulfan has been shown to be highly toxic to fish and marine invertebrates
and is readily absorbed in sediments. It therefore represents a potential hazard
in the aquatic environment (Sittig, 1980). However, data are currently insufficient
to assess nationwide endosulfan contamination (NAS, 1991). Endosulfan was
recommended for monitoring by the FDA and has been included in one national
fish contaminant monitoring program (U.S. EPA 301 (h) Program) evaluated by
the EPA Workgroup (Appendix C). No consumption advisories are currently in
effect for endosulfan I or II (RTI, 1993).
Endosulfan I and II should be considered for inclusion in all State fish and
shellfish contaminant monitoring programs in areas where its use is or has been
extensive. States should contact their appropriate State agencies to obtain
information on the historic and current uses of this pesticide.
4.3.2.6 Endrin—
Endrin is a chlorinated cyclodiene that historically was widely used as a broad
spectrum pesticide. Endrin was first registered for use in the United States in
1951. However, recognition of its long-term persistence in soil and its high levels
of mammalian toxicity led to restriction of its use beginning in 1964 (U.S. EPA,
1980a) and 1979 (44 FR 43632) and to final cancellation of its registration in
1984 (U.S. EPA, 1984a) (Appendix D).
Endrin is highly toxic to humans (Toxicity Class I), with acute exposures affecting
the central nervous system primarily (Sax, 1984). At present, evidence of both
animal and human carcinogenicity of endrin is considered inadequate (IRIS,
1992).
Although endrin has been included in six national fish contaminant monitoring
programs (Appendix C), it has not been found widely throughout the United
States. Endrin was detected in freshwater and marine species at 11 percent of
the 362 sites surveyed in the EPA National Study of Chemical Residues in Fish
(U.S. EPA, 1992c, 1992d) and was found in only 29 percent of 112 stations
sampled in the NCBP (Schmitt et al., 1990). No States have issued fish
consumption advisories for endrin (RTI, 1993).
Endrin should be considered for inclusion in all State fish and shellfish contami-
nant monitoring programs.
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4. TARGET ANALYTES
4.3.2.7 Heptachlor Epoxide—
Heptachlor epoxide is not a formulated pesticide, but is a metabolic degradation
product of the pesticide heptachlor. It is also found as a contaminant in
heptachlor and chlordane formulations (Appendix D). Heptachlor has been used
as a persistent, nonsystemic contact and ingested insecticide on soils
(particularly for termite control) and seeds and as a household insecticide
(Worthing, 1991). EPA suspended the major uses of heptachlor in 1978
(ATSDR, 1987b). Acute exposures to high doses of heptachlor epoxide in
humans can cause central nervous system effects (e.g., irritability, dizziness,
muscle tremors, and convulsions (U.S. EPA, 1986e). In animals, liver, kidney,
and blood disorders can occur (IRIS, 1989). Exposure to this compound
produced an increased incidence of liver carcinomas in rate and mice and
hepatomas in female rats (IRIS, 1989). Heptachlor epoxide has been classified
by EPA as a probable human carcinogen (B2) (IRIS, 1992).
Heptachlor epoxide has been included in seven national fish monitoring
programs (Appendix C) and has been detected widely in freshwater finfish
(Schmitt et al., 1990) but infrequently in bivalves and marine fish (NOAA, 1987,
1989a). Heptachlor epoxide was detected in fish tissue at 16 percent of the 362
sites where it was surveyed in the National Study of Chemical Residues in Fish
(U.S. EPA, 1992c, 1992d). One State (Nebraska) currently has fish advisories
for heptachlor epoxide contamination (RTI, 1993).
Heptachlor epoxide should be considered for inclusipn in all State fish and
shellfish monitoring programs.
4.3.2.8 Hexachlorobenzene—
Hexachlorobenzene is a fungicide that was widely used as a seed protectant in
the United States until 1985 (Appendix D). The use of hexachlorobenzene and
the presence of hexachlorobenzene residues in food are banned in many
countries including the United States (Worthing, 1991). Registration of
hexachlorobenzene as a pesticide was voluntarily canceled in 1984 (Morris and
Cabral, 1986).
The toxicity of this compound is minimal; it has been given a toxicity
classification of IV (i.e., oral LD50 greater than 5,000 ppm in laboratory animals
(Farm Chemicals Handbook, 1989). However, nursing infants are particularly
susceptible to hexachlorobenzene poisoning as lactational transfer can increase
infant tissue levels to levels two to five times maternal tissue levels (ATSDR,
1989b). Hexachlorobenzene is a known animal carcinogen (ATSDR, 1989b) and
has been classified by EPA as a probable human carcinogen (B2) (IRIS, 1992)
(Appendix D).
Of the chlorinated benzenes, hexachlorobenzene is the most widely monitored
(Worthing, 1991). It was included as a target analyte in seven of the major
monitoring programs reviewed (Appendix C). Hexachlorobenzene was detected
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4. TARGET ANALYTES
in fish tissue at 46 percent of the 362 sites where it was surveyed in the National
Study of Chemical Residues in Fish (U.S. EPA, 1992c, 1992d). Two States
(Louisiana and Ohio) have issued advisories for hexachlorobenzene
contamination in fish and shellfish (RTI, 1993).
Hexachlorobenzene should be considered for inclusion in all State fish and
shellfish monitoring programs.
4.3.2.9 Lindane—
Lindane is a mixture of isomers of hexachlorocyclohexane (C6H6CI6), whose
major component (>99 percent) is the gamma isomer. It is commonly referred
to as either Y^HCH (hexachlorocyclohexane) or y-BHC (benzene hexachloride).
Lindane is used primarily in seed treatments, soil treatments for tobacco
transplants, foliage applications on fruit and nut trees and vegetables, and wood
and timber protection. Since 1985, many uses of lindane have been banned or
restricted (see Appendix D). At present, its application is permitted only under
supervision of a certified applicator (U.S. EPA, 1985c).
Lindane is a neurotoxin (assigned to Toxicity Class II) and has been found to
cause aplastic anemia in humans (Worthing, 1991). Lindane has been classified
by EPA as a probable/possible human carcinogen (B2/C). Available data for this
pesticide need to be reviewed, but at a minimum the carcinogenicity
classification will be C (U.S. EPA, 1992a).
Lindane has been included in eight major fish contaminant monitoring programs
(Appendix C). This pesticide has been detected in freshwater fish (Schmitt et
al., 1990) and in marine fish and bivalves (NOAA, 1987, 1989a) nationwide.
Lindane was detected in fish tissue at 42 percent of 362 sites surveyed in the
National Study of Chemical Residues in Fish (U.S. EPA, 1992c, 1992d).
Although lindane has been widely monitored and widely detected, no
consumption advisories are currently in effect for lindane (RTI, 1993).
Lindane should be considered for inclusion in all State fish and shellfish
monitoring programs.
4.3.2.10 Mirex—
Mirex is a chlorinated cyclodiene pesticide that was used in large quantities in
the United States from 1962 through 1975 primarily for control of fire ants in the
Southeast and, more widely, under the name Dechlorane as a fire retardant and
polymerizing agent in plastics (Kaiser, 1978) (Appendix D).
Mirex has been assigned to Toxicity Class II and has been classified as a
probable human carcinogen by the International Agency for Research on Cancer
(IARC, 1987); however, the carcinogenicity data are currently under review by
EPA (IRIS, 1992). EPA instituted restrictions on the use of mirex in 1975, and,
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4. TARGET ANALYTES
shortly thereafter, the U.S. Department of Agriculture (USDA) suspended the fire
ant control program (Hodges, 1977).
Mirex has been included in eight major fish contaminant monitoring programs
(Appendix C). It has been found primarily in the Southeast and the Great Lakes
regions (NAS, 1991; Schmitt et al., 1990). Mirex was detected in fish tissue at
36 percent of 362 sites surveyed in the National Study of Chemical Residues in
Fish (U.S. EPA, 1992c, 1992d). Three States (New York, Ohio, and
Pennsylvania) currently have fish consumption advisories for mirex (RTI, 1993).
Mirex should be considered for inclusion in all State fish and shellfish monitoring
programs.
4.3.2.11 Toxaphene—
Toxaphene is a mixture of chlorinated camphenes. Historically, it was used in
the United States as an insecticide primarily on cotton (Hodges, 1977). Partly
as a consequence of the ban on the use of DDT imposed in 1972, toxaphene
was for many years the most heavily used pesticide in the United States (Eichers
et al., 1978). In 1982, toxaphene's registration for most uses was canceled (47
FR 53784).
Like many of the other organochlorine pesticides, toxaphene has been assigned
to Toxicity Class II (Appendix D). Unlike the other organochlorine pesticides,
toxaphene is fairly easily metabolized by mammals and is not stored in the fatty
tissue to any great extent. Toxaphene has been classified by EPA as a probable
human carcinogen (B2) (IRIS, 1992).
Toxaphene has been included in five major fish contaminant monitoring
programs (Appendix C). It has been detected frequently in both fresh (Schmitt
et al., 1990) and estuarine (NOAA, 1989a) waters but is only consistently found
in Georgia, Texas, and California (NAS, 1991). Note: A toxaphene-like
compound that is a byproduct of the paper industry has been identified in the
Great Lakes Region (J. Hesse, Michigan Department of Public Health, personal
communication, 1993). Two States (Arizona and Texas) currently have fish
advisories in effect for toxaphene (RTI, 1993).
Toxaphene should be considered for inclusion in all State fish and shellfish
monitoring programs.
4.3.3 Organophosphate Pesticides
The following organophosphate pesticides are recommended as target analytes
in screening studies: chlorpyrifos, diazinon, disulfoton, ethion, and terbufos
(Appendix D). Most of these organophosphate pesticides share two distinct
features. Organophosphate pesticides are generally more toxic to vertebrates
than organochlorine pesticides and exert their toxic action by inhibiting the
activity of cholinesterase (ChEj, one of the vital nervous system enzymes. In
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4. TARGET ANALYTES
addition, organophosphates are chemically unstable and thus are less persistent
in the environment. It is this latter feature that made them attractive alternatives
to the organochlorine pesticides that were used extensively in agriculture from
the 1940s to the early 1970s.
With the exception of chlorpyrifos, none of the organophosphates has been
included in any of the national fish contaminant monitoring programs evaluated
by the EPA Workgroup and none of these pesticides (including chlorpyrifos) has
triggered State fish consumption advisories. All of the compounds have active
pesticide registrations and have been recommended for monitoring because they
have a Toxicity Classification of I or II (Appendix D), have BCFs > 300, a half-life
of 30 days or more in the environment, and their use profiles suggest they could
be potential problems in some agricultural watersheds.
The reader should note that all of the organophosphate pesticides recommended
as target analytes have active registrations. States should contact their
appropriate State agencies to obtain information on both the historic and current
uses of these pesticides. In addition, if a State determines that use of these
pesticides may be occurring in its waters, sampling should be conducted during
late spring or early summer within 1 to 2 months following pesticide application
because these compounds are degraded and metabolized relatively rapidly by
fish species. Additional discussion of appropriate sampling times for fish
contaminant'monitoring programs is provided in Section 6.1.1.5.
4.3.3.1 Chlorpyrifos—
This organophosphate pesticide was first introduced in 1965 to replace the more
persistent organochlorine pesticides (e.g., DDT) (U.S. EPA, 1986e) and has
been used for a broad range of insecticide applications (Appendix D).
Chlorpyrifos is used primarily to control soil and foliar insects on cotton, peanuts,
and sorghum (Worthing, 1991; U.S. EPA, 1986e). Chlorpyrifos is also used to
control root-infesting and boring insects on a variety of fruits (e.g., apples,
bananas, citrus, grapes), nuts (e.g., almonds, walnuts), vegetables (e.g., beans,
broccoli, brussel sprouts, cabbage, cauliflower, peas, and soybeans), and field
crops (e.g., alfalfa and corn) (U.S. EPA, 1984c). As a household insecticide,
chlorpyrifos has been used to control ants, cockroaches, fleas, and mosquitoes
(Worthing, 1991) and is registered for use in controlling subsurface termites in
California (U.S. EPA, 1983a). Based on use application, 57 percent of all
chlorpyrifos manufactured in the United States is used on corn, while 22 percent
is used for pest control and lawn and garden services (U.S. EPA, 1993a).
Chlorpyrifos has a moderate mammalian toxicity and has been assigned to
Toxicity Class II based on oral feeding studies (Farm Chemicals Handbook,
1989). No teratogenic or fetotoxic effects were found in mice or rats (IRIS,
1989). No carcinogenicity was found in chronic feeding studies with rats, mice
and dogs (U.S. EPA, 1983a). EPA has assigned chlorpyrifos a carcinogenicity
classification of D—not classifiable based on inadequate evidence of carcino-
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4. TARGET ANALYTES
genicity or lack of data in at least two animal studies or in both epidemiologic
and animal studies (U.S. EPA, 1992a).
Chlorpyrifos was recommended for monitoring by the FDA and has been includ-
ed in one national monitoring program, the National Study of Chemical Residues
in Fish (see Appendix C). In this latter study, Chlorpyrifos was detected at 26
percent of sites sampled nationally (U.S. EPA, 1992c, 1992d). Eighteen percent
of the sites with relatively high concentrations (2.5 to 344 ppb) were scattered
throughout the East, Midwest, and in California; the highest concentrations
detected (60 to 344 ppb) were found either in agricultural areas or in urban areas
with a variety of nearby industrial sources. It should be noted that this national
study did not specifically target agricultural sites where this pesticide historically
had been used or is currently used. Chlorpyrifos residues in fish could be much
higher if sampling were targeted for pesticide runoff. Experimental evidence
indicates this compound bioaccumulates in rainbow trout (BCF from 1,280 to
3,903) (U.S. EPA, 1993a); however, no consumption advisories are currently in
effect for Chlorpyrifos (RTI, 1993).
Chlorpyrifos should be considered for inclusion in State fish and shellfish
contaminant monitoring programs in areas where its use is or has been
extensive. States should contact their appropriate State agencies to obtain
information on the historic and current uses of this pesticide.
4.3.3.2 Diazlnon—
Diazinon is a phosphorothiate insecticide and nematicide that was first registered
in 1952 for control of soil insects and pests of fruits, vegetables, tobacco, forage,
field crops, range, pasture, grasslands, and ornamentals; for control of
cockroaches and other household insects; for control of grubs and nematodes
in turf; as a seed treatment; and for fly control (U.S. EPA, 1986f).
Diazinon is moderately toxic to mammals and has been assigned to Toxicity
Class II based on oral toxicity tests (Appendix D). EPA has assigned diazinon
to carcinogenicity classification D—not classifiable based on a lack of data or
inadequate evidence of carcinogenicity in at least two animal tests or in both
epidemiologic and animal studies (U.S. EPA, 1992a). This compound is also
highly toxic to birds, fish, and other aquatic invertebrates (U.S. EPA, 1986f).
Diazinon has not been included in any national fish contaminant monitoring
program evaluated by the EPA Workgroup (Appendix C). Experimental evidence
indicates this compound accumulates in trout (BCF of 542) (U.S. EPA, 1993a);
however, no consumption advisories are currently in effect for diazinon (RTI,
1993).
Diazinon should be considered for inclusion in State fish and shellfish
contaminant monitoring programs in areas where its use is or has been
extensive. States should contact their appropriate State agencies to obtain
information on the historic and current uses of this pesticide.
4-23
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4. TARGET ANALYTES
4.3.3.3 Dlsulfoton—
Disulfoton is a multipurpose systemic insecticide and acaricide first registered in
1958 for use as a side dressing, broadcast, or foliar spray in the seed furrow to
control many insect and mite species and as a seed treatment for sucking
insects (Appendix D).
Disulfoton is highly toxic to all mammalian systems and has been assigned to
Toxicity Class I on the basis of all routes of exposure (Farm Chemicals
Handbook, 1989). All labeling precautions and use restrictions are based on
human health risk. Disulfoton and its major metabolites are potent
cholinesterase inhibitors primarily attacking acetylcholinesterase. Contradictory
evidence is available on the mutagenicity of this compound and the EPA has
concluded that the mutagenic potential is not adequately defined (U.S. EPA,
1984d). EPA has assigned disulfoton to carcinogenicity classification D—not
classifiable based on a lack of data or inadequate evidence of carcinogenicity in
at least two animal tests or in both epidemiologic and animal studies (U.S. EPA,
1992a).
Disulfoton has not been included in any national fish contaminant monitoring
program evaluated by the EPA Workgroup (Appendix C). Experimental evidence
indicates this compound accumulates in fish (BCF from 460 to 700) (U.S. EPA,
1993a); however, no consumption advisories are currently in effect for disulfoton
(RTI, 1993).
Disulfoton should be considered for inclusion in State fish and shellfish
contaminant monitoring programs in areas where its use is or has been
extensive. States should contact their appropriate State agencies to obtain
information on the historic and current uses of this pesticide.
4.3.3.4 Ethion—
Ethion is a multipurpose insecticide and acaricide that has been registered since
1965 for use on a wide variety of nonfood crops (turf, evergreen plantings, and
ornamentals), food crops (seed, fruit, nut, fiber, grain, forage, and vegetables),
and for domestic outdoor uses around dwellings and for lawns (Appendix D).
Application to citrus crops accounts for 86 to 89 percent of the ethion used in the
United States. The remaining 11 to 14 percent is applied to cotton and a variety
of fruit and nut trees and vegetables. Approximately 55 to 70 percent of all
domestically produced citrus fruits are treated with ethion (U.S. EPA, 1989e).
Acute oral toxicity studies have shown that technical-grade ethion is moderately
toxic to mammals (Toxicity Class II) (Farm Chemicals Handbook, 1989). In a
chronic rat toxicity study, a decrease in serum cholinesterase was observed in
both males and females. Ethion was not found to be carcinogenic in rats and
mice (U.S. EPA, 1989e). EPA has assigned ethion to carcinogenicity
classification D—not classifiable based on a lack of data or inadequate evidence
4-24
-------
4. TARGET ANALYTES
of carcinogenicity in at least two animal tests or in both epidemiologic and animal
studies (U.S. EPA, 1992a).
Ethion has not been included in any national fish contaminant monitoring
program evaluated by the EPA Workgroup (Appendix C). Experimental evidence
indicates this compound accumulates in Bluegill sunfish (BCF from 880 to 2,400)
(U.S. EPA, 1993a); however, no consumption advisories are currently in effect
forethion (RTI, 1993).
Ethion should be considered for inclusion in State fish and shellfish contaminant
monitoring programs in areas where its use is or has been extensive. States
should contact their appropriate State agencies to obtain information on the
historic and current uses of this pesticide.
4.3.3.5 Terbufos—
Terbufos is a systemic organophosphate insecticide and nematicide registered
in 1974 principally for use on corn, sugar beets, and grain sorghum. The
primary method of application involves direct soil incorporation of a granular
formulation (Farm Chemicals Handbook, 1989).
Terbufos is highly toxic to humans and has been assigned to Toxicity Class I
(Appendix D). Symptoms of acute cholinesterase inhibition have been reported
in all acute studies, and cholinesterase inhibition was reported in several chronic
mammalian feeding studies (U.S. EPA, 1985d). EPA has assigned terbufos to
carcinogenicity classification D—not classifiable based on a lack of data or
inadequate evidence of carcinogenicity in at least two animal tests or in both
epidemiologic and animal studies (U.S. EPA, 1992a). Terbufos is also highly
toxic to birds, fish, and other aquatic invertebrates (U.S. EPA, 1985d).
Terbufos has not been included in any national fish contaminant monitoring
program evaluated by the EPA Workgroup (Appendix C). Experimental evidence
indicates this compound accumulates in fish (BCF from 320 to 1,400) (U.S. EPA,
1993a); however no consumption advisories are currently in effect for terbufos
(RTI, 1993).
Terbufos should be considered for inclusion in State fish and shellfish
contaminant monitoring programs in areas where its use is or has been
extensive. States should contact their appropriate State agencies to obtain
information on the historic and current uses of this pesticide.
4.3.4 Chlorophenoxy Herbicides
Chlorophenoxy herbicides, which include oxyfluorfen, are nonselective foliar
herbicides that are most effective in hot weather (Ware, 1978).
4-25
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4. TARGET ANALYTES
4.3.4.1 Oxyfluorfen--
Oxyfluorfen is a pre- and postemergence herbicide that has been registered
since 1979 for use to control a wide spectrum of annual broadleaf weeds and
grasses in apples, artichokes, corn, cotton, jojoba, tree fruits, grapes, nuts,
soybeans, spearmint, peppermint, and certain tropical plantation and ornamental
crops (Appendix D).
Evidence suggests that oxyfiuorfen is moderately toxic to mammals and has
been assigned to Toxicity Class II based on a chronic mouse feeding study
(Farm Chemicals Handbook, 1989; IRIS, 1993). There is also evidence of
carcinogenicity (liver tumors) in mice (U.S. EPA, 1993a) and therefore
oxyfiuorfen has been classified by EPA as a possible human carcinogen (C)
(U.S. EPA, 1992c).
Although oxyfiuorfen has an active registration, it has not been included in any
national fish contaminant monitoring program evaluated by the EPA Workgroup
(Appendix C). Experimental evidence indicates this herbicide accumulates in
Bluegill sunfish (BCF from 640 to 1,800) (U.S. EPA, 1993a); however, no
consumption advisories are currently in effect for oxyfiuorfen (RTI, 1993).
Oxyfluorfen should be considered for inclusion in State fish and shellfish
contaminant monitoring programs in areas where its use is or has been
extensive. States should contact their appropriate State agencies to obtain
information on the historic and current uses of this pesticide.
4.3.5 Polycyclic Aromatic Hydrocarbons (PAHs)—
Polycyclic aromatic hydrocarbons are base/neutral organic compounds that have
a fused ring structure of two or more benzene rings. PAHs are also commonly
referred to as polynuclear aromatic hydrocarbons (PNAs). PAHs with two to five
benzene rings (i.e., 10 to 24 skeletal carbons) are.generally of greatest concern
for environmental and human health effects (Benkert, 1992). These PAHs
include those listed as priority pollutants (U.S. EPA, 1995a)
Acenaphthene
Acenaphthylene
Anthracene
Benz[a]anthracene
Benzo[a]pyrene
Benzo[d]fluoranthene
Benzo[/c]fluoranthene
Benzo[gr,/7,/]perylene
Chlorinated naphthalenes
Chrysene
Dibenz[a,/7]anthracene
Fluoranthene
Fluorene
lndeno[ /,2,3-ccQpyrene
Naphthalene
Phenanthrene
Pyrene.
The metabolites of many of the high-molecular-weight PAHs (e.g., benz[a]an-
thracene, benzo[a]pyrene, benzo[fc]fluoranthene, benzo^fluoranthene, chrysene,
4-26
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4. TARGET ANALYTES
dibenz[a,ft]anthracene, indeno[/,2,3-ccdpyrene) have been shown in laboratory
test systems to be carcinogens, cocarcinogens, teratogens, and/or mutagens
(Moore and Ramamoorthy, 1984; U.S. DHHS, 1990). Benzo[a]pyrene, one of
the most widely occurring and potent PAHs, and several other PAHs (e.g.,
benz[a]anthracene, benzo[6]fluoranthene, benzo[/]fluoranthene, benzo[AJfluor-
anthene, chrysene, cyclopenta[ccflpyrene, dibenz[a,/j]anthracene, dibenzo[a,e]
fluoranthene, dibenzo[a,e]pyrene, dibenzo[a,/7]pyrene, dibenzo[a,/]pyrene,
dibenzo[a,/lpyrene, indeno[7,2,3-ccflpyrene) have been classified by EPA as
probable human carcinogens (B2) (IRIS, 1992). Evidence for the carcinogenicity
of PAHs in humans comes primarily from epidemiologic studies that have shown
an increased mortality due to lung cancer in humans exposed to PAH-containing
coke oven emissions, roof-tar emissions, and cigarette smoke (U.S. DHHS,
1990).
PAHs are ubiquitous in the environment and usually occur as complex mixtures
with other toxic chemicals. They are components of crude and refined petroleum
products and of coal. They are also produced by the incomplete combustion of
organic materials. Many domestic and industrial activities involve pyrosynthesis
of PAHs, which may be released into the environment in airborne particulates or
in solid (ash) or liquid byproducts of the pyrolytic process. Domestic activities
that produce PAHs include cigarette smoking, home heating with wood or fossil
fuels, waste incineration, broiling and smoking foods, and use of internal
combustion engines. Industrial activities that produce PAHs include coal coking;
production of carbon blacks, creosote, and coal tar; petroleum refining; synfuel
production from coal; and use of Soderberg electrodes in aluminum smelters and
ferrosilicum and iron works (Neff, 1985). Historic coal gasification sites have
also been identified as significant sources of PAH contamination (J. Hesse,
Michigan Department of Public Health, personal communication, March 1991).
Major sources of PAHs found in marine and fresh waters include biosynthesis
(restricted to anoxic sediments), spillage and seepage of fossil fuels, discharge
of domestic and industrial wastes, atmospheric deposition, and runoff (Neff,
1985). Urban stormwater runoff contains PAHs from leaching of asphalt roads,
wearing of tires, deposition from automobile exhaust, and oiling of roadsides and
unpaved roadways with crankcase oil (MacKenzie and Hunter, 1979). Solid
PAH-containing residues from activated sludge treatment facilities have been
disposed of in landfills or in the ocean (ocean dumping was banned in 1989).
Although liquid domestic sewage contains <1 ng/L total PAH, the total PAH
content of industrial sewage is 5 to 15 ng/L (Borneff and Kunte, 1965) and that
of sewage sludge is 1 to 30 mg/kg (Grimmer et al., 1978; Nicholls et al., 1979).
In most cases, there is a direct relationship between PAH concentrations in river
water and the degree of industrialization and human activity in the surrounding
watersheds. Rivers flowing through heavily industrialized areas may contain 1
to 5 ppb total PAH, compared to unpolluted river water, ground water, or
seawater that usually contains less than 0.1 ppb PAH (Neff, 1979).
4-27
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4. TARGET ANALYTES
PAHs can accumulate in aquatic organisms from water, sediments, and food.
BCFs of PAHs in fish and crustaceans have frequently been reported to be in
the range of 100 to 2,000 (Eisler, 1987). In general, bioconcentration was
greater for the higher molecular weight PAHs than for the lower molecular weight
PAHs. Biotransformation by the mixed function oxidase system in the fish liver
can result in the formation of carcinogenic and mutagenic intermediates, and
exposure to PAHs has been linked to the development of tumors in fish (Eisler,
1987). The ability of fish to metabolize PAHs probably explains why
benzo[a]pyrene frequently is not detected or is found only at very low
concentrations in fish from areas heavily contaminated with PAHs (Varanasi and
Gmur, 1980, 1981).
Sediment-associated PAHs can be accumulated by bottom-dwelling invertebrates
and fish (Eisler, 1987). For example, Great Lakes sediments containing elevated
levels of PAHs were reported by Eadie et al. (1983) to be the source of the body
burdens of the compounds in bottom-dwelling invertebrates. Similarly, Varanasi
et al. (1985) found that benzo[a]pyrene was accumulated in fish, amphipod
crustaceans, shrimp, and clams when estuarine sediment was the source of the
compound. Approximate tissue-to-sediment ratios were 0.6 to 1.2 for
amphipods, 0.1 for clams, and 0.05 for fish and shrimp. Although fish and most
crustaceans evaluated to date have the mixed function oxidose system required
for biotransformation of PAHs, some molluscs lack this system and are unable
to .metabolize PAHs efficiently (Varanasi et al., 1985). Thus, bivalves are good
bioaccumulators of some PAHs. NAS (1991) reported that PAH contamination
in bivalves has been found in all areas of the United States. Varanasi et al.
(1985) ranked benzo[a]pyrene metabolism by aquatic organisms as follows: fish
> shrimp > amphipod crustaceans > clams. Half-lives for elimination of PAHs
in fish ranged from less than 2 days to 9 days (Niimi, 1987). If PAHs are
included as target analytes at a site, preference should be given to selection of
a bivalve mollusc as one of the target species (if available) and a finfish as the
other target species.
Three States (Massachusetts, Michigan, and Ohio) have issued advisories for
PAH contamination in finfish (RTI, 1993).
Although several PAHs have been classified as probable human carcinogens
(Group B2), benzo[a]pyrene is the only PAH for which an oral cancer slope
factor (SF) is currently available in IRIS (1995). It is recommended that, in both
screening and intensive studies, tissue samples be analyzed for benzo[a]pyrene,
benz[a]anthracene, benzo[b]fluoranthene, benzo[/cjfluoranthene, chrysene,
dibenz[a,/7]anthracene, and indeno/7,2,3-cd]pyrene, and that the relative
potencies given for these PAHs in the EPA provisional guidance for quantitative
risk assessment of PAHs (U.S. EPA, 1993c) be used to calculate a potency
equivalency concentration (PEC) for each sample for comparison with the
recommended SV for benzo[a]pyrene (see Section 5.3.2.3). At this time, EPA's
recommendation for risk assessment of PAHs (U.S. EPA, 1993c) is considered
provisional because quantitative risk assessment data are not available for all
PAHs. This approach is under Agency review and over the next year will be
4-28
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4. TARGET ANALYTES
evaluated as new health effects benchmark values are developed. Therefore,
the method provided in this guidance document is subject to change pending
results of the Agency's reevaluation.
4.3.6 Poiychlorlnated Blphenyls (Total)
PCBs are base/neutral compounds that are formed by the direct chlorination of
biphenyl. PCBs are closely related to many chlorinated hydrocarbon pesticides
(e.g., DDT, dieldrin, and aldrin) in their chemical, physical, and toxicologic
properties and in their widespread occurrence in the aquatic environment
(Nimmo, 1985). There are 209 different PCB compounds, termed congeners,
based on the possible chlorine substitution patterns. In the United States,
mixtures of various PCB congeners were formulated for commercial use under
the trade name Aroclor on the basis of their percent chlorine content. For
example, a common PCB mixture, Aroclor 1254, has an average chlorine content
of 54 percent by weight (Nimmo, 1985).
Unlike the organochlorine pesticides, PCBs were never intended to be released
directly into the environment; most uses were in industrial systems. Important
properties of PCBs for industrial applications include thermal stability, fire and
oxidation resistance, and solubility in organic compounds (Hodges, 1977). PCBs
were used as insulating fluids in electrical transformers and capacitors, as
plasticizers, as lubricants, as fluids in vacuum pumps and compressors, and as
heat transfer and hydraulic fluids (Hodges, 1977; Nimmo, 1985). Although use
of PCBs as a dielectric fluid in transformers and capacitors was generally
considered a closed-system application, the uses of PCBs, especially during the
1960s, were broadly expanded to many open systems where losses to the
environment were likely. Heat transfer systems, hydraulic fluids in die cast
machines, and uses in specialty inks are examples of more open-ended
applications that resulted in serious contamination in fish near industrial
discharge points (Hesse, 1976).
Although PCBs were once used extensively by industry, their production and use
in the United States were banned by the EPA in July 1979 (Miller, 1979). Prior
to 1979, the disposal of PCBs and PCB-containing equipment was not subject
to Federal regulation. Prior to regulation, of the approximately 1.25 billion
pounds purchased by U.S. industry, 750 million pounds (60 percent) were still
in use in capacitors and transformers, 55 million pounds (4 percent) had been
destroyed by incineration or degraded in the environment, and over 450 million
pounds (36 percent) were either in landfills or dumps or were available to biota
via air, water, soil, and sediments (Durfee et al., 1976).
PCBs are extremely persistent in the environment and are bioaccumulated
throughout the food chain (Eisler, 1986; Worthing, 1991). There is evidence that
PCB health risks increase with increased chlorination because more highly
chlorinated PCBs are retained more efficiently in fatty tissues (IRIS, 1992).
However, individual PCB congeners have widely varying potencies for producing
a variety of adverse biological effects including hepatotoxicity, developmental
4-29
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4. TARGET ANALYTES
toxicity, immunotoxicity, neurotoxicity, and carcinogenicity. The non-ortho-
substituted coplanar PCB congeners, and some of the mono-ortho-substituted
congeners, have been shown to exhibit "dioxin-like" effects (Golub et al., 1991;
Kimbrough and Jensen, 1989; McConnell, 1980; Poland and Knutson, 1982;
Safe, 1985, 1990; Tilson et al., 1990; U.S. EPA 1993c). The neurotoxic effects
of PCBs appear to be associated with some degree of ortho-chlorine substitution.
There is increasing evidence that many of the toxic effects of PCBs result from
alterations in hormonal function. However, because PCBs can act directly as
hormonal agonists or antagonists, PCB mixtures may have complex interactive
effects in biological systems (Korach et al., 1988; Safe et al., 1991; Shain et al.,
1991; U.S. EPA, 1993c). Because of the lack of sufficient toxicologic data, EPA
has not developed quantitative estimates of health risk for specific congeners.
PCB mixtures have been classified as probable human carcinogens (Group B2)
(IRIS, 1992; U.S. EPA, 1988a).
Of particular concern are several studies that have suggested that exposure to
PCBs may be damaging to the health of fetuses and children (Fein et al., 1984;
Jacobson et al., 1985, 1990). However, these studies are inconclusive due to
a failure to assess confounding variables (J. Hesse, Michigan Department of
Public Health, personal communication, 1992). In a more recent study of
prenatal exposure to PCBs and reproductive outcome, birth size was found to
be associated positively with PCB exposure, contrary to expectations (Dar et al.,
1992). The results of these investigations clearly indicate the need for further
study. Nevertheless, it may be appropriate for States in which PCBs are found
to be a problem contaminant in fish or shellfish tissue to assess the need to
issue consumption advisories, particularly for pregnant women, nursing mothers,
and children.
PCBs have been included in eight major fish contaminant monitoring programs
(Appendix C). A recent summary of the National Contaminant Biomonitoring
Program data from 1976 through 1984 indicated a significant downward trend in
total PCBs, although PCB residues in fish tissue remained widespread (Schmitt
et al., 1990). Total PCBs were detected at 91 percent of 374 sites surveyed in
the National Study of Chemical Residues in Fish (U.S. EPA, 1992c, 1992d).
Currently, PCB contamination in fish and shellfish has resulted in the issuance
of consumption advisories in 31 States (Figure 4-3) (RTI, 1993).
PCBs may be analyzed quantitatively as Aroclor equivalents or as individual
congeners. Historically, Aroclor analysis has been performed by most
laboratories. This procedure can, however, result in significant error in
determining total PCB concentrations (Schwartz et al., 1987) and in assessing
the toxicologic significance of PCBs, because it is based on the assumption that
distribution of PCB congeners in environmental samples and parent Aroclors is
similar.
The distribution of PCB congeners in Aroclors is, in fact, altered considerably by
physical, chemical, and biological processes after release into the environment,
particularly when the process of biomagnification is involved (Norstrom, 1988;
4-30
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4. TARGET ANALYTES
I
O
(A
O
O
at
1
in
w
•o
i
v>
o>
I
.i2
-------
4. TARGET ANALYTES
Oliver and Niimi, 1988; Smith et al., 1990). Recent aquatic environmental
studies indicate that many of the most potent, dioxin-like PCB congeners are
preferentially accumulated in higher organisms (Bryan et al., 1987; Kubiak et al.,
1989; Oliver and Niimi, 1988). This preferential accumulation probably results
in a significant increase in the total toxic potency of PCB residues as they move
up the food chain. Consequently, the congener-specific analysis of PCBs is
required for more accurate determination of total PCB concentrations and for
more rigorous assessment of the toxicologic effects of PCBs.
Even though the large number of congeners of PCBs and their similar chemical
and physical properties present serious analytical difficulties, analytical methods
for the determination of PCB congeners have been improved in recent years so
that it is now possible to determine essentially all PCB congeners in mixtures
(Huckins et al., 1988; Kannan et al., 1989; MacLeod et al., 1985; Maack and
Sonzogni, 1988; Mes and Weber, 1989; NOAA, 1989b; Smith et al., 1990;
Tanabe et al., 1987). Both NOAA (MacLeod et al., 1985; NOAA, 1989b) and the
EPA Narragansett Research Laboratory conduct PCB congener analyses and
have adopted the same 18 PCB congeners for monitoring fish contamination.
However, quantitation of individual PCB congeners is relatively time-consuming
and expensive and many laboratories do not have the capability or expertise to
perform such analyses. Some States currently conduct both congener and
Aroclor analysis; however, most States routinely perform only Aroclor analysis.
For the purposes of screening tissue residues against potential levels of public
health concern in fish and shellfish contaminant monitoring programs, the issue
of whether to determine PCB concentrations as Aroclor equivalents or as
individual congeners cannot be resolved entirely satisfactorily at this time,
primarily because of a lack of toxicologic data for individual congeners.
Ideally, congener analysis should be conducted. However, at present, only an
Aroclor-based quantitative risk estimate of carcinogenicity is available (IRIS,
1993) for developing SVs and risk assessment. Consequently, until adequate
congener-specific toxicologic data are available to develop quantitative risk
estimates for a variety of toxicologic endpoints, the EPA Office of Water
recommends, as an interim measure, that PCBs be analyzed as Aroclor
equivalents, with total PCB concentrations reported as the sum of Aroclors.
States are encouraged to develop the capability to perform PCB congener
analysis. When congener analysis is conducted, the 18 congeners
recommended by NOAA (shown in Table 4-3) should be analyzed and summed
to determine a total PCB concentration according to the approach used by
NOAA (1989b). States may wish to consider including additional congeners
based on site-specific considerations. PCB congeners of potential environmental
importance identified by McFarland and Clarke (1989) are listed in Table 4-3.
This interim recommendation is intended to (1) allow States flexibility in PCB
analysis until reliable congener-specific quantitative risk estimates are available,
and (2) encourage the continued development of a reliable database of PCB
4-32
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4. TARGET ANALYTES
Table 4-3. Polychlorlnated Blphenyl (PCB) Congeners Recommended for
Quantltatlon as Potential Target Analytes
PCB Congener"'*
2,4' diCB
2,2', 5 triCB
2,4,4' triCB
3,4,4' triCB
2,2'3,5' tetraCB
2,2*4,5' tetraCB
2,2',5,5' tetraCB
2,3',4,4' tetraCB
2,3',4',5 tetraCB
2,4,4>,5 tetraCB
3,3',4,4' tetraCB
3,4,4',5 tetraCB
2,2',3,4,5' pentaCB
2,2',3,4'I5 pentaCB
2,2',4,5,5' pentaCB
2,3,3',4.4' pentaCB
2,3,4,41,5 pentaCB
2,3',4,4I,5 pentaCB
2,3',4,4',6 pentaCB
2',3,4A',5perttaCB
3,3',4,4',5 pentaCB
2',3,3',4,4' hexaCB
2,2',3>4,4>,5' hexaCB
2,2',3,5,5',6 hexaCB
2,2',4,4',5I5' hexaCB
2,3,3',4,4',5 hexaCB
2,3,3>,4,4',5 hexaCB
2,3,3',4,4',6 hexaCB
2,3',4,4',5,5' hexaCB
2,3'.4I4',5',6 hexaCB
3,31,4,4',5,5' hexaCB
Recommended by
NOAAC
8
18
28
44
52
66
77
101
105
118
126
128
138
153
169
Recommended by McFariand
and Clarke (1989)
Highest
Priority"
77
87,
49
101
105
118
126
128
138
153
156
169
Second
Priority*
18
37
44
99
52
70
74
81
114
119
123
151
157
158
167
168
(continued)
4-33
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4. TARGET ANALYTES
Table 4-3 (continued)
Recommended by
PCB Congener*-" NOAA6
2,2',3,3',414',5 heptaCB 170
2,2' ,3 AA', 5,5' heptaCB 180
2,2',3,4,4t)51,6 heptaCB
2,2',3,4,4I,6,6' heptaCB
2,2',3,4>,5,5',6 heptaCB 187
2,3,3',4,4>I5,5f heptaCB
2,2',3,3f,414',5,6 octaCB
2,2',3,3',4,5,5',6' octaCB
2,2',3,3',4,4>,5,51,6 nonaCB
2,2',3,3',4,4>,5I5>,616> decaCB
Recommended by McFartand
and Clarke (1989)
Highest
Priority"
170
180
183
184
195
206
209
Second
Priority*
187
189
201
' PCB congeners recommended for quantitation, from dichlorobiphenyl (diCB) through
decachtorobiphenyl (decaCB).
b Congeners are identified in each column by their International Union of Pure and Applied
Chemistry (IUPAC) number, as referenced in Ballschmitter and Zell (1980) and Mullin et al.
(1984).
c EPA recommends that these 18 congeners be summed to determine total PCB concentration
(NOAA, 1989b).
d PCB congeners having highest priority for potential environmental importance based on potential
for toxicity, frequency of occurrence in environmental samples, and relative abundance in animal
tissues (McFartand and Clarke, 1989).
* PCB congeners having second priority for potential environmental importance based on potential
for toxicity, frequency of occurrence in environmental samples, and relative abundance in animal
tissues (McFartand and Clarke, 1989).
4-34
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4. TARGET ANALYTES
congener concentrations in fish and shellfish tissue in order to increase our
understanding of the mechanisms of action and toxicities of these chemicals.
The rationale for, and the uncertainties of, this recommended approach are
discussed further in Section 5.3.2.3.
4.3.7 Dloxlns and Dlbenzofurans
Note: At this time, the EPA Office of Research and Development is reevaluating
the potency of dioxins and dibenzofurans. Information provided below as well
as information in Section 5.3.2.4 related to calculating toxicity equivalent
concentrations (TECs) and SVs for dioxins/furans is subject to change pending
the results of this reevaluation.
The polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-
furans (PCDFs) are included as target analytes primarily because of the extreme
potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). Extremely low
doses of this isomer have been found to elicit a wide range of toxic responses
in animals, including carcinogenicity, teratogenicity, fetotoxicity, reproductive
dysfunction, and immunotoxicity (U.S. EPA, 1987d). This compound is the most
potent animal carcinogen evaluated by EPA, and EPA has determined that there
is sufficient evidence to conclude that 2,3,7,8-TCDD is a probable human car-
cinogen (B2) (IRIS, 1992). Concern over the health effects of 2,3,7,8-TCDD is
increased because of its persistence in the environment and its high potential to
bioaccumulate (U.S. EPA, 1987d).
Because dioxin/furan contamination is found almost exclusively in proximity to
industrial sites (e.g., bleached kraft paper mills or facilities handling 2,4,5-
trichlorophenoxyacetic acid [2,4,5-T], 2,4,5-trichlorophenol [2,4,5-TCP], and/or
silvex) (U.S. EPA, 1987d), it is recommended that each State agency
responsible for monitoring include these compounds as target analytes on a site-
specific basis based on the presence of industrial sites and results of any
environmental (water, sediment, soil, air) monitoring performed in areas adjacent
to these sites. All States should maintain a current awareness of potential
dioxin/furan contamination.
Fifteen dioxin and dibenzofuran congeners have been included in two major fish
contaminant monitoring programs; however, one congener, 2,3,7,8-TCDD, has
been included in seven national monitoring programs (Appendix C). Six dioxin
congeners and nine dibenzofuran congeners were measured in fish tissue and
shellfish samples in the National Study of Chemical Residues in Fish. The
various dioxin congeners were detected at from 32 to 89 percent of the 388 sites
surveyed, while the furan congeners were detected at from 1 to 89 percent of the
388 sites surveyed (U.S. EPA, 1992c, 1992d). The dioxin/furan congeners
detected at more than 50 percent of the sites are listed below:
• 1,2,3,4,6,7,8 HpCDD (89 percent)
• 2,3,7,8 TCDF (89 percent)
• 2,3,7,8 TCDD (70 percent)
4-35
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4. TARGET ANALYTES
• 1,2,3,6,7,8 HxCDD (69 percent)
• 2,3,4,7,8 PeCDF (64 percent)
• 1,2,3,4,6,7,8 HpCDF (54 percent)
• 1,2,3,7,8 PeCDD (54 percent).
Currently, 22 States have issued fish consumption advisories for dioxins/furans
(Figure 4-4) (RTI, 1993).
Dioxins/furans should be considered for analysis primarily at sites of pulp and
paper mills using a chlorine bleaching process and at industrial sites where the
following organic compounds have been or are currently formulated: herbicides
(containing 2,4,5-trichlorophenoxy acids and 2,4,5-trichlorophenol),
hexachlorophene, pentachlorophenol, and PCBs (U.S. EPA, 1987d). If
resources permit, it is recommended that the 17 2,3,7,8-substituted tetra- through
octa-chlorinated dioxin and dibenzofuran congeners shown in Table 4-4 be
included as target analytes. At a minimum, 2,3,7,8-TCDD and 2,3,7,8-tetra-
chlorodibenzofuran (2,3,7,8-TCDF) should be determined.
4.4 TARGET ANALYTES UNDER EVALUATION
At present, the EPA Office of Water is evaluating one metal (lead) for possible
inclusion as a recommended target analyte in State fish and shellfish
contaminant monitoring programs. A toxicologic profile for this metal and the
status of the evaluation are provided in this section. Other contaminants will be
evaluated and may be recommended as target analytes as additional toxicologic
data become available.
Table 4-4. Dibenzo-p-Dloxins and Dibenzofurans Recommended
as Target Analytes
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDF
Source: Barnes and Bellin, 1989.
4-36
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4. TARGET ANALYTES
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4-37
-------
4. TARGET ANALYTES
Note: Any time a State independently deems that the analyte currently under
evaluation and/or other contaminants are of public health concern within its
jurisdiction, the State should include these contaminants in its fish and shellfish
contaminant monitoring program.
4.4.1 Lead
Lead is derived primarily from the mining and processing of limestone and
dolomite deposits, which are often sources of lead, zinc, and copper (May and
McKinney, 1981). it is also found as a minor component of coal. Historically,
lead has had a number of industrial uses, including use in paints, in solder used
in plumbing and food cans, and as a gasoline additive. As recently as the mid-
1980s, the primary source of lead in the environment was the combustion of
gasoline; however, use of lead in U.S. gasoline has fallen sharply in recent
years. At present, lead is used primarily in batteries, electric cable coverings,
some exterior paints, ammunition, and sound barriers. Currently, the major
points of entry of lead into the environment are from mining and smelting
operations, from fly ash resulting from coal combustion, and from the leachates
of landfills (May and McKinney, 1981).
Lead has been included in six national monitoring programs (Appendix C). Lead
has been shown to bioaccumulate, with the organic forms, such as tetraethyl
lead, appearing to have the greatest potential for bioaccumulation in fish tissues.
High concentrations of lead have been found in marine bivalves and finfish from
both estuarine and marine waters (NOAA, 1987, 1989a). Lead concentrations
in freshwater fish declined significantly from a geometric mean concentration of
0.28 ppm in 1976 to 0.11 ppm in 1984. This trend has been attributed primarily
to reductions in the lead content of U.S. gasoline (Schmitt and Brumbaugh,
1990). Currently three States (Massachusetts, Missouri, and Tennessee) and
American Samoa have issued fish advisories for lead contamination (RTI, 1993).
Lead is particularly toxic to children and fetuses. Subtle neurobehavioral effects
(e.g., fine motor dysfunction, impaired concept formation, and altered behavior
profile) occur in children exposed to lead at concentrations that do not result in
clinical encephalopathy (ATSDR, 1988). A great deal of information on the
health effects of lead has been obtained through decades of medical observation
and scientific research. This information has been assessed in the development
of air and water quality criteria by the Agency's Office of Health and
Environmental Assessment (OHEA) in support of regulatory decisionmaking by
the Office of Air Quality Planning and Standards (OAQPS) and by the Office of
Drinking Water (ODW). By comparison to most other environmental toxicants,
the degree of uncertainty about the health effects of lead is quite low. It appears
that some of these effects, particularly changes in the levels of certain blood
enzymes and in aspects of children's neurobehavioral development, may occur
at blood lead levels so low as to be essentially without a threshold. The
Agency's Reference Dose (RfD) Work Group discussed inorganic lead (and lead
compounds) in 1985 and considered it inappropriate to develop an RfD for
4-38
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4. TARGET ANAL YTES
inorganic lead (IRIS, 1993). Lead and its inorganic compounds have been
classified as probable human carcinogens (B2) by EPA (IRIS, 1992). However,
at this time, a quantitative estimate of carcinogenic risk from oral exposure is not
available (IRIS, 1993).
Because of the lack of quantitative health risk assessment information for oral
exposure to inorganic lead, the EPA Office of Water has not included lead as a
recommended target analyte in fish and shellfish contaminant monitoring
programs at this time. Note: Because of the observation of virtually no-
threshold neurobehavioral developmental effects of lead in children, States
should include lead as a target analyte in fish and shellfish contaminant
programs if there is any evidence that this metal may be present at detectable
levels in fish or shellfish tissue. Additional information is provided on this issue
in Volume II—Risk Assessment and Fish Consumption Limits—in this guidance
series (U.S. EPA, 1994).
4-39
-------
-------
5. SCREENING VALUES FOR TARGET ANALVTES
SECTION 5
SCREENING VALUES FOR TARGET ANALYTES
For the purpose of this guidance document, screening values are defined as
concentrations of target analytes in fish or shellfish tissue that are of potential
public health concern and that are used as standards against which levels of
contamination in similar tissue collected from the ambient environment can be
compared. Exceedance of these SVs should be taken as an indication that more
intensive site-specific monitoring and/or evaluation of human health risk should
be conducted.
The EPA-recommended risk-based method for developing SVs (U.S. EPA,
1989d) is described in this section. This method is considered to be appropriate
for protecting the health of fish and shellfish consumers for the following reasons
(Reinert et al., 1991):
It gives full priority to protection of public health.
It provides a direct link between fish consumption rate and risk levels (i.e.,
between dose and response).
It generally leads to conservative estimates of increased risk.
It is designed for protection of consumers of locally caught fish and shellfish,
including susceptible subpopulations such as sport and subsistence
fishermen who are at potentially greater risk than the general adult
population because they tend to consume greater quantities of fish and
because they frequently fish the same sites repeatedly.
At this time, the EPA Office of Water is recommending use of this method
because it is the basis for developing current water quality criteria and was the
approach used in the National Study of Chemical Residues in Fish (U.S. EPA,
1992c, 1992d). EPA recognizes that there are many other approaches and
models currently in use. Further discussion of the EPA Office of Water risk-
based approach, including a detailed description of the four steps involved in risk
assessment (hazard identification, dose-response assessment, exposure
assessment, and risk characterization) will be discussed in greater detail in the
second guidance document in this series.
5-1
-------
5. SCREENING VALUES FOR TARGET ANALYTES
5.1 GENERAL EQUATIONS FOR CALCULATING SCREENING VALUES
Risk-based SVs are derived from the general model for calculating the effective
ingested dose of a chemical m (Em) (U.S. EPA, 1989d):
Enl = (Cm • CR • XJ / BW (5-1)
where
Em = Effective ingested dose of chemical m in the population of concern
averaged over a 70-yr lifetime (mg/kg/d)
Cm = Concentration of chemical m in the edible portion of the species of
interest (mg/kg; ppm)
CR = Mean daily consumption rate of the species of interest by the general
population or subpopulation of concern averaged over a 70-yr lifetime
(kg/d)
Xm = Relative absorption coefficient, or the ratio of human absorption
efficiency to test animal absorption efficiency for chemical m
(dimensionless)
BW = Mean body weight of the general population or subpopulation of
concern (kg).
Using this model, the SV for the chemical m (SVm) is equal to Cm when the
appropriate measure of toxicologic potency of the chemical m (Pm) is substituted
for Em. Rearrangement of Equation (5-1), with these substitutions, gives
svm =
BW) / (CR - Xm)
(5-2)
where
Pm = Toxicologic potency for chemical m; the effective ingested dose of
chemical m associated with a specified level of health risk as
estimated from dose-response studies; dose-response variable.
In most instances, relative absorption coefficients (Xm) are assumed to be 1.0
(i.e., human absorption efficiency is assumed to be equal to that of the test
animal), so that
(Pm.BW)/CR.
(5-3)
However, if Xm is known, Equation (5-2) should be used to calculate SVm.
Dose-response variables for noncarcinogens and carcinogens are defined in
Sections 5.1.1 and 5.1.2, respectively. These variables are based on an assess-
ment of the occurrence of a critical toxic or carcinogenic effect via a specific
route of exposure (i.e., ingestion, inhalation, dermal contact). Oral dose-
5-2
-------
5. SCREENING VALUES FOR TARGET ANALYTES
response variables for the recommended target analytes are given in Appendix
E. Because of the fundamental differences between the noncarcinogenic and
carcinogenic dose-response variables used in the EPA risk-based method, SVs
must be calculated separately for noncarcinogens and potential carcinogens as
shown in the following subsections.
5.1.1 Noncarcinogens
The dose-response variable for noncarcinogens is the Reference Dose (RfD).
The RfD is an estimate of a daily exposure to the human population (including
sensitive subpopulations) that is likely to be without appreciable risk of
deleterious effects during a lifetime. The RfD is derived by applying uncertainty
or modifying factors to a subthreshold dose (i.e., LOAEL if the NOAEL is
indeterminate) observed in chronic animal bioassays. These uncertainty or
modifying factors range from 1 to 10 for each factor and are used to account for
uncertainties in:
• Sensitivity differences among human subpopulations
Interspecies extrapolation from animal data to humans
Short-term to lifetime exposure extrapolation from less than chronic results
on animals to humans when no long-term human data are available
• Deriving an RfD from a LOAEL instead of a NOAEL
Incomplete or inadequate toxicity or pharmacokinetic databases.
The uncertainty (UF) and modifying (MF) factors are multiplied to obtain a final
UF«MF value. This factor is divided into the NOAEL or LOAEL to derive the RfD
(Barnes and Dawson, 1988; U.S. EPA, 1989d).
The following equation should be used to calculate SVs for noncarcinogens:
SVn s (RfD - BW)/CR (5-4)
where
SVn = Screening value for a noncarcinogen (mg/kg; ppm)
RfD = Oral reference dose (mg/kg/d)
and BW and CR are defined as in Equation (5-1).
5.1.2 Carcinogens
According to The Risk Assessment Guidelines of 1986 (U.S. EPA, 1987f), the
default model for low-dose extrapolation of carcinogens is a version (GLOBAL
86) of the linearized multistage no-threshold model developed by Crump et al.
(1976). This extrapolation procedure provides an upper 95 percent bound risk
estimate (referred to as a q1*), which is considered by some to be a
conservative estimate of cancer risk. Other extrapolation procedures may be
used when justified by the data.
5-3
-------
5. SCREENING VALUES FOR TARGET ANALYTES
Screening values for carcinogens are derived from: (1) a carcinogenicity potency
factor or slope factor (SF), which is generally an upper bound risk estimate; and
(2) a risk level (RL), an assigned level of maximum acceptable individual
lifetime risk (e.g., RL = 10~5 for a level of risk not to exceed one excess case of
cancer per 100,000 individuals exposed over a 70-yr lifetime) (U.S. EPA, 1989d).
The following equation should be used to calculate SVs for carcinogens:
SVC = [(RL / SF) - BW] / CR (5-5)
where
svc
RL
= Screening value for a carcinogen (mg/kg; ppm)
= Maximum acceptable risk level (dimensionless)
SF = Oral slope factor (mg/kg/d)"1
and BW and CR are defined as in Equation (5-1).
5.1.3 Recommended Values for Variables In Screening Value Equations
The recommended values in this section for variables used in Equations (5-4)
and (5-5) to calculate SVs are based upon assumptions for the general adult
population. For risk management purposes (e.g., to direct limited resources
toward protection of sensitive subpopulations), States may choose to use values
for consumption rate (CR), body weight (BW), and risk level (RL) different from
those recommended in this section.
5.1.3.1 Dose-Response Variables—
EPA has developed oral RfDs and/or SFs for all of the recommended target
analytes in Section 4 (see Appendix E). These are maintained in the EPA
Integrated Risk Information System (IRIS, 1992), an electronic database
containing health risk and EPA regulatory information on approximately 400
different chemicals. The IRIS RfDs and SFs are reviewed regularly and updated
as necessary when new or more reliable information on the toxic or carcinogenic
potency of chemicals becomes available.
When IRIS values for oral RFDs and SFs are available, they should be used to
calculate SVs for target analytes from Equations (5-4) and (5-5), respectively.
It is important that the most current IRIS values for oral RfDs and SFs be used
to calculate SVs for target analytes, unless otherwise recommended.
A summary description of IRIS and instructions for accessing information in IRIS
are found in U.S. EPA (1989d). Additional information can be obtained from
IRIS User Support (Tel: 513-569-7254). IRIS is also available on the National
Institutes of Health (NIH) National Library of Medicine TOXNET system (Tel:
301-496-6531).
5-4
-------
5. SCREENING VALUES FOR TARGET ANALYTES
In cases where IRIS values for oral RFDs or SFs are not available for calculating
SVs for target analytes, estimates of these variables should be derived from the
most recent water quality criteria (U.S. EPA, 1992e) according to procedures
described in U.S. EPA (1991 a, p. IV-12), or from the most current Reference
Dose List (U.S. EPA, 1993b) and the Classification List of Chemicals Evaluated
for Carcinogenicity Potential (U.S. EPA 1992a) from the Office of Pesticide
Programs Health Effects Division.
5.1.3.2 Body Weight (BW) and Consumption Rate (CR)—
Values for the variables BW and CR in Equations (5-4) and (5-5) are given in
Table 5-1 for the general adult population and various subpopulations. In this
document, the EPA Office of Water used a BW = 70 kg and a CR = 6.5 g/d to
calculate SVs for the general adult population. Note: The 6.5-g/d CR value that
is used to establish water quality criteria is currently under review by the EPA
Office of Water. This CR, which represents a consumption rate for the average
fish consumer in the general adult population (45 FR 231, Part V), may not be
appropriate for sport and subsistence fishermen who generally consume larger
quantities of fish and shellfish (U.S. EPA, 1990a).
With respect to consumption rates, EPA recommends that States always
evaluate any type of consumption pattern they believe could reasonably be
occurring at a site. Evaluating additional consumption rates only involves
calculating additional SVs and does not add to sampling or analytical costs.
The EPA has published detailed guidance on exposure factors (U.S. EPA,
1990a). In addition, EPA has published a review and analysis of survey
methods that can be used by States to determine fish and shellfish consumption
rates of local populations (U.S. EPA, 1992b). States should consult these
documents to ensure that appropriate values are selected to calculate SVs for
site-specific exposure scenarios.
5.1.3.3 Risk Level (RL)—
The EPA Office of Water recommends that an RL of 10"5 be used to calculate
screening values for the general adult population. However, States may choose
to use an appropriate RL value typically ranging from 10"* to 10'7. This is the
range of risk levels employed in various U.S. EPA programs. Selection of the
appropriate RL is a risk management decision that is made by the State.
5.2 RECOMMENDED SCREENING VALUES FOR TARGET ANALYTES
Recommended target analyte SVs, and the dose-response variables used to
calculate them, are given in Table 5-2. These SVs were calculated from
Equations (5-4) or (5-5) using the following values for BW, CR, and RL and the
most current IRIS values for oral RfDs and SFs (IRIS, 1992) unless otherwise
noted:
5-5
-------
5. SCREENING VALUES FOR TARGET ANALYTES
Table 5-1. Recommended Values for Mean Body Weights (BWs)
and Fish Consumption Rates (CRs) for Selected Subpopulatlons
Variable Recommended value Subpopulation
BW
CR«
70kg
78kg
65kg
12kg
17kg
25.kg
36kg
51 kg
61 kg
6.5 g/d (0.0065 kg/d)
14 g/d (0.014 kg/d)
15 g/d (0.015 kg/d)
30 g/d (0.030 kg/d)
140 g/d (0.140 kg/d)
All adults (U.S. EPA, 1990a)
Adult males (U.S. EPA, 1985b, 1990a)
Adult females (U.S. EPA, 1985b, 1990a)
Children <3 yr (U.S. EPA, 1985b, 1990a)
Children 3 to <6 yr (U.S. EPA, 19S5b, 1990a)
Children 6 to <9 yr (U.S. EPA, 1985b, 1990a)
Children 9 to <12 yr (U.S. EPA, 1985b, 1990a)
Children 12 to <15 yr (U.S. EPA, 1985b, 1990a)
Children 15 to <18 yr (U.S. EPA, 1985b, 1990a)
Estimate of the average consumption of fish and
shellfish from estuarine and fresh waters by the
general U.S. population (45 FR 231, Part V)
Estimate of the average consumption of fish and
shellfish from marine, estuarine, and fresh waters by
the general U.S. population (45 FR 231, Part V)
Estimate of the average consumption of fish from the
Great Lakes by the 95th percentile of the regional
population (fishermen and nonfishermen) (U.S. EPA,
1992e)
Estimate of the average consumption of fish and
shellfish from marine, estuarine, and fresh waters by
the 50th percentile of recreational fishermen (U.S.
EPA, 1990a)
Estimate of the average consumption of fish and
shellfish from marine, estuarine, and fresh waters by
the 90th percentile of recreational fishermen (i.e.,
subsistence fishermen) (U.S. EPA, 1990a)
* These are recommended consumption rates only. Note: EPA is currently evaluating the use of
6.5 g/d, 30 g/d, and 140 g/d as estimates of consumption rates for the general population, the 50th
percentile of recreational fishermen, and subsistence fishermen, respectively. When local
consumption rate data are available for these populations, they should be used to calculate SVs for
noncarcinogens and carcinogens, as described in Sections 5.1.1 and 5.1.2, respectively.
5-6
-------
5. SCREENING VALUES FOR TARGET ANALYTES
• For noncarclnogens:
BW = 70 kg, average adult body weight
CR = 6.5 g/d (0.0065 kg/d), estimate of average consumption of fish and
shellfish from estuarine and fresh waters by the general adult
population (45 FR 231, Part V).
• For carcinogens:
BW and CR, as above
RL = 10~5, a risk level corresponding to one excess case of cancer per
100,000 individuals exposed over a 70-yr lifetime.
Where both oral RfD and SF values are available for a given target analyte SVs
for, both noncarcinogenic and carcinogenic effects are listed in Table 5-2.
Unless otherwise indicated, the lower of the two SVs should be used. EPA
recommends that the SVs in the shaded boxes (Table 5-2) be used by States
when making the decision to implement Tier 2 intensive monitoring. However,
States may choose to adjust these SVs for specific target analytes for the
protection of sensitive subpopulations (e.g., pregnant women, children, and
recreational or subsistence fishermen). EPA recognizes that States may use
higher CRs that are more appropriate for recreational and subsistence fishermen
in calculating SVs for use in their jurisdictions rather than the 6.5-g/d CR for the
general adult population used to calculate the SVs shown in Table 5-2.
Note: States should use the same SV (i.e., either for the general adult
population or adjusted for other subpopulations) for a given target analyte for
both screening and intensive studies. Therefore, it is critical that States clearly
define their program objectives and accurately characterize the population or
subpopulation(s) of concern in order to ensure that appropriate SVs are selected.
If analytical methodology is not sensitive enough to reliably quantitate target
analytes at or below selected SVs (see Section 8.2.2 and Table 8-4), program
managers must determine appropriate fish consumption guidance based on
lowest detectable concentrations or provide justification for adjusting SVs to
values at or above achievable method detection limits. It should be emphasized
that when SVs are below method detection limits, the failure to detect a target
analyte cannot be assumed to indicate that there is no cause for concern for
human health effects.
For noncarcinogens, adjusted SVs should be calculated from Equation (5-4)
using appropriate alternative values of BW and/or CR. For carcinogens,
adjusted SVs should be calculated from Equation (5-5) using an RL ranging from
10"4 to 10~7 and/or sufficiently protective alternative values of BW and CR.
Examples of SVs calculated for selected subpopulations of concern and for RL
values ranging from 10 to 10"' are given in Table 5-3.
5-7
-------
5. SCREENING VALUES FOR TARGET ANALYTES
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5. SCREENING VALUES FOR TARGET ANALYTES
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lt|l|1ll
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5 s §ag « $ •
The SF value listed is for benzo[a]pyrene. Values for other P/
screening and intensive studies, tissue samples be analyzed f<
chrysene, dibenz[a,ft]anthracene, and indeno/"7,2,3-cc/]pyrene,
provisional guidance for quantitative risk assessment of PAHs
each sample for comparison with the recommended SV for be
assessment of PAHs (U.S. EPA 1993c) is considered provisioi
approach is under Agency review and over the next year will b
method provided in this guidance document is subject to charr
cr
1 8
1! g, a
•£ CD .£=
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t *^~ c
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»— CD O) n
C73 'tj -r §
~ CD ^ CO
S^' Q. . 0
CM-52^ o
The RfD for PCBs is based on the chronic toxicity of Aroclor 1
developmental toxicity of Aroclor 1016 (7x1 0'5) and, therefore,
Volume II (Section 5.6.19) of this guidance document series (I
in conducting quantitative risk assessments and determination
._
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is intended
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The SF is based on a carcinogenicity assessment of Aroclor 1
mixtures (IRIS, 1992).
e)
2
CO 3
_ o
1*. 1
i co -S g* |
§ 1 1 '|L?
•55 «f c m o
E > -o o>e\f
• '5 S •— ~o
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33 _o *" co
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LU • ca o,g
cq 1 -1 'I 1
= 8 g 3. 2=
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g=ni
The SF value listed is for 2,3,7,8-tetrachlorodibenzo-p-dioxin (
value of RfD = 1x1Q-9 for 2,3,7,8-TCDD from ATSDR (1987d).
substituted tetra- through octa-chlorinated dibenzo-p-dioxins a
calculated for each sample for comparison with the recommer
Concentrations (TECs) (Barnes and Bellin, 1989; U.S. EPA, 1
be determined at a minimum.
.-
5-12
-------
5. SCREENING VALUES FOR TARGET ANALYTES
Table 5-3. Example Screening Values (SVs) for Various
Subpopulatlons and Risk Levels (RLs)a
Chemical
Subpopulation" CRC BW
RfO
SF RL SV (ppm)
ChlorpyrHos
Cadmium
Standard adults
Children
Subsistence
fishermen
6.5 70 3x10'J
6.5 36d 3x10'3
140 70 3x10'3
Standard adults
Children
Subsistence
fishermen
6.5 70 1 x 10'3
6.5 36d 1 x 1Q-3
140 70 1 x 10'3
30
20
2
10
6
0.5
Lindane
Toxaphene
Standard adults
6.5 70
1.3
1.3
1.3
1.3
10"
10
10"
10
,-5
,-7
8x 101
8x 10'2
8 x 10'3
8 x 10'4
Children 6.5 36°
Subsistence 140 70
fishermen
,
Standard adults 6.5 70
Children 6.5 36d
Subsistence 1 40 70
fishermen
— 1.3
1.3
1.3
1.3
— 1.3
1.3
1.3
1.3
— 1.1
1.1
1.1
1.1
— 1.1
1.1
1.1
1.1
— 1.1
1.1
1.1
1.1
10'4
io-5
10'6
io-7
10'4
10'5
io-6
10"7
10'4
10'5
io-6
io-7
10'4
10"5
10'6
10'7
10'4
10'5
10'6
10'7
4X1Q-1
4 x 10'2
4x10'3
4x10"4
4x 10'2
4 x 10'3
4 x 10'4
4x 10'5
10X10'1
10x10'2
10 x 10'3
10x10'4
'5 x 10'1
5x 10"2
5x 10'3
5x10'4
5x 10"2
5 x 10'3
5 x 10'4
5x 10'5
OR
BW
RfD
SF
RL
° See Equations (5-4) and (5-5).
See Table 5-2 for definitions of subpopulations.
° To calculate SVs, the CRs given in this table must be divided by 1,000 to convert g/d to kg/d.
Mean daily fish or shellfish consumption rate, averaged over a 70-yr lifetime for the population of concern (g/d).
Mean body weight, estimated for the population of concern (kg).
Oral reference dose for noncarcinogens (mg/kg/d).
Oral slope factor for carcinogens (mg/kg/d) .
Maximum acceptable risk level for carcinogens (dimensionless).
BW used is for children 9 to <12 yr (see Table 5-2).
5-13
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5. SCREENING VALUES FOR TARGET ANALYTES
The need to accurately characterize the subpopulation of interest in order to
establish sufficiently protective SVs cannot be overemphasized. For example,
the recommended consumption rate of 140 g/d for subsistence fishermen may
be an underestimate of consumption rate for some subsistence populations. In
a recent study of Alaskan subsistence fishing economies (Wolf and Walker,
1987), daily consumption rates for subsistence fishermen were found to range
from 6 to 1,536 g/d, with an average daily consumption rate of 304 g/d. Using
this average consumption rate and an estimated average body weight of 70 kg,
the SV for cadmium (RfD = 1 x 10'3 mg/kg/d) is, from Equation (5-4),
SV = (0.001 mg/kg/d • 70 kg) / (0.304 kg/d) = 0.2 mg/kg (ppm) . (5-7)
This value is significantly lower than the SV of 0.5 ppm for cadmium based on
the recommended consumption rate of 140 g/d for subsistence fishermen, as
shown in Table 5-3.
5.3 COMPARISON OF TARGET ANALYTE CONCENTRATIONS WITH
SCREENING VALUES
As noted previously, the same SV for a specific target analyte should be used
in both the screening and intensive studies. The measured concentrations of
target analytes in fish or shellfish tissue should be compared with their
respective SVs in both screening and intensive studies to determine the need for
additional monitoring and risk assessment.
Recommended procedures for comparing target analyte concentrations with SVs
are provided below. Related guidance on data analysis is given in Section 9.1.
5.3.1 Metals
5.3.1.1 Arsenic—
Most of the arsenic present in fish and shellfish tissue is organic arsenic,
primarily pentavalent arsenobetaine, which has been shown in numerous studies
to be metabolically inert and nontoxic (Brown et al., 1990; Cannon et al., 1983;
Charbonneau et al., 1978; Jongen et al., 1985; Kaise et al. 1985; Luten et al.,
1982; Sabbioni et al., 1991; Siewicki, 1981; Tarn et al., 1982; Vahter et al., 1983;
Yamauchi et al., 1986). Inorganic arsenic, which is of concern for human health
effects (ATSDR, 1993; WHO, 1989), is generally found in seafood at concentra-
tions ranging from <1 to 20 percent of the total arsenic concentration (Edmonds
and Francesconi, 1993; Nraigu and Simmons, 1990). It is recommended that,
in both screening and intensive studies, total inorganic arsenic tissue
concentrations be determined for comparison with the recommended SV for
chronic oral exposure. This approach is more rigorous than the current FDA
method of analyzing for total arsenic and estimating inorganic arsenic concentra-
tions based on the assumption that 10 percent of the total arsenic in fish tissue
is in the inorganic form (U.S. FDA, 1993). Although the cost of analysis for
inorganic arsenic (see Table 8-5) may be three to five times greater than for total
5-14
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5. SCREENING VALUES FOR TARGET ANALYTES
arsenic, the increased cost is justified to ensure that the most accurate data are
obtained for quantitative assessment of human health risks.
5.3.1.2 Cadmium, Mercury, and Selenium—
For cadmium, mercury, and selenium, the total metal tissue concentration should
be determined for comparison with the appropriate SV. For mercury, the SV that
is calculated from the RfD for developmental effects of methylmercury (see Table
5-2) should be used because it is'most protective.
The determination of methylmercury is not recommended even though methyl-
mercury is the compound of greatest concern for human health (NAS, 1991;
Tollefson, 1989) and the recommended SV is for methylmercury (see Table 5-2).
Because most mercury in fish and shellfish tissue is present as methylmercury
(NAS, 1991; Tollefson, 1989), and because of the relatively high analytical cost
for methylmercury, it is recommended that total mercury be determined and the
conservative assumption be made that all mercury is present as methylmercury.
This approach is deemed to be most protective of human health and most cost-
effective.
Note: The EPA has recently reevaluated the RfD for methylmercury, primarily
because of concern about evidence that the fetus is at increased risk of adverse
neurological effects from exposure to methylmercury (Marsh et al., 1987;
Piotrowski and Inskip, 1981; NAS, 1991; WHO, 1976, 1990). On May 1, 1995,
IRIS was updated to include an oral RfD of 1x10"4 mg/kg/d based on
developmental neurological effects in human infants. An oral RfD of 3x10"4
mg/kg/d for chronic systemic effects of methylmercury among the general adult
population was available in IRIS until May 1,1995; however, it was not listed in
the IRIS update on that date. For the purposes of calculating an SV for
methylmercury that is protective of fetuses and nursing infants, the EPA Office
of Water has chosen to continue to use the general adult population RfD of
3x1 (T4 mg/kg/d for chronic systemic effects of methylmercury until a value is
relisted in IRIS, and to reduce this value by a factor of 5 to derive an RfD of
6x10"5 mg/kg/d for developmental effects among infants. This factor is based
on experimental results that suggest a possible fivefold increase in fetal
sensitivity to methylmercury exposure. This more protective approach
recommended by the EPA Office of Water was deemed to be most prudent at
this time. This approach should be considered interim until such time as the
Agency has reviewed new studies on the chronic and developmental effects of
methylmercury.
5.3.1.3 Tributyltln—
Tissue samples should be analyzed specifically for tributyltin for comparison with
the recommended SV for this compound.
5-15
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5. SCREENING VALUES FOR TARGET ANALYTES
5.3.2 Organlcs
For each of the recommended organic target analytes that are single
compounds, the determination of tissue concentration and comparison with the
appropriate SV is straightforward. However, for those organic target analytes
that include a parent compound and structurally similar compounds or metabo-
lites (i.e., total chlordane, total DDT), or that represent classes of compounds
(i.e., PAHs, PCBs, dioxins/furans), additional guidance is necessary to ensure
that a consistent approach is used to determine appropriate target analyte
concentrations for comparison with recommended SVs.
5.3.2.1 Chlordane—
The SV for total chlordane is derived from technical-grade chlordane. Oral slope
factors are not available in IRIS (1992) for cis- and trans-chlordane, cis- and
trans-nonachlor, and oxychlordane. At this time, as a conservative approach,
EPA recommends that, in both screening and intensive studies, the
concentrations of cis- and trans-chlordane, cis- and trans-nonachlor, and
oxychlordane be determined and summed to give a total chlordane concentration
for comparison with the recommended SV for total chlordane (see Table 5-2).
5.3.2.2 DDT—
DDT and its metabolites (i.e., the 4,4'- and 2,4'-isomers of DDE and ODD) are
all potent toxicants, DDE isomers being the most prevalent in the environment.
As a conservative approach, EPA recommends that, in both screening and
intensive studies, the concentrations of 4,4'- and 2,4'-DDT and their DDE and
DDD metabolites be determined and a total DDT concentration be calculated for
comparison with the recommended SV for total DDT (see Table 5-2).
5.3.2.3 PAHs—
Although several PAHs have been classified as B2 carcinogens (probable human
carcinogens), benzo[a]pyrene is the only PAH for which an SF is currently
available in IRIS (1995). As a result, EPA quantitative risk estimates for PAH
mixtures have often assumed that all carcinogenic PAHs are equipotent to
benzo[a]pyrene. The EPA Office of Health and Environmental Assessment has
recently issued provisional guidance for quantitative risk assessment of PAHs
(U.S. EPA, 1993c) in which an estimated order of potential potency for six Group
82 PAHs relative to benzo[a]pyrene is recommended, as shown in Table 5-4.
Based on this guidance, it is recommended that, in both screening and intensive
studies, tissue samples be analyzed for the seven PAHs shown in Table 5-4 and
that a potency-weighted total concentration be calculated for each sample for
comparison with the recommended SV for benzo[a]pyrene. This potency
equivalency concentration (PEC) should be calculated using the following
equation:
PEC = I (RPj
(5-8)
5-16
-------
5. SCREENING VALUES FOR TARGET ANAL YTES
where
RPj = Relative potency for the ith PAH (from Table 5-4)
C, = Concentration of the ith PAH.
At this time, EPA's recommendation for risk assessment of PAHs (U.S. EPA,
1993c) is considered provisional because quantitative risk assessment data are
not available for all PAHs. This approach is under Agency review and over the
next year will be evaluated as new health effects benchmark values are
developed. Therefore, the method provided in this guidance document is subject
to change pending results of the Agency's reevaluation.
5.3.2.4 PCBs—
Using the interim approach for PCB analysis recommended by the EPA Office
of Water (see Section 4.3.5), total PCB concentrations should be determined, in
both screening and intensive studies, as the sum of Aroclor equivalents. The
total PCB concentration should be compared with the recommended SV for
PCBs (see Table 5-2). Because this SV is based on the SF for Aroclor 1260,
the recommendation to use this SV for comparison with total Aroclor
concentration requires the assumption that Aroclor 1260 is representative of
Table 5-4. Estimated Order of Potential Potencies of Selected PAHs
Compound
Benzo[a]pyrene
Benz[a]anthracene
Benzo[b]fluoranthene
Benzo[/c]fluoranthene
Chrysene
Dibenz[a,/7]anthracene
lndeno[1 ,2,3-ccflpyrene
Relative
Potency8-"
1.0
0.1
0.1
0.01
0.001
1.0
0,1.
Reference
Bingham and Falk, 1969
Habs et al., 1980
Habs et al., 1980
Wynder and Hoffmann, 1959
Wynder and Hoffmann, 1959
Habs et al., 1980; Hoffmann
and Wynder, 1966
a Model was P(d)=1-exp[-a(1+bd)2J for all but indeno[1,2,3-cc/]pyrene.
b Values listed are order-of-magnitude potencies based on the following scheme for
rounding experimental values: 0.51-5.0=1.0; 0.051-0.50=0.1; 0:0051-Q.050=0.01.
Source: Modified from U.S. EPA, 1993c,
5-17
-------
5. SCREENING VALUES FOR TARGET ANALYTES
other PCB mixtures, i.e., that the SF for Aroclor 1260 is an upper limit risk
estimate for all other PCB mixtures as well (IRIS, 1992; U.S. EPA, 1988a). The
EPA Office of Water recognizes that this assumption has significant
uncertainties.
The comparison of total PCB concentrations (determined as the sum of Aroclor
equivalents) with the Aroclor 1260-based SV may be overly conservative. The
EPA Carcinogen Assessment Group has reported a much lower SF for Aroclor
1254 (SF = 2.6) and data from studies of Aroclor 1242 (Schaeffer et al., 1984)
indicate that there are no statistically significant increases in liver tumors
compared to controls. A recent reassessment of the results of five PCB studies
in rats found significant differences between Aroclor 1260 and other Aroclors in
the types and incidence of pathological effects on rats (IEHR, 1991). On the
other hand, Aroclor 1260 may not represent an upper bound risk estimate
because the PCB congener distribution in fish and shellfish tissue is usually
markedly altered from, and may be more potent than, the parent Aroclor mixture
(Bryan et al., 1987; Kubiak et al., 1989; Norstrom, 1988; Oliver and Niimi, 1988;
Smith et al., 1990). This underscores the need to move toward congener-
specific analysis based on (1) pharmacokinetics and (2) relative potency at
specific site(s) of action (NAS, 1991).
EPA also recognizes that the current recommended SV of 10 ppb for total PCBs
will result in widespread exceedance in waterbodies throughout the country and
will drive virtually all fish and shellfish contaminant monitoring programs into the
risk assessment phase for PCBs. The decision on whether to issue a
consumption advisory for PCBs at this level is one that must be made by risk
managers in each State.
EPA is currently giving high priority to addressing the unresolved issues related
to PCB analysis and risk assessment. A work group has been convened to
examine the feasibility of TEFs for PCB congeners similar to those developed
for PCDDs and PCDFs (U.S. EPA, 1991J) and two EPA-sponsored national
workshops have been held recently to identify problematic issues and areas for
future research (U.S. EPA, 1993d; U.S. EPA, 1993e). Additional guidance on
PCB analyses will be provided in addenda to this document and in subsequent
documents in this series.
5.3.2.4 Dloxlns and Dlbenzofurans—
Note: At this time, the EPA Office of Research and Development is reevaluating
the potency of dioxins/furans. Consequently, the following recommendation is
subject to change pending the results of this reevaluation.
It is recommended in both screening and intensive studies that the 17 2,3,7,8-
substituted tetra- through octa-chlorinated PCODs and PCDFs be determined
and that a toxicity-weighted total concentration be calculated for each sample for
comparison with the recommended SV for 2,3,7,8-TCDD (see Table 5-2).
5-18
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5. SCREENING VALUES FOR TARGET ANALYTES
The revised interim method for estimating toxicity equivalency concentrations
(Barnes and Bellin, 1989) should be used to estimate TCDD equivalent concen-
trations according to the following equation:
TEC = I (TEF, - C,)
(5-9)
where
TEFj = Toxicity equivalency factor for the ith congener (relative to 2,3,7,8-
TCDD)
C, = Concentration of the ith congener.
TEFs for the 2,3,7,8-substituted tetra- through octa-PCDDs and PCDFs are
shown in Table 5-5.
If resources are limited, the 2,3,7,8-TCDD and 2,3,7,8-TCDF congeners should
be determined and the calculated TEC compared with the recommended SV for
2,3,7,8-TCDD (see Table 5-2).
5-19
-------
5. SCREENING VALUES FOR TARGET ANALYTES
Table 5-5. Toxlclty Equivalency Factors (TEFs) for Tetra-
through Octa-Chlorlnated Dibenzo-p-Dloxins and Dlbenzofurans
Analyte
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDF
TEF"
1.00
0.50
0.10
0.10
0.10
0.01
0.001
0.10
0.05
0.50
0.10
0.10
0.10
0.10
0.01
0.01
0.001
Source: Barnes and Bellin, 1989.
aTEFs for all non-2,3,7,8-substituted congeners are zero.
5-20
-------
6. FIELD PROCEDURES
SECTION 6
FIELD PROCEDURES
This section provides guidance on sampling design of screening and intensive
studies and recommends field procedures for collecting, preserving, and shipping
samples to a processing laboratory for target analyte analysis. Planning and
documentation of all field procedures are emphasized to ensure that collection
activities are cost-effective and that sample integrity is preserved during all field
activities.
6.1 SAMPLING DESIGN
Prior to initiating a screening or intensive study, the program manager and field
sampling staff should develop a detailed sampling plan. As described in Section
2, there are seven major parameters that must be specified prior to the initiation
of any field collection activities:
Site selection
• Target species (and size class)
Target analytes
Target analyte screening values
• Sampling times
Sample type
Replicate samples.
In addition, personnel roles and responsibilities in all phases of the fish and
shellfish sampling effort should be defined clearly. All aspects of the final
sampling design for a State's fish and shellfish contaminant monitoring program
should be documented clearly by the program manager in a Work/QA Project
Plan (see Appendix F). Routine sample collection procedures should be
prepared as standard operating procedures (U.S. EPA, 1984b) to document the
specific methods used by the State and to facilitate assessment of final data
quality and comparability.
The seven major parameters of the sampling plan should be documented on a
sample request form prepared by the program manager for each sampling site.
The sample request form should provide the field collection team with readily
available information on the study objective, site location, site name/number,
target species and alternate species to be collected, target analytes to be
evaluated, anticipated sampling dates, sample type to be collected, number and
size range of individuals to be collected for each composite sample, sampling
6-1
-------
6. FIELD PROCEDURES
method to be used, and number of replicates to be collected. An example of a
sample request form is shown in Figure 6-1. The original sample request form
should be filed with the program manager and a copy kept with the field logbook.
The seven major parameters that must be specified in the sampling plan for
screening and intensive studies are discussed in Sections 6.1.1 and 6.1.2,
respectively.
6.1.1 Screening Studies (Tier 1)
The primary aim of screening studies is to identify frequently fished sites where
commonly consumed fish and shellfish species are contaminated and may pose
a risk to human health. Ideally, screening studies should include all waterbodies
where commercial, recreational, or subsistence fishing and shellfish harvesting
are practiced.
6.1.1.1 Site Selection-
Sampling sites should be selected to identify extremes of the bioaccumulation
spectrum, ranging from presumed undisturbed reference sites to sites where
existing data (or the presence of potential pollutant sources) suggest significant
contamination. Where resources are limited, States initially should target those
harvest sites suspected of having the highest levels of contamination and of
posing the greatest potential health risk to local fish and shellfish consumers.
Screening study sites should be located in frequently fished areas near
• Point source discharges such as
— Industrial or municipal dischargers
— Combined sewer overflows (CSOs)
— Urban storm drains
Nonpoint source inputs such as
— Landfills, Resource Conservation and Recovery Act (RCRA) sites, or
Superfund Comprehensive Environmental Response, Compensation,
and Liability Act (CERCLA) sites
— Areas of intensive agricultural, silvicultural, or resource extraction
activities or urban land development
— Areas receiving inputs through multimedia mechanisms such as
hydrogeologic connections or atmospheric deposition (e.g., areas
affected by acid rain impacts, particularly lakes with pH <6.0 since
elevated mercury concentrations in fish have been reported for such
sites)
6-2
-------
6. FIELD PROCEDURES
Sample Request Form
Project
Objective
Sample
Type
Target
Contaminants
CD Screening Study
D Fish fillets only
D Shellfish (edible portions)
(Specify portions if other than
whole )
CD Whole fish or portions other
than fillet (Specify tissues used
if other than whole
)
CD All target contaminants
CD Additional contaminants
(Specify )
CD Intensive Study
D Fish fillets only
D Shellfish (edible portions)
(Specify portions if other than whole
}
CD Whole fish or portions other than fillet (Specify
tissues used if other than whole
)
CD Contaminants exceeding screening study SVs
(Specify
)
INSTRUCTIONS TO SAMPLE COLLECTION TEAM
Project Number:
County/Parish:
Target Species:
CD Freshwater
Site (Name/Number):
LatAona:
Alternate Species: (in order of preference)
CH Estuarine
Proposed Sampling
Proposed Sampling
Dates:
Method:
ED Electrof ishing CD Mechanical grab or tongs
CD Seining CD Biological dredge
CD Trawling CD Hand collection
D Other (SoecHv )
Number of Sample Replicates: CD No field replicates (1
n
Number of Individua
per Composite:
(Specify number for t
Is
Fish per composii
Shellfish per com
composite sample only)
field replicates
9ach target species)
te
Dosite (specify number to obtain 200 grams of tissue)
Figure 6-1. Example of a sample request form.
6-3
-------
6. FIELD PROCEDURES
Areas acting as potential pollutant sinks where contaminated sediments
accumulate and bioaccumulation potential might be enhanced (i.e., areas
where water velocity slows and organic-rich sediments are deposited)
Areas where sediments are disturbed by dredging activities
Unpolluted areas that can serve as reference sites for subsequent intensive
studies. For example, Michigan sampled lakes that were in presumed
unpolluted areas but discovered mercury contamination in fish from many of
these areas and subsequently issued a fish consumption advisory for all of
its inland lakes.
The procedures required to identify candidate screening sites near significant
point source discharges are usually straightforward. It is often more difficult,
however, to identify clearly defined candidate sites in areas affected by pollutants
from nonpoint sources. For these sites, assessment information summarized in
State Section 305(b) reports should be reviewed before locations are selected.
State 305(b) reports are submitted to the EPA Assessment and Watershed
Protection Division biennially and provide an inventory of the water quality in
each State. The 305{b) reports often contain Section 319 nonpoint source
assessment information that may be useful in identifying major sources of
nonpoint source pollution to State waters. States may also use a method for
targeting pesticide hotspots in estuarine watersheds that employs pesticide use
estimates from NOAA's National Coastal Pollutant Discharge Inventory (Farrow
et al., 1'989).
It is important for States to identify and document at least a few unpolluted sites,
particularly for use as reference sites in subsequent monitoring studies.
Verification that targeted reference sites show acceptably low concentrations of
contaminants in fish or shellfish tissues also provides at least partial validation
of the methods used to select potentially contaminated sites. Clear differences
between the two types of sites support the site-selection methodology and the
assumptions about primary sources of pollution.
In addition to the intensity of subsistence, sport, or commercial fishing, factors
that should be evaluated (Versar, 1982) when selecting fish and shellfish
sampling sites include
Proximity to water and sediment sampling sites
Availability of data on fish or shellfish community structure
Bottom condition
• Type of sampling equipment
• Accessibility of the site.
The most important benefit of locating fish or shellfish sampling sites near sites
selected for water and sediment sampling is the possibility of correlating
contaminant concentrations in different environmental compartments (water,
sediment, and fish). Selecting sampling sites in proximity to one another is also
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more cost-effective in that it provides opportunities to combine sampling trips for
different matrices.
Availability of data on the indigenous fish and shellfish communities should be
considered in final site selection. Information on preferred feeding areas and
migration patterns is valuable in locating populations of the target species
(Versar, 1982). Knowledge of habitat preference provided by fisheries biologists
or commercial fishermen may significantly reduce the time required to locate a
suitable population of the target species at a given site.
Bottom condition is another site-specific factor that is closely related to the
ecology of a target fish or shellfish population (Versar, 1982). For example, if
only soft-bottom areas are available at an estuarine site, neither oysters
(Crassosfrea virginica) nor mussels (Mytilus edulis and M. californianus) would
likely be present because these species prefer hard substrates. Bottom
condition also must be considered in the selection and deployment of sampling
equipment. Navigation charts provide depth contours and the locations of large
underwater obstacles in coastal areas and larger navigable rivers. Sampling
staff might also consult commercial fishermen familiar with the candidate site to
identify areas where the target species congregates and the appropriate
sampling equipment to use.
Another factor closely linked to equipment selection is the accessibility of the
sampling site. For some small streams or land-locked lakes (particularly in
mountainous areas), it is often impractical to use a boat (Versar, 1982). In such
cases the sampling site should have good land access. If access to the site is
by land, consideration should be given to the type of vegetation and local
topography that could make transport of collection equipment difficult. If access
to the sampling site is by water, consideration should be given to the location of
boat ramps and marinas and the depth of water required to deploy the selected
sampling gear efficiently and to operate the boat safely. Sampling equipment
and use are discussed in detail in Section 6.2.1.
The selection of each sampling site must be based on the best professional
judgment of the field sampling staff. Once the site has been selected, it should
be plotted and numbered on the most accurate, up-to-date map available.
Recent 7.5-minute (1:24,000 scale) maps from the U.S. Geologic Survey or blue
line maps produced by the U.S. Army Corps of Engineers are of sufficient detail
and accuracy for sample site mapping. The type of sampling to be conducted,
water depth, and estimated time to the sampling site from an access point
should be noted. The availability of landmarks for visual or range fixes should
be determined for each site, and biological trawl paths (or other sampling gear
transects) and navigational hazards should be indicated. Additional information
on site-positioning methods, including Loran-C, VIEWNAV, TRANSIT (NAVSAT),
GEOSTAR, and the NAVSTAR Global Positioning System (GPS), is provided in
Battelle (1986), TetraTech (1986), and Puget Sound Estuary Program (1990a).
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6. FIELD PROCEDURES
Each sampling site must be described accurately because State fish and
shellfish contaminant monitoring data may be stored in a database available to
users nationwide (see Section 9.2). For example, a sampling site may be
defined as a 2-mile section of river (e.g., 1 mile upstream and 1 mile down-
stream of a reference point) or a 2-mile stretch of lake or estuarine/marine
shoreline (U.S. EPA, 1990d). Each sampler should provide a detailed descrip-
tion of each site using a 7.5-minute USGS map to determine the exact latitude
and longitude coordinates for the reference point of the site. This information
should be documented on the sample request form and field record sheets (see
Section 6.2.3).
6.1.1.2 Target Species and Size Class Selection—
After reviewing information on each sampling site, the field collection staff should
identify the target species that are likely to be found at the site. Target species
recommended for screening studies in freshwater systems are shown in Tables
3-1, 3-2, and 3-4. Tables 3-10 through 3-16 list recommended species for
estuarine/marine areas. In freshwater ecosystems, one bottom-feeding and one
predator fish species should be collected. In estuarine/marine ecosystems,
either one bivalve species and one finfish species or two finfish species should
be collected. Second and third choice target species should be selected in the
event that the recommended target species are not collected at the site. The
same criteria used to select the recommended target species (Section 3.2)
should be used to select alternate target species. In all cases, the primary
selection criterion should be that the target species is commonly consumed
locally and is of harvestable size.
EPA recognizes that resource limitations may influence the sampling strategy
selected by a State. If monitoring resources are severely limited, precluding
performance of any Tier 2 intensive studies (Phase I and Phase II), EPA
recommends three sampling options to States for collecting additional samples
during the screening studies. These options are:
1. Collecting one composite sample for each of three size (age) classes of
each target species
2. Collecting replicate composite samples for each target species
3. Collecting replicate composite samples for each of three size (age) classes
of each target species.
Option 1 (single composite analysis for each of three size classes) provides
additional information on size-specific levels of contamination that may allow
States to issue an advisory for only the most contaminated! size classes while
allowing other size classes of the target species to remain open to fishing. The
State could analyze the composite sample from the largest size class first. If
any SVs are exceeded, analysis of the smaller size class composite samples
could be conducted. This option, however, does not provide any additional
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6. FIELD PROCEDURES
information for estimating the variability of the contamination level in any specific
size class. To obtain information for estimating the variability of the contamina-
tion level in the target species, States could separately analyze each individual
fish specimen in any composite that exceeded the SVs. Note: This option of
analyzing individual fish within a composite sample is more resource-intensive
with respect to analytical costs but is currently used by some Great Lakes
States.
Option 2 (replicate analyses of one size class) provides additional statistical
power that would allow States to estimate the variability of contamination levels
within the one size class sampled; however, it does not provide information on
size-specific contamination levels.
Option 3 (replicate analyses of three size classes) provides both additional
information on size-specific contamination levels and additional statistical power
to estimate the variability of the contaminant concentrations in each of three size
classes of the target species. If resources are limited, the State could analyze
the replicate samples for the largest size class first; if the SVs are exceeded,
analysis of the smaller size class composite samples could then be conducted.
Note: The correlation between increasing size (age) and contaminant tissue
concentration observed for some freshwater finfish species (Voiland et al., 1991)
may be much less evident in estuarine/marine finfish species (G. Pollock,
California Environmental Protection Agency, personal communication, 1993).
The movement of estuarine and marine species from one niche to another as
they mature may change their exposure at a contaminated site. Thus, size-
based sampling in estuarine/marine systems should be conducted only when it
is likely to serve a potential risk management outcome.
6.1.1.3 Target Analyte Selection-
All 25 recommended target analytes listed in Table 4-1 should be included in
screening studies unless reliable historic tissue, sediment, or pollutant source
data indicate that an analyte is not present at a level of concern for human
health. Additional regional or site-specific target analytes should be included in
screening studies when there is indication or concern that such contaminants are
a potential health risk to local fish or shellfish consumers. Historic data on water,
sediment, and tissue contamination and priority pollutant scans from known point
source discharges or nonpoint source monitoring should be reviewed to
determine whether analysis of additional analytes is warranted.
6.1.1.4 Target Analyte Screening Values—
To enhance national consistency in screening study data, States should use the
target analyte screening values listed in Table 5-2 to evaluate tissue contaminant
data. Specific methods used to calculate SVs for noncarcinogenic and
carcinogenic target analytes, including examples of SVs calculated for selected
subpopulations, are given in Sections 5.1 and 5.2. If target analytes in addition
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6. FIELD PROCEDURES
to those recommended in Table 5-2 are included in a screening study, these
calculation procedures should be used to estimate SVs based on typical
exposure assumptions for the general population for the additional compounds.
Note: If the State chooses to use a different risk level or consumption rate to
address site-specific considerations, the corresponding SVs should be calculated
prior to initiation of chemical analyses to ensure that the detection limits of the
analytical procedures are sufficiently low to allow reliable quantitation at or below
the chosen SV. If analytical methodology is not sensitive enough to reliably
quantitate target analytes at or below selected SVs (see Section 8.2.2 and Table
8-4), program managers must determine appropriate fish consumption guidance
based on lowest detectable concentrations or provide justification for adjusting
SVs to values at or above achievable method detection limits. It should be
emphasized that when SVs are below method detection limits, the failure to
detect a target analyte can not be assumed to indicate that there is no cause for
concern for human health effects.
6.1.1.5 Sampling Times—
If program resources are sufficient, biennial screening of waterbodies is
recommended where commercial, recreational, or subsistence harvesting is
commonly practiced (as identified by the State). Data from these screenings can
then be used in the biennial State 305(b) reports to document the extent of
support of Clean Water Act goals. If biennial screening is not possible, then
waterbodies should be screened at least once every 5 years.
Selection of the most appropriate sampling period is very important, particularly
when screening studies may be conducted only once every 2 to 5 years. Note:
For screening studies, sampling should be conducted during the period when the
target species is most frequently harvested (U.S. EPA, 1989d; Versar, 1982).
In fresh waters, as a general rule, the most desirable sampling period is from
late summer to early fall (i.e., August to October) (Phillips, 1980; Versar, 1982).
The lipid content of many species (which represents an important reservoir for
organic pollutants) is generally highest at this time. Also, water levels are
typically lower during this time, thus simplifying collection procedures. This late
summer to early fall sampling period should not be used, however, if (1) it does
not coincide with the legal harvest season of the target species or (2) the target
species spawns during this period. Note: If the target species can be legally
harvested during its spawning period, however, then sampling to determine
contaminant concentrations should be conducted during this time.
A third exception to the late summer to early fall sampling recommendation
concerns monitoring for the organophosphate pesticides. Sampling for these
compounds should be conducted during late spring or early summer within 1 to
2 months following pesticide application because these compounds are
degraded and metabolized relatively rapidly compared to organochlorine
pesticides. Note: The target species should be sampled during the Spring only
if the species can be legally harvested at this time.
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In estuarine and coastal waters, the most appropriate sampling time is during the
period when most fish are caught and consumed (usually summer for
recreational and subsistence fishermen). For estuarine/marine shellfish (bivalve
molluscs and crustaceans), two situations may exist. The legal harvesting
season may be strictly controlled for fisheries resource management purposes
or harvesting may be open year round. In the first situation, shellfish
contaminant monitoring should be conducted during the legal harvest period. In
the second situation, monitoring should be conducted to correspond to the period
when the majority of harvesting is conducted during the legal season. State staff
may have to consider different sampling times for target shellfish species if
differences in the commercial and recreational harvesting period exist.
Ideally, the sampling period selected should avoid the spawning period of the
target species, including the period 1 month before and 1 month after spawning,
because many aquatic species are subject to stress during spawning. Tissue
samples collected during this period may not always be representative of the
normal population. For example, feeding habits, body fat (lipid) content, and
respiration rates may change during spawning and may influence pollutant
uptake and clearance. Collecting may also adversely affect some species, such
as trout or bass, by damaging the spawning grounds. Most fishing regulations
protect spawning periods to enhance propagation of important fishery species.
Species-specific information on spawning periods and other life history factors
is available in numerous sources (e.g., Carlander, 1969; Emmett et a!., 1991;
Pflieger, 1975; Phillips, 1980). In addition, digitized life history information is
available in many States through the Multistate Fish and Wildlife Information
System (1990).
Exceptions to the recommended sampling periods for freshwater and
estuarine/marine habitats will be determined by important climatic, regional, or
site-specific factors that favor alternative sampling periods. For many States,
budgetary constraints may require that most sampling be conducted during June,
July, and August when temporary help or student interns are available for hire.
The actual sampling period and the rationale for its selection should be
documented fully and the final data report should include an assessment of
sampling period effects on the results.
.6.1.1.6 Sample Type—
Composite samples of fish fillets or of the edible portions of shellfish are
recommended for analysis of target analytes in screening studies (U.S. EPA,
1987b; 1989d). For health risk assessments, a composite sample should consist
of that portion of the individual organism that is commonly consumed by the
population at risk. Skin-on fillets (with the belly flap included) are recommended
for most scaled finfish (see Sections 7.2.2.6 and 7.2.2.7). Other sample types
(e.g., skinless fillets) may be more appropriate for some target species (e.g.,
catfish and other scaleless finfish species). For shellfish, the tissue considered
to be edible will vary by target species (see Section 7.2.4.4) based on local food
preferences. A precise description of the sample type (including the number and
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6. FIELD PROCEDURES
size of the individuals in the composite) should be documented in the program
records for each target species. Note: For freshwater turtles, the tissues
considered to be edible vary based on the dietary and culinary practices of local
populations (see Section 7.2.3.3). The EPA recommends use of individual turtle
samples rather than composite samples for evaluating turtle tissue
contamination.
Note: Composite samples are homogeneous mixtures of samples from two or
more individual organisms of the same species collected at a particular site and
analyzed as a single sample. Because the costs of performing individual
chemical analyses are usually higher than the costs of sample collection and
preparation, composite samples are most cost-effective for estimating average
tissue concentrations of target analytes in target species populations. Besides
being cost-effective, composite samples also ensure adequate sample mass to
allow analyses for all recommended target analytes. A disadvantage of using
composite samples, however, is that extreme contaminant concentration values
for individual organisms are lost.
In screening studies, EPA recommends that States analyze one composite
sample for each of two target species at each screening site. Organisms used
in a composite sample
Must all be of the same species
Should satisfy any legal requirements of harvestable size or weight, or at
least be of consumable size if no legal harvest requirements are in effect
Should be of similar size so that the smallest individual in a composite is no
less than 75 percent of the total length (size) of the largest individual
Should be collected at the same time (i.e., collected as close to the same
time as possible but no more than 1 week apart) [Note: This assumes that
a sampling crew was unable to collect all fish needed to prepare the
composite sample on the same day. If organisms used in the same
composite are collected on different days (no more than 1 week apart), they
should be processed within 24 hours as described in Section 7.2 except that
individual fish may have to be filleted and frozen until all the fish to be
included in the composite are delivered to the laboratory. At that time, the
composite homogenate sample may be prepared.]
• Should be collected in sufficient numbers to provide a 200-g composite
homogenate sample of edible tissue for analysis of recommended target
analytes.
Individual organisms used in composite samples must be of the same species
because of the significant species-specific bioaccumulation potential. Accurate
taxonomic identification is essential in preventing the mixing of closely related
species with the target species. Note: Under no circumstance should indivi-
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6. FIELD PROCEDURES
duals from different species be used in a composite sample (U.S. EPA, 1989d,
1990d).
For cost-effectiveness, EPA recommends that States collect only one size class
for each target species and focus on the larger individuals commonly harvested
by the local population. Ideally, the individuals within each target species
composite should be of similar size within a target size range. For persistent
chlorinated organic compounds (e.g., DDT, PCBs, and toxaphene) and organic
mercury compounds, the larger (older) individuals within a population are
generally the most contaminated (Phillips, 1980; Voiland et al., 1991). As noted
earlier, this correlation between increasing size and increasing contaminant
concentration is most striking in freshwater finfish species but is less evident in
estuarine and marine species. Size is used as a surrogate for age, which
provides some estimate of the total time the individual organism has been at risk
of exposure. Therefore, the primary target size range ideally should include the
larger individuals harvested at each sampling site. In this way, the States will
maximize their chances of detecting high levels of contamination in the single
composite sample collected for each target species. If this ideal condition
cannot be met, the field sampling team should retain individuals of similar length
that fall within a secondary target size range.
Individual organisms used in composite samples should be of similar size
(WDNR, 1988). Note: Ideally, for fish or shellfish, the total length (or size) of
the smallest individual in any composite sample should be no less than 75
percent of the total length (or size) of the largest individual in the composite
sample (U.S. EPA, 1990d). For example, if the largest fish is 200 mm, then the
smallest individual included in the composite sample should be at least 150 mm.
In the California Mussel Watch Program, a predetermined size range (55 to 65
mm) for the target bivalves (Mytilus californianus and M. edulis) is used as a
sample selection criterion at all sampling sites to reduce size-related variability
(Phillips, 1988). Similarly, the Texas Water Commission (1990) specifies the
target size range for each of the recommended target fish species collected in
the State's fish contaminant monitoring program.
Individual organisms used in a composite sample ideally should be collected at
the same time so that temporal changes in contaminant concentrations
associated with the reproduction cycle of the target species are minimized.
Each composite sample should contain 200 g of tissue so that sufficient material
will be available for the analysis of recommended target analytes. A larger
composite sample mass may be required when the number of target analytes is
increased to address regional or site-specific concerns. However, the tissue
mass may be reduced in the Tier 2 intensive studies (Phase I and II) when a
limited number of specific analytes of concern have been identified (see Section
7.2.2.9). Given the variability in size among target species, only approximate
ranges can be suggested for the number of individual organisms to collect to
achieve adequate mass in screening studies (U.S. EPA, 1989d;.Versar, 1982).
For fish, 3 to 10 individuals should be collected for a composite sample for each
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6. FIELD PROCEDURES
target species; for shellfish, 3 to 50 individuals should be collected for a
composite sample. In some cases, however, more than 50 small shellfish (e.g.,
mussels, shrimp, crayfish) may be needed to obtain the recommended 200-g
sample mass. Note: The same number of individuals should be used in each
composite sample for a given target species at each sampling site.
As alluded to above, one limitation of using composite samples is that
information on extreme levels of contamination in individual organisms is lost.
Therefore, EPA recommends that the residual individual homogenates be saved
to allow for analyses of individual specimens if resources permit (Versar, 1982).
Analysis of individual homogenates allows States to estimate the underlying
population variance-which, as described in Section 6.1.2.6, facilitates sample
size determination for the intensive studies. Furthermore, individual
homogenates may also be used to provide materials for split and spike samples
for routine QC procedures either for composites or individual organisms (see
Section 8.3). The circumstances in which the analysis of individual fish samples
might be preferred over the analysis of composite samples is described in more
detail in Appendix A.
Recommended sample preparation procedures are discussed in Section 7.2.
6.1.1,7 Replicate Samples—
The collection of sufficient numbers of individual organisms from a target species
at a site to allow for the independent preparation of more than one composite
sample (i.e., sample replicates) is strongly encouraged but is optional in
screening studies. If resources and storage are available, single replicate (i.e.,
duplicate) composite samples should be collected at a minimum of 10 percent
of the screening sites (U.S. EPA, 1990d). The collection and storage of replicate
samples, even if not analyzed at the time due to inadequate resources, allow for
followup QC checks. These sites should be identified during the planning phase
and sample replication specifications noted on the sample request form. If
replicate field samples are to be collected, States should follow the guidance
provided: in Section 6.1.2.7. Note: Additional replicates must be collected at
each site for each target species if statistical comparisons with the target analyte
SVs are required in the State monitoring programs. The statistical advantages
of replicate sampling are discussed in detail in Section 6.1.2.7.
6.1.2 Intensive Studies (Tier 2)
The primary aim of intensive studies is to characterize the magnitude and
geographic extent of contamination in harvestable fish and shellfish species at
those screening sites where concentrations of target analytes in tissues were
found to be above selected SVs. Intensive studies should be designed to verify
results of the screening study, to identify specific fish and shellfish species and
size classes for which advisories should be issued, and to determine the geo-
graphic extent of the fish contamination. In addition, intensive studies should be
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designed to provide data for States to tailor their advisories based on the
consumption habits or sensitivities of specific local human subpopulations.
State staff should plan the specific aspects of field collection activities for each
intensive study site after a thorough review of the aims of intensive studies
(Section 2.2) and the fish contaminant data obtained in the screening study. All
the factors that influence sample collection activities should be considered and
specific aspects of each should be documented clearly by the program manager
on the sample request form for each site.
6.1.2.1 Site Selection-
Intensive studies should be conducted at all screening sites where the selected
SV for one or more target analytes was exceeded. The field collection staff
should review a 7.5-minute (1:24,000 scale) USGS hydrologic map of the study
site and all relevant water, sediment, and tissue contaminant data. The site
selection factors evaluated in the screening study (Section 6.1.1.1) must be
reevaluated before initiating intensive study sampling.
States should conduct Tier 2 intensive studies in two phases if program
resources allow. Phase I intensive studies should be more extensive
investigations of the magnitude of tissue contamination at suspect screening
sites. Phase II intensive studies should define the geographic extent of the
contamination around these suspect screening sites in a variety of size (age)
classes for each target species. The field collection staff must evaluate the
accessibility of these additional sites and develop a sampling strategy that is
scientifically sound and practicable.
Selection of Phase II sites may be quite straightforward where the source of
pollutant introduction is highly localized or if site-specific hydrologic features
create a significant pollutant sink where contaminated sediments accumulate and
the bioaccumulation potential might be enhanced (U.S. EPA, 1986f). For
example, upstream and downstream water quality and sediment monitoring to
bracket point source discharges, outfalls, and regulated disposal sites showing
contaminants from surface runoff or leachate can often be used to characterize
the geographic extent of the contaminated area. Within coves or small
embayments where streams enter large lakes or estuaries, the geographic extent
of contamination may also be characterized via multilocational sampling to
bracket the areas of concern. Such sampling designs are clearly most effective
where the target species are sedentary or of limited mobility (Gilbert, 1987). In
addition, the existence of barriers to migration, such as dams, should be taken
into consideration.
6.1.2.2 Target Species and Size Class Selection—
Whenever possible, the target species found in the screening study to have
elevated tissue concentrations of one or more of the target analytes should be
resampled in the intensive study. Recommended target species for freshwater
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sites are listed in Tables 3-1, 3-2, and 3-4; target species for estuarine/marine
waters are listed in Tables 3-10 through 3-12 for Atlantic Coast estuaries, in
Table 3-13 for Gulf Coast estuaries, and in Tables 3-14 through 3-16 for Pacific
Coast estuaries. If the target species used in the screening study are not
collected in sufficient numbers, alternative target species should be selected
using criteria provided in Section 3.2. The alternative target species should be
specified on the sample request form.
For Phase I intensive studies, States should collect replicate composite samples
of one size class for each target species and focus sampling on larger individ-
uals commonly harvested by the local population (as appropriate). If contamina-
tion of this target size class is high, Phase II studies should include collection of
replicate composite samples of three size classes within each target species.
EPA recognizes that resource limitations may influence the sampling strategy
selected by a State. If monitoring resources are limited for intensive studies,
States may determine that it is more resource-efficient to collect replicate
composite samples of three size classes (as required for Phase II studies) during
Phase I sampling rather than revisit the site at a later time to conduct Phase II
intensive studies. In this way, the State may save resources by reducing field
sampling costs associated with Phase II intensive studies.
By sampling three size (age) classes, States collect data on the target species
that may provide them with additional risk management options. If contaminant
concentrations are positively correlated with fish and shellfish size, frequent
consumption of smaller (less contaminated) individuals may be acceptable even
though consumption of larger individuals may be restricted by a consumption
advisory. In this way, States can tailor an advisory to protect human health and
still allow restricted use of the fishery resource. Many Great Lakes States have
used size (age) class data to allow smaller individuals within a given target
species to remain fishable while larger individuals are placed under an advisory.
6.1.2.3 Target Analyte Selection—
Phase I intensive studies should include only those target analytes found in the
screening study to be present in fish and shellfish tissue at concentrations
exceeding selected SVs (Section 5.2). Phase II studies should include only
those target analytes found in Phase I intensive studies to be present at
concentrations exceeding SVs. In most cases, the number of target analytes
evaluated in Phase I and II intensive studies will be significantly smaller than the
number evaluated in screening studies.
6.1.2.4 Target Analyte Screening Values-
Target analyte SVs used in screening studies should also be used in Phase I
and II intensive studies. Specific methods used to calculate SVs for
noncarcinogenic and carcinogenic target analytes, including examples of SVs
calculated for various exposure scenarios, are given in Section 5.1.
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6.1.2.5 Sampling Times—
To the extent that program resources allow, sampling in intensive studies should
be conducted during the same period or periods during which screening studies
were conducted (i.e., when the target species are most frequently harvested for
consumption) and should be conducted preferably within 1 year of the screening
studies. In some cases, it may be best to combine Phase I and Phase II
sampling to decrease both the time required to obtain adequate data for
issuance of specific advice relative to species, size classes, and geographic
extent and/or the monitoring costs entailed in revisiting the site (see Section
6.1.2.2).
States should follow the general guidance provided in Section 6.1.1.5 for
recommended sampling times. The actual sampling period and rationale for its
selection should be documented fully for Phase I and II studies.
6.1.2.6 Sample Type—
Composite samples of fish fillets or the edible portions of shellfish are
recommended for analysis of target analytes in intensive studies. The general
guidance in Section 6.1.1.6 should be followed to prepare composite samples
for each target species. In addition, separate composite samples may be
prepared for selected size (age) classes within each target species, particularly
in Phase II studies after tissue contamination has been verified in Phase I
studies. Because the number of replicate composite samples and the number
of fish and shellfish per composite required to test whether the site-specific mean
contaminant concentration exceeds an SV are intimately related, both will be
discussed in the next section.
Note: The same number of individual organisms should be used to prepare all
replicate composite samples for a given target species at a given site. If this
number is outside the recommended range, documentation should be provided.
Recommended sample preparation procedures are discussed in Section 7.2.
States interested in analyzing target analyte residues in individual fish or shellfish
samples should review information presented in Appendix A.
6.1.2.7 Replicate Samples
In intensive studies (Phases I and II), EPA recommends that States analyze
replicate composite samples of each target species at each sampling site.
Replicate composite samples should be as similar to each other as possible. In
addition to being members of the same species, individuals within each
composite should be of similar length (size) (see Section 6.1.1.6). The relative
difference between the average length (size) of individuals within any composite
sample from a given site and the average of the average lengths (sizes) of
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individuals in all composite samples from that site should not exceed 10 percent
(U.S. EPA, 1990d). In order to determine this, States should first calculate the
average length of the target species fish constituting each composite replicate
sample from a site. Then, States should take the average of these averages for
the site. In the following example, the average of the average lengths of
individuals (±10 percent) in five replicate composite samples is calculated to be
310 (±31) mm.
Replicate
1
2
3
4
5
Average of the average
Average Length of Individual
Fish In Composite Sample (mm)
300
320
330
280
320
length (±10%) = 310 (±31) mm.
Therefore, the acceptable range for the average length of individual composite
samples is 279 to 341 mm, and the average length of individual fish in each of
the five replicate composites shown above falls within the acceptable average
size range.
All replicate composite samples for a given sampling site should be collected
within no more than 1 week of each other so that temporal changes in target
analyte concentrations associated with the reproductive cycle of the target
species are minimized.
The remainder of this section provides general guidelines for estimating the
number of replicate composite samples per site (n) and the number of individuals
per composite (m) required to test the null hypothesis that the mean target
analyte concentration of replicate composite samples at a site is equal to the SV
versus the alternative hypothesis that the mean target analyte concentration is
greater than the SV. These guidelines are applicable to any target species and
any target analyte.
Note: It is not possible to recommend a single set of sample size requirements
(e.g., number of replicate composite samples per site and the number of
individuals per composite sample) for all fish and shellfish contaminant
monitoring studies. Rather, EPA presents a more general approach to sample
size determination that is both scientifically defensible and cost-effective. At
each site, States must determine the appropriate number of replicate composite
samples and of individuals per composite sample based on
• Site-specific estimations of the population variance of the target analyte
concentration
6-16
-------
6. FIELD PROCEDURES
• Fisheries management considerations
• Statistical power consideration.
If the population variance of the target analyte concentrations at a site is small,
fewer replicate composite samples and/or fewer individuals per composite
sample may be required to test the null hypothesis of interest with the desired
statistical power. In this case, using sample sizes that are larger than required
to achieve the desired statistical power would not be cost-effective.
Alternatively, suppose EPA recommended sample sizes based on an analyte
concentration with a population variance that is smaller than that of the target
analyte. In this case, the EPA-recommended sample size requirements may be
inadequate to test the null hypothesis of interest at the statistical power level
selected by the State. Therefore, EPA recommends an approach that provides
the flexibility to sample less in those waters where the target analyte
concentrations are less variable, thereby reserving sampling resources for those
site-specific situations where the population variance of the target analyte tissue
concentration is greater.
The EPA recommends the following statistical model, which assumes that zs is
the contaminant concentration of the ith replicate composite sample at the site
of interest where i=1,2,3,...,n and, furthermore, that each replicate composite
sample is comprised of m individual fish fillets of equal mass. Let z be the mean
target analyte concentration of observed replicate composite samples at a site.
Ignoring measurement error, the variance of z is
where
o2
n
m
Var(z) = o2/(nm)
Population variance
Number of replicate composite samples
Number of individual samples in each composite sample.
(6-1)
To test the null hypothesis that the mean target analyte concentration across the
.n replicate composite samples is equal to the SV versus the alternative
hypothesis that the mean target analyte concentration is greater than the SV, the
estimate of the Var(z), s2, is
s2 = [Z(Zj - z)2] / [n(n - 1)] (6-2)
where the summation occurs over the n composite samples. Under the null
hypothesis, the following statistic
(z - SV) / s
(6-3)
6-17
-------
6. FIELD PROCEDURES
has a Student-t distribution with (n - 1) degrees of freedom (Cochran, 1977;
Kish, 1965). The degrees of freedom are one less than the number of
composite samples.
An optimal sampling design would specify the minimum number of replicate
composite samples (n) and of individuals per composite (m) required to detect
a minimum difference between the SV and the mean target analyte concentration
of replicate composite samples at a site. Design characteristics necessary to
estimate the optimal sampling design include
• Minimum detectable difference between the site-specific mean target analyte
concentration and the SV
Power of the hypothesis test (i.e., the probability of detecting a true
difference when one exists)
• Level of significance (i.e., the probability of rejecting the null hypothesis of
no difference between the site-specific mean target analyte concentration
and the SV when a difference does not exist)
Population variance, o2 (i.e., the variance in target analyte concentrations
among individuals from the same species, which the statistician often must
estimate from prior information)
Cost components (including fixed costs and variable sample collection,
preparation, and analysis costs).
In the absence of such design specifications, guidance for selecting the number
of replicate composite samples at each site and the number of fish per
composite sample is provided. This guidance is based on an investigation of the
precision of the estimate of o2/nm and of statistical power.
Note: Under optimal field and laboratory conditions, at least two replicate
composite samples are required at each site for variance estimation. To
minimize the risk of a destroyed or contaminated composite sample precluding
the site-specific statistical analysis, a minimum of three replicate composite
samples should be collected at each site if possible. Because three replicate
composite samples provide only two degrees of freedom for hypothesis testing,
additional replicate composite samples are recommended.
The stability of the estimated standard error of z must also be considered
because this estimated standard error is the denominator of the statistic for
testing the null hypothesis of interest. A measure of the stability of an estimate
is its statistical precision. The assumption is made that the Zj's come from a
normal distribution, and then the standard error of a2/nm is defined as a product
of a2 and a function of n (the number of replicate composite samples) and m
(the number of fish per composite). A fortunate aspect of composite sampling
is that the composite target analyte concentrations tend to be normally
6-18
-------
6. FIELD PROCEDURES
distributed via the Central Limit Theorem. This formulation is used to determine
which combinations of n and m are associated with a more precise estimate of
c^/nm.
Modifying Cochran (1963) to reflect the normality assumption and the sampling
design of n replicate composite samples and m fish per composite sample, the
function of n and m of interest is shown in square brackets:
se
nm
1/2
.nm(n-1)_
(6-4)
Table 6-1 provides values of this function for various combinations of m and n.
The data presented in Table 6-1 suggest that, as either n or m increases, the
standard error of o^/nm decreases. The advantage of increasing the number of
replicate composite samples can be described in terms of this standard error.
For example, the standard error of o^/nm from a sample design of five replicate
composite samples and six fish per composite (0.024) will be more than 50
percent smaller than that from a sample design of three replicate composite
samples and six fish per composite (0.056). In general, holding the number of
fish per composite fixed, the standard error of o2/nm estimated from five
replicate samples will be about 50 percent smaller than that estimated from three
replicate samples.
Table 6-1. Values of
n2m2(n-1)
for Various Combinations of n and m
No. of
replicate
composite
samples (n)
3
4
5
6
• 7
10
15
Number of fish per composite sample (m)
3
0.111
0.068
0.047
0.035
0.027
0.016
0.008
4
0.083
0.051
0.035
0.026
0.021
0.012
0.006
5
0.067
0.041
0.028
0.021
0.016
0.009
0.005
6
0.056
0.034
0.024
0.018
0.014
0.008
0.004
7
0.048
0.029
0.020
0.015
0.012
0.007
0.004
8
0.042
0.026
0.018
0.013
0.010
0.006
0.003
9
0.037
0.023
0.016
0.012
0.009
0.005
0.003
10
0.033
0.020
0.014
0.011
0.008
0.005
0.003
12
0.028
0.017
0.012
0.009
0.007
0.004
0.002
15
0.022
0.014
0.009
0.007
0.005
0.003
0.002
6-19
-------
6. FIELD PROCEDURES
The data in Table 6-1 also suggest that greater precision in the estimated
standard error of z is gained by increasing the number of replicate samples (n)
than by increasing the number of fish per composite (m). If the total number of
individual fish caught at a site, for example, is fixed at 50 fish, then, with a
design of 10 replicate samples of 5 fish each, the value of the function of n and
m in Table 6-1 is 0.009; with 5 replicate samples of 10 fish each, the value is
0.014. Thus, there is greater precision in the estimated standard error of z
associated with the first design as compared with the second design.
Two assumptions are made to examine the statistical power of the test of the
null hypothesis of interest. First, it is assumed that the true mean of the site-
specific composite target analyte concentrations (M,) is either 10 percent or 50
percent higher than the screening value. Second, it is presumed that a factor
similar to a coefficient of variation, the ratio of the estimated population standard
deviation to the screening value (i.e., o/SV), is 50 to 100 percent. Four
scenarios result from joint consideration of these two assumptions. The power
of the test of the null hypothesis that the mean composite target analyte
concentration at a site is equal to the SV versus the alternative hypothesis that
the mean target analyte concentration is greater than the SV is estimated under
each set of assumptions. Estimates of the statistical power for two of the four
scenarios are shown in Table 6-2.
Power estimates for the two scenarios where the true mean of the site-specific
composite target analyte concentration was assumed to be only 10 percent
higher than the screening value are not presented. The power to detect this
small difference was very poor: for 125 of the resulting 140 combinations of n
and m, the power was less than 50 percent.
Several observations can be made concerning the data in Table 6-2. Note: The
statistical power increases as either n (number of replicate composite samples)
or m (number of fish per composite) increases. However, greater power is
achieved by increasing the number of replicate composite samples as opposed
to increasing the number of fish per composite. Furthermore, if the number of
replicate composite samples per site and the number of fish per composite are
held constant, then, as the ratio of the estimated population variance to the SV
increases (i.e., a/SV), the statistical power decreases.
States may use these tables as a starting point for setting the number of
replicate composite samples per site and the number of fish per composite in
their fish and shellfish contaminant monitoring studies. The assumption regard-
ing the ratio of the estimated population variance to the SV presented in Section
A of Table 6-2 is unrealistic for some fish and shellfish populations. Data in
Section B, which reflect more realistic assumptions concerning the estimated
population variance, show that States will be able to detect only large differences
between the site-specific mean target analyte concentrations and the SV.
Specifically, using five replicate composite samples and six to seven fish per
composite sample, the power to detect a 50 percent increase over the SV is
6-20
-------
6. FIELD PROCEDURES
Table 6-2. Estimates of Statistical Power of
Hypothesis of Interest Under Specified Assumptions
NO. Of
replicate
composite
samples
(n)
' Number of fish per composite (m)
3 4 5 6 7 8 9 10 12 15
A. Ratio of cr/SV = 0.5 and n = 1.5 x SV:
3
4
5
6
7
10
15
6
8
9
9
9
9
9
6
9
9
9
9
9
9
7
9
9
9
9
9
9
8
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
B. Ratio of c/SV = 1 .0 and
1 .5 x SV:
3
4
5
6
7
10
15
-
-
-
5
6
8
9
-
-
5
6
7
8
9
-
-
6
7
8
9
9
-
5
7
8
8
9
9
-
6
8
8
9
9
9
-
6
8
8
9
9
9
-
7
8
9
9
9
9
-
7
8
9
9
9
9
5
8
9
9
9
9
9
6
8
9
9
9
9
9
-: Power less than 50 percent.
5: Power between 50 and 60 percent.
6: Power between 60 and 70 percent.
7: Power between 70 and 80 percent.
8: Power between 80 and 90 percent
9: Power above 90 percent.
between 70 and 80 percent. However, when the number of fish per composite
increases to 8 to 10, the power increases by about 10 percentage points.
One final note on determining the number of replicate composite samples per
site and the number of fish per composite should be emphasized. According to
Section 6.1.2.3, Phase I intensive studies will focus on those target analytes that
exceeded the selected SV used in the screening study. Thus, multiple target
analytes may be under investigation during Phase I intensive studies, and the
population variances of these analytes are likely to differ. Note: States should
use the target analyte that exhibits the largest population variance when
selecting the number of replicate composite samples per site and the number of
fish per composite. This conservative approach supports use of the data in
6-21
-------
6. FIELD PROCEDURES
Section B of Table 6-2 where the ratio of oYSV is twice that of the data in Section
A. States may estimate population variances from historic fish contaminant data
or from composite data as described by EPA (1989d). This estimate of o2 can
be used to determine whether the sampling design (i.e., number of replicate
composite samples [n] and number of individuals per composite [m]) should be
modified to achieve a desired statistical power.
After States have implemented their fish and shellfish contaminant monitoring
program, collected data on cost and variance components, and addressed other
design considerations, they may want to consider using an optimal composite
sampling protocol as described in Rohlf et al. (1991) for refining their sampling
design. An optimal sampling design is desirable because it detects a specified
minimum difference between the site-specific mean contaminant concentration
and the SV at minimum cost.
6.2 SAMPLE COLLECTION
Sample collection activities should be initiated in the field only after an approved
sampling plan has been developed. This section discusses recommended
sampling equipment and its use, considerations for ensuring preservation of
sample integrity, and field recordkeeping and chain-of-custody procedures
associated with sample processing, preservation, and shipping.
6.2.1 Sampling Equipment and Use
In response to the variations in environmental conditions and target species of
interest, fisheries biologists have had to devise sampling methods that are
intrinsically selective for certain species and sizes of fish and shellfish (Versar,
1982). Although this selectivity can be a hindrance in an investigation of
community structure, it is not a problem where tissue contaminant analysis is of
concern because tissue contaminant data can best be compared only if factors
such as differences in taxa and size are minimized.
Collection methods can be divided into two major categories, active and passive.
Each collection method has advantages and disadvantages. Various types of
sampling equipment, their use, and their advantages and disadvantages are
summarized in Table 6-3 for fish and in Table 6-4 for shellfish. Note: Either
active or passive collection methods may be used as long as the methods
selected result in collection of a representative fish sample of the type consumed
by local sport and subsistence fishermen.
A basic checklist of field sampling equipment and supplies is shown in Table 6-5.
Safety considerations associated with the use of a boat in sample collection
activities are summarized in Table 6-6.
6-22
-------
6. FIELD PROCEDURES
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6-23
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6. FIELD PROCEDURES
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6-24
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6. FIELD PROCEDURES
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6-25
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6. FIELD PROCEDURES
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6-26
-------
6. FIELD PROCEDURES
Table 6-5. Checklist of Field Sampling Equipment and Supplies
for Fish and Shellfish Contaminant Monitoring Programs
Boat supplies
- Fuel supply (primary and auxiliary supply)
- Spare parts repair kit
- Life preservers
- First aid kit (including emergency phone numbers of local hospitals, family contacts for each
member of the sampling team)
- Spare oars
- Nautical charts of sampling site locations
Collection equipment (e.g., nets, traps, electroshocking device)
Recordkeeping/documentation supplies
- Field logbook
- Sample request forms
- Specimen identification labels
- Chain-of-Custody (COG) Forms and COC tags or labels
- Indelible pens
Sample processing equipment and supplies
- Holding trays
- Fish measuring board (metric units)
- Calipers (metric units)
- Shucking knife
- Balance to weigh representative specimens for estimating tissue weight (metric units)
- Aluminum foil (extra heavy duty)
- Freezer tape
- String
- Several sizes of plastic bags for holding individual or composite samples
- Resealable watertight plastic bags for storage of Field Records, COC Forms, and Sample
Request Forms
Sample preservation and shipping supplies
- Ice (wet ice, blue ice packets, or dry ice)
- Ice chests
- Filament-reinforced tape to seal ice chests for transport to the central processing laboratory
6-27
-------
6. FIELD PROCEDURES
Table 6-6. Safety Considerations for Field Sampling Using a Boat
Field collection personnel should not be assigned to duty alone in boats.
Life preservers should be worn at all times by field collection personnel near the water or
on board boats.
If electrofishing is the sampling method used, there must be two shutoff switches-one at
the generator and a second on the bow of the boat.
All deep water sampling should be performed with the aid of an experienced, licensed
boat captain.
All sampling during nondaylight hours, during severe weather conditions, or during
periods of high water should be avoided or minimized to ensure the safety of field
collection personnel.
All field collection personnel should be trained in CPR, water safety, boating safety, and
first aid procedures for proper response in the event of an accident. Personnel should
have local emergency numbers readily available for each sampling trip and know the
location of the hospitals or other medical facilities nearest each sampling site.
6.2.1.1 Active Collection-
Active collection methods employ a wide variety of sampling techniques and
devices. Devices for fish sampling include electroshocking units, seines, trawls,
and angling equipment (hook and line). Rotenone, a chemical piscicide, has
been used extensively to stun fish prior to their collection with seines, trawls, or
other sampling devices. Rotenone has not been found to interfere with the
analysis of the recommended organic target analytes (see Table 4-1) when the
recommended analysis procedures are used. See Section 8 for additional
information on appropriate analysis methods for the recommended organic target
analytes. Devices for shellfish sampling include seines, trawls, mechanical grabs
(e.g., pole- or cable-operated grab buckets and tongs), biological and hydraulic
dredges, scoops and shovels, rakes, and dip nets. Shellfish can also be
collected manually by SCUBA divers. Although active collection requires greater
fishing effort, it is usually more efficient than passive collection for covering a
large number of sites and catching the relatively small number of individuals
needed from each site for tissue analysis (Versar, 1982). Active collection
methods are particularly useful in shallow waters (e.g., streams, lake shorelines,
and shallow coastal areas of estuaries).
Active collection methods have distinct disadvantages for deep water sampling.
They require more field personnel and more expensive equipment than passive
collection methods. This disadvantage may be offset by coordinating sampling
6-28
-------
6. FIELD PROCEDURES
efforts with commercial fishing efforts. Purchasing fish and shellfish from
commercial fishermen using active collection devices is acceptable; however,
field sampling staff should accompany the commercial fishermen during the
collection operation to ensure that samples are collected and handled properly
and to verify the sampling site location. The field sampling staff then remove the
target species directly from the sampling device and ensure that sample
collection, processing, and preservation are conducted as prescribed in sample
collection protocols, with minimal chance of contamination. This is an excellent
method of obtaining specimens of commercially important target species,
particularly from the Great Lakes and coastal estuarine areas (Versar, 1982).
More detailed descriptions of active sampling devices and their use are provided
in Battelle (1975); Bennett (1970); Gunderson and Ellis (1986); Hayes (1983);
Mearns and Allen (1978); Pitt, Wells, and McKone (1981); Puget Sound Estuary
Program (1990b); Versar (1982); and Weber (1973).
6.2.1.2 Passive Collection—
Passive collection methods employ a wide array of sampling devices for fish and
shellfish, including gill nets, fyke nets, trammel nets, hoop nets, pound nets, and
d-traps. Passive collection methods generally require less fishing effort than
active methods but are usually less desirable for shallow water sample collection
because of the ability of many species to evade these entanglement and
entrapment devices. These methods normally yield a much greater catch than
would be required for a contaminant monitoring program and are time consuming
to deploy. In deep water, however, passive collection methods are generally
more efficient than active methods. Crawford and Luoma (1993) caution that
passive collection devices (e.g., gill nets) should be checked frequently to ensure
that captured fish do not deteriorate prior to removal from the sampling device.
Versar (1982, 1984) and Hubert (1983) describe passive sampling devices and
their use in more detail.
Purchasing fish and shellfish from commercial fishermen using passive collection
methods is acceptable; however, field sampling staff should accompany the
fishermen during both the deployment and collection operations to ensure that
samples are collected and handled properly and to verify the sampling site
location. The field sampling staff can then ensure that sample collection,
processing, and preservation are conducted as prescribed in sample collection
protocols, with minimal chance of contamination.
6.2.2 Preservation of Sample Integrity
The primary QA consideration in sample collection, processing, preservation, and
shipping procedures is the preservation of sample integrity to ensure the
accuracy of target analyte analyses. Sample integrity is preserved by prevention
of loss of contaminants already present in the tissues and prevention of
extraneous tissue contamination (Smith, 1985).
6-29
-------
6. FIELD PROCEDURES
Loss of contaminants already present in fish or shellfish tissues can be
prevented in the field by ensuring that the skin on fish specimens has not been
lacerated by the sampling gear or that the carapace of crustaceans or shells of
bivalves have not been cracked during sample collection resulting in loss of
tissues and/or fluids that may contain contaminants. Once the samples have
reached the laboratory, further care must be taken during thawing (if specimens
are frozen) to ensure that all liquids from the thawed specimens are retained with
the tissue sample as appropriate (see Sections 7.2.2, 7.2.3, and 7.2.4).
Sources of extraneous tissue contamination include contamination from sampling
gear, grease from ship winches or cables, spilled engine fuel (gasoline or diesel),
engine exhaust, dust, ice chests, and ice used for cooling. All potential sources
of contamination in the field should be identified and appropriate steps taken to
minimize or eliminate them. For example, during sampling, the boat should be
positioned so that engine exhausts do not fall on the deck. Ice chests should be
scrubbed clean with detergent and rinsed with distilled water after each use to
prevent contamination. To avoid contamination from melting ice, samples should
be placed in waterproof plastic bags (Stober, 1991). Sampling equipment that
has been obviously contaminated by oils, grease, diesel fuel, or gasoline should
not be used. All utensils or equipment that will be used directly in handling fish
or shellfish (e.g., fish measuring board or calipers) should be cleaned in the
laboratory prior to each sampling trip, rinsed in acetone and pesticide-grade
hexane, and stored in aluminum foil until use (Versar, 1982). Between sampling
sites, the field collection team should clean each measurement device by rinsing
it with ambient water and rewrapping it in aluminum foil to prevent contamination.
Note: Ideally, all sample processing (e.g., resections) should be performed at a
sample processing facility under cleanroom conditions to reduce the possibility
of sample contamination (Schmitt and Finger, 1987; Stober, 1991). However,
there may be some situations in which State staff find it necessary to fillet finfish
or resect edible turtle or shellfish tissues in the field prior to packaging the
samples for shipment to the processing laboratory. This practice should be
avoided whenever possible. If States find that filleting fish or resecting other
edible tissues must be performed in the field, a clean area should be set up
away from sources of diesel exhaust and areas where gasoline, diesel fuel, or
grease are used to help reduce the potential for surface and airborne
contamination of the samples from PAHs and other contaminants. Use of a
mobile laboratory or use of a portable resection table and enclosed hood would
provide the best environment for sample processing in the field. General
guidance for conducting sample processing under cleanroom conditions is
provided in Section 7.2.1. States should review this guidance to ensure that
procedures as similar as possible to those recommended for cleanroom
processing are followed. If sample processing is conducted in the field, a
notation should be made in the field records and on the sample processing
record (see Figure 7-2). Procedures for laboratory processing and resection are
described in Section 7.2. Procedures for assessing sources of sample
contamination through the analyses of field and processing blanks are described
in Section 8.3.3.6.
6-30
-------
6. FIELD PROCEDURES
6.2.3 Field Recordkeeplng
Thorough documentation of all field sample collection and processing activities
is necessary for proper interpretation of field survey results. For fish and
shellfish contaminant studies, it is advisable to use preprinted waterproof data
forms, indelible ink, and writing implements that can function when wet (Puget
Sound Estuary Program, 1990b). When multicopy forms are required, no-
carbon-required (NCR) paper is recommended because it allows information to
be forwarded on the desired schedule and retained for the project file at the
same time.
Four separate preprinted sample tracking forms should be used for each
sampling site to document field activities from the time the sample is collected
through processing and preservation until the, sample is delivered to the
processing laboratory. These are
Field record form
Sample identification label
• Chain-of-custody (COG) label or tag
COC form.
6.2.3.1 Field Record Fofm-
The following information should be included on the field record for each
sampling site in both Tier 1 screening (Figures 6-2 and 6-3) and Tier 2 intensive
studies as appropriate (Figures 6-4 and 6-5):
Project number
Sampling date and time (specify convention used, e.g., day/month/year and
24-h clock)
Sampling site location (including site name and number, county/parish,
latitude/longitude, waterbody name/segment number, waterbody type, and
site description)
Sampling depth
Collection method
Collectors' names and signatures
Agency (including telephone number and address)
6-31
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6. FIELD PROCEDURES
Reid Record for Fish Contaminant Monitoring Program — Screening Study
Project Number.
SITE LOCATION
Site Name/Number
County/Parish:.
Sampling Date and Time:.
. LatVLong.:.
Waterbody Name/Segment Number.
Waterbody Type: D RIVER
Site Description:
D LAKE
D ESTUARY
Collection Method:
Collector Name:
(print *nd sign)
Agency: _
Address:
Phone: ( ).
FISH COLLECTED r^-V^'
Bottom Feeder—Species'Name: _
Composite Sample #:
Fish» Length (mm) Sex
_ Number of Individuals:
Fish * Length (mm)
Sex
001
002
003
004
005
Minimum size
006
007
008
009
010
x100
Maximum size
Notes (e.g., morphological anomalies):
. >75% Composite mean length.
mm
Predator—Species Name:
Composite Sample #:
Fish * Length (mm) Sex
_ Number of Individuals:
Fish # Length (mm)
Sex
001
002
003
004
005
Minimum size
006
007
008
009
010
xi(JO
>75%
Maximum size
Motes (e.g., morphological anomalies):
Composite mean length.
. mm
Figure 6-2. Example of a field record for fish contaminant monitoring
program—screening study.
6-32
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6. FIELD PROCEDURES
Field Record for Shellfish Contaminant Monitoring Program — Screening Study
Project Number: Sampling Date and Time:
SITE LOCATION
Site Name/Number:
County/Parish:
Waterbody Name/Segment Number:
Waterbody Type: D RIVER
Site Description:
. LatAong.:.
D LAKE
D ESTUARY
Collection Method:
Collector Name: _
(print and sign)
Agency: _
Address:
Phone: (.
SHELLFISH COLLECTED [
Bivalve Species Name:
Composite Sample #:
Bivalve # Size (mm)
Bivalve #
Number of Individuals:
Size (mm) Bivalve # Size (mm)
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015 .
016
017
Minimum size
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
X100 =
Maximum size
Notes (e.g., morphological anomalies):
2.75%
Composite mean size.
mm
L
Figure 6-3. Example of a field record for shellfish contaminant monitoring
program—screening study.
6-33
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6. FIELD PROCEDURES
Reid Record for Fish Contaminant Monitoring Program — Intensive Study
Project Number:
SITE LOCATION
WaterbodyTypa: D RIVER D LAKE
Collection Method*
(print and sign)
Sampling Date and Time:
I at ./Long.: _
D ESTUARY
Phone: ( )
ppgv,, t> — .^ .„•. " -: — •— TT-TT'T , ,0, „ -, , • .,*'*• -, "" - .^j
Composite Sample #*
Fish # Length (mm) Sex (M, F, or 1)
OQ1
DO?
003
no4
OO5
Minimum length ifim_ „_
Maximum length
Rah # Length (mm) Sex (M, F, or I)
nni
nrw
ooa
004
O05
Minimum length inn_ >7«5%
Maximum length
Radicate Numbar
fvlnmhar of Individuals:
Fish * Length (mm) Sex (M, F, or I)
006
007
008
009
010
Composite mean length __ mm
Replicate Numbar
Number of individuals": _ _,.
Fish # Length (mm) Sex (M, F, or I)
006
007
008
009
010
Composite mean length mm
page 1 of 2
Figure 6-4. Example of a field record for fish contaminant monitoring
program—intensive study.
6-34
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6. FIELD PROCEDURES
Reid Record for Fish Contaminant Monitoring Program — Intensive Study (con.)
Project Number:
SITE LOCATION:
Sampling Date and Time:
Site Name/Number:
County/Parish:
FISH COLLECTED »;,.. ,» ^ „...,...>. ,..
Species Name:
Composite Sample #:
Fish # Length (mm) Sex (M, F, or 1)
001
002
003
004
005
Minimum length jt10Q_ %
Maximum length
Notes (e.g., morphological anomalies):
Species Name:
Composite Sample #:
Fish # Length (mm) Sex (M, F, or 1)
001
002
003
004
005
Minimum length
— x 1 00 - %
Maximum length
Notes (e.g.. morphological anomalies):
Species Name:
Composite Sample #:
Fish # Length (mm) Sex (M, F, or I)
001 •
002
003
004
005
Minimum length
Maximum lengtn
Notes (e.g.. morphological anomalies):
LatAong.:
,,. " :.^.<-..'..^:"*.t.?.?. •• ."..-'......' s...\...; \. '• .Ill
Replicate Number
Number of Individuals:
Fish # Length (mm) Sex (M, F, or I)
006
007
008
009
010
Composite mean length mm
Replicate Number
Number of Individuals:
Fish # Length (mm) Sex (M, F, or I)
006
007
008
009
010
Composite mean length mm
Replicate Number
Number of Individuals:
Fish # Length (mm) Sex (M, F, or I)
006
007
008
009
010
Composite mean length mm
page 2 of 2
Figure 6-4 (continued)
6-35
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6. FIELD PROCEDURES
Reid Record for Shellfish Contaminant Monitoring Program — Intensive Study
Project Number:
Sampling Date and Time:
SITE LOCATION
Site Name/Number
County/Parish:
Waterbody Name/Segment Number:
Waterbody Type: D RIVER
Site Description:
LatAona:
D LAKE D ESTUARY
Collection Method:
Collector Name:
(print and sign)
Agency:
Address:
Phone: ( )
SHELLJHSH roLL£CTED nZx":::1::1:"::::::^
Species Name:
Composite Sample #:
Shellfish # Size (mm) Sex Shellfish #
001
002
003
004
005
006
007
008
009
01O
011
012
013
014
015
016
017
Minimum size
x100= >
Maximum size
Notes (e.g., morphological anomalies]
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
75%
Replicate Number:
Number of Individuals:
Size (mm) Sex Shellfish # Size (mm) Sex
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
Composite mean size mm
Figure 6-5. Example of a field record for shellfish contaminant monitoring
program—intensive study.
6-36
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6. FIELD PROCEDURES
Species collected (including species scientific name, composite sample
number, individual specimen number, number of individuals per composite
sample, number of replicate samples, total length/size [mm], sex [male,
female, indeterminate])
Note: States should specify a unique numbering system to track samples for
their own fish and shellfish contaminant monitqring programs.
• .Percent difference in size between the smallest and largest specimens to be
composited (smallest individual length [or size] divided by the largest
individual length [or size] x 100; should be >75 percent) and mean
composite length or size (mm)
Notes (including visible morphological abnormalities, e.g., fin erosion, skin
ulcers, cataracts, skeletal and exoskeletal anomalies, neoplasms, or
parasites).
6.2.3.2 Sample Identification Label—
A sample identification label should be completed in indelible ink for each
individual fish or shellfish specimen after it is processed to identify each sample
uniquely (Figure 6-6). The following information should be included on the
sample identification label:
Species scientific name or code number
Total length/size of specimen (mm)
Specimen number
Sample type: F (fish fillet analysis only)
S (shellfish edible portion analysis only)
W (whole fish analysis)
O (other fish tissue analysis)
• Sampling site—waterbody name and/or identification number
Sampling date/time (specify convention, e.g., day/month/year and 24-h
clock).
A completed sample identification label should be taped to each alumi-
num-foil-wrapped specimen and the specimen should be placed in a waterproof
plastic bag.
6.2.3.3 Chaln-of-Custody Label or Tag—
A COC label or tag should be completed in indelible ink for each individual fish
specimen. The information to be completed for each fish is shown in Figure 6-7.
6-37
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6. FIELD PROCEDURES
Species Name or Code
Total Length or Size (cm)
Sample Type
Sampling Site (name/number)
Specimen Number
Sampling Date (d/mo/yr)
Time (24-h clock)
Figure 6-6. Example of a sample Identification label.
Project Number
Sampling Site (name and/or ID number)
Collecting Agency (name, address, phone)
Sampler (name and signature)
Composite Number/Specimen Number(s)
Sampling Date (d/m/yr)/Time (24-hr clock)
Species Name or Code
Chemical Analyses
Q All target analytes
l~l Others (specify)
Processing
Whole Body
Comments
Resection
Study Type
Screening
Intensive
Phase! D
Phase II Q
Type of Ice
Wet
Dry
Figure 6-7. Example of a chaln-of-custody tag or label.
6-38
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6. FIELD PROCEDURES
After all information has been completed, the COC label or tag should be taped
or attached with string to the outside of the waterproof plastic bag containing the
individual fish sample. Information on the COC label/tag should also be
recorded on the COC form (Figure 6-8).
Because of the generally smaller size of shellfish, several individual aluminum-
foil-wrapped shellfish specimens (within the same composite sample) may be
placed in the same waterproof plastic bag. A COC label or tag should be
completed in indelible ink for each shellfish composite sample. If more than 10
individual shellfish are to be composited, several waterproof plastic bags may
have to be used for the same composite. It is important not to place too many
individual specimens in the same plastic bag to ensure proper preservation
during shipping, particularly during summer months. Information on the COC
label/tag should also be recorded on the COC form.
6.2.3.4 Chaln-of-Custody Form—
A COC form should be completed in indelible ink for each shipping container
(e.g., ice chest) used. Information recommended for documentation on the COC
form (Figure 6-8) is necessary to track all samples from field collection to receipt
at the processing laboratory. In addition, this form can be used for tracking
samples through initial laboratory processing (e.g., resection) as described in
Section 7.2.
Prior to sealing the ice chest, one copy of the COC form and a copy of the field
record sheet should be sealed in a resealable waterproof plastic bag. This
plastic bag should be taped to the inside cover of the ice chest so that it is
maintained with the samples being tracked. Ice chests should be sealed with
reinforced tape for shipment.
6.2.3.5 Field Logbook—
In addition to the four sample tracking forms discussed above, the field collection
team should document in a field logbook any additional information on sample
collection activities, hydrologic conditions (e.g., tidal stage), weather conditions,
boat or equipment operations, or any other unusual activities observed (e.g.,
dredging) or problems encountered that would be useful to the program manager
in evaluating the quality of the fish and shellfish contaminant monitoring data.
6.3 SAMPLE HANDLING
6.3.1 Sample Selection
6.3.1.1 Species Identification—
As soon as fish, shellfish, and turtles are removed from the collection device,
they should be identified by species. Nontarget species or specimens of target
species that do not meet size requirements (e.g., juveniles) should be returned
6-39
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6. FIELD PROCEDURES
Chain-of-Custody Record
Collecting Agency (name, address, phone)
Samplers (print and sign)
Composite
Number
Spodmsr
Has.
Sampling
Time
Study Type
Scr
Int
Sampling Date
Container
ol
Sampling Site (name/number)
Chwn
Analy
/
/•
/ *
leal / / /
"•/ /& /
//
-------
6. FIELD PROCEDURES
to the water. Species identification should be conducted only by experienced
personnel knowledgeable of the taxonomy of species in the waterbodies included
in the contaminant monitoring program. Taxonomic keys, appropriate for the
waters being sampled, should be consulted for species identification. Because
the objective of both the screening and intensive monitoring studies is to
determine the magnitude of contamination in specific fish, shellfish, and turtle
species, it is necessary that all individuals used in a composite sample be of a
single species. Note: Correct species identification is important and different
species should never be combined in a single composite sample.
When sufficient numbers of the target species have been identified to make up
a composite sample, the species name and all other appropriate information
should be recorded on the field record forms (Figures 6-2 through 6-5).
Note: EPA recommends that, when turtles are used as the target species,
target analyte concentrations be determined for each turtle rather than for a
composite turtle sample.
6.3.1.2 Initial Inspection and Sorting-
Individual fish of the selected target species should be rinsed in ambient water
to remove any foreign material from the external surface. Large fish should be
stunned by a sharp blow to the base of the skull with a wooden club or metal
rod. This club or rod should be used solely for the purpose of stunning fish, and
care should be taken to keep it reasonably clean to prevent contamination of the
samples (Versar, 1982). Small fish may be placed on ice immediately after
capture to stun them, thereby facilitating processing and packaging procedures.
Once stunned, individual specimens of the target species should be grouped by
species and general size class and placed in clean holding trays to prevent
contamination. All fish should be inspected carefully to ensure that their skin
and fins have not been damaged by the sampling equipment and damaged
specimens should be discarded (Versar, 1982).
Freshwater turtles should be rinsed in ambient water and their external surface
scrubbed if necessary to remove any foreign matter from their carapace and
limbs. Each turtle should be inspected carefully to ensure that the carapace and
extremities have not been damaged by the sampling equipment, and damaged
specimens should be discarded (Versar, 1982). Care should be taken when
handling large turtles, particularly snapping turtles; many can deliver severe
bites. Particularly during procedures that place fingers or hands within striking
range of the sharp jaws, covering the turtle's head, neck, and forelimbs with a
cloth towel or sack and taping it in place is often sufficient to prevent injury to the
field sampling crew (Frye, 1994).
After inspection, each turtle should be placed individually in a heavy burlap sack
or canvas bag tied tightly with a strong cord and then placed in an ice-filled
cooler. Placing turtles on ice will slow their metabolic rate, making them easier
to handle. Note: It is recommended that each turtle be analyzed as an individual
6-41
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6. FIELD PROCEDURES
sample, especially if the target turtle species is not abundant in the waterbody
being sampled or if the collected individuals differ greatly in size or age.
Analysis of individual turtles can provide an estimate of the maximum contam-
inant concentrations to which recreational or subsistence fishermen are
exposed. Target analyte concentrations in composite samples represent
averages for a specific target species population. The use of these values in risk
assessment is appropriate if the objective is to estimate the average
concentration to which consumers of the target species are exposed over a long
period of time. The use of long exposure periods (e.g., 70 years) is typical for
the assessment of carcinogenic effects, which may be manifest over an entire
lifetime (see Volume II of this guidance series). Noncarcinogenic effects, on the
other hand, may cause acute health effects over a relatively short period of time
(e.g., hours or days) after consumption. The maximum target analyte contam-
inant concentration may be more appropriate than the average target analyte
concentration for use with noncarginogenic target analytes (U.S. EPA, 1989d).
This is especially important for those target analytes for which acute exposures
to very high concentrations may be toxic to consumers.
Stone et al. (1980) reported extremely high concentrations of PCBs in various
tissues of snapping turtles from a highly contaminated site on the Hudson River.
Contaminant analysis of various turtle tissues showed mean PCB levels of 2,991
ppm in fatty tissue, 66 ppm in liver tissue, and 29 ppm in eggs as compared to
4 ppm in skeletal muscle. Clearly, inclusion of the fatty tissue, liver, and eggs
with the muscle tissues as part of the edible tissues will increase observed
residue concentrations over those detected in muscle tissue only. States
interested in using turtles as target species should review Appendix A for
additional information on the use of individual samples in contaminant monitoring
programs.
Bivalves (oysters, clams, scallops, and mussels) adhering to one another should
be separated and scrubbed with a nylon or natural fiber brush to remove any
adhering detritus or fouling organisms from the exterior shell surfaces (NOAA,
1987). All bivalves should be inspected carefully to ensure that the shells have
not been cracked or damaged by the sampling equipment and damaged
specimens should be discarded (Versar, 1982). Crustaceans, including shrimp,
crabs, crayfish, and lobsters, should be inspected to ensure that their
exoskeletons have not been cracked or damaged during the sampling process,
and damaged specimens should be discarded (Versar, 1982). After shellfish
have been rinsed, individual specimens should be grouped by target species and
placed in clean holding trays to prevent contamination.
A few shellfish specimens may be resected (edible portions removed) to
determine wet weight of the edible portions. This will provide an estimate of the
number of individuals required to ensure that the recommended sample weight
(200 g) is attained. Note: Individuals used to determine the wet weight of the
edible portion should not be used for target analyte analyses.
6-42
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6. FIELD PROCEDURES
6.3.1.3 Length or Size Measurements-
Each fish within the selected target species should be measured to determine
total body length (mm). To be consistent with the convention used by most
fisheries biologists in the United States, maximum body length should be
measured as shown in Figure 6-9. The maximum body length is defined as the
length from the anterior-most part of the fish to the tip of the longest caudal fin
ray (when the lobes of the caudal fin are compressed dorsoventrally) (Anderson
and Gutreuter, 1983).
Each turtle within the selected target species should be measured to determine
total carapace length (mm). To be consistent with the convention used by most
herpetologists in the United States, carapace length should be measured as
shown in Figure 6-9. The maximum carapace length is defined as the straight
line distance from the anterior edge of the carapace to the posterior edge of the
carapace (Conant and Collins, 1991).
For shellfish, each individual specimen should be measured to determine the
appropriate body size (mm). As shown in Figure 6-9, the recommended body
measurements differ depending on the type of shellfish being collected. Height
is a standard measurement of size for oysters, mussels, clams, scallops, and
other bivalve molluscs (Abbott, 1974; Galtsoff, 1964). The height is the distance
from the umbo to the anterior (ventral) shell margin. For crabs, the lateral width
of the carapace is a standard size measurement (U.S. EPA, 1990c); for shrimp
and crayfish, the standard measurement of body size is the length from the ros-
trum to the tip of the telson {Texas Water Commission, 1990); and for lobsters,
two standard measurements of body size are commonly used. For clawed and
spiny lobsters, the standard size is the length of the carapace. For spiny lob-
sters, the length of the tail is also used as a standard size measurement.
6.3.1.4 Sex Determination (Optional)—
An experienced fisheries biologist can often make a preliminary sex
determination for fish by visual inspection. The body of the fish should not be
dissected in the field to determine sex; sex can be determined through internal
examination of the gonads during laboratory processing (Section 7.2.2.4).
An experienced herpetologist can often make a preliminary sex determination of
a turtle by visual inspection in the field. The plastron (ventral portion of the
carapace) is usually flatter in the female and the tail is less well developed than
in the male. The plastron also tends to be more concave in the male (Holmes,
1984). For the common snapping turtle (Chelydra serpentina), the cloaca of the
female is usually located inside or at the perimeter of the carapace, while the
icloaca of the male extends slightly beyond the perimeter of the carapace. The
carapace of the turtle should never be resected in the field to determine sex; sex
can be determined through internal examination of the gonads during laboratory
processing (Section 7.2.3.4.).
6-43
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6. FIELD PROCEDURES
Fish
Maximum body length3
Crab
Carapace widthb
I Umbo
\L
Bivalve
Height0
Rostrum
Shrimp, Crayfish
Body lengthd
a Maximum body length is the length from the anterior-most part of the fish to the tip of the
longest caudal fin ray (when the lobes of the caudal fin are compressed dorso ventrally
{Anderson and Gutreuter, 1983).
Carapace width is the lateral distance across the carapace (from tip of spine to tip of spine)
(U.S. EPA, 1990c).
° Height is the distance from the umbo to the anterior (ventral) shell margin (Galtsoff, 1964).
Body length is the distance from the tip of the rostrum to the tip of the telson (Texas Water
Commission, 1990).
6 Carapace length is distance from top of rostrum to the posterior margin of the carapace.
Figure 6-9. Recommended measurements of body length and
size for fish, shellfish, and turtles.
6-44
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6. FIELD PROCEDURES
Spiny Lobster
Clawed Lobster
Carapace
length®
Tail
length'
Carapace
length9
Turtle
Carapace lengthh
e Carapace length is the distance from the anterior-most edge of the groove between the horns
directly above the eyes, to the rear edge of the top part of the carapace as measured along the
middorsal line of the back (Laws of Florida Chapter 46-24.003).
T Tail length is the distance measured lengthwise along the top middorsal line of the entire tail
to the rear-most extremity (this measurement shall be conducted with the tail in a flat straight
position with the tip of the tail closed (Laws of Florida Chapter 46-24.003).
9 Carapace length is the distance from the rear of the eye socket to the posterior margin of the
carapace (New York Environmental Conservation Law 13-0329.5.a and Massachusetts General
Laws Chapter 130).
h Carapace length is the straight-line distance from the anterior margin to the posterior margin
of the shell (Conant and Collins, 1991).
Figure 6-9 (continued)
6-45
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6. FIELD PROCEDURES
For shellfish, a preliminary sex determination can be made by visual inspection
only for crustaceans. Sex cannot be determined in bivalve molluscs without
shucking the bivalves and microscopically examining gonadal material. Bivalves
should not be shucked in the field to determine sex; sex determination through
examination of the gonads can be performed during laboratory processing if
desired (Section 7.2.4.2).
6.3.1.5 Morphological Abnormalities {Optional)—
If resources allow, States may wish to consider documenting external gross
morphological conditions in fish from contaminated waters. Severely polluted
aquatic habitats have been shown to produce a higher frequency of gross
pathological disorders than similar, less polluted habitats (Krahn et at., 1986;
Malins et al., 1984, 1985; Mix, 1986; Sinderman, 1983; and Sinderman et a!.,
1980).
Sinderman et al. (1980) reviewed the literature on the relationship of fish
pathology to pollution in marine and estuarine environments and identified four
gross morphological conditions acceptable for use in monitoring programs:
Fin erosion
Skin ulcers
Skeletal anomalies
Neoplasms (i.e., tumors).
Fin erosion is the most frequently observed gross morphological abnormality in
polluted areas and is found in a variety of fishes (Sinderman, 1983). In demersal
fishes, the dorsal and anal fins are most frequently affected; in pelagic fishes,
the caudal fin is primarily affected.
Skin ulcers have been found in a variety of fishes from polluted waters and are
the second most frequently reported gross abnormality. Prevalence of ulcers
generally varies with season and is often associated with organic enrichment
(Sinderman, 1983).
Skeletal anomalies include abnormalities of the'head, fins, gills, and spinal
column (Sinderman, 1983). Skeletal anomalies of the spinal column include
fusions, flexures, and vertebral compressions.
Neoplasms or tumors have been found at a higher frequency in a variety of
polluted areas throughout the world. The most frequently reported visible tumors
are liver tumors, skin tumors (i.e., epidermal papillomas and/or carcinomas), and
neurilemmomas (Sinderman, 1983).
The occurrence of fish parasites and other gross morphological abnormalities
that are found at a specific site should be noted on the field record form. States
interested in documenting morphological abnormalities in fish should review the
6-46
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6. FIELD PROCEDURES
protocols for fish pathology studies recommended in the Puget Sound Estuary
Program (1990c) and those described by Goede and Barton (1990).
6.3.2 Sample Packaging
6.3.2.1 Fish-
After initial processing to determine species, size, sex, and morphological
abnormalities, each fish should be individually wrapped in extra heavy duty
aluminum foil. Spines on fish should be sheared to minimize punctures in the
aluminum foil packaging (Stober, 1991). The sample identification label shown
in Figure 6-6 should be taped to the outside of each aluminum foil package,
each individual fish should be placed into a waterproof plastic bag and sealed,
and the COG tag or label should be attached to the outside of the plastic bag
with string or'tape. All of the packaged individual specimens in a composite
sample should be kept together (if possible) in one large waterproof plastic bag
in the same shipping container (ice chest) for transport. Once packaged,
samples should be cooled on ice immediately.
6.3.2.2 Turtles
After inital processing to determine the species, size (carapace length), and sex,
each turtle should be placed on ice in a separate burlap or canvas bag and
stored on ice for transport to the processing laboratory. A completed sample
identification label (Figure 6-6) should be attached with string around the neck
or one of the turtle's extremities and the COG tag or label should be attached to
the outside of the bag with string or tape. Note: Bagging each turtle should not
be undertaken until the specimen has been sufficiently cooled to induce a mild
state of torpor, thus facilitating processing. The samplers should work rapidly to
return each turtle to the ice chest as soon as possible after packaging as the
turtle may suddenly awaken as it warms thus becoming a danger to samplers
(Frye, 1994). As mentioned in Section 6.3.1, States should analyze turtles
individually rather than compositing samples. This is especially important when
very few specimens are collected at a sampling site or when specimens of
widely varying size/age are collected.
Note: When a large number of individual specimens in the same composite
sample are shipped together in the same waterproof plastic bag, the samples
must have adequate space in the bag to ensure that contact with ice can occur,
thus ensuring proper preservation during shipping. This is especially important
when samples are collected during hot weather and/or when the time between
field collection and delivery to the processing laboratory approaches the
maximum shipping time (Table 6-7).
6.3.2.3 Shellfish-
After initial processing to determine species, size, sex, and morphological
abnormalities, each shellfish specimen should be wrapped individually in extra
6-47
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6. FIELD PROCEDURES
heavy duty aluminum foil. A completed sample identification label (Figure 6-6)
should be taped to the outside of each aluminum foil package. Note: Some
crustacean species (e.g., blue crabs and spiny lobsters) have sharp spines on
their carapace that might puncture the aluminum foil wrapping. Carapace spines
should never be sheared off because this would destroy the integrity of the
carapace. For such species, one of the following procedures should be used to
reduce punctures to the outer foil wrapping:
Double-wrap the entire specimen in extra heavy duty aluminum foil.
Place clean cork stoppers over the protruding spines prior to wrapping the
specimen in aluminum foil.
• Wrap the spines with multiple layers of foil before wrapping the entire
specimen in aluminum foil.
All of the individual aluminum-foil-wrapped shellfish specimens (in the same
composite sample) should be placed in the same waterproof plastic bag for
transport. In this case, a COG tag or label should be completed for the
composite sample and appropriate information recorded on the field record sheet
and COC form. The COG label or tag should then be attached to the outside of
the plastic bag with string or tape. For composite samples containing more than
10 shellfish specimens or especially large individuals, additional waterproof
plastic bags may be required to ensure proper preservation. Once packaged,
composite samples should be cooled on ice immediately. Note: When a large
number of individual specimens in the same composite sample are shipped
together in the same waterproof plastic bag, the samples must have adequate
space in the bag to ensure that contact with ice can occur; thus ensuring proper
preservation during shipping. This is especially important when samples are
collected during hot weather and/or when the time between field collection and
delivery to the processing laboratory approaches the maximum shipping time
(Table 6-7).
6.3.3 Sample Preservation
The type of ice to be used for shipping should be determined by the length of
time the samples will be in transit to the processing laboratory and the sample
type to be analyzed (Table 6-7).
6.3.3.1 Fish, Turtles, or Shellfish To Be Resected—
Note: Ideally fish, turtles, and shellfish specimens should not be frozen prior to
resection if analyses will include edible tissue only because freezing may cause
some internal organs to rupture and contaminate fillets or other edible tissues
(Stober, 1991; U.S. EPA, 1986b). Wet ice or blue ice (sealed prefrozen ice
packets) is recommended as the preservative of choice when the fish fillet, turtle
meat, or shellfish edible portions are the primary tissues to be analyzed.
Samples shipped on wet or blue ice should be delivered to the processing
6-48
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6. FIELD PROCEDURES
Table 6-7. Recommendations for Preservation of Fish, Shellfish, and Turtle
Samples from Time of Collection to Delivery at the Processing Laboratory
Sample
typo
Number per
composite
Container
Preservation
Maximum
shipping
time
Fish"
Whole fish
(to be filleted)
3-10 Extra heavy duty
aluminum foil wrap of
each fish.b Each fish
is placed in a
waterproof plastic
bag.
Cool on wet ice or
blue ice packets
(preferred method)
or
Freeze on dry ice
only if shipping
time will exceed 24
hours
24 hours
48 hours
Whole fish
3-10
Same as above.
Cool on wet ice or
blue ice packets
or
Freeze on dry ice
24 hours
48 hours
Shellfish* ,mT1-
Whole shellfish
(to be resected for
edible tissue)
Whole shellfish
Whole turtles
(to be resected for
edible tissue)
••
3-50c Extra heavy duty
aluminum foil wrap of
each specimen.6
Shellfish in the same
composite sample
may be placed in the
same waterproof
plastic bag.
3-50° Same as above.
1d Heavy burlap or
canvas bags.
. •.
Cool on wet ice or
blue ice packets
(preferred method)
or
Freeze on dry ice
if shipping time
will exceed 24 hours
Cool on wet ice or
blue ice packets
or
Freeze on dry ice
Cool on wet ice or
blue ice packets
(preferred method)
or
Freeze on dry ice if
shipping time to
exceed 24 hours
. •••• v ]
24 hours
48 hours
24 hours
48 hours
24 hours
48 hours
Use only individuals that have attained at least legal harvestable or consumable size.
b Aluminum foil should not be used for long-term storage of any sample (i.e., whole organisms, fillets, or
hompgenates) that will be analyzed for metals.
c Species and size dependent. For very small shellfish species, more than 50 individuals may be required to
achieve the 200-g composite sample mass recommended for screening studies.
d Turtles should be analyzed as individual rather than as composite samples.
6-49
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6. FIELD PROCEDURES
laboratory within 24 hours (Smith, 1985; U.S. EPA, 1990d). If the shipping time
to the processing laboratory will exceed 24 hours, dry ice should be used.
Note: One exception to the use of dry ice for long-term storage is if fish or
shellfish are collected as part of extended offshore fieldsurveys. States involved
in these types of field surveys may employ shipboard freezers to preserve
samples for extended periods rather than using dry ice. Ideally, all fish should
be resected in cleanrooms aboard ship prior to freezing.
6.3.3.2 Fish, Turtles, or Shellfish for Whole-Body Analysis—
At some sites, States may deem it necessary to collect fish, turtles, or shellfish
for whole-body analysis if a local subpopulation of concern typically consumes
whole fish, turtles, or shellfish. If whole fish, turtles, or shellfish samples are to
be analyzed, either wet ice, blue ice, or dry ice may be used; however, if the
shipping time to the processing laboratory will exceed 24 hours, dry ice should
be used.
Dry ice requires special packaging precautions before shipping by aircraft to
comply with U.S. Department of Transportation (DOT) regulations. The Code of
Federal Regulations (49 CFR 173.217) classifies dry ice as Hazard Class 9
UN1845 (Hazardous Material). These regulations specify the amount of dry ice
that may be shipped by air transport and the type of packaging required. For
each shipment by air exceeding 5 pounds of dry ice per package, advance
arrangements must be made with the carrier. Not more than 441 pounds of dry
ice may be transported in any one cargo compartment on any aircraft unless the
shipper has made special written arrangements with the aircraft operator.
The regulations further specify that the packaging must be designed and
constructed to permit the release of carbon dioxide gas to prevent a buildup of
pressure that could rupture the package. If samples are transported in a cooler,
several vent holes should be drilled to allow carbon dioxide gas to escape. The
vents should be near the top of the vertical sides of the cooler, rather than in the
cover, to prevent debris from falling into the cooler. Wire screen or cheesecloth
should be installed in the vents to keep foreign materials from contaminating the
cooler. When the samples are packaged, care should be taken to keep these
vents open to prevent the buildup of pressure.
Dry ice is exempted from shipping certification requirements if the amount is less
than 441 pounds and the package meets design requirements. The package
must be marked "Carbon Dioxide, Solid" or "Dry Ice" with a statement indicating
that the material being refrigerated is to be used for diagnostic or treatment
purposes (e.g., frozen tissue samples).
6.3.4 Sample Shipping
The fish, turtle, and shellfish samples should be hand-delivered or shipped to the
processing laboratory as soon as possible after collection. The time the samples
6-50
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6. FIELD PROCEDURES
were collected and time of their arrival at the processing laboratory should be
recorded on the GOC form (Figure 6-8).
If the sample is to be shipped rather than hand-delivered to the processing
laboratory, field collection staff must ensure the samples are packed properly
with adequate ice layered between samples so that sample degradation does not
occur. In addition, a member of the field collection staff should telephone ahead
to the processing laboratory to alert them to the anticipated delivery time of the
samples and the name and address of the carrier to be used. Field collection
staff should avoid shipping samples for weekend delivery to the processing
laboratory unless prior plans for such a delivery have been agreed upon with the
processing laboratory staff.
6-51
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
SECTION 7
LABORATORY PROCEDURES I — SAMPLE HANDLING
This section provides guidance on laboratory procedures for sample receipt,
chain-of-custody, processing, distribution, analysis, and archiving. Planning,
documentation, and quality assurance and quality control of all laboratory
activities are emphasized to ensure that (1) sample integrity is preserved during
all phases of sample handling and analysis, (2) chemical analyses are performed
cost-effectively and meet program data quality objectives, and (3) data produced
by different States and Regions are comparable.
Laboratory procedures should be documented in a Work/QA Project Plan (U.S.
EPA, 1980b) as described in Appendix F. Routine sample processing and
analysis procedures should be prepared as standard operating procedures
(SOPs) (U.S. EPA, 1984b).
7.1 SAMPLE RECEIPT AND CHAIN-OF-CUSTODY
Fish, shellfish, and turtle samples may be shipped or hand-carried from the field
according to one or more of the following pathways:
From the field to a State laboratory for sample processing and analysis
From the field to a State laboratory for sample processing arid shipment of
composite sample aliquots to a contract laboratory for analysis
From the field to a contract laboratory for sample processing and analysis.
Sample processing and distribution for analysis ideally should be performed by
one processing laboratory. Transportation of samples from the field should be
coordinated by the sampling team supervisor and the laboratory supervisor
responsible for sample processing and distribution (see Section 6.3.4). An
accurate written custody record must be maintained so that possession and
treatment of each sample can be traced from the time of collection through
analysis and final disposition.
Fish, shellfish, and turtle samples should be brought or shipped to the sample
processing laboratory in sealed containers accompanied by a copy of the sample
request form (Figure 6-1), a chain-of-custody form (Figure 6-8), and the field
records (Figures 6-2 through 6-5). Each time custody of a sample or set of
samples is transferred, the Personnel Custody Record of the COC form must be
7-1
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
completed and signed by both parties. Corrections to the COC form should be
made in indelible ink by drawing a single line through the original entry, entering
the correct information and the reason for the change, and initialing and dating
the correction. The original entry should never be obscured.
When custody is transferred from the field to the sample processing laboratory,
the following procedure should be used:
Note the shipping time. If samples have been shipped on wet or blue ice,
check that the shipping time has not exceeded 24 hours.
Check that each shipping container has arrived undamaged and that the
seal is intact.
Open each shipping container and remove the copy of the sample request
form, the COC form, and the field records.
Note the general condition of the shipping container (samples iced properly
with no leaks, etc.) and the accompanying documentation (dry, legible, etc.).
Locate individuals in each composite sample listed on the COC form and
note the condition of their packaging. Individual specimens should be
properly wrapped and labeled. Note any problems (container punctured,
illegible labels, etc.) on the COC form.
If individuals in a composite are packaged together, check the contents of
each composite sample container against the field record for that sample to
ensure that the individual specimens are properly wrapped and labeled.
Note any discrepancies or missing information on the COC form.
Initial the COC form and record the date and time of sample receipt.
Enter the following information for each composite sample into a permanent
laboratory record book and, if applicable, a computer database:
— Sample identification number (specify conventions for the composite
sample number and the specimen number) Note: EPA recommends
processing and analysis of turtles as individual samples.
— Receipt date (specify convention, e.g., day/month/year)
— Sampling date (specify convention, e.g., day/month/year)
— Sampling site (name and/or identification number)
— Fish, turtle, and shellfish species (scientific name or code number)
7-2
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
— Total length of each fish, carapace length of each turtle, or size of each
shellfish (mm)
If samples have been shipped on wet or blue ice, distribute them
immediately to the technician responsible for resection (see Section 7.2).
See Section 7.2.3 for the procedure for processing turtle samples as
individual samples. If samples have been shipped on dry ice, they may be
distributed immediately to the technician for processing or stored in a freezer
at <-20 °C for later processing. Once processed, fillets or edible portions of
fish, turtles or shellfish, or tissue homogenates, should be stored according
to the procedures described in Section 7.2 and in Table 7-1. Note: Holding
times in Table 7-1 are maximum times recommended for holding samples
from the time they are received at the laboratory until they are analyzed.
These holding times are based on guidance that is sometimes administrative
rather than technical in nature; there are no promulgated holding time criteria
for tissues (U.S. EPA, 1995k). If States choose to use longer holding times,
they must demonstrate and document the stability of the target analyte
residues over the extended holding times.
7.2 SAMPLE PROCESSING
This section includes recommended procedures for preparing composite
homogenate samples of fish fillets and edible portions of shellfish and individual
samples of edible portions of freshwater turtles as required in screening and
intensive studies. Recommended procedures for preparing whole fish composite
homogenates are included in Appendix G for use by States in assessing the
potential risk to local subpopulations known to consume whole fish or shellfish.
7.2.1 General Considerations
All laboratory personnel performing sample processing procedures (see Sections
7.2.2, 7.2.3, and 7.2.4) should be trained or supervised by an experienced
fisheries biologist. Care must be taken during sample processing to avoid
contaminating samples. Schmitt and Finger (1987) have demonstrated that
contamination of fish flesh samples is likely unless the most exacting clean
dissection procedures are used. Potential sources of contamination include dust,
instruments, utensils, work surfaces, and containers that may contact the
samples. All sample processing (i.e., filleting, removal of other edible tissue,
homogenizing, compositing) should be done in an appropriate laboratory facility
under cleanroom conditions (Stober, 1991). Cleanrooms or work areas should
be free of metals and organic contaminants. Ideally, these areas should be
under positive pressure with filtered air (HEPA filter class 100) (California
Department of Fish and Game, 1990). Periodic wipe tests should be conducted
in clean areas to verify the absence of significant levels of metal and organic
contaminants. All instruments, work surfaces, and containers used to process
samples must be of materials that can be cleaned easily and that are not
themselves potential sources of contamination. More detailed guidance on
establishing trace metal cleanrooms is provided in U.S. EPA (1995b).
7-3
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Table 7-1. Recommendations for Container Materials, Preservation, and Holding
Times for Fish, Shellfish, and Turtle Tissues from Receipt at Sample
Processing Laboratory to Analysis
Storage
Other
metals
Organics
Metals and
organics
Analyte
Mercury
Matrix
Tissue (fillets and edible
portions, homogenates)
Sample
container
Plastic, borosilicate
glass, quartz,
PTFE
Preservation
Freeze at <-20 °C
Holding time'
28 days"
Lipids
Tissue (fillets and edible
portions, homogenates)
Tissue (fillets and edible
portions, homogenates)
Tissue (fillets and edible
portions, homogenates)
Tissue (fillets and edible
portions, homogenates)
Plastic, borosilicate
glass, quartz,
PTFE
Borosilicate glass,
PTFE, quartz,
aluminum foil
Borosilicate glass,
quartz, PTFE
Plastic, borosilicate
glass, quartz,
PTFE
Freeze at <-20 °C 6 months0
Freeze at ^-20 °C 1 year"
Freeze at <.-20 °C 28 days
(for mercury);
6 months
(for other
metals); and 1
year (for
organics)
Freeze at <-20 °C 1 year
PTFE « Polytetrafluoroethylene (Teflon).
a Maximum holding times recommended by EPA (1995k).
b This maximum holding time is also recommended by the Puget Sound Estuary Program (1990e).
The California Department of Fish and Game (1990) and the USGS National Water Quality
Assessment Program (Crawford and Luoma, 1993) recommend a maximum holding time of 6
months for all metals, including mercury.
0 This maximum holding time is also recommended by the California Department of Fish and Game
(1990), the 301(h) monitoring program (U.S. EPA, 1986b), and the USGS National Water Quality
Assessment Program (Crawford and Luoma, 1993). The Puget Sound Estuary Program (1990e)
recommends a maximum holding time of 2 years.
d This maximum holding time is also recommended by the Puget Sound Estuary Program (1990e).
The California Department of Fish and Game (1990) and the USGS National Water Quality
Assessment Program (Crawford and Luoma, 1993) recommend a more conservative maximum
holding time of 6 months. The EPA (1995c) recommends a maximum holding time of 1 year at
£-10 °C for dioxins/furans.
7-4
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
To avoid cross-contamination, all equipment used in sample processing (i.e.,
resecting, homogenizing, and compositing) should be cleaned thoroughly before
each composite sample is prepared. Verification of the efficacy of cleaning
procedures should be documented through the analysis of processing blanks or
rinsates (see Section 8.3.3.6).
Because sources of organic and metal contaminants differ, it is recommended
that duplicate samples be collected, if time and funding permit, when analyses
of both organics and metals are required (e.g., for screening studies). One
sample can then be processed and analyzed for organics and the other can be
processed independently and analyzed for metals (Batelle, 1989; California
Department of Fish and Game, 1990; Puget Sound Estuary Program, 1990c,
1990d). If fish are of adequate size, separate composites of individual fillets may
be prepared and analyzed independently for metals and organics. If only one
composite sample is prepared for the analyses of metals and organics, the
processing equipment must be chosen and cleaned carefully to avoid
contamination by both organics and metals.
Suggested sample processing equipment and cleaning procedures by analysis
type are discussed in Sections 7.2.1.1 through 7.2.1.3. Other procedures may
be used if it can be demonstrated, through the analysis of appropriate blanks,
that no contamination is introduced (see Section 8.3.3.6).
7.2.1.1 Samples for Organics Analysis—
Equipment used in processing samples for organics analysis should be of
stainless steel, anodized aluminum, borosilicate glass, polytetrafluoroethylene
(PTFE), ceramic, or quartz. Polypropylene and polyethylene (plastic) surfaces,
implements, gloves, and containers are a potential source of contamination by
organics and should not be used. If a laboratory chooses to use these materials,
there should be clear documentation that they are not a source of contamination.
Filleting should be done on glass or PTFE cutting boards that are cleaned
properly between fish or on cutting boards covered with heavy duty aluminum
foil that is changed after each filleting. Tissue should be removed with clean,
high-quality, corrosion-resistant stainless steel or quartz instruments or with
knives with titanium blades and PTFE handles (Lowenstein and Young, 1986).
Fillets or tissue homogenates may be stored in borosilicate glass, quartz, or
PTFE containers with PTFE-lined lids or in heavy duty aluminum foil (see Table
7-1).
Prior to preparing each composite sample, utensils and containers should be
washed with detergent solution, rinsed with tap water, soaked in pesticide-grade
isopropanol or acetone, and rinsed with organic-free, distilled, deionized water.
Work surfaces should be cleaned with pesticide-grade isopropanol or acetone,
washed with distilled water, and allowed to dry completely. Knives, fish sealers,
measurement boards, etc., should be cleaned with pesticide-grade isopropanol
7-5
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
or acetone followed by a rinse with contaminant-free distilled water between
each fish sample (Stober, 1991).
7.2.1.2 Samples for Metals Analysis-
Equipment used in processing samples for metals analyses should be of quartz,
PTFE, ceramic, polypropylene, or polyethylene. The predominant metal
contaminants from stainless steel are chromium and nickel. If these metals are
not of concern, the use of high-quality, corrosion-resistant stainless steel for
sample processing equipment is acceptable. Quartz utensils are ideal but
expensive. For bench liners and bottles, borosilicate glass is preferred over
plastic (Stober, 1991). Knives with titanium blades and PTFE handles are
recommended for performing tissue resections (Lowenstein and Young, 1986).
Borosilicate glass bench liners are recommended. Filleting may be done on
glass or PTFE cutting boards that are cleaned properly between fish or on
cutting boards covered with heavy duty aluminum foil that is changed after each
fish. Fillets or tissue homogenates may be stored in plastic, borosilicate glass,
quartz, or PTFE containers (see Table 7-1).
Prior to preparing each composite sample, utensils and containers should be
cleaned thoroughly with a detergent solution, rinsed with tap water, soaked in
acid, and then rinsed with rnetal-free water. Quartz, PTFE, glass, or plastic
containers should be soaked in 50% HN03, for 12 to 24 hours at room
temperature. Note: Chromic acid should not be used for cleaning any
materials. Acids used should be at least reagent grade. Stainless steel parts
may be cleaned as stated for glass or plastic, omitting the acid soaking step
(Stober, 1991).
7.2.1.3 Samples for Both Organics and Metals Analyses—
As noted above, several established monitoring programs, including the Puget
Sound Estuary Program (1990c, 1990d), the NOAA Mussel Watch Program
(Battelle, 1989), and the California Mussel Watch Program (California
Department of Fish and Game, 1990), recommend different procedures for
processing samples for organics and metals analyses. However, this may not
be feasible if fish are too small to allow for preparing separate composites from
individual fillets or if resources are limited. If a single composite sample is
prepared for the analyses of both organics and metals, precautions must be
taken to use materials and cleaning procedures that are noncontaminating for
both organics and metals.
Quartz, ceramic, borosilicate glass, and PTFE are recommended materials for
sample processing equipment. If chromium and nickel are not of concern, high-
quality, corrosion-resistant stainless steel utensils may be used. Knives with
titanium blades and PTFE handles are recommended for performing tissue
resections (Lowenstein and Young, 1986). Borosilicate glass bench liners are
recommended. Filleting should be done on glass or PTFE cutting boards that
are cleaned properly between fish or on cutting boards covered with heavy duty
7-6
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
aluminum foil that is changed after each filleting. Fillets or tissue homogenates
should be stored in clean borosilicate glass, quartz, or PTFE containers with
PTFE-lined lids.
Prior to preparing each composite sample, utensils and containers should be
cleaned thoroughly with a detergent solution, rinsed with tap water, soaked in
50% HNO3, for 12 to 24 hours at room temperature, and then rinsed with
organics- and metal-free water. Note: Chromic acid should not be used for
cleaning any materials. Acids used should be at least reagent grade. Stainless
steel parts may be cleaned using this recommended procedure with the acid
soaking step method omitted (Stober, 1991).
Aliquots of composite homogenates taken for metals analysis (see Section 7.3.1)
may be stored in plastic containers that have been cleaned according to the
procedure outlined above, with the exception that aqua regia must not be used
for the acid soaking step.
7.2.2 Processing Fish Samples
Processing in the laboratory to prepare fish fillet composite homogenate samples
for analysis (diagrammed in Figure 7-1) involves
Inspecting individual fish
Weighing individual fish
Removing scales and/or otoliths for age determination (optional)
Determining the sex of each fish (optional)
Examining each fish for morphological abnormalities (optional)
Scaling all fish with scales (leaving belly flap on); removing skin of scaleless
fish (e.g., catfish)
Filleting (resection)
Weighing fillets
Homogenizing fillets
Preparing a composite homogenate
Preparing aliquots of the composite homogenate for analysis
Distributing frozen aliquots to one or more analytical laboratories.
7-7
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Log in fish samples using COC procedures
Unwrap and inspect individual fish
Weigh individual fish
Remove and archive scales and/or otolittis for age determination (optional)
Determine sex (optional); note morphological abnormalities (optional)
Remove scales from all scaled fish
Remove skin from scalelsss fish (e.g., catfish)
Fillet fish
Weigh fillets (g)
Homogenize fillets
Divide homogenized sample into quarters, mix opposite
quarters, and then mix halves (3 times)
Optional
Composite equal weights (g) of »^k-§ Save remainder of fillet
homogenized fillet tissues from the «*Sjj^:* homogenate from each fish
selected number of fish (200-g)
Seal and label (200-g) composite
homogenate in appropriate container(s)
and store at £-20 °C until analysis (see
Table 7-1 for recommended container
materials and holding times).
Seal and label individual fillet
homogenates in appropriate
container(s) and archive at
S-20 "C (see Table 7-1 for
recommended container
materials and holding times).
COC - Chain of custody.
Figure 7-1. Preparation of fish fillet composite homogenate samples.
7-8
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Whole fish should be shipped or brought to the sample processing laboratory
from the field on wet or blue ice within 24 hours of sample collection. Fillets
should be resected within 48 hours of sample collection. Ideally, fish should not
be frozen prior to resection because freezing may cause internal organs to
rupture and contaminate edible tissue (Stober, 1991; U.S. EPA, 1986b).
However, if resection cannot be performed within 48 hours, the whole fish should
be frozen at the sampling site and shipped to the sample processing laboratory
on dry ice. Fish samples that arrive frozen (i.e., on dry ice) at the sample
processing laboratory should be placed in a <-20 °C freezer for storage until
filleting can be performed. The fish should then be partially thawed prior to
resection. Note: If the fillet tissue is contaminated by materials released from
the rupture of the internal organs during freezing, the State may eliminate the
fillet tissue as a sample or, alternatively, the fillet tissues should be rinsed in
contaminant-free, distilled deionized water and blotted dry. Regardless of the
procedure selected, a notation should be made in the sample processing record.
Sample processing procedures are discussed in the following sections. Data
from each procedure should be recorded directly in a bound laboratory notebook
or on forms that can be secured in the laboratory notebook. An example sample
processing record for fish fillet composites is shown in Figure 7-2.
7.2.2.1 Sample Inspection-
Individual fish received for filleting should be unwrapped and inspected carefully
to ensure that they have not been compromised in any way (i.e., not properly
preserved during shipment). Any specimen deemed unsuitable for further
processing and analysis should be discarded and identified on the sample
processing record.
7.2.2.2 Sample Weighing—
A wet weight should be determined for each fish. All samples should be
weighed on balances that are properly calibrated and of adequate accuracy and
precision to meet program data quality objectives. Balance calibration should be
checked at the beginning and end of each weighing session and after every 20
.weighings in a weighing session.
Fish shipped on wet or blue ice should be weighed directly on a foil-lined
balance tray. To prevent cross contamination between individual fish, the foil
lining should be replaced after each weighing. Frozen fish (i.e., those shipped
on dry ice) should be weighed in clean, tared, noncontaminating containers if
they will thaw before the weighing can be completed. Note: Liquid from the
thawed whole fish sample will come not only from the fillet tissue but from the
gut and body cavity, which are not part of the final fillet sample. Consequently,
inclusion of this liquid with the sample may result in an overestimate of target
analyte and lipid concentrations in the fillet homogenate. Nevertheless, it is
recommended, as a conservative approach, that all liquid from the thawed whole
fish sample be kept in the container as part of the sample.
7-9
-------
7. LABORATORY PROCEDURES I — SAMPLE HANDLDNG
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7-10
-------
7. LABORATORY PROCEDURES I — SAMPLE HANDLING
All weights should be recorded to the nearest gram on the sample processing
record and/or in the laboratory notebook.
7.2.2.3 Age Determination (Optional)—
Age provides a good indication of the duration of exposure to pollutants (Versar,
1982). A few scales or otoliths (Jearld, 1983) should be removed from each fish
and delivered to a fisheries biologist for age determination. For most warm
water inland gamefish, 5 to 10 scales should be removed from below the lateral
line and behind the pectoral fin. On soft-rayed fish such as trout and salmon,
the scales should be taken just above the lateral line (WDNR, 1988). For catfish
and other scaleless fish, the pectoral fin spines should be clipped and saved
(Versar, 1982). The scales, spines, or otoliths may be stored by sealing them
in small envelopes (such as coin envelopes) or plastic bags labeled with, and
cross-referenced by, the identification number assigned to the tissue specimen
(Versar, 1982). Removal of scales, spines, or otoliths from each fish should be
noted (by a check mark) on the sample processing record.
7.2.2.4 Sex Determination (Optional)—
Fish sex should be determined before filleting. To determine the sex of a fish,
an incision should be made on the ventral surface of the body from a point
immediately anterior to the anus toward the head to a point immediately posterior
to the pelvic fins. If necessary, a second incision should be made on the left
side of the fish from the initial point of the first incision toward the dorsal fin. The
resulting flap should be folded back to observe the gonads. Ovaries appear
whitish to greenish to golden brown and have a granular texture. Testes appear
creamy white and have a smooth texture (Texas Water Commission, 1990). The
sex of each fish should be recorded on the sample processing form.
7.2.2.5 Assessment of Morphological Abnormalities (Optional)—
Assessment of gross morphological abnormalities in finfish is optional. This
assessment may be conducted in the field (see Section 6.3.1.5) or during initial
inspection at the processing laboratory prior to filleting. States interested in
documenting morphological abnormalities should consult Sinderman (1983) and
review recommended protocols for fish pathology studies used in the Puget
Sound Estuary Program (1990c) and those described by Goede and Barton
(1990).
7.2.2.6 Scaling or Skinning—
To control contamination, separate sets of utensils and cutting boards should be
used for skinning or scaling fish and for filleting fish. Fish with scales should be
scaled and any adhering slime removed prior to filleting. Fish without scales
(e.g., catfish) should be skinned prior to filleting. These fillet types are
recommended because it is believed that they are most representative of the
7-11
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
edible portions of fish prepared and consumed by sport anglers. However, it is
the responsibility of each program manager, in consultation with State fisheries
experts, to select the fillet or sample type most appropriate for each target
species based on the dietary customs of local populations of concern.
A fish is scaled by laying it flat on a clean glass or PTFE cutting board or on one
that has been covered with heavy duty aluminum foil and removing the scales
and adhering slime by scraping from the tail to the head using the blade edge
of a clean stainless steel, ceramic, or titanium knife. Cross-contamination is
controlled by rinsing the cutting board and knife with contaminant-free distilled
water between fish. If an aluminum foil covered cutting board is used, the foil
should be changed between fish. The skin should be removed from fish without
scales by loosening the skin just behind the gills and pulling it off between knife
blade and thumb or with pliers as shown in Figure 7-3.
Once the scales and slime have been scraped off or the skin removed, the
outside of the fish should be washed with contaminant-free distilled water and
it should be placed on a second clean cutting board for filleting.
7.2.2.7 Filleting—
Filleting should be conducted only by or under the supervision of an experienced
fisheries biologist. If gloves are worn, they should be talc- or dust-free, and of
non- contaminating materials. Prior to filleting, hands should be washed with
Ivory soap and rinsed thoroughly in tap water, followed by distilled water (U.S.
EPA, 1991d). Specimens should come into contact with noncontaminating
surfaces only. Fish should be filleted on glass or PTFE cutting boards that are
cleaned properly between fish or on cutting boards covered with heavy duty
aluminum foil that is changed between fish (Puget Sound Estuary Program,
1990d, 1990e). Care must be taken to avoid contaminating fillet tissues with
material released from inadvertent puncture of internal organs. Note: If the fillet
tissue is contaminated by materials released from the inadvertent puncture of the
internal organs during resection, the State may eliminate the fillet tissue as a
sample or, alternatively, the fillet tissue should be rinsed in contaminant-free,
d§ionized distilled water and blotted dry. Regardless of the procedure selected,
a notation should be made in the sample processing record.
Ideally, fish should be filleted while ice crystals are still present in the muscle
tissue. Therefore, if fish have been frozen, they should not be allowed to thaw
completely prior to filleting. Fish should be thawed only to the point where it
becomes possible to make an incision into the flesh (U.S. EPA, 1991d).
Clean, high-quality stainless steel, ceramic, or titanium utensils should be used
to remove one or both fillets from each fish, as necessary. The general
procedure recommended for filleting fish is illustrated in Figure 7-3 (U.S. EPA,
1991d).
7-12
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Scaled Fish
After removing the scales (by
scraping with the edge of a
knife) and rinsing the fish:
Scaleless Fish
Grasp the skin at the base of the head
(preferably with pliers) and pull toward
the tail.
Note: This step
applies only for
catfish and
other scaleless
species.
Make a shallow cut through the
sMn (on either side of the dorsal
fin) from the top of the head to
the base of the tail.
Make a cut behind the entire
length of the gill cover, cutting
through the skin and flesh to the
bone.
Make a shallow cut along the belly
from the base of the pectoral fin to
the tail. A single cut is made from
behind the gill cover to the anus
and then a cut is made on both
sides of the anal fin. Do not cut into
the gut cavity as this may
contaminate fillet tissues.
Remove the fillet.
Source: U.S. EPA, 1991d.
Figure 7-3. Illustration of basic fish filleting procedure.
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
The belly flap should be included in each fillet. Any dark muscle tissue in the
vicinity of the lateral line should not be separated from the light muscle tissue
that constitutes the rest of the muscle tissue mass. Bones still present in the
tissue after filleting should be removed carefully (U.S. EPA, 1991d).
If both fillets are removed from a fish, they can be combined or kept separate for
duplicate QC analysis, analysis of different analytes, or archival of one fillet.
Fillets should be weighed (either individually or combined, depending on the
analytical requirements) and the weight(s) recorded to the nearest gram on the
sample processing record.
If fillets are to be homogenized immediately, they should be placed in a properly
cleaned glass or PTFE homogenization container. If samples are to be analyzed
for metals only, plastic homogenization containers may be used. To facilitate
homogenization it may be necessary or desirable to chop each fillet into smaller
pieces using a titanium or stainless steel knife prior to placement in the
homogenization container.
If fillets are to be homogenized later, they should be wrapped in heavy duty
aluminum foil and labeled with the sample identification number, the sample type
(e.g., "F" for fillet), the weight (g), and the date of resection. If composite
homogenates are to be prepared from only a single fillet from each fish, fillets
should be wrapped separately and the designation "F1" and "F2" should be
added to the sample identification number for each fillet. The individual fillets
from each fish should be kept together. All fillets from a composite sample
should be placed in a plastic bag labeled with the composite identification
number, the individual sample identification numbers, and the date of resection
and stored at <-20 °C until homogenization.
7.2.2.8 Preparation of Individual Homogenates—
To ensure even distribution of contaminants throughout tissue samples and to
facilitate extraction and digestion of samples, the fillets from individual fish must
be ground and homogenized prior to analysis. The fillets from an individual fish
may be ground and homogenized separately, or combined, depending on the
analytical requirements and the sample size.
Fish fillets should be ground and homogenized using an automatic grinder or
high-speed blender or homogenizer. Large fillets may be cut into 2.5-cm cubes
with high-quality stainless steel or titanium knives or with a food service band
saw prior to homogenization. Parts of the blender or homogenizer used to grind
the tissue (i.e., blades, probes) should be made of tantalum or titanium rather
than stainless steel. Stainless steel blades and/or probes have been found to
be a potential source of nickel and chromium contamination (due to abrasion at
high speeds) and should be avoided.
7-14
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Grinding and homogenization of tissue is easier when it is partially frozen
(Stober, 1991). Chilling the grinder/blender briefly with a few chips of dry ice will
also help keep the tissue from sticking to it (Smith, 1985).
The fillet sample should be ground until it appears to be homogeneous. The
ground sample should then be divided into quarters, opposite quarters mixed
together by hand, and the two halves mixed together. The grinding, quartering,
and hand-mixing steps should be repeated at least two more times. If chunks
of tissue are present at this point, the grinding and homogenization should be
repeated. Note: Skin-on fillets are the fish fillet sample type recommended for
use in State fish contaminant monitoring programs. However, skin-on fillets of
some finfish species are especially difficult to homogenize completely. No
chunks of tissue or skin should remain in the sample homogenate because these
may not be extracted or digested efficiently and could bias the analytical results.
If complete homogenization of skin-on fillets for a particular target species is a
chronic problem or if local consumers are likely to prepare skinless fillets of the
species, the State should consider analyzing skinless fillet samples. If the
sample is to be analyzed for metals only, the ground tissue may be mixed by
hand in a polyethylene bag (Stober, 1991). The preparation of each individual
homogenate should be noted (marked with a check) on the sample processing
- record. At this time, individual homogenates may be either processed further to
prepare composite homogenates or frozen separately and stored at <-20 °C (see
Table 7-1).
7.2.2.9 Preparation of Composite Homogenates—
Composite homogenates should be prepared from equal weights of individual
homogenates. The same type of individual homogenate (i.e., either single fillet
or combined fillet) should always be used in a given composite sample.
If individual homogenates have been frozen, they should be thawed partially and
rehomogenized prior to weighing and compositing. Any associated liquid should
be kept as a part of the sample. The weight of each individual homogenate
used in the composite homogenate should be recorded, to the nearest gram, on
the sample processing record.
Each composite homogenate should be blended as described for individual
homogenates in Section 7.2.2.8. The composite homogenate may be processed
immediately for analysis or frozen and stored at <-20 °C (see Table 7-1).
The remainder of each individual homogenate should be archived at <-20 °C with
the designation "Archive" and the expiration date recorded on the sample label.
The location of the archived samples should be indicated on the sample
processing record under "Notes."
It is essential that the weights of individual homogenates yield a composite
homogenate of adequate size to perform all necessary analyses. Weights of
individual homogenates required for a composite homogenate, based on the
7-15
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
number of fish per composite and the weight of composite homogenate
recommended for analyses of all screening study target analytes (see Table 4-1),
are given in Table 7-2. The total composite weight required for intensive studies
may be less than that for screening studies if the number of target analytes is
reduced significantly.
The recommended sample size of 200 g for screening studies is intended to
provide sufficient sample material to (1) analyze for all recommended target
analytes (see Table 4-1) at appropriate detection limits; (2) meet minimum QC
requirements for the analyses of laboratory duplicate, matrix spike, and matrix
spike duplicate samples (see Sections 8.3.3.4 and 8.3.3.5); and (3) allow for
reanalysis if the QC. control limits are not met or if the sample is lost. However,
sample size requirements may vary among laboratories and the analytical
methods used. Each program manager must consult with the analytical
laboratory supervisor to determine the actual weights of composite homogenates
required to analyze for all selected target analytes at appropriate detection limits.
Table 7-2. Weights (g) of Individual Homogenates
Required for Screening Study Composite Homogenate Samplea>b
Number of
fish per
sample
3
4
5
6
7
8
9
10
Total composite weight
100 g
(minimum)
33
25
20
17
14
13
11
10
200 g
(recommended)
67
50
40
33
29
25
22
20
500 g
(maximum)
167
125
100
84
72
63
56
50
aBased on total number of fish per composite and the total composite weight required for
analysis in screening studies. The total composite weight required in intensive studies may
be less if the number of target analytes is reduced significantly.
Individual homogenates may be prepared from one or both fillets from a fish. A composite
homogenate should be prepared only from individual homogenates of the same type (i.e.,
either from individual homogenates each prepared from a single fillet or from individual
homogenates each prepared from both fillets).
7-16
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
7.2.3 Processing Turtle Samples
Processing in the laboratory to prepare individual turtle homogenate samples for
analysis (diagrammed in Figure 7-4) involves
• Inspecting individual turtles
Weighing individual turtles
Removing edible tissues
Determining the sex of each turtle (optional)
• Determining the age of each turtle (optional)
Weighing edible tissue or tissues
Homogenizing tissues
Preparing individual homogenate samples
Preparing aliquots of the individual homogenates for analysis
Distributing frozen aliquots to one or more analytical laboratories.
Whole turtles should be shipped or brought to the sample processing laboratory
from the field on wet or blue ice within 24 hours of sample collection. The
recommended euthanizing method for turtles is freezing (Frye, 1994) and a
minimum of 48 hours or more may be required for large specimens. Turtles that
arrive on wet or blue ice or frozen (i.e., on dry ice) at the sample processing
laboratory should be placed in a <-20 °C freezer for storage until resection can
be performed. If rupture of internal organs is noted for an individual turtle, the
specimen may be eliminated as a sample or, alternatively, the edible tissues
should be rinsed in distilled deionized water and blotted dry.
Sample processing procedures are discussed in the following sections. Data
from each procedure should be recorded directly in a bound laboratory notebook
or on forms that can be secured in the laboratory notebook. An example sample
processing record for individual turtle samples is shown in Figure 7-5.
7.2.3.1 Sample Inspection-
Turtles received for resection should be removed from the canvas or burlap
collection bags and inspected carefully to ensure that they have not been
compromised in any way (i.e., not properly preserved during shipment). Any
specimen deemed unsuitable for further processing and analysis should be
discarded and identified on the sample processing record.
7-17
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Log in turtle samples using COC procedures
Remove turtle from bag and inspect turtle
Weigh individual turtle
Sever bony bridges on ventral side; remove plastron
Resect forelimbs, hindlimbs, neck, and tail muscle tissue from the body.
Skin all muscle tissue, remove daws and bones. Also resect muscle
tissue inside carapace. NOTE: Depending on dietary practices of
population of concern, add heart, liver, fatty tissues, and eggs to
muscle sample or, alternatively, retain these other tissues for separate
analysis.
Determine the sex of each turtle (optional)
Retain bones for age determination (optional)
Weigh edible tissue (g)
(muscle with or without other internal tissues added)
Homogenize edible tissue sample
Divide homogenized sample into quarters, mix opposite
quarters, and then mix halves (3 times)
Seal and label remaining
individual homogenate in
appropriate container(s)
and store at £-20 °C until
analysis (see Table 7-1 for
recommended container
materials and holding
times).
Seal and label (200-g)
individual homogenate in
appropriate containers)
and store at £-20 °C until
analysis (see, Table 7-1 for
recommended container
materials and holding
times).
Weigh heart, liver, fatty deposits, and eggs
separately (g)
Homogenize individual tissue types separately
Divide homogenized sample of each tissue type
into quarters, mix opposite quarters, and then
mix halves (3 times)
Seal and label individual tissue homogenates in
appropriate container(s) and archive at £-20 °C
until analysis (see Table 7-1 for recommended
container materials and holding times).
COC « Chain of custody.
Figure 7-4. Preparation of individual turtle homogenate samples.
7-18
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
I
"
5* I
7-19
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
7.2.3.2 Sample Weighing—
A-wet weight should be determined for each turtle. All samples should be
weighed on balances that are properly calibrated and of adequate accuracy and
precision to meet program data quality objectives. Balance calibration should be
checked at the beginning and end of each weighing session and after every 20
weighings in a weighing session.
Turtles euthanized by freezing should be weighed in clean, tared,
noncontaminating containers if they will thaw before the weighing can be
completed. Note: Liquid from the thawed whole turtle sample will come not only
from the muscle tissue but from the gut and body cavity, which may not be part
of the desired edible tissue sample. Consequently, inclusion of .this liquid with
the sample may result in an overestimate of target analyte and lipid
concentrations in the edible tissue homogenate. Nevertheless, it is
recommended, as a conservative approach, that all liquid from the thawed whole
turtle be kept in the container as part of the sample.
All weights should be recorded to the nearest gram on the sample processing
record and/or in the laboratory notebook.
7.2.3.3 Removal of Edible Tissues—
Edible portions of a turtle should consist only of those tissues that the population
of concern might reasonably be expected to eat. Edible tissues should be
clearly defined in site-specific sample processing protocols. A brief description
of the edible portions used should also be provided on the sample processing
record. General procedures for removing edible tissues from a turtle are
illustrated in Appendix I.
Resection should be conducted only by or under the supervision of an
experienced fisheries biologist. If gloves are worn, they should be talc- or dust-
free, and of noncontaminating materials. Prior to resection, hands should be
washed with soap and rinsed thoroughly in tap water, followed by distilled water
(U.S. EPA, 1991d). Specimens should come into contact with noncontaminating
surfaces only. Turtles should be resected on glass or PTFE cutting boards that
are cleaned properly between each turtle or on cutting boards covered with
heavy duty aluminum foil that is changed between each turtle (Puget Sound
Estuary Program, 1990d, 1990e). A turtle is resected by laying it flat on its back
and removing the plastron by severing the two bony ridges between the fore and
hindlimbs. Care must be taken to avoid contaminating edible tissues with
material released from the inadvertent puncture of internal organs.
Ideally, turtles should be resected while ice crystals are still present in the
muscle tissue. Thawing of frozen turtles should be kept to a minimum during
tissue removal to avoid loss of liquids. A turtle should be thawed only to the
point where it becomes possible to make an incision into the flesh (U.S. EPA,
1991d).
7-20
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Clean, high-quality stainless steel, ceramic, or titanium utensils should be used
to remove the muscle tissue and, depending on dietary or culinary practices of
the population of concern, some of the other edible tissues from each turtle. The
general procedure recommended for resecting turtles is illustrated in Figure 7-6.
Skin on the forelimbs, hindlimbs, neck, and tail should be removed. Claws
should be removed from the fore and hindiimbs. Bones still present in the
muscle tissue after resection should be removed carefully (U.S. EPA, 1991 d) and
may be used in age determination (see Section 7.2.3.5).
To control contamination, separate sets of utensils and cutting boards should be
used for skinning muscle tissue and resecting other internal tissues from the
turtle (e.g., heart, liver, fatty deposits, and eggs). These other tissue types are
recommended for inclusion with the muscle tissue as part of the edible tissue
sample because it is believed that they are most representative of the edible
portions of turtles that are prepared and consumed by sport anglers and
subsistence fishers. Alternatively, States may choose to analyze some of these
other lipophilic tissues separately. It is the responsibility of each program
manager, in consultation with State fisheries experts, to select the tissue sample
type most appropriate for each target species based on the dietary customs of
local populations of concern.
The edible turtle tissues should be weighed and the weight recorded to the
nearest gram on the sample processing record. If the State elects to analyze the
heart, liver, fatty deposits, or eggs separately from the muscle tissue, these other
tissues should be weighed separately and the weights recorded to the nearest
gram in the sample processing record.
If the tissues are to be homogenized immediately, they should be placed in a
properly cleaned glass or PTFE homogenization container. If samples are to be
analyzed for metals only, plastic homogenization containers may be used. To
facilitate homogenization it may be necessary or desirable to chop each of the
large pieces of muscle tissue into smaller pieces using a titanium or stainless
steel knife prior to placement in the homogenization container.
If the tissues are to be homogenized later, they should be wrapped in heavy duty
aluminum foil and labeled with the sample identification number, the sample type
(e.g., "M" for muscle, "E" for eggs, or "FD" for fatty deposits), the weight (g), and
the date of resection. The individual muscle tissue samples from each turtle
should be packaged together and given an individual sample identification
number. The date of resection should be recorded and the sample should be
stored at <-20 °C until homogenization. Note: State staff may determine that
the most appropriate sample type is muscle tissue only, with internal organ
tissues analyzed separately (liver, heart, fatty deposits, or eggs). Alternatively,
State staff may determine that the most appropriate sample type is muscle tissue
with several other internal organs included as the turtle tissue sample. This
latter sample type typically will provide a more conservative estimate of
7-21
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Source: Hamerstrom, 1989.
Figure 7-6. Illustration of basic turtle resection procedure.
7-22
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
contaminant residues, particularly with respect to lipophilic target analytes (e.g.,
PCBs, dioxins, and organochlorine pesticides).
7.2.3.4 Sex Determination (Optional)—
Turtle sex should be determined during resection if it has not already been
determined in the field. Once the plastron is removed, the ovaries or testes can
be observed posterior and dorsal to the liver. Each ovary is a large egg-filled
sac containing yellow spherical eggs in various stages of development (Ashley,
1962) (see Appendix I). Each testes is a spherical organ, yellowish in color,
attached to the ventral side of each kidney. The sex of each turtle should be
verified and recorded on the sample processing form.
7.2.3.5 Age Determination (Optional)—
Age provides a good indication of the duration of exposure to pollutants (Versar,
1982). Several methods have been developed for estimating the age of turtles
(Castanet, 1994; Frazer et al., 1993; Gibbons, 1976). Two methods are
appropriate for use in contaminant monitoring programs where small numbers
of animals of a particular species are to be collected and where the animals
must be sacrificed for tissue residue analysis. These methods include (1) the
use of external annuli (scute growth marks) on the plastron and (2) the use of
growth rings on the bones.
The surface of epidermal keratinous scutes on the plastron of turtle shells
develops successive persistent grooves or growth lines during periods of slow
or arrested growth (Zangerl, 1969). Because these growth rings are fairly
obvious, they have been used extensively for estimating age in various turtle
species (Cagle, 1946,1948,1950; Gibbons, 1968; Legler, 1960; Sexton, 1959).
This technique is particularly useful for younger turtles where the major growth
rings are more definitive and clear cut than in older individuals (Gibbons, 1976).
However, a useful extension of the external annuli method is presented by
Sexton (1959) showing that age estimates can be made for adults on which all
annuli are not visible. This method may be performed by visually examining the
plastron of the turtle during the resection, or the plastron may be tagged with the
sample identification number of the turtle and retained for later analysis.
The use of bone rings is the second method that may be used to estimate age
in turtles (Enlow and Brown, 1969; Peabody, 1961). Unlike the previous visual
method, this method requires that the bones of the turtle be removed during
resection and retained for later analysis. The growth rings appear at the surface
or inside primary compacta of bone tissues. There are two primary methods for
observing growth marks: either directly at the surface of the bone as in flat bones
using transmitted or reflected light or inside the long bones using thin sections
(Castanet, 1994; Dobie, 1971; Galbraith and Brooks, 1987; Hammer, 1969;
Gibbons, 1976; Mattox, 1935; Peabody, 1961). The methods of preparation of
whole bones and histological sections of fresh material for growth mark
determinations are now routinely performed. Details of these methods can be
7-23
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
found in Castanet (1974 and 1987), Castanet et al. (1993), and Zug et al. (1986).
State staff interested in using either of these methods for age determination of
turtles should read the review articles by Castanet (1994) and Gibbons (1976)
for discussions of the advantages and disadvantages of each method, and the
associated literature cited in these articles on turtle species of particular interest
within their jurisdictions.
7.2.3.6 Preparation of Individual Homogenates—
To ensure even distribution of contaminants throughout tissue samples and to
facilitate extraction and digestion of samples, the edible tissues from individual
turtles must be ground and homogenized prior to analysis. The various tissues
from an individual turtle may be ground and homogenized separately, or
combined, depending on the sampling program's definition of edible tissues.
Turtle tissues should be ground and homogenized using an automatic grinder or
high-speed blender or homogenizer. Large pieces of muscle or organ tissue
(e.g., liver or fatty deposits) may be cut into 2.5-cm cubes with high-quality
stainless steel or titanium knives or with a food service band saw prior to
homogenization. Parts of the blender or homogenizer used to grind the tissue
(i.e., blades, probes) should be made of tantalum or titanium rather than
stainless steel. Stainless steel blades and/or probes,have been found to be a
potential source of nickel and chromium contamination (due to abrasion at high
speeds) and should be avoided.
Grinding and homogenization of tissue is easier when it is partially frozen
(Stober, 1991). Chilling the grinder/blender briefly with a few chips of dry ice will
also help keep the tissue from sticking to it (Smith, 1985).
The tissue sample should be ground until it appears to be homogeneous. The
ground sample should then be divided into quarters, opposite quarters mixed
together by hand, and the two halves mixed together. The grinding, quartering,
and hand-mixing steps should be repeated at least two more times. If chunks
of tissue are present at this point, the grinding and homogenization should be
repeated. No chunks of tissue should remain because these may not be
extracted or digested efficiently and could bias the analytical results. This is
particularly true when lipophilic tissues (e.g., fatty deposits, liver, or eggs) are not
completely homogenized throughout the sample. Portions of the tissue sample
that retain unhomogenized portions of tissues may exhibit higher or lower
residues of target analytes than properly homogenized samples.
If the sample is to be analyzed for metals only, the ground tissue may be mixed
by hand in a polyethylene bag (Stober, 1991). The preparation of each
individual homogenate should be noted (marked with a check) on the sample
processing record. At this time, individual homogenates may be frozen
separately and stored at <-20 °C (see Table 7-1).
7-24
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
The remainder of each individual homogenate should be archived at <-20 °C with
the designation "Archive" and the expiration date recorded on the sample label.
The location of the archived samples should be indicated on the sample
processing record under "Notes."
It is essential that the weight of individual homogenate samples is of adequate
size to perform all necessary analyses. The recommended sample size of 200
g for screening studies is intended to provide sufficient sample material to (1)
analyze for all recommended target analytes (see Table 4-1) at appropriate
detection limits; (2) meet minimum QC requirements for the analyses of
laboratory duplicate, matrix spike, and matrix spike duplicate samples (see
Sections 8.3.3.4 and 8.3.3.5); and (3) allow for reanaiysis if the QC control limits
are not met or if the sample is lost. However, sample size requirements may
vary among laboratories and the analytical methods used. Each program
manager must consult with the analytical laboratory supervisor to determine the
actual weights of homogenates required to analyze for all selected target
analytes at appropriate detection limits. The total sample weight required for
intensive studies may be less than that for screening studies if the number of
target analytes is reduced significantly.
7.2.4 Processing Shellfish Samples
Laboratory processing of shellfish to prepare edible tissue composite
homogenates for analysis (diagrammed in Figure 7-7) involves
• Inspecting individual shellfish
• Determining the sex of each shellfish (optional)
Examining each shellfish for morphological abnormalities (optional)
• Removing the edible parts from each shellfish in the composite sample (3
to 50 individuals, depending upon the species)
Combining the edible parts in an appropriate noncontaminating container
Weighing the composite sample
Homogenizing the composite sample
Preparing aliquots of the composite homogenate for analysis
Distributing frozen aliquots to one or more analytical laboratories.
Sample aliquotting and shipping are discussed in Section 7.3; all other
processing steps are discussed in this section. Shellfish samples should be
processed following the general guidelines in Section 7.2.1 to avoid
contamination. In particular, it is recommended that separate composite
7^25
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Log in shellfish samples using COG procedures
Unwrap and inspect individual shellfish
Determine sex (optional); note morphological
abnormalities (optional)
Remove edible tissue from each shellfish in composite
Combine edible tissue from individual shellfish in
composite in a tared container (g)
Weigh the filled container (g)
Homogenize the composite sample
Divide homogenized sample into quarters, mix opposite
quarters and then mix halves (3 times)
Seal and label (200-g) composite
homogenate in appropriate
containers) and store at £-20 °C
until analysis (see Table 7-1 for
recommended container materials
and holding times).
Seal and label remaining
composite homogenate in
appropriate container(s) and
archive at £-20 °C (see Table 7-1
for recommended container
materials and holding times).
COC m Chain of custody.
Figure 7-7. Preparation of shellfish edible tissue composite homogenate samples.
7-26
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
homogenates be prepared for the analysis of metals and organics if resources
allow. An example sample processing record for shellfish edible tissue
composite samples is shown in Figure 7-8.
Shellfish samples should be shipped or brought to the sample processing
laboratory either on wet or blue ice (if next-day delivery is assured) or on dry ice
(see Section 6.3.3). Shellfish samples arriving on wet ice or blue ice should
have edible tissue removed and should be frozen to <-20 °C within 48 hours
after collection. Shellfish samples that arrive frozen (i.e., on dry ice) at the
processing laboratory should be placed in a <-20 °C freezer for storage until
edible tissue is removed.
7.2.4.1 Sample Inspection—
Individual shellfish should be unwrapped and inspected carefully to ensure that
they have not been compromised in any way (i.e., not properly preserved during
shipment). Any specimen deemed unsuitable for further processing and analysis
should be discarded and identified on the sample processing record.
7.2.4.2 Sex Determination (Optional)—
The determination of sex in shellfish species is impractical if large numbers of
individuals of the target species are required for each composite sample.
For bivalves, determination of sex is a time-consuming procedure that must be
performed after shucking but prior to removal of the edible tissues. Once the
bivalve is shucked, a small amount of gonadal material can be removed using
a Pasteur pipette. The gonadal tissue must then be examined under a
microscope to identify egg or sperm cells.
For crustaceans, sex also should be determined before removal of the edible
tissues. For many species, sex determination can be accomplished by visual
inspection. Sexual dimorphism is particularly striking in many species of
decapods. In the blue crab, Callinectes sapidus, the female possesses a broad
abdomen suited for retaining the maturing egg mass or sponge, while the
abdomen of the male is greatly reduced in width. For shrimp, lobsters, and
crayfish, sexual variations in the structure of one or more pair of pleopods are
common.
States interested in determining the sex of shellfish should consult taxonomic
keys for specific information on each target species.
7.2.4.3 Assessment of Morphological Abnormalities (Optional)—
Assessment of gross morphological abnormalities in shellfish is optional. This
assessment may be conducted in the field (see Section 6.3.1.5) or during initial
inspection at the processing laboratory prior to removal of the edible tissues.
States interested in documenting morphological abnormalities should consult
7-27
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Sample Processing Record for Shellfish Con
Project Number
STUDY PHASE: Screening Study
SITE LOCATION
Site Name/Number
Countv/Parish:
Waterbody Name/Segment Number
_J;
Samolina Date and Time:
Intensive Study: Phase 1 1 I Phase II I I
Lat./Lona.:
Waterbodv Tvpe:
SHELLFISH COLLECTED
Species Name:
Descriotion of Edible Tissue
Composite Sample #:
Shellfish Included in
* Composite (•/) Shellfish f
001
002
003
004
005
006
007
008
009
010
011
012
013
014
015
016
017
Preparation of Composite:
Weight of container + shellfish
Weight of container (tare weight)
Total weight of composite
Notes:
018
019
020
021
022
023
024
025
026
027
028
029
030
031
032
033
034
Number of Individuals:
Included in Included in
Composite (/) Shellfish f Composite (/)
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
g
g
a + =
# of specimens Average weight
of specimen
Analyst
Date
Figure 7-8. Example of a sample processing record for shellfish contaminant
monitoring program—edible tissue composites.
7-28
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Sinderman and Rosenfield (1967), Rosen (1970), and Murchelano (1982) for
detailed information on various pathological conditions in shellfish and review
recommended protocols for pathology studies used in the Puget Sound Estuary
Program (1990c).
7.2.4.4 Removal of Edible Tissue-
Edible portions of shellfish should consist only of those tissues that the
population of concern might reasonably be expected to eat. Edible tissues
should be clearly defined in site-specific sample processing protocols. A brief
description of the edible portions used should also be provided on the sample
processing record. General procedures for removing edible tissues from a
variety of shellfish are illustrated in Appendix I.
Thawing of frozen shellfish samples should be kept to a minimum during tissue
.removal to avoid loss of liquids. Shellfish should be rinsed well with organics-
and metal-free water prior to tissue removal to remove any loose external debris.
Bivalve molluscs (oysters, clams, mussels, and scallops) typically are prepared
by severing the adductor muscle, prying open the shell, and removing the soft
tissue. The soft tissue includes viscera, meat, and body fluids (Smith, 1985).
Byssal threads from mussels should be removed with a knife before shucking
and should not be included in the composite sample.
Edible tissue for crabs typically includes all leg and claw meat, back shell meat,
and body cavity meat, internal organs generally are removed. Inclusion of the
hepatopancreas should be determined by the eating habits of the local
population or subpopulations of concern. If the crab is soft-shelled, the entire
crab should be used in the sample. Hard- and soft-shelled crabs must not be
combined in the same composite (Smith, 1985).
Typically, shrimp and crayfish are prepared by removing the cephalothorax and
then removing the tail meat from the shell. Only the tail meat with the section
of intestine passing through the tail muscle is retained for analysis (Smith, 1985).
Edible tissue for lobsters typically includes the tail and claw meat. If the
tomalley (hepatopancreas) and gonads or ovaries are consumed by local
populations of concern, these parts should also be removed and analyzed
separately (Duston et al., 1990).
7.2.4.5 Sample Weighing-
Edible tissue from all shellfish in a composite sample (3 to 50 individuals) should
be placed in an appropriate preweighed and labeled noncontaminating container.
The weight of the empty container (tare weight) should be recorded to the
nearest gram on the sample processing record. All fluids accumulated during
removal of edible tissue should be retained as part of the sample. As the edible
portion of each shellfish is placed in the container, it should be noted on the
7-29
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
sample processing record. When the edible tissue has been removed from ail
shellfish in the composite, the container should be reweighed and the weight
recorded to the nearest gram on the sample processing record. The total
composite weight should be approximately 200 g for screening studies. If the
number of target analytes is significantly reduced in intensive studies, a smaller
composite homogenate sample may suffice (see Section 7.2.2.9). At this point,
the composite sample may be processed for analysis or frozen and stored at
£-20 °C (see Table 7-1).
7.2.4.6 Preparation of Composite Homogenates--
Composite samples-of the edible portions of shellfish should be homogenized in
a grinder, blender, or homogenizer that has been cooled briefly with dry ice
(Smith, 1985). For metals analysis, tissue may be homogenized in 4-oz
polyethylene jars (California Department of Fish and Game, 1990) using a
Polytron equipped with a titanium generator. If the tissue is to be analyzed for
organics only, or if chromium and nickel contamination are not of concern, a
commercial food processor with stainless steel blades and glass container may
be used. The composite should be homogenized to a paste-like consistency.
Larger samples may be cut into 2.5-cm cubes with high-quality stainless steel
or titanium knives before grinding. If samples were frozen after dissection, they
can be cut without thawing with either a knife-and-mallet or a clean bandsaw.
The ground samples should be divided into quarters, opposite quarters mixed
together by hand, and the two halves mixed together. The quartering and mixing
should be repeated at least two more times until a homogeneous sample is
obtained. No chunks should remain in the sample because these may not be
extracted, or digested efficiently. At this point, the composite homogenates may
be processed for analysis or frozen and stored at £-20 °C (see Table 7-1).
7.3 SAMPLE DISTRIBUTION
The sample processing laboratory should prepare aliquots of the composite
homogenates for analysis, distribute the aliquots to the appropriate laboratory (or
laboratories), and archive the remainder of each composite homogenate.
7.3.1 Preparing Sample Aliquots
Note: Because lipid material tends to migrate during freezing, frozen composite
homogenates must be thawed and rehomogenized before aliquots are prepared
(U.S. EPA, 1991d). Samples may be thawed overnight in an insulated cooler or
refrigerator and then homogenized. Recommended aliquot weights and
appropriate containers for different types of analyses are shown in Table 7-3.
The actual sample size required will depend on the analytical method used and
the laboratory performing the analysis. Therefore, the exact sample size
required for each type of analysis should be determined in consultation with the
analytical laboratory supervisor.
7-30
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7. LABORATORY PROCEDURES I — SAMPLE HANOLfMG
The exact quantity of tissue required for each digestion or extraction and
analysis should be weighed and placed in an.appropriate container that has
been labeled with the aliquot identification number, sample weight (to the nearest
0.1 g), and the date aliquots were prepared (Stober, 1991). The analytical
laboratory can then recover the entire sample, including any liquid from thawing,
by rinsing the container directly into the digestion or extraction vessel with the
appropriate solvent. It is also the responsibility of the processing laboratory to
provide a sufficient number of aliquots for laboratory duplicates, matrix spikes,
and matrix spike duplicates so that the QC requirements of the program can be
met (see Sections 8.3.3.4 and 8.3.3.5), and to provide extra aliquots to allow for
reanalysis if the sample is lost or if QC control limits are not met.
It is essential that accurate records be maintained when aliquots are prepared
for analysis. Use of a carefully designed form is recommended to ensure that
all the necessary information is recorded. An example of a sample aliquot
record is shown in Figure 7-9. The composite sample identification number
should be assigned to the composite sample at the time of collection (see
Section 6.2.3.1) and carried through sample processing (plus T1," "F2," or "C"
if the composite homogenate is comprised of individual or combined fillets). The
aliquot identification number should indicate the analyte class (e.g., MT for
metals, OR for organics, DX for dioxins) and the sample type (e.g., R for routine
sample; RS for a routine sample that is split for analysis by a second laboratory;
MS1 and MS2 for sample pairs, one of which will be prepared as a matrix spike).
For example, the aliquot identification number may be of the form WWWWW-XX-
YY-ZZZ, where WWWWW is a 5-digit sample composite identification number;
XX indicates individual (F1 or F2), or combined (C) fillets; YY is the analyte
code; and 777 is the sample type.
Blind laboratory duplicates should be introduced by preparing two separate
aliquots of the same composite homogenate and labeling one aliquot with a
"dummy" composite sample identification. However, the analyst who prepares
the laboratory duplicates must be careful to assign a "dummy" identification
Table 7-3. Recommended Sample Aliquot Weights and Containers
for Various Analyses
Analysis
Metals
Organics
Dioxins/furans
Aliquot weight (g)
1-5
20-50
20-50
Shipping/storage container
Polystyrene, •borosilicate glass, or PTFE
jar with PTFE-lined lid
Glass or PTFE jar with PTFE-lined lid
Glass or PTFE jar with PTFE-lined lid
PTFE = Polytetrafluoroethylene (Teflon).
7-31
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
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7-32
-------
7. LABORATORY PROCEDURES I — SAMPLE HANDLING
number that has not been used for an actual sample and to indicate clearly on
the processing records that the samples are blind laboratory duplicates. The
analytical laboratory should not receive this information.
When the appropriate number of aliquots of a composite sample have been
prepared for all analyses to be performed on that sample, the remainder of the
composite sample should be labeled with "ARCHIVE" and the expiration date
and placed in a secure location at <-20 °C in the sample processing laboratory.
The location of the archived samples should be indicated on the sample aliquot
record. Unless analyses are to be performed immediately by the sample
processing laboratory, aliquots for sample analysis should be frozen at <-20 °C
before they are transferred or shipped to the appropriate analytical laboratory.
7.3.2 Sample Transfer
The frozen aliquots should be transferred on dry ice to the analytical laboratory
(or laboratories) accompanied by a sample transfer record such as the one
shown in Figure 7-10. Further details on Federal regulations for shipping
biological specimens in dry ice are given in Section 6.3.3.2. The sample transfer
record may include a section that serves as the analytical laboratory COC
record. The COC record must be signed each time the samples change hands
for preparation and analysis.
7-33
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7. LABORATORY PROCEDURES I — SAMPLE HANDLING
Fish and Shellfish Monitoring Program
Sample Transfer Record
Date
Time.
DD MM YY
Released by:
HH MM
(24-h clock)
(name)
At:
(location)
Shipment Method.
Shipment Destination
Date
Time
00 MM
Received by:
YY
HH MM
(24-h clock)
(name)
At:
Comments
(location)
Study Typa: D Screening—Analyze for: D Trace metals D Organics D LJpid
Intensive Phase 1 D Phase II D — Analyze for (specify)
Sample IDs:
Laboratory Chain of Custody
Relinquished by
Received by
Purpose
Location
Figure 7-10. Example of a fish and shellfish monitoring program
sample transfer record.
7-34
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
SECTIONS
LABORATORY PROCEDURES II — SAMPLE ANALYSES
Sample analyses may be conducted by one or more State or private contract
laboratories. Because of the toxicity of dioxins/furans and the difficulty and cost
of these analyses, relatively few laboratories currently have the capability of
performing them. Table 8-1 lists contract laboratories experienced in dioxin/furan
analyses. This list is provided for information purposes only and is not an
endorsement of specific laboratories.
8.1 RECOMMENDED ANALYTES
8.1.1 Target Analytes
All recommended target analytes listed in Table 4-1 should be included in
screening studies unless reliable historic tissue, sediment, or pollutant source
data indicate that an analyte is not present at a level of concern for human
health. Additional target analytes should be included in screening studies if
States have site-specific information (e.g., historic tissue or sediment data,
discharge monitoring reports from municipal and industrial sources) that these
contaminants may be present at levels of concern for human health.
8.1.2 Llpld
Intensive studies should include only those target analytes found to exceed
screening values in screening studies (see Section 5.2).
A lipid analysis should also be performed and reported (as percent lipid by wet
weight) for each composite tissue sample in both screening and intensive
studies. This measurement is necessary to ensure that gel permeation
chromatography columns are not overloaded when used to clean up tissue
extracts prior to analysis of organic target analytes. In addition, because
bioconcentration of nonpolar organic compounds is dependent upon lipid content
(i.e., the higher the lipid content of the individual organism, the higher the residue
in the organism), lipid analysis is often considered essential by users of fish and
shellfish monitoring data. Consequently, it is important that lipid data are
obtained for eventual inclusion in a national database of fish and shellfish
contaminant data.
8-1
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Table 8-1. Contract Laboratories Conducting Dloxln/Furan
Analyses in Fish and Shellfish Tissues"
Alta Analytical Laboratory13
5070 Robert J. Matthews Parkway, Suite 2
Eldorado Hills, CA 95630
916/933-1640
FAX: 916/933-0940
Bill Luksemburg
Battalia-Columbus Laboratories0
505 King Avenue
Columbus, OH 43201
614/424-7379
Karen Riggs/Gerry Pitts
Enseco-Calrfomia Analytical Labs0
2544 Industrial Blvd.
West Sacramento, CA 95691
916/372-1393
916/372-1059
Kathy Gill/Michael Rligenzi/Mike Millie
IT Corporation
Technology Development Laboratory"
304 Directors Drive
Knoxville, TN 37923
615/690-3211
Duane Root/Nancy Conrad/Bruce Wagner
Midwest Research Institute0
425 Volker Boulevard
Kansas City, MO 64110
816/753-7600 ext. 190/ext. 160
Paul Kramer/John Stanley
New York State Department of Health0
Wadsworth Laboratories
Empire State Plaza
P.O. Box 509 •
Albany, NY 12201-0509
518/474-4151
Arthur Richards/Kenneth Aldous
Pacific Analytical Inc.0
1989-B Patomar Oaks Way
Carlsbad, CA 92009
619/931-1766
Phil Ryan/Bruce Colby
Seakem Analytical Services0
P.O. Box 2219
2045 Mills Road
Sidney, BC V8L 351
Canada
604/656-0881
Valerie Scott/Allison Peacock/Coreen Hamilton
TMS Analytical Services0
7726 Moller Road
Indianapolis, IN 46268
317/875-5894
FAX: 317/872-6189
Dan Denlinger/Don Eickhoff/
Kelly Mills/Janet Sachs
Triangle Laboratories'3
Alston Technical Park
801 Caprtola Drive, Suite 10
Research Triangle Park, NC 27713
919/544-5729
Laurie White
Twin City Testing Corporation0
662 Cromwell Avenue
St. Paul, MN 55114
612/649-5502
Chuck Sueper/Fred DeRoos
University of Nebraska
Mid-West Center for Mass Spectromotry
12th and T Street
Lincoln, NE 68588
402/472-3507
Michael Gross
Wellington Environmental Consultants0
395 Laird Road
Guelph, Ontario N1G 3X7
Canada
519/822-2436
Judy Sparling/Brock Chittin
Wright State University0
175 Brehm Laboratory
3640 Colonel Glen Road
Dayton, OH 45435
513/873-2202
Thomas Tiernan/Garrett Van Ness
aThis list should not be construed as an endorsement of these laboratories, but is provided for information
purposes only.
DLaboratory participating in Method 1613 interlaboratory (round-robin) dioxin study (May 1991).
8-2
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Note: Because the concentrations of contaminants, particularly nonpolar
organics, are often correlated with the percentage of lipid in a tissue sample,
contaminant data are often normalized to the lipid concentration before statistical
analyses are performed. This procedure can, in some instances, improve the
power of the statistical tests. States wishing to examine the relationship between
contaminant concentrations and percentage of lipid should refer to Hebert and
Keenleyside (1995) for a discussion of the possible statistical approaches.
8.2 ANALYTICAL METHODS
This section provides guidance on selecting methods for analysis of
recommended target analytes. Analytical methods should include appropriate
procedures for sample preparation (i.e., for digestion of samples to be analyzed
for metals and for extraction and extract cleanup of samples to be analyzed for
organics).
8.2.1 Lipid Method
It is recommended that a gravimetic method be used for lipid analysis. This
method is easy to perform and is commonly used by numerous laboratories,
employing various solvent systems such as chloroform/methanol (Bligh and
.Dyer, 1959), petroleum ether (California Department of Fish and Game, 1990;
U.S. FDA, 1990), and dichloromethane (NOAA, 1993a; Schmidt et al., 1985).
The results of lipid analyses may vary significantly (i.e., by factors of 2 or 3),
however, depending on the solvent system used for lipid extraction (Randall et
al., 1991; D. Swackhamer, University of Minesota, personal communication,
1993; D. Murphy, Maryland Department of the Environment, Water Quality
Toxics Division, personal communication, 1993). Therefore, to ensure consis-
tency of reported results among fish contaminant monitoring programs, it is
recommended that dichloromethane be used as the extraction solvent in all lipid
analyses.
In addition to the effect of solvent systems on lipid analysis, other factors can
also increase the inter- and intralaboratory variation of results if not adequately
controlled (Randall et al., 1991). For example, high temperatures have been
found to result in decomposition of lipid material and, therefore, should be
avoided during extraction. Underestimation of total lipids can also result from
denaturing of lipids by solvent contaminants, lipid decomposition from exposure
to oxygen or light, and lipid degradation from changes in pH during cleanup.
Overestimation of total lipids may occur if a solvent such as alcohol is used,
which results in substantial coextraction of nonlipid material. It is essential that
these potential sources of error be considered when conducting and evaluating
results of lipid analyses.
8-3
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Table 8-2. Current References for Analytical Methods for
Contaminants in Fish and Shellfish Tissues
Analytical Chemistry of PCBs (Erickson, 1991)
Analytical Methods for Pesticides and Plant Growth Regulators, Vol. 11 (Zweig and Sherma, 1980)
Analytical Procedures and Quality Assurance Plan for the Determination of Mercury in Fish (U.S.
EPA, 1989a)
Analytical Procedures and Quality Assurance Plan for the Determination of Xenobiotic Chemical
Contaminants in Fish (U.S. EPA, 1989c)
Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish (U.S.
EPA, 1989D)
Arsenic Speciation by Coupling High-performance Liquid Chromatography with Inductively Coupled
Plasma Mass Spectrometry (Demesmay et al., 1994)
Assessment and Control of Bioconcentratable Contaminants in Surface Water (U.S. EPA, 1991 a).
Bioaccumulation Monitoring Guidance: 4. Analytical Methods for U.S. EPA Priority Pollutants and
301 (h) Pesticides in Tissues from Marine and Estuarine Organisms (U.S. EPA, 1986b)
Determination of Arsenic Species by High-performance Liquid Chromatography - Inductively Coupled
Plasma Mass Spectrometry (Beauchemin et al., 1989)
Determination of Arsenic Species in Fish by Directly Coupled High-performance Liquid
Chromatography-lnductively Coupled Plasma Mass Spectrometry (Branch et al., 1994)
Determination of Butyltin and Cyclohexyltin Compounds in the Marine Environment by
High-performance Liquid Chromatography-Graphite Furnace Atomic Absorption Spectrometry with
Confirmation by Mass Spectrometry (Cullen et al., 1990)
Determination of Butyltin, Methyltin and Tetraalkyltin in Marine Food Products with Gas
Chromatography-Atomic Absorption Spectrometry (Forsyth and Cleroux, 1991)
Determination of Tributyltin Contamination in Tissues by Capillary Column Gas Chromatography-
Flame Photometric Detection with Confirmation by Gas Chromatography-Mass Spectroscopy (Wade
et al., 1988)
Determination of Tributyltin in Tissues and Sediments by Graphite Furnace Atomic Absorption
Spectrometry (Stephenson and Smith, 1988)
Environmental Monitoring and Assessment Program (EMAP) Near Coastal Virginian Province Quality
Assurance Project Plan (Draft) (U.S. EPA, 1991e)
Guidelines for Studies of Contaminants in Biological Tissues for the National Water-Quality
Assessment Program (Crawford and Luoma, 1993)
Interim Methods for the Sampling and Analysis of Priority Pollutants in Sediments and Fish Tissue
(U.S. EPA, 1981b)
Laboratory Quality Assurance Program Plan (California Department of Fish and Game, 1990)
Methods for Organic Analysis of Municipal and Industrial Wastewater (40 CFR 136, Appendix A).
Methods for the Chemical Analysis of Water and Wastes (U.S. EPA, 1979b)
Methods for the Determination of Metals in Environmental Samples (U.S. EPA, 1991g)
Official Methods of Analysis of the Association of Official Analytical Chemists (Williams, 1984)
Pesticide Analytical Manual (PAM Vols. I and II) (U.S. FDA, 1990)
Puget Sound Estuary Program Plan (1990d, 1990e)
Quality Assurance/Quality Control (QA/QC) for 301 (h) Monitoring Programs: Guidance on Field and
Laboratory Methods (U.S. EPA, 1987e) ,
(continued)
8-4
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Table 8-2 (continued)
Sampling and Analytical Methods of the National Status and Trends Program National Benthic
Surveillance and Mussel Watch Projects 1984-92. Volume II. Comprehensive Descriptions of
Complementary Measurements (NOAA, 1993a)
Sampling and Analytical Methods of the National Status and Trends Program National Benthic
Surveillance and Mussel Watch Projects 1984-92. Volume III. Comprehensive Descriptions of
Elemental Analytical Methods (NOAA, 1993b)
Sampling and Analytical Methods of the National Status and Trends Program National Benthic
Surveillance and Mussel Watch Projects 1984-92. Volume IV. Comprehensive Descriptions of Trace
Organic Analytical Methods (NOAA, 1993c)
Separation of Seven Arsenic Compounds by High-performance Liquid Chromatography with On-line
Detection by Hydrogen-Argon Flame Atomic Absorption Spectrometry and Inductively Coupled
Plasma Mass Spectrometry (Hansen et a)., 1992)
Speciation of Selenium and Arsenic in Natural Waters and Sediments by Hydride Generation
Followed by Atomic Absorption Spectroscopy (Crecelius et al., 1986)
Standard Analytical Procedures of the NOAA National Analytical Facility (Krahn et al., 1988; MacLeod
et al., 1985)
Standard Methods for the Examination of Water and Wastewater (Greenburg et al., 1992)
Test Methods for the Chemical Analysis of Municipal and Industrial Wastewater (U.S. EPA, 1982)
Test Methods for the Evaluation of Solid Waste, Physical/Chemical Methods (SW-846) (U.S. EPA,
1986f)
U.S. EPA Contract Laboratory Program Statement of Work for Inorganic Analysis (U.S. EPA, 1991b)
U.S. EPA Contract Laboratory Program Statement of Work for Organic Analysis (U.S. EPA, 1991c)
U.S. EPA Method 1613B: Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution
HRGC/HRMS (U.S. EPA, 1995c)
U.S. EPA Method 1625; Semivolatile Organic Compounds by Isotope Dilution GC/MS (40 CFR 136,
Appendix A)
U.S. EPA Method 1631: Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic
Fluorescence Spectrometry (U.S. EPA, 1995d)
U.S. EPA Method 1632: Determination of Inorganic Arsenic in Water by Hydride Generation Flame
Atomic Absorption (U.S. EPA, 1995e)
U.S. EPA Method 1637: Determination of Trace Elements in Ambient Waters by Chelation
Preconcentration with Graphite Furnace Atomic Absorption (U.S. EPA, 1995f)
U.S. EPA Method 1638: Determination of Trace Elements in Ambient Waters by Inductively Coupled
Plasma-Mass Spectrometry (U.S. EPA, 1995g)
U.S. EPA Method 1639: Determination of Trace Elements in Ambient Waters by Stabilized
Temperature Graphite Furnace Atomic Absorption (U.S. EPA, 1995h)
U.S. EPA Method 625: Base/Neutrals and Acids by GC/MS (40 CFR 136, Appendix A).
U.S. EPA Method 8290: Polychlorinated Dibenzodioxins (PCDDs) and Polychlorinated Dibenzofurans
(PCDFs) by High Resolution Gas Chromatography/High Resolution Mass Spectrometry
(HRGC/HRMS) (U.S. EPA, 1990b)
8-5
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
8.2.2 Target Analyte Methods
EPA has published interim procedures for sampling and analysis of priority
pollutants in fish tissue (U.S. EPA, 1981b); however, at present, official EPA-
approved methods are available only for the analysis of low parts-per-billion
concentrations of some metals in fish and shellfish tissues (U.S. EPA, 1991g).
Because of the lack of official EPA-approved methods for all recommended
target analytes, and to allow States and Regions flexibility in developing their
analytical programs, specific analytical methods for recommended target
analytes in fish and shellfish monitoring programs are not included in this
guidance document.
Note: A performance-based analytical program is recommended for the analysis
of target analytes. This recommendation is based on the assumption that the
analytical results produced by different laboratories and/or different methods will
be comparable if appropriate QC procedures are implemented within each
laboratory and if comparable analytical performance on round-robin comparative
analyses of standard reference materials or split sample analyses of field
samples can be demonstrated. This approach is intended to allow States to use
cost-effective procedures and to encourage the use of new or improved
analytical methods without compromising data quality. Performance-based
analytical programs currently are used in several fish and shellfish monitoring
programs, including the NOAA Status and Trends Program (Battelle, 1989b;
Cantillo, 1991; NOAA, 1987), the EPA Environmental Monitoring and Assess-
ment Program (EMAP) (U.S. EPA, 1991e), and the Puget Sound Estuary
Program (1990d, 1990e).
Analytical methods used in fish and shellfish contaminant monitoring programs
should be selected using the following criteria:
Technical merit—Methods should be technically sound; they should be
specific for the target analytes of concern and based on current, validated
analytical techniques that are widely accepted by the scientific community.
Sensitivity—Method detection and quantitation limits should be sufficiently
low to allow reliable quantitation of the target analytes of concern at or below
selected Screening Values (SVs). Ideally, the method detection limit (in
tissue) should be at least five times lower than the selected SV for a given
target analyte (Puget Sound Estuary Program, 1990e).
Data quality—The accuracy and precision should be adequate to ensure that
analytical data are of acceptable quality for program objectives.
Cost-efficiency—Resource requirements should not be unreasonably high.
A review of current EPA guidance for chemical contaminant monitoring programs
and of analytical methods currently used or recommended in several of these
programs (as shown in Table 8-2) indicates that a limited number of analytical
8-6
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
techniques are most commonly used for the determination of the recommended
target analytes. These techniques are listed in Table 8-3. As shown in Table
8-4 and Appendix J, analytical methods employing these techniques have
typically achievable detection and/or quantitation limits that are well below the
recommended SVs for most target analytes, with the possible exception of
dieldrin, heptachlor epoxide, toxaphene, PCBs, and dioxins/furans. Recom-
mended procedures for determining method detection and quantitation limits are
given in Section 8.3.3.3.
If lower SVs are used in a study (e.g., for susceptible populations), it is the
responsibility of program managers to ensure that the detection and quantitation
limits of the analytical methods are sufficiently low to allow reliable quantitation
of target analytes at or below these SVs. If analytical methodology is not
sensitive enough to reliably quantitate target analytes at or below selected SVs
(e.g., dieldrin, heptachlor epoxide, toxaphene, PCBs, dioxins/furans), program
managers must determine appropriate fish consumption guidance based on
lowest detectable concentrations or provide justification for adjusting SVs to
values at or above achievable method detection limits. It should be emphasized
that when SVs are below detection limits, the failure to detect a target analyte
cannot be assumed to mean that there is no cause for concern for human health
effects.
The analytical techniques identified in Table 8-3 are recommended for use in
State fish and shellfish contaminant monitoring programs. However, alternative
techniques may be used if acceptable detection limits, accuracy, and precision
can be demonstrated. Note: Neither rotenone, the most widely used piscicide
in the United States, nor its biotransformation products (e.g., rotenolone, 6',7'-
dihydro-6',7'-dihydroxyretonone, 6',7'-dihydro-6',7'-dihydroxyretonolone) would
be expected to interfere with the analyses of organic target analytes using the
recommended gas chromatographic methods of analysis. Furthermore, rotenone
has a relatively short half-life in water (3.7,1.3, and 5.2 days for spring, summer,
and fall treatments, respectively) (Dawson et al., 1991) and does not bioaccumu-
late significantly in fish (bioconcentration factor [BCFj = 26 in fish carcass)
(Gingerich and Rach, 1985), so that tissue residues should not be significant.
Laboratories should select analytical methods for routine analyses of target
analytes that are most appropriate for their programs based on available
resources, experience, program objectives, and data quality requirements. A
recent evaluation of current methods for the analyses of organic and trace metal
target analytes in fish tissue provides useful guidance on method selection,
validation, and data reporting procedures (Capuzzo et al., 1990).
The references in Table 8-2 should be consulted in selecting appropriate analyti-
cal methods. Note: Because many laboratories may have limited experience
in determining inorganic arsenic, a widely accepted method for this analysis is
included in Appendix K. An additional resource for method selection is the EPA
Environmental Monitoring Methods Index System (EMMI), an automated
inventory of information on environmentally significant analytes and methods for
8-7
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Table 8-3. Recommended Analytical Techniques for Target Analytes
Target analyte
Analytical technique
Metals
Arsenic (inorganic)
Cadmium
Mercury
Selenium
Tributyttin
Organlcs
PAHs
PCBs (total Arochtors)*
Organochlorine pesticides
Organophosphate pesticides
Chlorophenoxy herbicides
Dioxins/dibenzofurans
HAA, or HPLC with ICP-MS
GFAA or ICPa
CVAA
GFAA, ICP, or HAAa-b
GFAA or GC/FPD0
GC/MS or HRGC/HRMSd
GC/ECDfl9-h
GC/ECD'-9
GC/MS, GC/FPD, or GC/NPD1
GC/ECDf-9
HRGC/HRMSjlk
CVAA - Cold vapor atomic absorption spectrophotometry.
GC/ECD - Gas chromatography/electron capture detection.
GC/FPD - Gas chromatography/flame photometric detection.
GC/MS - Gas chromatography/mass spectrometry.
GC/NPD - Gas chromatography/nitrogen-phosphorus detection.
GFAA - Graphite furnace atomic absorption spectrophotometry.
HAA m Hydride generation atomic absorption spectrophotometry.
HPLC » High-performance liquid chromatography.
HRGC/HRMS « High-resolution gas chromatography/high-resolution mass spectrometry.
ICP » Inductively coupled plasma emission spectrometry.
ICP-MS « Inductively coupled plasma mass spectrometry.
PAHs - Polycyclic aromatic hydrocarbons.
PCBs - Polychtorinated biphenyls.
* Atomic absorption methods require a separate determination for each element, which increases the time
and cost relative to the broad-scan ICP method. However, GFAA detection limits are typically more than an
order of magnitude lower than those achieved with ICP.
b Use of HAA can tower detection limits for selenium by a factor of 10-100 (Crecelius, 1978; Skoog, 1985).
0 GC/FDP is specific for tributyltin and the most widely accepted analytical method. GFAA is less expensive
(see Table 8-5) but is not specific for tributyltin. Depending on the extraction scheme, mono-, di-, and
tetrabutyltin and other alkyltins may be included in the analysis. Contamination of samples with tin may
also be a potential problem, resulting in false positives (E. Crecelius, Battelle Pacific Northwest
Laboratories, Marine Sciences Laboratory, Sequim, WA, personal communication, 1995).
d GC/MS is also recommended for base/neutral organic target analytes (except organochtorine pesticides and
PCBs) that may be included in a study. Detection limits of less than 1 ppb can be achieved for PAHs using
HRGC/HRMS. It is recommended that, in both screening and intensive studies, tissue samples be
analyzed for benzo[a]pyrene, benz[a]anthracene, benzo[6]fluoranthene, benzo[/c]fluoranthene, chrysene,
dibenz[a,/j]anthracene, and indeno/X2,3-cd]pyrene, and that the relative potencies given for these PAHs in
the EPA provisional guidance for quantitative risk assessment of PAHs (U.S. EPA, 1993c) be used to
calculate a potency equivalency concentration (PEC) for each sample for comparison with the
recommended SV for benzo[a]pyrene (see Section 5.3.2.3). At this time, EPA's recommendation to use the
PEC approach for risk assessment of PAHs (U.S. EPA 1993c) is considered provisional because
quantitative risk assessment data are not available for all PAHs. This approach is under Agency review
and over the next year will be evaluated as new health effects benchmark values are developed.
Therefore, the method provided in this guidance document is subject to change pending results of the
Agency's revaluation.
(continued)
8-8
-------
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Table 8-3 (continued)
• Analysis of total PCBs, as the sum of Aroclor equivalents, is recommended in both screening and
intensive studies because of the lack of adequate toxicologic data to develop screening values (SVs) for
individual PCB congeners (see Section 4.3.5). However, because of the wide range of toxicfties among
different PCB congeners and the effects of metabolism and degradation on Aroclor composition in the
environment, congener analysis is deemed to be a more scientifically sound and accurate method for
determining total PCB concentrations. Consequently, States are encouraged to develop the capability to
conduct PCB congener analysis.
' GC/ECD does not provide definitive compound identification, and false positives due to interferences are
commonly reported. Confirmation by an alternative GC column phase (with ECD), or by GC/MS with
selected ion monitoring, is required for positive identification of PCBs, organochlorine pesticides, and
chlorophenoxy herbicides.
9 GC/MS with selected ion monitoring may be used for quantitative analyses of these compounds if
acceptable detection limits can be achieved.
h If PCB congener analysis is conducted, capillary GC columns are recommended (NOAA, 1989b; Dunn et
al., 1984; Schwartz et a)., 1984; Mullin et al., 1984; Stalling et al., 1987). An enrichment step, employing
an activated carbon column, may also be required to separate and quantify coeluting congeners or
congeners present at very tow concentrations (Smith, 1981; Schwartz et al., 1993).
1 Some of the chlorinated organophosphate pesticides (i.e., chlorpyrifos, diazinon, ethion) may be
analyzed by GC/ECD (USGS, 1987).
i The analysis of the 17 2,3,7,8-substituted congeners of tetra- through octa-chlorinated dibenzo-p-dioxins
(PCDDs) and dibenzofurans (PCDFs) using isotope dilution is recommended. Note: If resources are
limited, at a minimum, 2,3,7,8-TCDD and 2,3,7,8-TCDF should be analyzed.
k Because of the toxicfty of dioxins/furans and the difficulty and cost of these analyses, relatively few
laboratories currently have the capability of performing these analyses. Contract laboratories
experienced in conducting dioxin/furan analyses are listed in Table 8-1.
their analysis (U.S. EPA, 1991f). At present, the EMMI database includes infor-
mation on more than 2,600 analytes from over 80 regulatory and nonregulatory
lists and more than 900 analytical methods in a variety of matrices, including
tissue. When fully implemented, this database will provide a comprehensive
cross-reference between analytes and analytical methods with detailed informa-
tion on each analytical method, including sponsoring organization, sample matrix,
and estimates of detection limits, accuracy, and precision.
EMMI is available from the EPA Sample Control Center for all EPA personnel
and from National Technical Information Service (NTIS) for all other parties. As
of September 1995, a new version of EMMI will be available through the EPA
Local Area Network (LAN).
8-9
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
The private sector may purchase EMMI Version 2.0 through the:
National Technical Information Service (NTIS)
5285 Port Royal Road
Springfield, VA 22161
USA
Phone: (703)487-4650
Fax: (703)321-8547
Rush Orders: (800) 553-NTIS
The order number is PB95-50174B for a single user, PB95-502399B for a 5-user
LAN package, and PB95-502407B for an unlimited user LAN package. Further
information may be obtained by contacting:
EEMI User Support
U.S. EPA Sample Control Center
Operated by DynCorp EENSP
P.O. Box 1407
Alexandria, VA 22313
USA
Phone: (703)519-1222
Fax: (703)684-0610
Monday—Friday 8:00 a.m. to 5:00 p.m.
Internet: EMMIUSER@USVA5.DYNCORP.COM
Because chemical analysis is frequently one of the most expensive components
of a sampling and analysis program, the selection of an analytical method often
will be influenced by its cost. In general, analytical costs may be expected to
increase with increased sensitivity (i.e., lower detection limits) and reliability (i.e.,
accuracy and precision). Analytical costs will also be dependent on the number
of samples to be analyzed, the requested turnaround time, the number and type
of analytes requested, the level of QC effort, and the amount of support
documentation requested (Puget Sound Estuary Program, 1990d). However,
differences in protocols, laboratory experience, and pricing policies of
laboratories often introduce large variation into analytical costs. Approximate
costs per sample for the analysis of target analytes by the recommended
analytical techniques are provided in Table 8-5.
8.3 QUALITY ASSURANCE AND QUALITY CONTROL CONSIDERATIONS
Quality assurance and quality control must be integral parts of each chemical
analysis program. The QA process consists of management review and
oversight at the planning, implementation, and completion stages of the
analytical data collection activity to ensure that data provided are of the quality
required. The QC process includes those activities required during data
collection to produce the data quality desired and to document the quality of the
collected data.
8-10
-------
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
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8-11
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
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8-12
-------
8. LABORATORY PROCEDURES It — SAMPLE ANALYSES
Table 8-5. Approximate Range of Costs per Sample for
Analysis of Recommended Target Anaiytes*
Target analyte
Metals"
Arsenic (inorganic)6
Cadmium
Mercury
Selenium
Tributyltind
Organochlorlne pesticides'1'
Organophosphate pesticides9
Chlorophenoxy herbicides'1
PAHs1
PCBs*
Total Aroctors
Dloxlns/furans1
TCDD/TCDF only
TCDD/TCDF through
OCDD/OCDF isomers
Llpld
Approximate cost range (1992 $)
150 - 300
25-50
35-50
25-50
150 - 350
285 - 500
250 - 500
250 - 500
250 - 525
210 - 500
200- 1,000
450-1,600
30-40
OCDD - Octachlorodibenzo-p-dioxin. PCBs » Polychlorinated biphenyls.
OCDF - Octachlorodibenzofuran. TCDD * 2,3,7,8-Tetrachlorodibenzo-p-dioxin.
PAHs - Polycyclfc aromatic hydrocarbons. TCDF - 2,3,7,8-Tetrachlorodibenzofuran.
These costs include sample digestion or extraction and cleanup, but not sample preparation (i.e., resection,
grinding, homogenization, compositing). Estimated cost of sample preparation for a composite
homogenate of five fish is $200 to $500.
Analysis of inorganic arsenic by hydride generation atomic absorption spectroscopy (HAA) or high-
performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP/MS).
Analysis of cadmium by graphite furnace atomic absorption spectrophotometry (GFAA). Analysis of
selenium by GFAA or HAA. Analysis of mercury by cold vapor atomic absorption spectrophotometry
^ (CVAA). Analysis of tributyftin by GFAA or gas chromatography/flame photometric detection (GC/FPD).
Estimated costs are for total inorganic arsenic. Estimated cost of analysis by HAA is $150 to $200.
Estimated cost of analysis by HPLC-ICP/MS is $250 to $300.
Estimated cost of analysis by GFAA is $150 to $200. Estimated cost of analysis by GC/FPD is $400.
Note: Analysis by GFAA is not specific for tributyftin. Depending on the extraction procedure, other butyl-
and alkyftin species may be detected.
• Analysis by gas chromatography/electron capture detection (GC/ECD).
Estimated costs are for analysis of all recommended target analyte organochlorine pesticides (see
Table 4-1).
8 Analysis by GC/FPD or gas chromatography/nitrogen-phosphorus detection (GC/NPD). Some of the
chlorinated organophosphate pesticides (i.e., chlorpyrifos, diazinon, ethion) may be analyzed as
organochlorine pesticides by GC/ECD (USGS, 1987)
h Analysis by GC/ECD.
Costs are for analysis by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/flame
lonizatfon detection (GC/FID). Cost for analysis by high-resolution gas chromatography/high resolution
mass spectrometry (HRGC/HRMS) is approximately $800 per sample
1 Analysis by HRGC/HRMS.
8-13
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
During the planning of a chemical analysis program, QA activities focus on
defining data quality criteria and designing a QC system to measure the quality
of data being generated. During the implementation of the data collection effort,
QA activities ensure that the QC system is functioning effectively and that the
deficiencies uncovered by the QC system are corrected. After the analytical data
are collected, QA activities focus on assessing the quality of data obtained to
determine its suitability to support decisions for further monitoring, risk
assessments, or issuance of advisories.
The purpose of this section is to describe the general QA and QC requirements
for chemical analysis programs.
8.3.1 QA Plans
Each laboratory performing chemical analyses in fish and shellfish contaminant
monitoring programs must have an adequate QA program (U.S. EPA, 1984b).
The QA program should be documented fully in a QA plan or in a combined
Work/QA Project Plan (U.S. EPA, 1980b). (See Appendix E.) Each QA and QC
requirement or procedure should be described clearly. Documentation should
clearly demonstrate that the QA program meets overall program objectives and
data quality requirements. The QA guidelines in the Puget Sound Estuary
Program (1990d, 1990e), the NOAA Status and Trends Program (Battelle,
1989b; Cantillo, 1991; NOAA, 1987), the EPA 301 (h) Monitoring Programs (U.S.
EPA, 1987e), the EPA EMAP Near Coastal (EMAP-NC) Program (U.S. EPA,
1991e), and the EPA Contract Laboratory (CLP) Program (U.S. EPA, 1991b,
1991c) are recommended as a basis for developing program-specific QA
programs. Additional method-specific QC guidance is given in references in
Table 8-2.
8.3.2 Method Documentation
Methods used routinely for the analyses of contaminants in fish and shellfish
tissues must be documented thoroughly, preferably as formal standard operating
procedures (U.S. EPA, 1984b). Recommended contents of an analytical SOP
are shown in Figure 8-1. Analytical SOPs must be followed exactly as written.
A published method may serve as an analytical SOP only if the analysis is
performed exactly as described. Any significant deviations from analytical SOPs
must be documented in the laboratory records (signed and dated by the
responsible person) and noted in the final data report. Adequate evidence must
be provided to demonstrate that an SOP deviation did not adversely affect
method performance (i.e., detection or quantitation limits, accuracy, precision).
Otherwise, the effect of the deviation on data quality must be assessed and
documented and all suspect data must be identified.
8-14
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Scope and application
Method performance characteristics (accuracy, precision,
method detection and quantitation limits) for each analyte
Interferences
Equipment, supplies, and materials
Sample preservation and handling procedures
Instrument calibration procedures
Sample preparation (i.e., extraction, digestion, cleanup)
procedures
Sample analysis procedures
Quality control procedures
Corrective action procedures
Data reduction and analysis procedures (with example
calculations)
Recordkeeping procedures (with standard data forms, if
applicable)
Safety procedures and/or cautionary notes
Disposal procedures
References
Figure 8-1. Recommended contents of analytical
standard operating procedures (SOPs).
8.3.3 Minimum QA and QC Requirements for Sample Analyses
The guidance provided in this section is derived primarily from the protocols
developed for the Puget Sound Estuary Program (1990d, 1990e). These
protocols have also provided the basis for the EPA EMAP-NC QA and QC
requirements (U.S. EPA, 1991e). QA and QC recommendations specified in this
document are intended to provide a uniform performance standard for all
analytical protocols used in State fish and shellfish contaminant monitoring
8-15
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
programs and to enable an assessment of the comparability of results generated
by different laboratories and different analytical procedures. These recommen-
dations are intended to represent minimum QA and QC procedures for any given
analytical method. Additional method-specific QC procedures should always be
followed to ensure overall data quality.
For sample analyses, minimum QA and QC requirements consist of (1) initial
demonstration of laboratory capability and (2) routine analyses of appropriate QA
and QC samples to demonstrate continued acceptable performance and to
document data quality.
Initial demonstration of laboratory capability (prior to analysis of field samples)
should include
Instrument calibration
• Documentation of detection and quantitation limits
Documentation of accuracy and precision
• Analysis of an accuracy-based performance evaluation sample provided by
an external QA program.
Ongoing demonstration of acceptable laboratory performance and documentation
of data quality should include
Routine calibration and calibration checks
Routine assessment of accuracy and precision
• Routine monitoring of interferences and contamination
• Regular assessment of performance through participation in external QA
interlaboratory comparison exercises, when available.
The QA and QC requirements for the analyses of target analytes in tissues
should be based on specific performance criteria (i.e., warning or control limits)
for data quality indicators such as accuracy and precision. Warning limits are
numerical criteria that serve to alert data reviewers and data users that data
quality may be questionable. A laboratory is not required to terminate analyses
when a warning limit is exceeded, but the reported data may be qualified during
subsequent QA review. Control limits are numerical data criteria that, when
exceeded, require suspension of analyses and specific corrective action by the
laboratory before the analyses may resume.
Typically, warning and control limits for accuracy are based on the historical
mean recovery plus or minus two or three standard deviation units, respectively.
Warning and control limits for precision are typically based on the historical
8-16
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
standard deviation or coefficient of variation (or mean relative percent difference
for duplicate samples) plus two or three standard deviation units, respectively.
Procedures incorporating control charts (ASTM, 1976; Taylor, 1985) and/or
tabular presentations of historical data should be in place for routine monitoring
of analytical performance. Procedures for corrective action in the event of
excursion outside warning and control limits should also be in place.
The results for the various QC samples analyzed with each batch of samples
should be reviewed by qualified laboratory personnel immediately following the
analysis of each sample batch to determine when warning or control limits have
been exceeded. When established control limits are exceeded, appropriate
corrective action should be taken and, if possible, all suspect samples
reanalyzed before resuming routine analyses. If reanalyses cannot be
performed, all suspect data should be identified clearly. Note: For the purposes
of this guidance manual, a batch is defined as any group of samples from the
same source that is processed at the same time and analyzed during the same
analytical run.
Recommended QA and QC samples (with definitions and specifications),
frequencies of analyses, control limits, and corrective actions are summarized
in Table 8-6.
Note: EPA recognizes that resource limitations may prevent some States from
fully implementing all recommended QA and QC procedures. Therefore, as
additional guidance, the minimum numbers of QA and QC samples recom-
mended for routine analyses of target analytes are summarized in Table 8-7. It
is the responsibility of each program manager to ensure that the analytical QC
program is adequate to meet program data quality objectives for method
detection limits, accuracy, precision, and comparability.
Recommended QA and QC procedures and the use of appropriate QA and QC
samples are discussed in Sections 8.3.3.2 through 8.3.3.8. Recommended
procedures for documenting and reporting analytical and QA and QC data are
given in Section 8.4. Because of their importance in assessing data quality and
interlaboratory comparability, reference materials are discussed separately in the
following section.
8.3.3.1 Reference Materials—
The appropriate use of reference materials is an essential part of good QA and
QC practices for analytical chemistry. The following definitions of reference
materials (Puget Sound Estuary Program, 1990d) are used in this guidance
document:
A reference material is any material or substance of which one or more
properties have been sufficiently well established to allow its use for
instrument calibration, method evaluation, or characterization of other
materials.
8-17
-------
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
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8. LABORATORY PROCEDURES If — SAMPLE ANALYSES
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
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8-26
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Table 8-7. Minimum Recommended QA and QC Samples for
Routine Analysis of Target Analytes"
Sample Type
Target analyte
Metals
Organics
Accuracy-based performance
evaluation sample13
Method blank
Once prior to routine
analysis of field samples,
plus one exercise (four
to six samples) per year.
1
Once prior to routine
analysis of field samples,
plus one exercise (four
to six samples) per year.
1
Laboratory duplicate
1
1
Matrix spike/matrix spike replicate
Laboratory control sample
(SRM or CRM, if available)
1
1
Calibration check standard
Surrogate spike (isotopically labeled
target analyte or other surrogate
compound added prior to extraction)
2°
NA
2c
Each sample
Instrument (injection) internal standard;
added prior to injection
NA
Each calibration or
calibration check
standard and each
sample or blank
analyzed by GC/MSd
CRM = Certified reference material (see Section 8.3.3.1).
GC/MS = Gas chromatography/mass spectroscopy.
NA = Not applicable.
QA = Quality assurance.
QC = Quality control.
SRM = Standard reference material (see Section 8.3.3.1).
a Unless otherwise specified, the number given is the recommended number of QC samples per
20 samples or per batch, whichever is more frequent. Additional method-specific QC
requirements should always be followed provided these minimum requirements have been met.
b QA samples from National Oceanic and Atmospheric Administration interlaboratory comparison
program (see Section 8i3.3.8.1).
0 One every 10 samples (plus one at beginning and end of each analytical run).
d Optional for analyses by GC/electron capture detection (ECD), GC/f lame ionization detection
(FID), or GC with other nonspecific detectors.
8-27
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
• A certified reference material (CRM) is a reference material of which the
value(s) of one or more properties has (have) been certified by a variety of
technically valid procedures. CRMs are accompanied by or traceable to a
certificate or other documentation that is issued by the certifying organization
(e.g., U.S. EPA, NIST, National Research Council of Canada [NRCC]).
• A standard reference material (SRM) is a CRM issued by the NIST.
Reference materials may be used to (1) provide information on method accuracy
and, when analyzed in replicate, on precision, and (2) obtain estimates of
intermethod and/or interlaboratory comparability. An excellent discussion of the
use of reference materials in QA and QC procedures is given in Taylor (1985).
The following general guidelines should be followed to ensure proper use off
reference materials (NOAA, 1992):
• When used to assess the accuracy of an analytical method, the matrix of the
reference material should be as similar as possible to that of the samples of
Interest. If reference materials in matrices other than fish or shellfish tissue
are used, possible matrix effects should be addressed in the final data
analysis or interpretation.
• Concentrations of reference materials should cover the range of possible
concentrations in the samples of interest. Note: Because of a lack of low-
and high-concentration reference materials for most analytes in fish and
shellfish tissue matrices, potential problems at low or high concentrations
often cannot be documented.
i
• Reference materials should be analyzed prior to beginning the analyses of
field samples to assess laboratory capability and regularly thereafter to
detect and document any changes in laboratory performance over time.
Appropriate corrective action should be taken whenever changes are
observed outside specified performance limits (e.g., accuracy, precision).
If possible, reference material samples should be introduced into the sample
stream as double blinds, that is, with identity and concentration unknown to
the analyst. However, because of the limited number of certified fish and
shellfish tissue reference materials available, the results of analyses of these
materials may be biased by an analyst's increasing ability to recognize these
materials with increased use.
Results of reference material analyses are essential to assess interlaboratory
or intermethod comparability. However, the results of sample analyses
should not be corrected based on percent recoveries of reference materials.
Final reported results should include both uncorrected sample results and
percent recoveries of reference materials.
8-28
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Sources of EPA-certified and other recommended reference materials for the
analysis of priority pollutants and selected related compounds in fish and
shellfish tissues are given in Appendix L. Currently available marine or estuarine
tissue reference materials that may be appropriate for use by analytical
laboratories in fish and shellfish contaminant monitoring programs are given in
Table 8-8.
8.3.3.2 Calibration and Calibration Checks-
General guidelines for initial calibration and routine calibration checks are
provided in this section. Method-specific calibration procedures are included in
the references in Table 8-2. It is the responsibility of each program manager to
ensure that proper calibration procedures are developed and followed for each
analytical method to ensure the accuracy of the measurement data.
All analytical instruments and equipment should be maintained and calibrated
properly to ensure optimum operating conditions throughout a measurement
program. Calibration and maintenance procedures should be performed
according to SOPs based on the manufacturers' specifications and the
requirements of specific analytical procedures. Calibration procedures must
include provisions for documenting calibration frequencies, conditions, standards,
and results to describe adequately the calibration history of each measurement
system. Calibration records should be inspected regularly to ensure that these
procedures are being performed at the required frequency and according to
established SOPs. Any deficiencies in the records or deviations from estab-
lished procedures should be documented and appropriate corrective action
taken.
Calibration standards of known and documented accuracy must be used to
ensure the accuracy of the analytical data. Each laboratory should have a
program for verifying the accuracy and traceabillty of calibration standards
against the highest quality standards available. If possible, NIST-SRMs or
EPA-certified standards should be used for calibration standards (see Section
8.3.3.4 and Appendix I). A log of all calibration materials and standard solutions
should be maintained. Appropriate storage conditions (i.e., container specifica-
tions, shelf-life, temperature, humidity, light condition) should be documented and
maintained.
8.3.3.2.1 Initial and routine calibration
Prior to beginning routine analyses of samples, a minimum of three (and
preferably five) calibration standards should be used to construct a calibration
curve for each target anaiyte, covering the normal working range of the
instrument or the expected target anaiyte concentration range of the samples to
be analyzed. The lowest-concentration calibration standard should be at or near
the estimated method detection limit (see Section 8.3.3.3.1). Calibration
standards should be prepared in the same matrix (i.e., solvent) as the final
sample extract or digestate. Criteria for acceptable calibration (e.g., acceptable
8-29
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Table 8-8. Fish and Shellfish Tissue Reference Materials
Identification
cod*
DOLT-1
DORM-1
LUTS-1
TORT-1
GBW-08571
GBW-08572
MA-A-1/OC
MA-A-3/OC
MA-B-3/OC
MA-M-2/OC
MA-A-1/TM
MA-A-2/TM
MA-B-3/TM
MA-B-3/RN
IAEA-350
IAEA-351
IAEA-352
CRM-278
CRM-422
EPA-FISH
EPA-SRS903
EPA-0952
EPA-2165
RM-50
SRM-1566a
SRM-1974
SRM-1974a*
SRM-2974"
NIES-6
Analytes
Elements
Elements
Elements
Elements
Elements
Elements
Organic compounds
Organic compounds
Organic compounds
Organic compounds
Elements
Elements
Elements
Isotopes
Elements
Organic compounds
Isotopes
Elements
Elements
Pesticides
Chlordane
Mercury
Mercury
Elements
Elements
Organic compounds
Organic compounds
Organic compounds
Elements
Source
NRCC
NRCC
NRCC
NRCC
NRCCRM
NRCCRM
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
IAEA
BCR
BCR
EPA1
EPA2
EPA1
EPA1
NIST
NIST
NIST
NIST
NIST
NIES
Matrix
Dogfish liver (freeze-dried)
Dogfish muscle (freeze-dried)
Non-defatted lobster hepatopancreas
Lobster hepatopancreas
Mussel tissue (freeze-dried)
Prawn tissue
Copepod homogenate (freeze-dried)
Shrimp homogenate (freeza-dried)
Fish tissue (freeze-dried)
Mussel tissue
Copepod homogenate (freeze-dried)
Fish flesh homogenate
Fish tissue (freeze-dried)
Fish tissue (freeze-dried)
Tuna homogenate (freeze-dried)
Tuna homogenate (freeze-dried)
Tuna homogenate (freeze-drisd)
Mussel tissue (freeze-dried)
Cod muscle (freeze-dried)
Fish tissue
Fish tissue
Fish tissue
Fish tissue
Albacore tuna (freeze-dried)
Oyster tissue (freeze-dried)
Mussel tissue (frozen)
Mussel tissue (frozen)
Mussel tissue (freeze-dried)
Mussel tissue
• Certification in progress as of June 1995. SRM-1974a is a renewal of SRM-1974, which was issued m 1990
Sources!
BCR = Community Bureau of Reference, Commission of the European Communities, Directorate General for
Science, Research and Development, 200 rue de la Loi, B-1049 Brussels, Belgium.
EPA = U.S. Environmental Protection Agency, Quality Assurance Branch, EMSL-Cindnnati, Cincinnati, OH,
45268. USA. (EPA1: Material available from Supelco, Inc., Supelco Park, Bellefonte, PA, 16823-
0048, USA. EPA2: Material available from Fisher Scientific, 711 Forbes Ave., Pittsburgh, PA
15219.)
IAEA = International Atomic Energy Agency, Analytical Quality Control Service. Laboratory Seibersdorf. P. O.
Box 100, A-1400 Vienna, Austria.
NRCCRM « National Research Center for CRMs. Office of CRMs, No. 7, District 11, Hepingjie. Chaoyangqu,
Beijing, 100013. China.
NRCC » National Research Council of Canada, Institute for Environmental Chemistry. Marine Analytical
Chemistry Standards Program, Division of Chemistry, Montreal Road, Ottawa, Ontario K1A OR9,
Canada.
NIST = National Institute of Standards and Technology, Office of Standard Reference Materials.
Gaithersburg, MD, 20899, USA.
NIES = National Institute for Environmental Studies. Yatabe-machi. Tsukuba. Ibaraki, 305, Japan.
8-30
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
limits for r2, slope, intercept, percent recovery, response factors) should be
established for each analytical method. If these control limits are exceeded, the
source of the problem (e.g., inaccurate standards, instrument instability or
malfunction) should be identified and appropriate corrective action taken. No
analyses should be performed until acceptable calibration has been achieved
and documented.
In addition to the initial calibration, an established schedule for the routine
calibration and maintenance of analytical instruments should be followed, based
on manufacturers' specifications, historical data, and specific procedural
requirements. At a minimum, calibration should be performed each time an
instrument is set up -for analysis, after any major disruption or failure, after any
major maintenance, and whenever a calibration check exceeds the recom-
mended control limits (see Table 8-6).
Two types of calibration procedures are used in the analytical methods
recommended for the quantitation of target analytes: external calibration and
internal standard calibration.
External calibration
In external calibration, calibration standards with known concentrations of target
analytes are analyzed, independent of samples, to establish the relationship
between instrument response and target analyte concentration. External
calibration is used for the analyses of metals and, at the option of the program
manager, for the analyses of organics by gas chromatography/electron capture
detection (GC/ECD), gas chromatography/flame ionization detection (GC/FID),
or GC methods using other nonspecific detectors.
External calibration for metals analysis is considered acceptable if the percent
recovery of all calibration standards is between 95 and 105 percent; external
calibration for organic analyses is considered acceptable if the relative standard
deviation (RSD) of the response factors (RFs) is <20 percent (see Table 8-6).
If these limits are exceeded, the initial calibration should be repeated.
Internal standard calibration
Calibration of GC/mass spectrometry (MS) systems used for the analysis of
organic target analytes requires the addition of an internal standard to each
calibration standard and determination of the response of the target analyte of
interest relative to that of the internal standard. Internal standard calibration may
also be used with nonspecific detector GC methods such as GC/ECD and
GC/FID. Internal standards used to determine the relative response factors
(RRFs) are termed instrument or injection internal standards (Puget Sound
Estuary Program, 1990d; U.S. EPA, 1991e). The addition of instrument internal
standards to both calibration standards and sample extracts ensures rigorous
quantitation, particularly accounting for shifts in retention times of target analytes
in complex sample extracts relative to calibration standards. Recommended
8-31
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
instrument internal standards for semivolatile organic compounds are included
• in analytical methods for these compounds (see references in Table 8-2).
The RRF for each target analyte is calculated for each calibration standard as
follows:
RRF, = (A,) (Cis) / (Ais) (Ct)
(8-1)
where
A, = Measured response (integrated peak area) for the target analyte
Cjs = Concentration of the instrument internal standard in the calibration
standard
AJS = Measured response (integrated peak area) for the instrument internal
standard
Ct = Concentration of the target analyte in the calibration standard.
If the relative standard deviation (RSD) of the average RRFt for all calibration
standards (RRFt) is £30 percent, RRFt can be assumed to be constant across
the working calibration range and RRFtcan be used to quantitate target analyte
concentrations in the samples as follows:
Ct (ppm or ppb, wet weight) = (A,) (Cis) (Ve) / (A,.) (RRFt) (W) (8-2)
where
Ct = Concentration of the target analyte in the sample
Cis = Concentration of the instrument internal standard in the sample
extract
Ve - Volume of the final sample extract (mL)
W = Weight of sample extracted (g)
and A,, AJS, and RRF, are defined as in Equation (8-1).
If the RSD of RRF, for all calibration standards is >30 percent, the initial
calibration should be repeated (see Table 8-6).
8-32
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
8.3.3.2.2 Routine calibration checks
After initial calibration has been achieved and prior to the routine analyses of
samples, the accuracy of the calibration should be verified by the analysis of a
calibration check standard. A calibration check standard is a mid-range
calibration standard that has been prepared independently (i.e., using a different
stock) from the initial calibration standards. When internal standard calibration
is being used, an instrument internal standard must be added to each calibration
check standard.
Routine calibration checks should be conducted often enough throughout each
analysis run to ensure adequate maintenance of instrument calibration (see
Table 8-6). A calibration check should always be performed after analyzing the
last sample in a batch and at the end of each analysis run.
If a calibration check does not fall within specified calibration control limits, the
source of the problem should be determined and appropriate corrective action
taken (see Table 8-6). After acceptable calibration has been reestablished, all
suspect analyses should be repeated. If resources permit, it is recommended
that all samples after the last acceptable calibration check be reanalyzed.
Otherwise, the last sample analyzed before the unacceptable calibration check
should be reanalyzed first and reanalysis of samples should continue in reverse
order until the difference between the reanalysis and initial results is within the
control limits specified in Table 8-6. If reanalysis is not possible, all suspect data
(i.e., since the last acceptable calibration check) should be identified clearly in
the laboratory records and the data report.
8.3.3.2.3 Calibration range and data reporting
As noted in Section 8.3.2.1, the lowest-concentration calibration standard should
be at or near the method detection limit. The highest-concentration calibration
standard should be selected to cover the full range of expected concentrations
of the target analyte in fish and shellfish tissue samples. If a sample
concentration occurs outside the calibration range, the sample should be diluted
or concentrated as appropriate and reanalyzed or the calibration range should
be extended. Extremely high concentrations of organic compounds may indicate
that the extraction capabilities of the method have been saturated and extraction
of a smaller sample or modification of the extraction procedure may be required.
All reported concentrations must be within the upper limit of the demonstrated
working calibration range. Procedures for reporting data, with appropriate
qualifications for data below method detection and quantitation limits, are given
in Section 8.3.3.3.3.
8-33
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
8.3.3.3 Assessment of Detection and Quantltatlon Limits-
It is the responsibility of each laboratory to determine appropriate detection and
quantitation limits for each analytical method for each target analyte in a fish or
shellfish tissue matrix. When available scientific literature demonstrates that the
selected SVs are analytically attainable, the laboratory is responsible for
ensuring that these limits are sufficiently low to allow reliable quantitation of the
analyte at or below the selected SVs (see Section 5.2). Detection and
quantitation limits must be determined prior to the use of any method for routine
analyses and after any significant changes are made to a method during routine
analyses. Several factors influence achievable detection and quantitation limits
regardless of the specific analytical procedure. These include amount of sample
available, matrix interferences, and stability of the instrumentation. The limits of
detection given in Table 8-4 and Appendix H are considered to be representative
of typically attainable values. Depending upon individual laboratory capabilities
and fish tissue matrix properties, it should be noted that SVs for some
recommended target analytes (e.g., dieldrin, heptachlor epoxide, toxaphene,
RGBs, and dioxins/furans) may not always be analytically attainable quantitation
limits. In these instances, all historic and current data on contaminant sources
and on water, sediment, and fish and shellfish contaminant tissue data should
be reviewed to provide additional information that could aid in the risk
assessment process and in making risk management decisions'.
The EPA has previously issued guidance on detection limits for trace metal and
organic compounds for analytical methods used in chemical contaminant
monitoring programs (U.S. EPA, 1985a). However, at present there is no clear
consensus among analytical chemists on a standard procedure for determining
and reporting the limits of detection and quantitation of analytical procedures.
Furthermore, detection and quantitation limits reported in the literature are
seldom clearly defined. Appendix H clearly illustrates the widespread
inconsistency in defining and reporting limits of detection and quantitation.
Reported detection limits may be based on instrument sensitivity or determined
from the analyses of method blanks or low-level matrix spikes; quantitation limits
may be determined from the analyses of method blanks or low-level matrix
spikes (Puget Sound Estuary Program, 1990d).
8.3.3.3.1 Detection limits
The EPA recommends that the method detection limit (MDL) defined below and
determined according to 40 CFR 136, Appendix B, be used to establish the limits
of detection for the analytical methods used for analyses of all target analytes:
• Method Detection Limit (MDL): The minimum concentration of an analyte
in a given matrix (i.e., fish or shellfish tissue homogenates for the purposes
of this guidance) that can be measured and reported with 99 percent
confidence that the concentration is greater than zero. The MDL is
determined by multiplying the appropriate (i.e., n-1 degrees of freedom)
one-sided 99 percent Student's t-statistic (t0 99) by the standard deviation (S)
8-34
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8. LABORATORY PROCEDURES H — SAMPLE ANALYSES
obtained from a minimum of seven replicate analyses of a spiked matrix
sample containing the analyte of interest at a concentration three to five
times the estimated MDL (Glaser et al., 1981; 40 CFR 136, Appendix B):
MDL = (to.99) (S). (8-3)
It is important to emphasize that all sample processing steps of the analytical
method (e.g., digestion, extraction, cleanup) must be included in the
determination of the MDL.
In addition to the MDL, three other types of detection limits have been defined
by the American Chemical Society Committee on Environmental Improvement
(Keith, 1991 a):
• Instrument Detection Limit (IDL): The smallest signal above background
noise that an instrument can detect reliably.
• Limit of Detection (LOD): The lowest concentration that can be determined
to be statistically different from a method blank at a specified level of
confidence. The recommended value for the LOD is three times the
standard deviation of the blank in replicate analyses, corresponding to a 99
percent confidence level.
• Reliable Detection Limit (RDL): The concentration level of an analyte in
a given matrix at which a detection decision is extremely likely. The RDL is
generally set higher than the MDL. When RDL=MDL, the risk of a false
positive at 3o from zero is <1 percent, whereas the corresponding risk of a
false negative is 50 percent. When RDL=2MDL, the risk of either a false
positive or a false negative at 3o from zero is <1 percent.
Each of these estimates has its practical limitations. The IDL does not account
for possible blank contaminants or matrix interferences. The LOD accounts for
blank contaminants but not for matrix effects or interferences. In some
instances, the relatively high value of the MDL or RDL may be too stringent and
result in the rejection of valid data; however, these are the only detection limit
estimates that account for matrix effects and interferences and provide a high
level of statistical confidence in sample results. The MDL is the recommended
detection limit in the EPA EMAP-NC Program (U.S. EPA, 1991 e).
The MDL, expressed as the concentration of target analyte in fish tissue, is
calculated from the measured MDL of the target analyte in the sample extract
or digestate according to the following equation:
MDL,issue (ppm or ppb) = (MDLextract • V) /W
(8-4)
8-35
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
where
V = Final extract or digestate volume, after dilution or concentration (ml_)
W = Weight of sample digested or extracted (g).
Equation (8-4) clearly illustrates that the MDL in tissue may be improved
(reduced) by increasing the sample weight (W) and/or decreasing the final
extract or digestate volume (V).
The initial MDL is a statistically derived empirical value that may differ in actual
samples depending on several factors, including sample size, matrix effects, and
percent moisture. Therefore, it is recommended that each laboratory reevaluate
annually all MDLs for the analytical methods used for the sample matrices
typically encountered (U.S. EPA, 1991e).
Experienced analysts may use their best professional judgment to adjust the
measured MDL to a lower "typically achievable" detection limit (Puget Sound
Estuary Program, 1990e; U.S. EPA, 1985a) or to derive other estimates of
detection limits. For example, EPA recommends the use of lower limits of
detection (LLDs) for GO/MS methods used to analyze organic pollutants in
bioaccumulation monitoring programs (U.S. EPA, 1986b). Estimation of the LLD
for a given analyte involves determining the noise level in the retention window
for the quantitation mass of the analyte for at least three field samples in the
sample set being analyzed. The LLD is then estimated as the concentration
corresponding to the signal required to exceed the average noise level observed
by at least a factor of 2. Based on the best professional judgment of the analyst,
this LLD is applied to samples in the set with comparable or lower interference;
samples with significantly higher interferences (i.e., by at least a factor of 2) are
assigned correspondingly higher LLDs. LLDs are greater than IDLs but usually
are less than the more rigorously defined MDLs. Thus, data quantified between
the LLD and the MDL have a lower statistical confidence associated with them
than data quantified above the MDL. However, these data are considered valid
and useful in assessing low-level environmental contamination.
If estimates of detection limits other than the MDL are developed and used to
qualify reported data, they should be clearly defined in the analytical SOPs and
in all data reports, and their relationship to the MDL should be clearly described.
8.3.3.3.2 Quantitation limits
In addition to the MDL, a method quantitation limit (MQL), or minimum
concentration allowed to be reported at a specified level of confidence without
qualifications, should be derived for each analyte. Ideally, MQLs should account
for matrix effects and interferences. The MQL can be greater than or equal to
the MDL. At present, there is no consistent guidance in the scientific literature
for determining MQLs; therefore, it is not possible to provide specific
recommendations for determining these limits at this time.
8-36
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
The American Chemical Society Committee on Environmental Improvement
(Keith, 1991b; Keith et al., 1983) has defined one type of quantitation limit:
• Limit of Quantitation (LOQ): The concentration above which quantitative
results may be obtained with a specified degree of confidence. The
recommended value for the LOQ is 10 times the standard deviation of a
method blank in replicate analyses, corresponding to an uncertainty of ±30
percent in the measured value (1 Oo ± 3o) at the 99 percent confidence level.
The LOQ is the recommended quantitation limit in the EPA EMAP-NC Program
(U.S. EPA, 1991e). However, the LOQ does not account for matrix effects or
interferences.
The U.S. EPA (1986d) has defined another type of quantitation limit:
• Practical Quantitation Limit (PQL): The lowest concentration that can be
reliably reported within specified limits of precision and accuracy under
routine laboratory operating conditions.
The Puget Sound Estuary Program (1990d) and the National Dioxin Study (U.S.
EPA, 1987d) used a PQL based on the lowest concentration of the initial
calibration curve (C, in \ig/mL), the amount of sample typically analyzed (W, in
g), and the final extract volume (V, in mL) of that method:
PQL (ng/gjppm) = C(ng/mL).V(mL)
W(g)
(8-5)
However, this PQL is also applicable only to samples without substantial matrix
effects or interferences.
A reliable detection limit (RDL) equal to 2 MDL may also be used as an estimate
of the MQL (see Section 8.3.3.3.1). The RDL accounts for matrix effects and
provides a high level of statistical confidence in analytical results.
Analysts must use their expertise and professional judgment to determine the
best estimate of the MQL for each target analyte. MQLs, including the estimated
degree of confidence in analyte concentrations above the quantitation limit,
should be clearly defined in the analytical SOPs and in all data reports.
8.3.3.3.3 Use of detection and quantitation limits in reporting data
The analytical laboratory does not have responsibility or authority to censor data.
Therefore, all data should be reported with complete documentation of limitations
and problems. Method detection and quantitation limits should be used to
qualify reported data for each composite sample as follows (Keith, 1991b):
• "Zero" concentration (no observed response) should be reported as not
detected (ND) with the MDL noted, e.g., "ND(MDL=X)".
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Concentrations below the MDL should be reported with the qualification that
they are below the MDL.
Concentrations between the MDL and the MQL should be reported with the
qualification that they are below the quantitation limit.
• Concentrations at or above the MQL may be reported and used without
qualification.
The use of laboratory data for comparing target analyte concentrations to SVs
in screening and intensive studies is discussed in Sections 9.1.1 and 9.1.2.
8.3.3.4 Assessment of Method Accuracy—
The accuracy of each analytical method should be assessed and documented
for each target analyte of interest, in a fish or shellfish tissue matrix, prior to
beginning routine analyses and on a regular basis during routine analyses.
Method accuracy may be assessed by analysis of appropriate reference
materials (i.e., SRMs or CRMs prepared from actual contaminated fish or
shellfish tissue, see Table 8-8 and Appendix I), laboratory control samples
(i.e., accuracy-based samples consisting of fish and shellfish tissue
homogenates spiked with compounds representative of the target analytes of
interest), and/or matrix spikes. If possible, laboratory control samples should
be SRMs or CRMs. Note: Only the analysis of fish or shellfish tissue SRMs or
CRMS prepared from actual contaminated fish or shellfish tissue allows rigorous
assessment of total method accuracy, including the accuracy with which an
extraction or digestion procedure isolates the target analyte of interest from
actual contaminated fish or shellfish. The analysis of spiked laboratory control
samples or matrix spikes provides an assessment of method accuracy including
sample handling and analysis procedures, but does not allow rigorous
assessment of the accuracy or efficiency of extraction or digestion procedures
for actual contaminated fish or shellfish. Consequently, these samples should
not be used for the primary assessment of total method accuracy unless SRMs
or CRMs prepared from actual contaminated fish or shellfish tissue are not
available.
The concentrations of target analytes in samples used to assess accuracy
should fall within the range of concentrations found in the field samples;
however, this may not always be possible for reference materials or laboratory
control samples because of the limited number of these samples available in fish
and shellfish tissue matrices (see Table 8-8 and Appendix I). Matrix spike
samples should be prepared using spike concentrations approximately equal to
the concentrations found in the unspiked samples. An acceptable range of spike
concentrations is 0.5 to 5 times the expected sample concentrations (U.S. EPA,
1987e). Spikes should always be added to the sample homogenates prior to
digestion or extraction.
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Accuracy is calculated as percent recovery from the analysis of reference
materials, or laboratory control samples, as follows:
% Recovery = 100 (M/T)
(8-6)
where
M = Measured value of the concentration of target analyte
T = "True" value of the concentration of target analyte.
Accuracy is calculated as percent recovery from the analysis of matrix spike
samples as follows:
% Recovery = [(Ms - MU)/TJ x 100
(8-7)
where
Mu
= Measured concentration of target analyte in the spiked sample
= Measured concentration of target analyte in the unspiked sample
Ts = "True" concentration of target analyte added to the spiked sample.
When sample concentrations are less than the MDL, the value of one-half the
MDL should be used as the concentration of the unspiked sample (Mu) in
calculating spike recoveries.
8.3.3.4.1 Initial assessment of method accuracy
As discussed above, method accuracy should be assessed initially by analyzing
appropriate SRMs or CRMs that are prepared from actual contaminated fish or
shellfish tissue. The number of reference samples required to be analyzed for
the initial assessment of method accuracy should be determined by each
laboratory for each analytical procedure with concurrence of the program
manager. If such SRMs or CRMs are not available, laboratory control samples
or matrix spikes may be used for initial assessment of method accuracy.
8.3.3.4.2 Routine assessment of method accuracy
Laboratory control samples and matrix spikes should be analyzed for continuous
assessment of accuracy during routine analyses. It is recommended that one
laboratory control sample and one matrix spike sample be analyzed with every
20 samples or with each sample batch, whichever is more frequent (Puget
Sound Estuary Program, 1990d, 1990e). Ideally, CRMs or SRMs should also
be analyzed at this recommended frequency; however, limited availability and
cost of these materials may make this impractical.
For organic compounds, isotopically labeled or surrogate recovery standards
which must be added to each sample to monitor overall method performance
also provide an assessment of method accuracy (see Section 8.3.3.7.1).
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Percent recovery values for spiked samples must fall within established control
limits (see Table 8-6). If the percent recovery falls outside the control limit, the
analyses should be discontinued, appropriate corrective action taken, and, if
possible, the samples associated with the spike reanalyzed. If reanalysis is not
possible, all suspect data should be clearly identified.
Note: Reported data should not be corrected for percent recoveries. Recovery
data should be reported for each sample to facilitate proper evaluation and use
of analytical results.
Poor performance on the analysis of reference materials or poor spike recovery
may be caused by inadequate mixing of the composite homogenate sample
before aliquotting, inconsistent digestion or extraction procedures, matrix
interferences, or instrumentation problems. If replicate analyses are acceptable
(see Section 8.3.3.5), matrix interferences or loss of target analytes during
sample preparation are indicated. To check for loss of target analytes during
sample preparation, a step-by-step examination of the procedure using spiked
blanks should be conducted. For example, to check for loss of metal target
analytes during digestion, a postdigestion spike should be prepared and
analyzed and the results compared with those from a predigestion spike. If the
results are significantly different, the digestion technique should be modified to
obtain acceptable recoveries. If there is no significant difference in the results
of pre- and postdigestion spikes, the sample should be diluted by at least a
factor of 5 and reanalyzed. If spike recovery is still poor, then the method of
standard additions or use of a matrix modifier is indicated (U.S. EPA, 1987e).
8.3.3.5 Assessment of Method Precision—
The precision of each analytical method should be assessed and documented
for each target analyte prior to the performance of routine analyses and on a
regular basis during routine analysis.
Precision is defined as the agreement among a set of replicate measurements
without assumption of knowledge of the true value. Method precision (i.e., total
variability due to sample preparation and analysis) is estimated by means of the
analyses of duplicate or replicate tissue homogenate samples containing
concentrations of the target analyte of interest above the MDL All samples used
for assessment of total method precision must be carried through the complete
analytical procedure, including extraction or digestion.
The most commonly used estimates of precision are the relative standard
deviation (RSD) or coefficient of variation (CV) for multiple samples, and the
relative percent difference (RPD) when only two samples are available. These
are defined as follows:
RSD = CV =
(8-8)
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
where
S = Standard deviation of the x; measurements
xj = Arithmetic mean of the xf measurements, and
RPD = 100 {(x, - x2)/[(x1 + x2)/2]} .
8.3.3.5.1 Initial assessment of method precision
(8-9)
Method precision should be assessed prior to routine sample analyses by
analyzing replicate samples of the same reference materials, laboratory control
samples, and/or matrix spikes that are used for initial assessment of method
accuracy (see Section 8.3.3.4.1). The number of replicates required to be
analyzed for the initial assessment of method precision should be determined by
each laboratory for each analytical procedure with concurrence of the program
manager. Because precision may be concentration-dependent, initial assess-
ments of precision across the estimated working range should be obtained.
8.3.3.5.2 Routine assessment of method precision
Ongoing assessment of method precision during routine analysis should be
performed by analyzing replicate aliquots of tissue hbmogenate samples taken
prior to sample extraction or digestion (i.e., laboratory replicates) and matrix
spike replicates. Matrix spike concentrations should approximate unspiked
sample concentrations; an acceptable range for spike concentrations is 0.5 to
5 times the sample concentrations (U.S. EPA, 1987e).
For ongoing assessment of method precision, it is recommended that one
laboratory duplicate and one matrix spike duplicate be analyzed with every 20
samples or with each sample batch, whichever is more frequent. In addition, it
is recommended that a laboratory control sample be analyzed at the above
frequency to allow an ongoing assessment of method performance, including an
estimate of method precision over time. Specific procedures for estimating
method precision by laboratory and/or matrix spike duplicates and laboratory
control samples are given in ASTM (1983). This reference also includes
procedures for estimating method precision from spike recoveries and for testing
for significant change in method precision over time.
Precision estimates obtained from the analysis of laboratory duplicates, matrix
spike duplicates, and repeated laboratory control sample analyses must fall
within specified control limits (see Table 8-7). If these values fall outside the
control limits, the analyses should be discontinued, appropriate corrective action
taken, and, if possible, the samples associated with the duplicates reanalyzed.
If reanalysis is not possible, all suspect data should be clearly identified.
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Unacceptable precision estimates derived from the analysis of duplicate or
replicate samples may be caused by inadequate mixing of the sample before
aliquotting; inconsistent contamination; inconsistent digestion, extraction, or
cleanup procedures; or instrumentation problems (U.S. EPA, 1987e).
8.3.3.5.3 Routine assessment of analytical precision
The analysis of replicate aliquots of final sample extracts or digestates
(analytical replicates) provides ah estimate of analytical precision only; it does
not provide an estimate of total method precision. For organic target analytes,
analytical replicates may be included at the discretion of the program manager
or laboratory supervisor. For the analysis of target metal analytes by graphite
furnace atomic absorption spectrophotometry (GFAA) and cold vapor atomic
absorption spectrophotometry (CVAA), it is recommended that duplicate
injections of each sample be analyzed and the mean concentration be reported.
The RPD should be within control limits established by the program manager or
laboratory supervisor, or the sample should be reanalyzed (U.S. EPA, 1987e).
8.3.3.5.4 Assessment of overall variability
Estimates of the overall variability of target analyte concentrations in a sample
fish or shellfish population and of the sampling and analysis procedures can be
obtained by collecting and analyzing field replicates. Replicate field samples
are optional in screening studies; however, if resources permit, it is
recommended that duplicate samples be collected at 10 percent of the screening
sites as a minimal QC check. Analysis of replicate field samples provides some
degree of variability in that the samples themselves are typically collected and
exposed to the same environmental conditions and contaminants. There are
many points of potential dissimilarity between samples of the type described
here; however, this variability is reduced when well-homogenized composite
samples are analyzed. In intensive studies, replicate samples should be
collected at each sampling site (see Section 6.1.2.7). Although the primary
purpose of replicate field samples in intensive studies is to allow more reliable
estimates of the magnitude of contamination, extreme variability in the results of
these samples may also indicate that sampling and/or analysis procedures are
not adequately controlled.
8.3.3.6 Routine Monitoring of Interferences and Contamination—
Because contamination can be a limiting factor in the reliable quantitation of
target contaminants in tissue samples, the recommendations for proper materials
and handling and cleaning procedures given in Sections 6.2.2 and 7.2 should be
followed carefully to avoid contamination of samples in the field and laboratory.
Many metal contamination problems are due to airborne dust. High zinc blanks
may result from airborne dust or galvanized iron, and high chromium and nickel
blanks often indicate contamination from stainless steel. Mercury thermometers
should not be used in the field because broken thermometers can be a source
8-42
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
of significant mercury contamination. In the laboratory, samples to be analyzed
for mercury should be isolated from materials and equipment (e.g., polarographs)
that are potential sources of mercury contamination. Cigarette smoke is a
source of cadmium. Consequently, care should be taken to avoid the presence
of cigarette smoke during the collection, handling, processing, and analysis of
samples for cadmium. In organic analyses, phthalates, methylene chloride, and
toluene are common laboratory contaminants that are often detected in blanks
at concentrations above the MDL (U.S. EPA, 1987e).
Cross-contamination between samples should be avoided during all steps of
analysis of organic contaminants by GC-based methods. Injection micro-
syringes must be cleaned thoroughly between uses. If separate syringes are
used for the injection of solutions, possible differences in syringe volumes should
be assessed and, if present, corrected for. Particular care should be taken to
avoid carryover when high- and low-level samples are analyzed sequentially.
Analysis of an appropriate method blank (see next page) may be required
following the analysis of a high-level sample to assess carryover (U.S. EPA,
1987e).
To monitor for interferences and contamination, the following blank samples
should be analyzed prior to beginning sample collection and analyses and on a
routine basis throughout each study (U.S. EPA, 1987e):
Field blanks are rinsates of empty field sample containers (i.e., aluminum
foil packets and plastic bags) that are prepared, shipped, and stored as
actual field samples. Field blanks should be analyzed to evaluate field
sample packaging materials as sources of contamination. Each rinsate
should be collected and the volume recorded. The rinsate should be
analyzed for target analytes of interest and the total amount of target analyte
in the rinsate recorded. It is recommended that one field blank be analyzed
with every 20 samples or with each batch of samples, whichever is more
frequent.
Processing blanks are rinsates of utensils and equipment used for
dissecting and homogenizing fish and shellfish. Processing blanks should
be analyzed, using the procedure described above for field blanks, to
evaluate the efficacy of the cleaning procedures used between samples. It
is recommended that processing blanks be analyzed at least once at the
beginning of a study and preferably once with each batch of 20 or fewer
samples.
• Bottle blanks are rinsates of empty bottles used to store and ship sample
homogenates. Bottle blanks should be collected after the bottles are
cleaned prior to use for storage or shipment of homogenates. They should
be analyzed, using the procedure described above for field blanks, to
evaluate their potential as sources of contamination. It is recommended that
one bottle blank be analyzed for each lot of bottles or with each batch of 20
or fewer samples, whichever is more frequent.
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
• Method blanks are samples of extraction or digestion solvents that are
carried through the complete analytical procedure, including extraction or
digestion; they are also referred to as procedural blanks. Method blanks
should be analyzed to evaluate contaminants resulting from the total
analytical method (e.g., contaminated glassware, reagents, solvents, column
packing materials, processing equipment). It is recommended that one
method blank be analyzed with every 20 samples or with each batch of
samples, whichever is more frequent.
Reagent blanks are samples of reagents used in the analytical procedure.
It is recommended that each lot of analytical reagents be analyzed for target
analytes of interest prior to use to prevent a potentially serious source of
contamination. For organic analyses, each lot of alumina, silica gel, sodium
sulfate, or Florasil used in extract drying and cleanup should also be
analyzed for target analyte contamination and cleaned as necessary.
Surrogate mixtures used in the analysis of organic target analytes have also
been found to contain contaminants and the absence of interfering impurities
should be verified prior to use (U.S. EPA, 1987e).
Because the contamination in a blank sample may not always translate into
contamination of the tissue samples, analysts and program managers must use
their best professional judgment when interpreting blank analysis data. Ideally,
there should be no detectable concentration of any target analyte in any blank
sample (i.e., the concentration of target analytes in all blanks should be less than
the MDL). However, program managers may set higher control limits (e.g.,
:SMQL) depending on overall data quality requirements of the monitoring
program. If the concentration of a target analyte in any blank is greater than the
established control limit, all steps in the relevant sample handling, processing,
and analysis procedures should be reviewed to identify the source of
contamination and appropriate corrective action should be taken. If there is
sufficient sample material, all samples associated with the unacceptable blank
should be reanalyzed. If reanalysis is not possible, all suspect data should be
identified clearly.
Note: Analytical data should not be corrected for blank contamination by the
reporting laboratory; however, blank concentrations should always be reported
with each associated sample value.
8.3.3.7 Special QA and QC Procedures for the Analysis of Organic Target Analytes—
8.3.3.7.1 Routine monitoring of method performance
To account for losses during sample preparation (i.e., extraction, cleanup) and
to monitor overall method performance, a standard compound that has chemical
and physical properties as similar as possible to those of the target analyte of
interest should be added to each sample prior to extraction and to each
calibration standard. Such compounds may be termed surrogate recovery
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
standards. A stable, Isotoplcally labeled analog of the target analyte is an
ideal surrogate recovery standard for GC/MS analysis.
If resources permit, an isotope dilution GC/MS technique such as EPA Method
1625 (40 CFR 136, Appendix A) is recommended for the analysis of organic
target analytes for which isotopically labeled analogs are available. In this
technique, RRFs used for quantitation may be calculated from measured isotope
ratios in calibration standards and not from instrument internal standards.
However, an instrument internal standard still must be added to the final sample
extract prior to analysis to determine the percent recoveries of isotopically
labeled recovery standards added prior to extraction. Thus, in isotope dilution
methods, instrument internal standards may be used only for QC purposes (i.e.,
to assess the quality of data) and not to quantify analytes. Control limits for the
percent recovery of each isotopically labeled recovery standard should be
established by the program manager, consistent with program data quality
requirements. Control limits for percent recovery and recommended corrective
actions given in EPA Method 1625 (40 CFR 136, Appendix A) should be used
as guidance.
If isotopically labeled analogs of target analytes are not available or if the isotope
dilution technique cannot be used (e.g., for chlorinated pesticides and PCBs
: analyzed by GC/ECD), other surrogate compounds should be added as recovery
standards to each sample prior to extraction and to each calibration standard.
These surrogate recovery standards should have chemical and physical
properties similar to the target analytes of interest and should not be expected
to be present in the original samples. Recommended surrogate recovery
standards are included in the methods referenced in Table 8-2 and in EMMI
(U.S. EPA, 1991f).
Samples to which surrogate recovery standards have been added are termed
surrogate spikes. The percent recovery of each surrogate spike (% Rs) should
be determined for all samples as follows:
where
%Rs=100(Cm/Ca)
(8-10)
% Rs = Surrogate spike percent recovery
Cm = Measured concentration of surrogate recovery standard
Ca = Actual concentration of surrogate recovery standard added to the
sample.
Control limits for the percent recovery of each surrogate spike should be
established by the program manager consistent with program data quality
requirements. The control limits in the most recent EPA CLP methods (U.S.
EPA, 1991c) are recommended for evaluating surrogate recoveries.
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Note: Reported data should not be corrected for percent recoveries of surrogate
recovery standards. Recovery data should be reported for each sample to
facilitate proper evaluation and use of the analytical results.
8.3.3.7.2 Other performance evaluation procedures
The following additional procedures are required to evaluate the performance of
GC-based analytical systems prior to the routine analysis of field samples (U.S.
EPA, 1989c; U.S. EPA, 1991c). It is the responsibility of each program manager
to determine specific evaluation procedures and control limits appropriate for
their data quality requirements.
Evaluation of the GC System
GC system performance should be evaluated by determining the number of
theoretical plates of resolution and the relative retention times of the internal
standards.
Column Resolution: The number of theoretical plates of resolution, N,
should be determined at the time the calibration curve is generated (using
chrysene-d10) and monitored with each sample set. The value of N should
not decrease by more than 20 percent during an analysis session. The
equation for N is given as follows:
where
N = 16 (RT/W)'
(8-11)
RT = Retention time of chrysene-d10 (s)
W = Peak width of chrysene-d10 (s).
Relative Retention Time: Relative retention times of the internal standards
should not deviate by more than ±3 percent from the values calculated at the
time the calibration curve was generated.
If the column resolution or relative retention times are not within the specified
control limits, appropriate corrective action (e.g., adjust GC parameters, flush GC
column, replace GC column) should be taken.
Evaluation of the MS System
The performance of the mass spectrometer should be evaluated for sensitivity
and spectral quality.
Sensitivity: The signal-to-noise value should be at least 3.0 or greater for
m/z 198 from an injection of 10 ng decafluorotriphenylphosphine (DFTPP).
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Spectral Quality: The intensity of ions in the spectrum of a 50-ng injection
of DFTPP should meet the following criteria (U.S. EPA, 1991c):
m/z Criteria
51 30-80% mass 198
68 <2% mass 69
69 present
70 <2% mass 69
127 25-75% mass 198
197 <1% mass 198
198 base peak, 100% relative abundance
199 5-9% mass 198
275 10-30% mass 198
365 >0.75% mass 198
441 present and
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Two types of external QA programs are recommended: round-robin Interlabor-
atory comparisons (often referred to as Interlaboratory calibration programs)
and split-sample Interlaboratory comparisons.
8.3.3.8.1 Round-robin analysis Interlaboratory comparison program
At present, the only external round-robin QA program available for analytical
laboratories conducting fish and shellfish tissue analyses for environmental
pollutants is administered by NOAA in conjunction with its National Status and
Trends (NS&T) Program (Cantillo, 1991). This QA program has been designed
to ensure proper documentation of sampling and analysis procedures and to
evaluate both the individual and collective performance of participating
laboratories. Recently, NOAA and the EPA have agreed to conduct the NS&T
Program and the EMAP-NC Program as a coordinated effort. As a result,
EMAP-NC now cosponsors and cooperatively funds the NS&T QA Program, and
the Interlaboratory comparison exercises include all EMAP-NC laboratories (U.S.
EPA, 1991e).
Note: Participation in the NS&T QA program by all laboratories performing
chemical analyses for State fish and shellfish contaminant monitoring programs
is recommended to enhance the credibility and comparability of analytical data
among the various laboratories and programs.
Each laboratory participating in the NS&T QA program is required to
demonstrate its analytic capability prior to the analysis of field samples by the
blind analysis of a fish and shellfish tissue sample that is uncompromiseci,
homogeneous, and contains the target analytes of interest at concentrations of
interest. A laboratory's performance generally will be considered acceptable if
its reported results are within ±30 percent (for organics) and ±15 percent (for
metals) of the actual or certified concentration of each target analyte in the
sample (U.S. EPA, 1991e). If any of the results exceed these control limits, the
laboratory will be required to repeat the analysis until all reported results are
within the control limits. Routine analysis of field samples will not be allowed
until initial demonstration of laboratory capability is acceptable.
Following the initial demonstration of laboratory capability, each participating
laboratory is required to participate in one intercomparison exercise per year as
a continuing check on performance. This intercomparison exercise includes both
organic and inorganic (i.e., trace metals) environmental and standard reference
samples. The organic analytical intercomparison program is coordinated by
NIST, and the inorganic analytical intercomparison program is coordinated by the
NRCC. Sample types and matrices vary yearly. Performance evaluation
samples used in the past have included accuracy-based solutions, sample
extracts, and representative matrices (e.g., tissue or sediment samples).
Laboratories are required to analyze the performance evaluation samples blind
and to submit their results to NIST or NRCC, as instructed. Individual laboratory
performance is evaluated against the consensus values (i.e., grand means) of
the results reported by all participating laboratories. Laboratories that fail to
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
achieve acceptable performance must take appropriate corrective action. NIST
and NRCC will provide technical assistance to participating laboratories that have
problems with the intercomparison analyses. At the end of each calendar year,
the results of the intercomparison exercises are reviewed at a workshop
sponsored by NIST and NRCC. Representatives from each laboratory are
encouraged to participate in these workshops, which provide an opportunity for
discussion of analytical problems encountered in the intercomparison exercises.
• Note: Nonprofit laboratories (e.g'., EPA and other Federal laboratories, State,
municipal, and nonprofit university laboratories) may participate in the NS&T QA
program at no cost on a space-available basis. In 1993, the estimated cost of
participation in the NIST Intercomparison Exercise Program for Organic Contami-
nants jn the Marine Environment will be $2,000 and $2,300 for private labora-
tories within and outside the United States, respectively. This cost covers
samples for one exercise per year. Samples may be obtained directly from NIST
by contacting Ms. Reenie Parris, NIST, Chemistry B158, Gaithersburg, MD
20899; Tel:301 -975-3103, FAX:301 -926-8671. At present, the cost of participa-
tion in trace inorganic exercises by private laboratories has not been established.
Once this cost has been set, trace inorganic samples will be available directly
from NRCC.
To obtain additional information about participation in the NS&T QA program,
contact Dr. Adriana Cantiilo, QA Manager, NOAA/National Status and Trends
Program, N/ORCA21, Rockville, MD 20852, Tel: 301-443-8655.
8.3.3.8.2 Split sample analysis Intel-laboratory comparison programs
Another useful external QA procedure for assessing interlaboratory comparability
of analytical data is a split-sample analysis program in which a percentage
(usually 5 to 10 percent) of all samples analyzed by each State or Region are
divided and distributed for analyses among laboratories from other States or
Regions. Because actual samples are used in a split-sample analysis program,
the results of the split-sample analyses provide a more direct assessment of the
comparability of the reported results from different States or Regions.
The NS&T QA program does not include an interlaboratory split-sample analysis
program. However, it is recommended that split-sample analysis programs be
established by States and/or Regions that routinely share results.
8.4 DOCUMENTATION AND REPORTING OF DATA
The results of all chemical analyses must be documented adequately and
reported properly to ensure the correct evaluation and interpretation of the data.
8.4.1 Analytical Data Reports
The documentation of analytical data for each sample should include, at a
minimum, the following information: '<
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Study identification (e.g., project number, title, phase)
Description of the procedure used, including documentation and justification
of any deviations from the standard procedure
Method detection and quantitation limits for each target analyte
Method accuracy and precision for each target analyte
Discussion of any analytical problems and corrective action taken
Sample identification number
Sample weight (wet weight)
Final dilution volume/extract volume
Date(s) of analysis
Identification of analyst
Identification of instrument used (manufacturer, model number, serial
number, location)
Summary calibration data, including identification of calibration materials,
dates of calibration and calibration checks, and calibration range(s); for
GC/MS analyses, include DFTPP spectra and quantitation report
Reconstructed ion chromatograms for each sample analyzed by GC/MS
Mass spectra of detected target compounds for each sample analyzed by
GC/MS
Chromatograms for each sample analyzed by GC/ECD and/or GC/FID
Raw data quantitation reports for each sample
Description of all QC samples associated with each sample (e.g., reference
materials, field blanks, rinsate blanks, method blanks, duplicate or replicate
samples, spiked samples, laboratory control samples) and results of all QC
analyses. QC reports should include quantitation of all target analytes in
each blank, recovery assessments for all spiked samples, and replicate
sample summaries. Laboratories should report all surrogate and matrix spike
recovery data for each sample; the range of recoveries should be included
in any reports using these data.
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8. LABORATORY PROCEDURES II — SAMPLE ANALYSES
Analyte concentrations with reporting units identified (as ppm or ppb wet
weight, to two significant figures unless otherwise justified). Note: Reported
data should not be recovery- or blank-corrected.
Lipid content (as percent wet weight)
Specification of all tentatively identified compounds (if requested) and any
quantitation data.
• Data qualifications (including qualification codes and their definitions, if
applicable, and a summary of data limitations).
To ensure completeness and consistency of reported data, standard forms
should be developed and used by each laboratory for recording and reporting
data from each analytical method. Standard data forms used in the EPA
Contract Laboratory Program (U.S. EPA, 1991b, 1991c) may serve as useful
examples for analytical laboratories.
All analytical data should be reviewed thoroughly by the analytical laboratory
supervisor and, ideally, by a qualified chemist who is independent of the
laboratory. In some cases, the analytical laboratory supervisor may conduct the
full data review, with a more limited QA review provided by an independent
chemist. The purpose of the data review is to evaluate the data relative to data
quality specifications (e.g., detection and quantitation limits, precision, accuracy)
and other performance criteria established in the Work/QA Project Plan. In many
instances, it may be necessary to qualify reported data values; qualifiers should
always be defined clearly in the data report. Recent guidance on the
documentation and evaluation of trace metals data collected for Clean Water Act
compliance monitoring (U.S. EPA, 1995i) provides additional useful information
on data review procedures.
8.4.2 Summary Reports
Summaries of study data should be prepared for each target species at each
sampling site. Specific recommendations for reporting data for screening and
intensive studies are given in Section 9.2.
8-51
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9. DATA ANALYSIS AND REPORTING
SECTION 9
DATA ANALYSIS AND REPORTING
This section provides guidance on (1) analysis of laboratory data for both
screening and intensive studies that should be included in State data reports and
(2) data reporting requirements for a national database (National Fish Tissue
Data Repository) for fish and shellfish contaminant monitoring programs.
All data analysis and reporting procedures should be documented fully as part
of the Work/QA Project Plan for each study, prior to initiating the study (see
Appendix E). All routine data analysis and reporting procedures should be
described in standard operating procedures. In particular, the procedures to be
used to determine if the concentration of a target analyte in fish or shellfish
tissue differs significantly from the selected Screening Value (SV) must be clearly
documented.
9.1 DATA ANALYSIS
9.1.1 Screening Studies
The primary objective of Tier 1 screening studies is to assist States in identifying
potentially contaminated harvest areas where further investigation of fish and
shellfish contamination may be warranted. The criteria used to determine
whether the measured target analyte concentration in a fish or shellfish tissue
composite sample is different from the SV (greater than or less than) should be
clearly documented. If a reported target analyte concentration exceeds the SV
in the screening study, a State should initiate a Tier 2, Phase I, intensive study
(see Section 6.1.2.1) to verify the level of contamination in the target species.
Because of resource limitations, some States may choose to conduct a risk
assessment using screening study data; however, this approach is not
recommended because a valid statistical analysis cannot be performed on a
single composite sample. If a reported analyte concentration is close to the SV
but does not exceed the SV, the State should reexamine historic data on water,
sediment, and fish tissue contamination at the site, and evaluate data on
laboratory performance. If these data indicate that further examination of the site
is warranted, the State should initiate a Tier 2, Phase I, intensive study to verify
the magnitude of the contamination.
Because replicate composite samples are not required as part of a screening
study, estimating the variability of the composite target analyte concentration at
any site is precluded. The following procedure is recommended for use by
9-1
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9. DATA ANALYSIS AND REPORTING
States for analysis of the individual target analyte concentration for each
composite sample from reported laboratory data (see Section 8.3.3.3)
• A datum reported below the method detection limit (MDL), including a datum
reported as not detected (i.e., ND, no observed response) should be
assigned a value of one-half the MDL.
• A datum reported between the MDL and the method quantitation limit (MQL)
should be assigned a value of the MDL plus one-half the difference between
the MQL and the MDL
• A datum reported at or above the MQL should be used as reported.
This approach is similar to that published in 40 CFR Parts 122, 123, 131, and
132—Proposed Water Quality Guidance for the Great Lakes System.
If resources permit and replicate composite samples are collected.at a suspected
site of contamination, then a State may conduct a statistical analysis of
differences between the mean target analyte concentration and the SV, as
.described in Section 9.1.2.
9.1.2 Intensive Studies
The primary objectives of Tier 2 intensive studies are to confirm the findings of
the screening study by assessing the magnitude and geographic extent of the
contamination in various size classes of selected target species. The EPA Office
of Water recommends that States collect replicate composite samples of three
size classes of each target species in the study area to verify whether the mean
target analyte concentration of replicate composite samples for any size class
exceeds the SV for any target analyte identified in the screening study. The
statistical approach for this comparison is described in Section 6.1.2.7.
The following procedure is recommended for use by States in calculating the
mean arithmetic target analyte concentration from reported laboratory data (see
Section 8.3.3.3.3).
• Data reported below the MDL, including data reported as not detected (i.e.,
ND, no observed response) should be assigned a value of one-half the MDL.
• Data reported between the MDL and the MQL should be assigned a value
of the MDL plus one-half the difference between the MQL and the MDL.
• Data reported at or above the MQL should be used as reported.
This approach is similar to that published in 40 CFR Parts 122, 123, 131, and
132—Proposed Water Quality Guidance for the Great Lakes System.
9-2
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9. DATA ANALYSIS AND REPORTING
Secondary objectives that may be assessed as part of Tier 2 intensive studies
can include defining the geographical region where fish contaminant concentra-
tions exceed screening values (SVs); identifying geographical distribution of
contaminant concentrations; and, in conjunction with historical data or future data
collection, assessing changes in fish contaminant concentrations over time. The
statistical considerations involved in comparing fish contaminant levels measured
at different locations or times are discussed in Appendix M.
State staff should consult a statistician in interpreting intensive study tissue
residue results to determine the need for additional monitoring, risk assessment,
and issuance of a fish or shellfish consumption advisory. Additional information
on risk assessment, risk management, and risk communication procedures will
be provided in later volumes in this guidance series.
9.2 DATA REPORTING
9.2.1 State Data Reports
State data reports should be prepared by the fish contaminant monitoring
program manager responsible for designing the screening and intensive studies.
Summaries of Tier 1 screening study data should be prepared for each target
species sampled at each screening site. For Tier 2 intensive studies (Phase I
and Phase II), data reports should be prepared for each target species (by size
class, as appropriate) at each sampling site within the waterbody under
investigation (see Section 6.1.2). Screening and intensive study data reports
should include, at a minimum, the information shown in Figure 9-2.
9.2.2 Reports to the National Fish Tissue Data Repository
The EPA Office of Science and Technology within the Office of Water has estab-
lished a NFTDR. The NFTDR is a collection of fish and shellfish contaminant
monitoring data gathered by various Federal, State, and local agencies. The
objectives of the NFTDR are to:
Facilitate the exchange of fish and shellfish contaminant monitoring data
nationally by improving the comparability and integrity of the data
• Encourage greater cooperation among regional and State fish advisory
programs
• Assist States in their data collection efforts by providing ongoing technical
assistance.
The NFTDR is currently part of the EPA's Ocean Discharge Evaluation System
(ODES) database, a primary source for maintaining, retrieving, and analyzing
freshwater, estuarine, and marine data. The EPA Office of Water selected the
ODES database to serve as a national repository for fish and shellfish
contaminant monitoring data for both inland and coastal waters. Unfortunately,
9-3
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9. DATA ANALYSIS AND REPORTING
Study identification (e.g., project number, title, and study type)
Program manager
Sampling site name
Latitude (in degrees, minutes, and seconds)
Longitude (in degrees, minutes, and seconds)
Type of waterbody (lake, river, estuary, etc.)
Name of waterbody
Sampling date (e.g., DD, MM, YY)
Sampling time (e.g., HH, MM in a 24-h format)
Sampling gear type used (e.g., dredge, seine, trawl)
Sampling depth
Scientific name of target species
Common name of target species
Composite sample numbers
Number of individuals in each composite sample
Number of replicate composite samples
Predominant characteristics of specimens used in each composite sample
- Predominant life stage of individuals in composite
- Predominant sex of individuals in composite (if applicable)
- Average age of individuals in composite (if applicable)
- Average body length or size (mm)
- Description of edible portion (tissue type)
(continued)
Figure 9-1. Recommended data reporting requirements for screening
and intensive studies.
9-4
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9. DATA ANALYSIS AND REPORTING
Analytical methods used (including method for lipid analysis)
Method detection and quantitation limits for each target analyte
Sample cleanup procedures (e.g., additional steps taken to further purify the
sample extracts or digestates)
Data qualifiers (e.g., additional qualifying information about the
measurement)
Percent lipid (wet weight basis) in each composite sample
For each target analyte in each composite sample:
- Total wet weight of composite sample (g) used in analysis
- Measured concentration (wet weight basis) as reported by the laboratory
(see Section 8.3.3.3.3)
- Units of measurement for target analyte concentration
- Evaluation of laboratory performance (i.e., description of all QA and QC
samples associated with the sample(s) and results of all QA and QC
analyses)
In screening studies with only one composite sample for each target
species, the State should provide for each target analyte a comparison of
reported concentration with selected SV and indication of whether SV was
exceeded (see Section 9.1.1).
In intensive studies, for each target analyte in each set of replicate
composite samples, the State should provide
- Range of target analyte concentrations for each set of replicate
composite samples
- Mean (arithmetic) target analyte concentration for each set of replicate
composite samples (see Section 9.1.2)
- Standard deviation of mean target analyte concentration
- Comparison of target analyte arithmetic mean concentration with selected
SV and indication of whether SV was exceeded.
Figure 9-1 (continued)
9-5
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9. DATA ANALYSIS AND REPORTING
ODES has not evolved into a widely used database and there is relatively little
fish and shellfish contaminant monitoring data currently stored in the NFTDR.
To make this database more accessible, EPA intends to modify the existing
NFTDR and incorporate it as a major prototype during the modernization (Phase
III) of the STORET database. During prototype development, EPA will use
actual fish contaminant monitoring data in ODES to identify needed data fields,
to test the data structure, and to develop the necessary data analysis programs
in the STORET database. During 1996, EPA intends to completely convert the
NFTDR to a STORET-based fish contaminant monitoring database. The primary
benefit of including the NFTDR as a subset of STORET is that one platform will
be able to store both water quality data and biological data, such as fish and
shellfish contaminant monitoring data. Existing data sets would be able to easily
migrate to the new STORET system when it is completed in 1997.
State, regional, and local agency staff may obtain more information by writing to
National Fish Tissue Data Repository
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
9-6
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10. LITERATURE CITED
SECTION 10
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10-35
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APPENDIX A
USE OF INDIVIDUAL SAMPLES IN FISH
CONTAMINANT MONITORING PROGRAMS
-------
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APPENDIX A
APPENDIX A
USE OF INDIVIDUAL SAMPLES IN FISH
CONTAMINANT MONITORING PROGRAMS
The use of composite samples is often the most cost-effective method for esti-
mating average tissue concentrations of analytes in target species populations
to assess chronic human health risks. However, there are some situations in
which individual sampling can be more appropriate from both ecological and risk
assessment perspectives. Individual sampling provides a direct measure of the
range and variability of contaminant levels in target fish populations. Information
on maximum contaminant concentrations in individual fish is useful in evaluating
acute human health risks. Estimates of the variability of contaminant levels
among individual fish can be used to ensure that studies meet desired statistical
objectives. For example, the population variance of a contaminant can be used
to estimate the sample size needed to detect statistically significant differences
in the mean contaminant concentration compared to the contaminant screening
values. Finally, the analysis of individual samples may be desirable, or
necessary, when the objective is to minimize the impacts of sampling on certain
vulnerable target populations, such as predators in headwater streams and
aquatic turtles, and in cases where the cost of collecting enough individuals for
a composite sample is excessive.
Analyzing individual fish incurs additional expenses, particularly when one
considers that a number of individual analyses are required to achieve measure-
ments of a reasonable statistical power. However, the recommendation that
States archive the individual fish homogenates from which composite samples
are prepared for both screening and intensive studies (see Section 6.1.1.6)
would make it possible to perform individual analyses where needed without
incurring additional sampling costs.
Individual analysis is especially well-suited for intensive studies, in which results
from multiple stations and time periods are to be compared. The remainder of
this appendix discusses how the sampling design might be affected by analyzing
individual rather than composite samples and how contaminant data from
individuals versus composites might be used in risk assessments.
A.1 SAMPLING DESIGN
There are seven major components of the sampling design for a fish or shellfish
monitoring program: site selection, target species, target analytes, target analyte
screening values (SVs), sampling time, sampling type and size class, and repli-
cate samples. Of these, only the number of replicate samples and possibly the
A-3
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APPENDIX A
target species would be expected to differ if individual samples were analyzed
rather than composites. Target species becomes a limiting factor when individ-
uals of the target species are not large enough to provide adequate tissue mass
for all the required chemical analyses.
The five factors that determine the optimal number of fish or shellfish to analyze
are presented in Section 6.1.2.7. Briefly, the five factors are:
• Cost components
• Minimum detectable difference between site-specific mean target analyte con-
centration and SV
• Level of significance
• Population variance
• Power of the hypothesis test
Each of these characteristics will be examined in detail for the collection and
analysis of individual samples.
A.1.1 Cost Components
The cost of obtaining contaminant data from individual fish or shellfish is
compared to the cost of obtaining contaminant data from composite samples in
Table A-1. These costs are dependent on the separate costs of collecting,
preparing, and analyzing the samples.
Typically, the cost of collecting individual samples will be less than that of
collecting composite samples when the target species is scarce or difficult to
capture. The cost of collecting individuals may not be a factor if the sample
Table A-1 Relative Cost of Obtaining Contaminant Data from
Individual Versus Composite Samples
Relative cost
Cost component
Collection
Preparation
Analysis
Composite samples
Moderate to high
Very low to moderate
Low to moderate
Individual samples
Low to moderate
Very low to low
Moderate to high
A-4
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APPENDIX A
collection method used typically allows for the collection of a large number of
individuals in a short period of time. In some situations, seines or gill nets might
have this characteristic. Also, in estuaries, coastal water, or large lakes where
productivity is high, the additional cost of collecting large numbers of individuals
for composite sampling may be minimal compared to the effort expended for col-
lecting individual samples.
The cost of preparing individual samples for analysis is typically lower than either
the costs of collection or analysis'. Generally, the cost of preparing composite
samples for analysis will be greater than that of preparing individual samples.
Sample preparation procedures can range in complexity from the grinding of
whole fish to delicate and time-consuming operations to resect specific tissues.
Costs of composite sampling depend largely on the number of individuals
required per composite sample and the number of replicate composite samples
required to achieve the desired statistical power; however, these costs can be
somewhat controlled (see Section 6.1.2.7).
The cost of analyzing individual samples is also typically higher than the cost of
analyzing composite samples. The cost differential between the two approaches
is directly correlated to the cost for the analysis of a single sample. For some
intensive studies, the number of target analytes exceeding the SV is small, so
few analyses are required. In these cases, the relative costs between the two
approaches may not differ greatly if the number of samples analyzed using the
two different approaches is similar (e.g., three to five samples). A sampling
design with such a small number of individual samples would be appropriate only
if the expected mean target analyte concentration was much greater than the
SV.
A.1.2 Minimum Detectable Difference
The difference between the mean target analyte concentration at a site and the
SV will not often be known before the screening study has been performed. The
minimum detectable difference between the mean concentration and the SV will
depend on the level of significance (see Section A.1.3), population variance
(Section A.1.4), and the number of replicates collected. In practice, the sample
size is often determined by establishing the minimum detectable difference prior
to the study according to the objectives of the project. For an SV that has not
been multiplied by an uncertainty factor, the cost of detecting a 10 percent
difference may be warranted. The issue of minimum detectable difference is
discussed in greater detail in Section A.1.5.
A.1.3 Level of Significance
The level of significance (LS) refers to the probability of incorrectly rejecting the
null hypothesis, that there is no difference between the mean target analyte con-
centration and the SV. This probability is also called Type I error. The LS can
be thought of as the chance of a "false positive" or of detecting a difference that
does not exist. The LS affects the sampling design by modifying the required
A-5
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APPENDIX A
power (thus impacting the sample size) of the statistical test to detect a signifi-
cant difference between the mean target analyte concentration and the SV (see
Section A.1.5). A typical LS used in biological sampling is 0.05. In some cases,
an LS other than 0.05 could be appropriate. If the ramifications of a statistically
significant difference are severe, a more conservative LS (e.g. 0.01) might be
used. On the other hand, if the statistical test is being conducted to identify
whether additional sampling should be performed (i.e., a screening survey), then
a less conservative LS (e.g. 0.10) might be used.
A.1.4 Population Variance
The variability in target analyte concentrations within a given fish or shellfish
population is a critical factor in determining how many individual samples to
collect and analyze. The population variance directly affects the power of the
statistical test to detect a significant difference between the mean target analyte
concentration and the SV (see Section A.1.5) by impacting the sample size. The
population variance may not be known prior to sampling, but it can be estimated
from similar data sets from the same target species, which could in many cases
be obtained by analyzing individual fish homogenates if these have been
archived as recommended in Section 6.1.1.6. In using historical data to estimate
population variance, it is important to consider contaminant data only from
individual fish or shellfish of the same species. By its very nature, a data set
consisting of replicate composite samples tends to smooth out the variability
• inherent in a group of individual organisms. An extreme example of this
phenomenon was presented by Fabrizio et al. (1995) in a study on procedures
for compositing fish samples. They used computer simulations to predict PCB
concentrations in composite samples of striped bass that had previously been
analyzed individually. The predicted variance in these concentrations in the
composite samples was approximately 20 percent of the variance obtained from
individual analyses.
A.1.5 Power of Statistical Test
Another critical factor in determining the sample size is the power of the statis-
tical test, that is, the probability of detecting a true difference between the mean
target analyte concentration and the SV. Because of its profound influence on
sample size, it is the power of the test that may ultimately control whether the
objectives of the survey are met. The effect of joint consideration of the desired
power, the population variance, and the minimum detectable difference on the
sample size is described by the following formula (Steel and Torrie, 1980):
A-6
-------
APPENDIX A
(Za+Zp)22o2
II —"mil .11 Him ^^^^^^
82
where
n = sample size
Za = Z statistic for Type I error (a)
Zg = Z statistic for Type II error (P)
cr = population variance (estimated from historical data)
8 = minimum detectable difference between mean target analyte
concentration and SV.
Recall that the Type I error is equal to the LS, and the value is generally
between 0.01 and 0.10. Type II error is the probability of accepting the null
hypothesis (that there is no difference between the mean target population
concentration and the SV) when it is actually false. This type of error can be
thought of as the chance of a "false negative," or not detecting a difference that
does in fact exist. The complement of Type II error (1-p) is the power of the
statistical test.
The above equation for determining sample size was solved for powers ranging
from 0.5 to 0.9 (50 to 90 percent; Figure A-1) assuming an LS of 0.05. The
values for a (standard deviation) and 6 were set relative to the SV. A similar
exercise was performed in Section 6.1.2.7 and two examples were provided. In
example A, both the standard deviation and minimum detectable difference were
set to 0.5 SV. Example A corresponds to a ratio of 1 on the x-axis of Figure
A-1. Applying example A to the collection of individual fish, the recommended
sample size would range from approximately 6 individual samples for a power
of 50 percent to 18 individual samples for a power of 90 percent (Figure A-1).
In example B, the standard deviation was set to 1.0 SV, while the minimum
detectable difference was kept at 0.5 SV. Example B corresponds to a ratio of
2 on the x-axis of Figure A-1. Applying example B to the collection of individual
samples, the sample size would have to be almost 40 individual samples to
achieve even a modest statistical power (i.e., 70 percent).
It is common to set the power of the statistical test to at least 80 percent
(Fairweather, 1991). Figure A-1 indicates that, to achieve a statistical power of
80 percent using the variability assumptions in examples A and B, 13 and 50 fish
would have to be collected, respectively. The estimated sample sizes for
individual fish or shellfish is similar to those calculated for composite samples
(see Section 6.1.2.7). For example A as applied to composite samples, 12 to
18 fish would have to be collected. For example B as applied to composite
samples, 30 to 50 fish would have to be collected. Thus, the cost of collecting
the fish to achieve a power of 80 percent would not be significantly different for
composite versus individual samples (see Section A.1.1). The number of
A-7
-------
APPENDIX A
(u)
A-8
-------
APPENDIX A
analyses, however, would be considerably less for composite samples (3 to 10
analyses of composite samples versus 13 or 50 analyses of individual samples).
Figure A-1 also indicates that 10 or fewer individual fish or shellfish should be
analyzed only if the ratio of the standard deviation to the minimum detectable
difference is 0.85 or less. For ratios less than 0.5, the effect of sample size on
the statistical power is minor. If the expected mean target analyte concentration
is many times greater than the SV, it may not be necessary to allocate resources
toward the collection and analysis of more than a minimum number (e.g., three
to five samples) of individual fish or shellfish.
A.2 USE OF CONTAMINANT DATA FROM INDIVIDUAL FISH/SHELLFISH
IN RISK ASSESSMENTS
Target analyte concentrations in composite samples represent averages for
specific target species populations. The use of these values in risk assessments
is appropriate if the objective is to estimate the average concentration to which
consumers of the target species might be exposed over a long period of time.
The use of long exposure durations (e.g., 30 to 70 years) is typical of the
assessment of carcinogenic target analytes, the health effects of which may be
manifested over an entire lifetime (see Volume II of this series). Target analytes
that produce noncarcinogenic effects, on the other hand, may cause acute
effects to human health over a relatively short period of time on the order of
hours or days. The use of average contaminant concentrations derived from the
analysis of composite samples may not be protective against acute health effects
because high concentrations in an individual organism may be masked by lower
concentrations in other individuals in the composite sample. Contaminant data
from individual samples permits the use of alternative estimates of contaminant
concentration for a group of fish or shellfish (e.g., maximum). Therefore, the
decision whether to collect and analyze individual fish or shellfish may depend
on the target analytes included in the monitoring program.
EPA has recommended that 25 target analytes be included in screening studies
(see Section 4). All of the target analytes except PCBs, PAHs, and dioxins/
furans have reference doses for noncarcinogenic health effects, although the
carcinogenic risk is likely to be greater than the noncarcinogenic risk for eight
other target analytes (see Table 5-2). EPA's draft reassessment of the health
effects of 2,3,7,8-TCDD (dioxin) indicated that this chemical may also pose a
significant noncarcinogenic health risk in some cases (U.S. EPA, 1994).
A.3 EXAMPLE CASE STUDY
The presentation of a case study will illustrate some of the sample size and data
interpretation issues discussed in Sections A.1 and A.2, respectively. A State
has prepared a composite sample of target species A from a particular water-
body of concern. This composite sample was analyzed for all 25 target analytes
listed in Table 4-1. Of the 25 target analytes, only cadmium was detected at a
concentration exceeding the SV (10 ppm) for cadmium listed in Table 5-2.
A-9
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APPENDIX A
Cadmium was detected at 20 ppm, twice the SV calculated for cadmium.
Because the SV for at least one target analyte was exceeded, an intensive study
was warranted. The State decided to collect and analyze individual fish in the
intensive study for the following reasons: (1) the cost of collecting individual fish
is less than the cost of collecting fish for composites, (2) the analytical costs for
analyzing cadmium are relatively low (<$50 sample), and (3) the cadmium
concentrations in individual fish should more accurately reflect the potential acute
(noncarcinogenic) health risk from cadmium than the mean cadmium
concentration derived from composite samples.
The first issue the State must decide is how many individual fish to collect and
analyze. The important factors in this decision are the minimum detectable
difference the State wishes to test and the variability in cadmium concentrations
within the target species population. The first factor can be obtained from the
results of the screening survey. The Slate wishes to test whether the difference
between the concentration detected in the single composite sample (20 ppm)
and the SV (10 ppm) is significant. This assumes that the mean cadmium
concentration for the individual is also 20 ppm. The expected standard deviation
(8 ppm) was obtained from a previous investigation performed on individuals of
the target species and was equal to 0.8 of the SV (10 ppm). Using Figure A-1,
it can be seen that, for a ratio of standard deviation (0.8 x SV) to detectable
difference (1.0 x SV) of 0.8, the sample size necessary to achieve a statistical
power of 80 percent would be eight fish.
The State determines that the mean cadmium concentration of eight individual
fish of the target species is 30 ppm and the standard deviation is equal to the
predicted value of 8 ppm. The State performs a Mest to determine if the mean
concentration is significantly greater than the SV. As described in Section
6.1.2.7, the statistic
(mean - SV)/standard deviation
has a f-distribution with n-1 degrees of freedom. For this example, the t statistic
is 2.5 ([(30-10)78] with 7 degrees of freedom. This value exceeds the critical
t-statistic (1.895) for a one-tailed LS of 0.05. Therefore, the State determines
that the mean cadmium concentration for these eight individual fish of the target
species is significantly greater than the SV and a risk assessment is performed.
A.4 REFERENCES
Fabrizio, M.C., A.M. Frank, and J.F. Savino. 1995. Procedures for formation of
composite samples from segmented populations. Environmental Science
and Technology 29(5):1137-1144.
Fairweather, P.G. 1991. Statistical power and design requirements for environ-
mental monitoring. Aust. J. Freshwater Res. 42:555-567.
A-10
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APPENDIX A
Steel, R.G.D., and J.H. Torrie.
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EPA/540/1-89/002.
U.S. EPA (U.S. Environmental Protection Agency). 1994. Health Assessment
for 2,3,7,8-TCDD and Related Compounds. Public Review Draft.
EPA/600/EP-92/001.
A-11
-------
-------
APPENDIX B
FISH AND SHELLFISH SPECIES FOR WHICH STATE
CONSUMPTION ADVISORIES HAVE BEEN ISSUED
-------
-------
APPENDIX B
APPENDIX B
FISH AND SHELLFISH SPECIES FOR WHICH STATE CONSUMPTION
ADVISORIES HAVE BEEN ISSUED
FRESHWATER FINFISH SPECIES FOR WHICH STATE
CONSUMPTION ADVISORIES HAVE BEEN ISSUED
AL catfish (unspecified), fish species (unspecified), bigmouth buffalo, brown
bullhead, channel catfish, white bass
AK no consumption advisories
AS no consumption advisories
AZ fish species (unspecified)
AR fish species (unspecified)
CA goldfish, Sacramento blackfish, brown bullhead, crappie (unspecified),
hitch, common carp, largemouth bass, smallmouth bass, channel catfish,
white catfish, rainbow trout, croaker (unspecified), orangemouth corvina,
sargo, tilapia (unspecified), squawfish, sucker (unspecified), trout (unspeci-
fied), fish species (unspecified)
CO rainbow trout, yellow perch, northern pike, walleye, smallmouth bass,
iargemouth bass, black crappie, kokanee salmon, channel catfish, trout
(unspecified), fish species (unspecified)
CT common carp and fish species (unspecified)
DE white catfish, channel catfish, fish species (unspecified)
DC . channel catfish, common carp, American eel
FL largemouth bass, gar, bowfin, warmouth, yellow bullhead, Mayan cichlid,
oscar, spotted sunfish
GA common carp, largemouth bass, catfish (unspecified), fish species (unspe-
cified)
GU no consumption advisories
B-3
-------
APPENDIX B
HI no consumption advisories
ID no consumption advisories
IL lake trout, coho salmon, Chinook salmon, brown trout, common carp,
catfish (unspecified), bigmouth buffalo, channel catfish, flathead catfish,
smallmouth buffalo, shovelnose sturgeon, bluegill, crappie (unspecified),
freshwater drum, largemouth bass, spotted bass, alewife
IN common carp, catfish (unspecified), coho salmon, brown trout, lake trout,
Chinook salmon, channel catfish, fish species (unspecified)
IA channel catfish, common carp, carpsucker (unspecified), fish species
(unspecified)
KS buffalo (unspecified), catfish (unspecified), common carp, freshwater drum,
sturgeon (unspecified), carpsucker (unspecified)
KY channel catfish, paddlefish, white bass, common carp, fish species
(unspecified)
LA bass (unspecified), fish species (unspecified)
ME fish species (unspecified)
MD channel catfish, American eel, black crappie, common carp, bullhead
(unspecified), sunfish (unspecified)
MA brown trout, yellow perch, white sucker, American eel, smallmouth bass,
largemouth bass, lake trout, channel catfish, brown bullhead, common
carp, white catfish, fish species (unspecified)
Ml common carp, rock bass, crappie (unspecified), yellow perch, largemouth
bass, smallmouth bass, walleye, northern pike, muskellunge, sauger, white
bass, longnose sucker, white perch, carpsucker (unspecified), brown
bullhead, bullhead (unspecified), bluegill, freshwater drum, sturgeon
(unspecified), brown trout, ciscowet, lake trout, coho salmon, Chinook
salmon, splake, catfish (unspecified), rainbow trout, brook trout, sucker
(unspecified), gizzard shad, freshwater drum, white sucker, lake whitefish
MN yellow perch, brown bullhead, black bullhead, yellow bullhead, quillback
carpsucker, brown trout, brook trout, lake trout, Chinook salmon, ciscowet,
walleye, northern pike, brook trout, muskellunge, splake, smallmouth bass,
largemouth bass, rock bass, white bass, rainbow trout, white sucker, tulli-
bee, bluegill, black crappie, white crappie, shorthead redhorse, silver
redhorse, common carp, smallmouth buffalo, redhorse sucker, sauger,
bigmouth buffalo, channel catfish, lake whitefish, freshwater drum,
pumpkinseed, chub bloater, lake herring, flathead catfish, bowfin
B-4
-------
APPENDIX B
MS fish species (unspecified), catfish (unspecified), buffalo (unspecified)
MO sturgeon (unspecified), common carp, channel catfish, buffalo (unspeci-
fied), flathead catfish, sucker (unspecified), paddlefish, catfish (unspeci-
fied), redhorse, freshwater drum
MT fish species (unspecified)
NE common carp, channel catfish
NV fish species (unspecified)
NH fish species (unspecified)
NJ striped bass, American eel, white perch, white catfish, fish species (unspe-
cified)
NM white crappie, channel catfish, common carp, brown trout, river
carpsucker, kokanee salmon, largemouth bass, bluegill, white bass,
walleye, white sucker, yellow perch, black bullhead, black crappie, bass
(unspecified), crappie (unspecified), rainbow trout, longnose dace, walleye,
northern pike, trout (unspecified), carpsucker (unspecified), bullhead
(unspecified), black bass
NY common carp, lake trout, brown trout, yellow perch, smallmouth bass,
splake, American eel, goldfish, striped bass, white perch, bluefish,
largemouth bass, brown bullhead, white catfish, walleye, rainbow smelt,
tiger muskellunge, white sucker, northern pike, Chinook salmon, coho
salmon, rainbow trout
NC largemouth bass, fish species (unspecified)
ND walleye, white bass, yellow perch, northern pike, bigmouth buffalo,
common carp, crappie (unspecified), bullhead (unspecified), white sucker,
channel catfish, goldeye, Chinook salmon, sauger, carpsucker (unspeci-
fied), sunfish (unspecified), smallmouth bass
OH common carp, catfish (unspecified), white bass, sucker (unspecified), fish
species (unspecified)
OK channel catfish, largemouth bass, fish species (unspecified)
OR fish species (unspecified), crayfish
PA white sucker, white perch, common carp, American eel, channel catfish,
goldfish, largemouth bass, green sunfish, quillback carpsucker, white bass,
lake trout, walleye, smallmouth bass, shorthead redhorse, sucker (unspeci-
fied), fish species (unspecified)
-------
APPENDIX B
PR no fish consumption advisories
Rl striped bass
SC fish and shellfish species (unspecified)
SD no fish consumption advisories
TN catfish (unspecified), largemouth bass, crappie (unspecified), common
carp, rainbow trout, striped bass, sauger, white bass, smallmouth buffalo,
fish species (unspecified)
TX catfish (unspecified), fish species (unspecified)
UT fish species (unspecified)
VT brown trout, lake trout, walleye
VA fish species (unspecified)
VI no fish consumption advisories
WA no fish consumption advisories
WV channel catfish, brown bullhead, common carp, sucker (unspecified), fish
species (unspecified)
Wl lake trout, coho salmon, Chinook salmon, brown trout, common carp,
catfish (unspecified), splake, rainbow trout, brook trout, lake trout,
ciscowet, northern pike, white bass, white sucker, walleye, yellow perch,
mu,skellunge, flathead catfish, freshwater drum, channel catfish, bullhead
(unspecified), bluegill, black crappie, crappie (unspecified), rock bass,
smallmouth bass, redhorse (unspecified), largemouth bass, lake sturgeon,
buffalo (unspecified), fish species (unspecified)
WY no fish consumption advisories
Source: RTI, 1993. National Listing of State Fish and Shellfish Consumption
Advisories and Bans. (Current as of July 22,1993.) Research Triangle
Institute, Research Triangle Park, NC.
B-6
-------
APPENDIX B
ESTUARINE/MARINE FISH AND SHELLFISH SPECIES FOR WHICH STATE
CONSUMPTION ADVISORIES HAVE BEEN ISSUED
AL no consumption advisories
AK no consumption advisories
AS fish and shellfish species (unspecified)
CA white croaker, black croaker, corbina, surf perch, queenfish, sculpin,
rockfish, kelp bass, striped bass, fish and shellfish species (unspecified)
CT striped bass, bluefish
DE no consumption advisories
DC channel catfish, American eel
FL shark (unspecified)
QA no consumption advisories
GU no consumption advisories
HI no consumption advisories
LA fish and shellfish species (unspecified)
ME no consumption advisories
MD channel catfish, American eel
f
MA American eel, flounder, American lobster, bivalves (unspecified), fish
species (unspecified)
MS no consumption advisories
NH no consumption advisories
NJ striped bass, bluefish, American eel, white perch, white catfish, blue crab,
fish and shellfish species (unspecified)
NY American eel, striped bass, bluefish, white perch, white catfish, rainbow
smelt, Atlantic needlefish, blue crab
NC fish species except herring, shad, striped bass, and shellfish species
(unspecified)
—
-------
APPENDIX B
OR no consumption advisories
PA white perch, channel catfish, American eel
PR no consumption advisories
Rl striped bass, bluefish
SC fish and shellfish species (unspecified)
TX blue crab, catfish (unspecified), fish species (unspecified)
VA fish species (unspecified)
VI no consumption advisories
WA no consumption advisories
Source: RTI, 1993. National Listing of State Fish and Shellfish Consumption
Advisories and Bans. (Current as of July 22,1993.) Research Triangle
Institute, Research Triangle Park, NC.
B-8
-------
APPENDIX C
TARGET ANALYTES ANALYZED IN NATIONAL OR
REGIONAL MONITORING PROGRAMS
-------
-------
APPENDIX C
Table C-1. Target Analytes Analyzed In National or
Regional Monitoring Programs
Analyte
Monitoring program
c d1
f g h
Aluminum (Al) _ • _ _ _
Antimony lib) _*_ _ _ _ *
Arsenic"(As)"(totaO _*_*_ _*"_*_ _*
Barium (Ba) _ _ _ _ _ _ ___ .
Beryllium (Be) • _ _ _
15admium~(Cdj~ ~ _"*_*_ _~* _ * _____ ]*
Chromium (Cr) • •_ _*_*_ _ _
Copper~(Cu)~ " _"• _ _ _• _ • _____ '•
Cyanide • _ _ _
ironTFeJ ..... _ _ _ ___•
Lead"(Pb) _*_*_ _"*_*_ _*
Manganese (Mn) • •
"Mercury ^Hg) _•_ _•_*_*_ _*_
Methylmercury • • .
Molybdenum _
Nrckei"(Ni)" '" _"•_*_ _____ *
") _*_*_ _ _*_ _*_
" _ _ _ _ _•_ __
sver (Ag) __*_ _ _ _^_ _ _
fhaiiium~(fi)" _*_ __ __*___
~fin"(Sn)^ __ ___*___
TributylTin __*______
Vanadium _
" ~' _ V _ J» _ •
-' j. ;, _ :. r ' ' - ' "" . '' ''
Aldrin _•_ _ _•_•_ _*_*
Butachlor
Chlordane (cis & trans) • • •• *2 *_*
Chlorpyrifos • •
Danitol *
(continued)
C-3
-------
APPENDIX C
Table C-1 (continued)
Monitoring program
Analyte
8
i
DCPA (chlorthal) • *
~ flotaT)V ¥ V
i^'-f DEJ" ~"" ~ ~"" ~ V "" 9: V V
(M'-TDE)" ~ V ~~lT~" " ~ V ~" • V ~ V ~ V
"I""""~~"~~~" I"" I I ~_ "•" I" •"" - - - v ~"" ~ "•'
4,4'-DDE _"*_"•" *" •" *"•"•"""•'
I" l±??iZ~I]~""""~I~~~] I"" I I"" I "• I" •"!"" I "• I"" I "•
f:l:9p][ _V "•" "" V "• V "V~V"
Demeton Q ""
Dicofol V V~V
Dieldrin 9 V V ~ V ¥ '^~'^"~'^"
Diphenyl disulfide V
^^^••••••^^•.•.•.^^•..••..^•._ __^^—>»,^^_.^^^^^_^_ ^^ — ... _^ __ ^^^ ^^ ^^ ^^
Endosulfan ~ ~~
a-Endosulfan (endosulfan I) • •
S-Endosulfan (endosulfan II) V ~9
Endosulfan sulfate O 0
_ Jndrin__ ~ V —J~ ^ ~ -0~ -^ ^"
Endrin aldehyde 9 r"~
Ethyl-p-nitrophenylphenylphosphorothioate (EPN) V
Fonofos ~¥
Guthion 9
Heptachlor • V~~V ¥ V~V~V~
Heptachlor epoxide O ~V V ~9 V~V~V"
Hexachlorocyclohexane (HCH) also known
as Benzene hexachloride (BHC)
a-Hexachlorocyclohexane 9 @ • ~ V V~V~V~
B-Hexachlorocyclohexane 9 V V~V~
'————"——*————————————.—_—.«—__«^ ^ —M. _ _ __ ^
8-Hexachlorocyclohexane • 9 - -- -^-_
Y-Hexachlorocyclohexane (lindane) 9 • • • ""• V~®"~V~
Technical-hexachlorocyclohexane ~Q
Hexachlorophene 0"
Isopropalin , V ~@~
Kepone ®" ^"~
Malathion @ ~
(continued)
C-4
-------
APPENDIX C
Table C-1 (continued)
Analyte
Monitoring program
a
* 9
Methoxychlor • • • •
Mirex " •••"•• • V 0
Nitrofen •
cis-Nonachlor • • • •
trans-Nonachlor • • • 9 9
Oxychlordane • • 99
Parathion •
Toxaphene (mixture) • • • 99
Triazine herbicides 93
Trichloronate •
friiiuralin • • V
Sjage/Neofr»i Organic Compoiinds
Acenaphthene • 9 9
Acenaphthylene 9 9 9
Anthracene 9 9 9
Benzidine •
Benzo(a)anthracene 999
Benzo(a)pyrene 999
Benzo(e)pyrene 9
Benzo(b)fluoranthene 999
Benzo(k)fluoranthene 999
Benzo(g,h,i)perylene 999
Benzyl butyl phthalate 9
Biphenyl • 9 9
4-Bromophenyl ether 9
bis(2-Chloroethoxy)methane 9
bis(2-Chloroethyl)ether 9
bis(2-Chloroisopropyl)ether •
Yis(2-~EthyThexyl]phFhTlate"(B¥^ V
Chlorinated benzenes •
2-Chloronaphthalene 9
4-Chlorophenyl ether •
Chrysene 999
(continued)
C-5
-------
APPENDIX C
Table C-1 (continued)
Monitoring program
Analyte
a b c d1 e f g h i
Dibenzo(a,h)anthracene
Di-n-butyl phthalate
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
3,3'-Dichlorobenzidine
Siethyl phthalate
2,6-Dimethylnaphthalene • •
2,3,5-Trimethylnaphthalene G
Dimethyl phthalate •
2,4-Dinitrotoluene •
2,6-Dinitrotoluene •
Di-n-octyl phthalate •
1,2-Diphenylhydrazine •
bis(2-EthyThexyi) phthaTate •
Fluoranthene • • •
Fluorene • • •
Heptachlorostyrene •
Hexachlorostyrene •
Hexachlorobenzene • • • • • • •
Hexachlorobutadiene • •
Hexachlorocyclopentadiene • •
Hexachloroethane * •
lndeno(1,2,3-cd)pyrene • c
Isophorone •
4,4'-Methylene bis(N,N'-dimethyl)aniline •
1-Methylnaphthalene •
2-MethylnaphthaFene •
1 -Methylphenanthrene •
Naphthalene • • •
Nitrobenzene •
N-Nitroso-di-n-butylamine •
N-Nitrosodimethylamine •
(continued)
C-6
-------
APPENDIX C
Table C-1 (continued)
Monitoring program
Analyte
a b c d1 e f
h i
N-Nitrosodiphenylamine •
N-Nitrosodipropylamine •
Octachlorostyrene • • "
PAHs (polycyctic aromatic hydrocarbons) *3
PBBs (polybrominated biphenyls) •
RGBs (polychlorinated biphenyls) • • • •
Aroclor 1016 (mixture) •
Aroclor 1221 (mixture) • _ _ _
Aroclor 1232 (mixture) • _
Aroclor 1242 (mixture) •
Aroclor 1248 (mixture) •
Aroclor 1254 (mixture) • _
Aroclor 1260 (mixture) • _ _ _
Selected individual congeners •
Pentachloroanisole (PCA) • _
Pentachlorobenzene •
Pentachloronitrobenzene (PCNB) •
—.«. «»_— — ——
Pentachlorophenyl methyl ether •
Pentachlorophenyl methyl sulfide •
Pentachlorostyrene •
Perthane •
Perylene •
Phenanthrene • •
Pyrene • •
..*.» — •••-«—MM«W_>»MW«—•••«—«••.»• — ——«— — M« •..•••— — —.» — ••.••••••. .MM• — — —— — —^— i— —' ^— —• — ——.— ^^-
Terphenyl •
1,2,3,4-Tetrachlorobenzene •
1,2,3,5-Tetrachlorobenzene •
1,2,4,5-Tetrachlorobenzene •
1,2,3-Trichlorobenzene •
1,2,4-Trichlorobenzene • •
1,3,5-Trichlorobenzene •
Triphenyl phosphate
• •
__
„
._
(continued)
C-7
-------
APPENDIX C
Table C-1 (continued)
Monitoring program
Analyte
e d1
f g
1,2,3,7,8-Pentachlorodibenzodioxin (PeCDD) •
2,3,7,8^trachlorodibenzodioxin ffcDD) ~ V ~9 V ~ V V
1,2.3,4,6,7,8-Heptachlorodibenzodioxin (HpCDD) V
1,2,3,4,7,8-Hexachlorodibenzodioxin (HxCDD) 9
1,2,3,6,7,8-Hexachlorodibenzodioxin (HxCDD) •
1,2,3,7',8,»-Hexachlorodib"enzodioxin (HxCDD) V
i>^ttibfcirati<* ^l^^lfv -''-"', '-'•''- %, — ^'"-' , ,
--^i*-i'-:o--:i.-V;|_i,.ai|tj|Llxtjj.r _-1±j~-5~&?>- ^1.— — --- f ' •" .•!*-•;. —^
1,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCDF) •
1,2,3,4,7,8,9-HeptachForodibenzofuran (HpCDF) •
1,2,3,4,7,8-Hexachlorodibenzofuran (Hxcb~F) V
1,2,3,6,7,8-Hexachlorodibenzofuran (HxCDF) ~ V
1,2,3,7,8,9-Hexachlorodibenzofuran (HxCDF) O
2,3,4,6,7,8-Hexachlorodibenzofuran (HxCDF) •
•••••••^••^•••..•••^^•.•«_^^^^^_ ^_^^_ _ _ _ _ | ^ _ ^_ ^_ ^^ __ ^^ •^•B
1,2,3,7,8-Pentachlorodibenzofuran (PeCDF) •
2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) V
2,3,7,8-Tetrachlorodibenzofuran (TCDF) •
^y^t^pft^^tTpup^^; ' ' •• . '"'"„". '",, -",/
Chlorinated phenols e3
4-Chloro-3-cresol •
2-Chlorophenol •
2,4-Dichlorophenol O
2,4-DimethyIphenol ~ V
4,6-Dinitro-2-cresol 9
2-4-Dinitrophenol •
2-Nrtrophenol •
4-Nitrophenol 9
Pentachlorophenol (PCP) •
Phenol 9,
2,4,6-Triohlorophenol e .
^^|»O
Acrolein
Acrylonitrile
9
V
(c»ntinued)
Co
~o
-------
APPENDIX C
Table C-1 (continued)
Analyte
Monitoring program
a b c d1 e f g h i
Benzene •
Bromodichloromethane •
Bromoform •
Bromomethane •
Carbon tetrachloride •
Chlorobenzene •
Chloroethane •
2-Chtoroethylvinyl ether •
Chloroform •
Chloromethane •
Dibromochloromethane •
1,1 -Dichloroethane 9
1,2-Dichloroethane 9
1,1 -Dichloroethene 9
trans-1,2-Dichloroethene 9
1,2-Dichloropropane 9
cis-1,3-Dichloropropene 9
trans-1,3-Dichloropropene •
Ethylbenzene 9
Methylene chloride •
1,1,2,2-Tetrachloroethane 9
TetrachloroQthene •
Toluene •
1,1,1 -Trichloroethane •
1,1,2-Trichloroethane •
Trfchloroethene 9
Vinyl chloride •
1 Contaminants listed were monitored by at least one Great Lakes State. NOTE: Contaminants monitored
exclusively by the Canadian Province of Ontario were not included.
2 Only the cis-isomer is monitored.
3 FDA recommends method development/improvement for this analysis.
a 301 (h) Monitoring Program. Source: U.S. EPA. 1985. Bioaccumulation Monitoring Guidance: 1. Estimating
the Potential for Bioaccumulation of Priority Pollutants and 301(h) Pesticides Discharged into Marine and
Estuarine Waters. EPA 503/3-90-001. Office of Marine and Estuarine Protection, Washington, DC.
C-9
-------
APPENDIX C
Table C-1 (continued)
b Food and Drug Administration recommendations. Source: Michael Boiger, FDA, personal communication, 1990.
c National Study of Chemical Residues in Fish. Source: U.S. EPA. 1992. National Study of Chemical Residu&s
in Fish. Volumes I and II. EPA 823/R-92-008a and OOSb. Office of Science and Technology, Washington, DC.
d Great Lakes Sport Rsh Contaminant Advisory Program. Source: Hesse, J. L 1990. Summary and Analyses
of Existing Sportfish Consumption Advisory Programs in the Great Lakes Basin—the Great Lakes. Fish
Consumption Advisory Task Force, Michigan Department of Health, Lansing, Ml.
* NOAA Status and Trends Program. Source: NOAA. 1989. National Status and Trends Program for Marine
Environmental Quality-Progress Report: A Summary of Selected Data on Tissue Contamination from the First
Three Years (1986-1988) of the Mussel Watch Project. NOAA Technical Memorandum NOS OMA 49. U.S.
Department of Commerce, Rockville, MD.
' EPA National Dioxin Study. Source: U.S. EPA. 1987. National Dioxin Study. Tiers 3, 5, 6 and 7. EPA
440/4-87-003. Office of Water Regulations and Standards, Washington, DC.
0 U.S. Rsh and Wildlife Service National Contaminant Biomonitoring Program. Sources: C. J. Schmitt, J. L.
Zajicak, and P. H. Peterman, 1990, National Contaminant Biomonitoring Program: Residues of organochlorine
chemicals in U.S. freshwater fish, 1976-1984, Arch. Environ. Contam. Toxicol. 19:748-781; and T. P. Lowe, T.
W. May, W. G. Brumbaugh, and D. A. Kane, 1985, National Contaminant Biomonitoring Program: Concentrations
of seven elements in freshwater fish, 1978-1981, Arch. Environ. Contam. Toxicol. 14:363-388.
h U.S. EPA. 1991. Assessment and Control of Bioconcentratable Contaminants in Surface Waters. Draft. Office
of Water, Office of Research and Development, Washington, DC.
1 U.S. Geological Survey National Water-Quality Assessment Program. Source: J.K. Crawford and S.N. Luoma.
1993. Guidelines for Studies of Contaminants in Biological tissues for the National Water-Quality Assessment
Program. USGS Open-File Report 92-494. U.S. Geological Survey, Lemoyne, PA.
C-10
-------
APPENDIX D
PESTICIDES AND HERBICIDES RECOMMENDED
AS TARGET ANALYTES
-------
-------
APPENDIX D
5 co
tin compounds have
registered since the
Several registrations
anceled or
Some organo
been actively
mid-19
have
activ
960s
been
manufacturers discontinued
production (U.S. EPA, 1988a
f inorganic arsenic
s are used as
fungicides, inse
and icides, but regist
uses of some were supers
because of their hazard to
and other nontarget specie
I
as
.a
a
c
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1 1 _ J8 3
o ro a> . T5 •=
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-------
APPENDIX D
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D-4
-------
APPENDIX 0
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b
D-5
-------
APPENDIX D
1
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APPENDIX D
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APPENDIX D
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APPENDIX F
QUALITY ASSURANCE AND
QUALITY CONTROL GUIDANCE
-------
-------
APPENDIX F
APPENDIX F
QUALITY ASSURANCE(QA) AND
QUALITY CONTROL (QC) GUIDANCE
F.1 GENERAL QA AND QC CONSIDERATIONS
The primary objective of the specific QA and QC guidance provided in this
document is to ensure that
Appropriate data quality objectives or requirements are established prior
to sample collection and analysis
Samples are collected, processed, and analyzed according to scientifically
valid, cost-effective, standardized procedures
The integrity and security of samples and data are maintained at all times
Recordkeeping and documentation procedures are adequate to ensure the
traceability of all samples and data from initial sample collection through
final reporting and archiving, and to ensure the verifiability and defensibility
of reported results
Data quality is assessed, documented, and reported properly
Reported results are complete, accurate, and comparable with those from
other similar monitoring programs.
F.2 QA PLAN REQUIREMENTS
To ensure the quality, defensibility, and comparability of the data used to
determine exposure assessments and fish consumption advisories, it is essential
that an effective QA program be developed as part of the overall design for each
monitoring program. The QA program should be documented in a written QA
plan or in a combined Work/QA Project Plan and should be implemented strictly
throughout all phases of the monitoring program. The QA plan should include
the following information either in full or by reference to appropriate standard
operating procedures (SOPs):
1. A clear statement of program objectives
F-3
-------
APPENDIX F
2. A description of the program organization and personnel roles and
responsibilities, including responsibility for ensuring adherence to the QA
plan
3. Specification of data quality objectives in terms of accuracy, precision,
representativeness, and completeness, for data generated from each type
of measurement system
4. Detailed descriptions of field sample collection and handling procedures,
including documentation of
• Target species and size (age) class
• Sampling site locations
Target contaminants
Sampling times/schedules
• Numbers of samples and sample replication strategy
• Sample collection procedures
• Sample processing procedures, including sample identification, labeling,
preservation, and storage conditions
Sample shipping procedures
5. A detailed description of chain-of-custody procedures, including specifi-
cation of standard chain-of-custody forms and clear assignment of field and
laboratory personnel responsibilities for sample custody
6. Detailed descriptions of laboratory procedures for sample receipt, storage,
and preparation, including specification of the kinds of samples to be
prepared for analyses (e.g., composite vs. individual, whole body vs. fillet,
replicates)
7. Detailed descriptions of the analytical methods used for quantitation of
target contaminants, and percent lipid determination including
• Specification and definition of method detection limits
Method validation procedures for verification of specifications for
method accuracy, precision, and detection limits prior to analysis of field
samples
8. Detailed descriptions of methods routinely used to assess data accuracy,
precision, and completeness, including
F-4
-------
APPENDIX F
• Internal QC checks using field, reagent, or method blanks; spiked
samples; split samples; QC samples prepared from standard reference
materials; and replicate analyses
• Calibration checks
• Data quality assessments
9. Detailed descriptions of calibration procedures for all measurement
instruments, including specification of reference materials used for
calibration standards and calibration schedules
10. Detailed descriptions of preventive maintenance procedures for sampling
and analysis equipment
11. Detailed description of health and safety procedures
12. Detailed descriptions of recordkeeping and documentation procedures,
including requirements for
• Maintaining field and laboratory logs and notebooks
• Use of standard data collection and reporting forms
Making changes to original records
• Number of significant figures to be recorded for each type of data
• Units of reporting
• Routine procedures to assess the accuracy and completeness of
records
13. Detailed descriptions of data analysis procedures, including
• Statistical treatment of data
• Data summary formats (e.g., plots, tables)
14. Detailed descriptions of data management and reporting procedures,
including requirements for
• Technical reports
• QA and QC reports
Data coding procedures
Database specifications
• QA review of reported data
Data storage and archiving procedures
F-5
-------
APPENDIX F
15. Detailed descriptions of procedures for internal QC performance and/or
systems audits for sampling and analysis programs.
16. Detailed descriptions of procedures for external QA performance and/or
systems audits for sampling and analysis programs, including participation
in certified QA proficiency testing or interlaboratory comparison programs.
17. Detailed descriptions of corrective action procedures in both sampling and
analysis programs, including
• Criteria and responsibility for determining the need for corrective action
• Procedures for ensuring that effective corrective action has been taken
• Procedures for documenting and reporting corrective actions
18. A description of procedures for documenting deviations from standard
procedures, including deviations from QA or QC requirements
19. A description of the procedure for obtaining approval for substantive
changes in the monitoring program.
Guidance for addressing each of the QA or QC elements outlined above,
including a list of recommended standard reference materials and external QA
or interlaboratory comparison programs for the analyses of target analytes, is
incorporated in the appropriate sections of this guidance document.
F-6
-------
APPENDIX G
RECOMMENDED PROCEDURES FOR PREPARING
WHOLE FISH COMPOSITE HOMOGENATE SAMPLES
-------
-------
APPENDIX G
APPENDIX G
RECOMMENDED PROCEDURES FOR PREPARING WHOLE
FISH COMPOSITE HOMOGENATE SAMPLES
G.1 GENERAL GUIDELINES
Laboratory processing to prepare whole fish composite samples (diagrammed
in Figure G-1) involves
• Inspecting individual fish for foreign material on the surface and rinsing if
necessary
Weighing individual fish
Examining each fish for morphological abnormalities (optional)
Removing scales or otoliths for age determination (optional)
Determining the sex of each fish (optional)
Preparing individual whole fish homogenates
Preparing a composite whole fish homogenate.
Whole fish should be shipped on wet or blue ice from the field to the sample
processing laboratory if next-day delivery is assured. Fish samples arriving in
this manner (chilled but not frozen) should be weighed, scales and/or otoliths
removed, and the sex of each fish determined within 48 hours of sample
collection. The grinding/homogenization procedure may be carried out more
easily and efficiently if the sample has been frozen previously (Stober, 1991).
Therefore, the samples should then be frozen (<-20 °C) in the laboratory prior
to being homogenized.
If the fish samples arrive frozen (i.e., on dry ice) at the sample processing
laboratory, precautions should be taken during weighing, removal of scales
and/or otoliths, and sex determination to ensure that any liquid formed in thawing
remains with the sample. Note: The liquid will contain target analyte
contaminants and lipid material that should be included in the sample for
analysis.
G-3
-------
APPENDIX G
Log in fish samples using COC procedures
Unwrap and inspect individual fish
Weigh individual fish
Remove and archive scales and/or otolrths for age determination (optional)
Determine sex (optional); note morphological abnormalities (optional)
Remove scales from all scaled fish
Remove skin from stateless fish (e.g., catfish)
Fillet fish
Weigh fillets (g)
Homogenize fillets
Divide homogenized sample into quarters, mix opposite
quarters, and then mix halves (3 times)
Optional
Composite equal weights (g) of
homogenized fillet tissues from the
selected number of fish (200-g)
Seal and label (200-g) composite
homogenate in appropriate container(s)
and store at £-20 °C until analysis (see
Table 7-1 for recommended container
materials and holding times).
Save remainder of fillet
homogenate from each fish
Seal and label individual fillet
homogenates in appropriate
containers) and archive at
£-20 °C (see Table 7-1 for
recommended container
materials and holding times).
COC = Chain of custody.
Figure G-1. Laboratory sample preparation and handling for
whole fish composite homogenate samples.
G-4
-------
APPENDIX G
Table G-1. Recommendations for Container Materials,
Preservation, and Holding Times for Fish, Shellfish, and Turtle
Tissues from Receipt at Sample Processing Laboratory to Analysis
Analyte
Mercury
Other metals
Organics
Metals and
organics
Matrix
Tissue (whole
specimens,
homogenates)
Tissue (whole
specimens,
homogenates)
Tissue (whole
specimens,
homogenates)
Tissue (whole
specimens,
homogenates)
Sample
container
Plastic,
borosilicate
glass, quartz,
and PTFE
Plastic,
borosilicate
glass, quartz,
and PTFE
Borosilicate
glass, quartz,
PTFE, and
aluminum foil
Borosilicate
glass, quartz,
and PTFE
Storage
Holding
Preservation tlmea
Freeze at <-20 °C 28 days6
Freeze at <-20 °C 6 months0
Freeze at <-20 °C 1 year4*
Freeze at <-20 °C 28 days
(mercury; 6
months; (for
Lipids Tissue (whole
specimens,
homogenates)
Plastic,
borosilicate
glass, quartz,
PTFE
other met-
als); and 1
year (for
organics)
Freeze at <.-20 °C 1 year
PTFE = polytetrafluoroethylene; Teflon.
a Maximum holding times recommended by U.S. EPA (1995b).
b This maximum holding time is also recommended by the Puget Sound Estuary Program (1990e).
The California Department of Fish and Game (1990) and the USGS National Water Quality
Assessment Program (Crawford and Luoma, 1993) recommend a maximum holding time of 6
months for all metals, including mercury.
0 This maximum holding time is also recommended by the California Department of Fish and Game
(1990), the 301 (h) monitoring program (U.S. EPA, 1986), and the USGS National Water Quality
Assessment Program (Crawford and Luoma, 1993). The Puget Sound Estuary Program (1990)
recommends a maximum holding time of 2 years.
d This maximum holding time is also recommended by the Puget Sound Estuary Program (1990).
The California Department of Fish and Game (1990) and the USGS National Water Quality
Assessment Program (Crawford and Luoma, 1993) recommend a more conservative maximum
holding time of 6 months. The EPA (1995a) recommends a maximum holding time of 1 year at
<-10 °C for dioxins and dibenzofurans.
G-5
-------
APPENDIX G
The thawed or partially thawed whole fish should then be homogenized
individually, and equal weights of each homogenate should be combined to form
the composite sample. Individual homogenates and/or composite homogenates
may be frozen; however, frozen individual homogenates must be rehomogenized
before compositing, and frozen composite homogenates must be rehomogenized
before aliquotting for analysis. The maximum holding time from sample
collection to analysis for mercury is 28 days at <-20 °C; for all other analytes, the
holding time is 1 year at <-20 °C (Stober, 1991). Recommended container
materials, preservation temperatures, and holding times are given in Table G-1.
Note: Holding times in Table G-1 are maximum times recommended for holding
samples from the time they are received at the laboratory until they are
anaylzed. These holding times are based on guidance that is sometimes
administrative rather than technical in nature; there are no promulgated holding
time criteria for tissues (U.S. EPA, 1995b). If States choose to use longer
holding times, they must demonstrate and document the stability of the target
analyte residues over the extended holding times.
G.2 SAMPLE PROCESSING PROCEDURES
Fish sample processing procedures are discussed in more detail in the sections
below. Each time custody of a sample or set of samples is transferred from one
person to another during processing, the Personal Custody Record of the chain-
of-custody (COC) form that originated in the field (Figure 6-8) must be completed
and signed by both parties so that possession and location of the samples can
be traced at all times (see Section 7.1). As each sample processing procedure
is performed, it should be documented directly in a bound laboratory notebook
or on standard forms that can be taped or pasted into the notebook. The use
of a standard form is recommended to ensure consistency and completeness of
the record. Several existing programs have developed forms similar to the
sample processing record for whole fish composite samples shown in Figure
G-2.
G.2.1 Sample Inspection
Individual fish received for filleting should be unwrapped and inspected carefully
to ensure that they have not been compromised in any way (i.e., not properly
preserved during shipment). Any specimen deemed unsuitable for further
processing and analysis should be discarded and identified on the sample
processing record.
G.2.2 Sample Weighing
A wet weight should be determined for each fish. All samples should be
weighed on balances that are properly calibrated and of adequate accuracy and
precision to meet program data quality objectives. Balance calibration should be
checked at the beginning and end of each weighing session and after every 20
weighings in a weighing session.
G-6
-------
APPENDIX G
Sample Processing Record for Fish Contaminant Monitoring Program — Whole Fish Composites
Project No.
STUDY PHASE: Screening | |;
SITE LOCATION
Site Name/Number
County/Parish:
Slate WatAibody Segment Number
Bottom Foarfor - Spoclon Nama:
Composite Sample #:
Scales/Otolfths
Fish* Weight (g) Removed (/)
001
002
003
001
005
006
007
008
009
010
Anatyst , .
Initials/Date / /
Pradator — Species Mama:
Composite Sample #:
Scales/Otoliths
Flsh# Weight (g) Removed (/)
001
002
003
004
005
006
007
008
009
01O
Analyst ,
Initials/Data / /
Notes:
• Sampling Date and Time:
Intensive: Phase 1 1 | Phase II | |
LatAong.:
WaterbodyType:
Number of Individuals:
Sax Homogenate Weight of homogenate
(M, F) Prepared (/) taken for composite (g)
/ / /
Total Composite Homogenate Weight
Number of Individuals:
Sex Homogenate Weight of homogenate
(M, F) Prepared (/) taken for composite (g)
/ / /
Total Composite Homogenate Weight
Figure G-2. Example of a sample processing record for fish contaminant monitoring
program—whole fish composites.
G-7
-------
APPENDIX G
Fish shipped on wet or blue ice should be weighed directly on a foil-lined
balance tray. To prevent cross contamination between individual fish, the foil
lining should be replaced after each weighing. Frozen fish (i.e., those shipped
on dry ice) should be weighed in clean, tared, noncontaminating containers if
they will thaw before the weighing can be completed. Liquid from the thawed
sample must be kept in the container as part of the sample because it will
contain lipid material that has separated from the tissue (Stober, 1991).
All weights should be recorded to the nearest gram on the sample processing
record and/or in the laboratory notebook.
G.2.3 Age Determination
Age provides a good indication of the duration of exposure to pollutants (Versar,
1982). A few scales or otoliths (Jearld, 1983) should be removed from each fish
and delivered to a fisheries biologist for age determination. For most warm
water inland gamefish, 5 to 10 scales should be removed from below the lateral
line and behind the pectoral fin. On softrayed fish such as trout and salmon, the
scales should be taken just above the lateral line (WDNR, 1988). For catfish
and other scaleless fish, the pectoral fin spines should be clipped and saved
(Versar, 1982). The scales, spines, or otoliths may be stored by sealing them
in small envelopes (such as coin envelopes) or plastic bags labeled with, and
cross-referenced by, the identification number assigned to the tissue specimen
(Versar, 1982). Removal of scales, spines, or otoliths from each fish should be
noted (by a check mark) on the sample processing record.
G.2.4 Sex Determination (Optional)
To determine the sex of a. fish, an incision should be made on the ventral
surface of the body from a point immediately anterior to the anus toward the
head to a point immediately posterior to the pelvic fins. If necessary, a second
incision-should be made on the left side of the fish from the initial point of the
first incision toward the dorsal fin. The resulting flap should be folded back to
observe the gonads. Ovaries appear whitish to greenish to golden brown and
have a granular texture. Testes appear creamy white and have a smooth texture
(Texas Water Commission, 1990). The sex of each fish should be recorded on
the sample processing record.
G.2.5 Assessment of Morphological Abnormalities (Optional)
Assessment of gross morphological abnormalities in finfish is optional. This
assessment may be conducted in the field (see Section 6.3.1.5) or during initial
inspection at the central processing laboratory prior to filleting. States interested
in documenting morphological abnormalities should consult Sinderman (1983)
and review recommended protocols for fish pathology studies used in the Puget
Sound Estuary Program (1990).
G-8
-------
APPENDIX G
G.2.6 Preparation of Individual Homogenates
To ensure even distribution of contaminants throughout tissue samples, whole
fish must be ground and homogenized prior to analyses.
Smaller whole fish may be ground in a hand crank meat grinder (fish < 300 g)
or a food processor (fish 300-1,000 g). Larger (>1,000 g) fish may be cut into
2.5-cm cubes with a food service band saw and then ground in either a small or
large homogenizer. To avoid contamination by metals, grinders, and
homogenizers used to grind and blend tissue should have tantalum or titanium
blades and/or probes. Stainless steel blades and probes have been found to be
a potential source of nickel and chromium contamination (due to abrasion at high
speeds) and should be avoided.
Grinding and homogenization of biological tissue, especially skin from whole fish
samples, is easier when the tissue is partially frozen (Stober, 1991). Chilling the
gririder/homogenizer briefly with a few chips of dry ice will reduce the tendency
of the tissue to stick to the grinder.
The ground sample should be divided into quarters, opposite quarters mixed
together by hand, and the two halves mixed back together. The grinding,
quartering, and hand mixing should be repeated two more times. If chunks of
tissue are present at this point, the grinding/homogenizing should be repeated.
No chunks of tissue should remain because these may not be extracted or
digested efficiently. If the sample is to be analyzed for metals only, the ground
tissue may be mixed by hand in a polyethylene bag (Stober, 1991). Homogeni-
zation of each individual fish should be noted on the sample processing record.
At this time, individual whole fish homogenates may be either composited or
frozen and stored at <-20 °C in cleaned containers that are noncontaminating for
the analyses to be performed (see Table G-1).
G.2.7 Preparation of Composite Homogenates
Composite homogenates should be prepared from equal weights of individual
homogenates. If individual whole fish homogenates have been frozen, they
should be thawed partially and rehomogenized prior to compositing. Any
associated liquid should be maintained as a part of the sample. The weight of
each individual homogenate that is used in the composite homogenate should
be recorded, to the nearest gram, on the sample processing record.
Each composite homogenate should be blended by dividing it into quarters,
mixing opposite quarters together by hand, and mixing the two halves together.
The quartering and mixing should be repeated at least two more times. If the
sample is to be analyzed only for metals, the composite homogenate may be
mixed by hand in a polyethylene bag (Stober, 1991). At this time, the composite
homogenate may be processed for analysis or frozen and stored at <-20 °C (see
Table G-1).
G-9
-------
APPENDIX G
The remainder of each individual homogenate should be archived at <-20 °C with
the designation "Archive" and the expiration date recorded on the sample label.
The location of the archived samples should be indicated on the sample
processing record under "Notes."
It is essential that the weights of individual homogenates yield a composite
homogenate of adequate size to perform all necessary analyses. Weights of
individual homogenates required for a composite homogenate, based on the
number of fish per composite and the weight of composite homogenate
recommended for analyses of all screening study target analytes (see Table 4-1)
are given in Table G-2. The total composite weight required for intensive studies
may be less than in screening studies if the number of target analytes is reduced
significantly.
The recommended sample size of 200 g for screening studies is intended to
provide sufficient sample material to (1) analyze for all recommended target
analytes (see Table 4-1) at appropriate detection limits, (2) meet minimum QA
and QC requirements for the analyses of replicate, matrix spike, and duplicate
matrix spike samples (see Section 8.3.3.4), and (3) allow for reanalysis if the QA
and QC control limits are not met or if the sample is lost. However, sample size
requirements may vary among laboratories and the analytical methods used.
Table G-2. Weights (g) of Individual Homogenates
Required for Screening Study Composite Homogenate Sample8
Number of
fish per
sample
3
4
5
6
7
8
9
10
Total composite weight
100 g
(minimum)
33
25
20
17
14
13
11
10
200 g
(recommended)
67
50
40
33
29
25
22
20
500 g
(maximum)
167
125
100
84
72
63
56
50
a Based on total number of fish per composite and the total composite weight required for
analysis in screening studies. The total composite weight required in intensive studies
may be less if the number of target analytes is reduced significantly.
G-10
-------
APPENDIX G
Therefore, it is the responsibility of each program manager to consult with the
analytical laboratory supervisor to determine the actual weights of composite
homogenates required to analyze for all selected target analytes at appropriate
detection limits.
G.3 REFERENCES
California Department of Fish and Game. 1990. Laboratory Quality Assurance
Program Plan. Environmental Services Division, Sacramento, CA.
Crawford, J.K., and S.N. Luoma. 1993. Guidelines for Studies of Contaminants
in Biological Tissues for the National Water-Quality Assessment Program.
USGS Open-File Report 92-494. U.S. Geological Survey, Lemoyne, PA.
Jearld, A. 1983. Age determination, pp. 301-324. In: Fisheries Techniques.
L.A. Nielsen and D. Johnson (eds.). American Fisheries Society, Bethesda
MD.
Puget Sound Estuary Program. 1990 (revised). Recommended protocols for
fish pathology studies in Puget Sound. Prepared by PTI Environmental
Services, Bellevue, WA. In: Recommended Protocols and Guidelines for
Measuring Selected Environmental Variables in Puget Sound. Region 10,
U.S. Environmental Protection Agency, Seattle, WA. (Looseleaf)
Sinderman, C. J. 1983. An examination of some relationships between pollution
and disease. Rapp. P. V. Reun. Cons. Int. Explor. Mer. 18237-43.
Stober, Q. J. 1991. Guidelines for Fish Sampling and Tissue Preparation for
Bioaccumulative Contaminants. Environmental Services Division, Region 4,
U.S. Environmental Protection Agency, Athens, GA.
Texas Water Commission. 1990. Texas Tissue Sampling Guidelines. Texas
Water Commission, Austin, TX.
U.S. EPA (U.S. Environmental Protection Agency). 1986. Bioaccumulation
Monitoring Guidance: 4. Analytical Methods for U.S. EPA Priority Pollutants
and 301 (h) Pesticides in Tissues from Marine and Estuarine Organisms.
EPA-503/6-90-002. Office of Marine and Estuarine Protection, Washington,
DC.
U.S. EPA (U.S. Environmental Protection Agency). 1995a. Method 1613b.
Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution
HRGC/HRMS. Final Draft. Office of Water, Office of Science and
Technology, Washington, DC.
G-11
-------
APPENDIX G
U.S. EPA (Environmental Protection Agency). 1995b. QA/QC Guidance for
Sampling and Analysis of Sediments, Water, and Tissues for Dredged
Material Evaluations—Chemical Evaluations. EPA 823-B-95-001. Office of
Water, Washington, DC, and Department of the Army, U.S. Army Corps of
Engineers, Washington, DC.
Versar, Inc. 1982. Sampling Protocols for Collecting Surface Water, Bed
Sediment, Bivalves and Fish for Priority Pollutant Analysis-Final Draft
Report. EPA Contract 68-01-6195. Prepared for U.S. EPA Office of Water
Regulations and Standards. Versar, Inc., Springfield, VA.
WDNR (Wisconsin Department of Natural Resources). 1988. Fish Contaminant
Monitoring Program—Field and Laboratory Guidelines (1005.1). Madison,
Wl.
G-12
-------
APPENDIX H
GENERAL PROCEDURES FOR REMOVING
EDIBLE TISSUES FROM FRESHWATER TURTLES
-------
-------
APPENDIX H
APPENDIX H
GENERAL PROCEDURES FOR REMOVING EDIBLE TISSUES
FROM FRESHWATER TURTLES
4.
Turtles brought to the processing laboratory on wet, blue, or dry ice should
be placed in a freezer for a minimum of 48 hours prior to resection.
Profound hypothermia can be employed to induce death (Frye, 1994)
Decapitation of alert animals is not recommended because there is
evidence that decapitation does not produce instantaneous loss of
consciousness (Frye, 1994).
The turtle should be placed on its back with the plastron (ventral plate)
facing upwards. The carapace and plastron are joined by a bony bridge
on each side of the body extending between the fore and hindlimbs
(Figure H-1). Using a bone shears, pliers, or sharp knife, break away the
two sides of the carapace from the plastron between the fore and hind
legs on each side of the body.
Remove the plastron to view the interior of the body cavity. At this point,
muscle tissue from the forelimbs, hindlimbs, tail (posterior to the anus),
and neck can be resected from the body. The muscle tissue should be
skinned and the bones should be removed prior to homogenization of the
muscle tissue. Typically, the muscle tissue is the primary tissue
consumed and turtle meat sold in local markets usually contains lean meat
and bones only (Liner, 1978).
Dietary and culinary habits with regard to which turtle tissues are edible,
however, differ greatly among various populations. In some populations,
the liver, heart, eggs, fatty deposits, and skin are also used (Liner, 1978).
Therefore only general information on the types of turtle tissues most
frequently considered edible can be presented here. State staff familiar
with the dietary and culinary habits of the turtle-consuming populations
within their jurisdictions are the best judge of which edible tissues should
be included as part of the tissue samples used to assess the health risks
to the turtle-consuming public.
Several of the tissue types that are considered edible include the fatty
deposits found in various parts of the body, the heart, liver (usually with
the gall bladder removed), and the eggs (if the specimen is a female).
These edible tissues are shown in Figure H-2.
H-3
-------
APPENDIX H
External Anatomy
Carapace
Bony Bridge
connecting carapace
and plastron
Plastron
Source: Ashley, 1962.
Figure H-1.
H-4
-------
APPENDIX H
Forelimb fatty
tissue deposits
(yellowish green)
Gall bladder
Hindlimb fatty
tissue deposits
(yellowish green)
Neck fatty
tissue deposits
(yellowish green)
Heart
Liver
(dark brown)
Ovary with eggs
(deep yellowish)
Internal Anatomy
Source: Ashley, 1962.
Figure H-2.
H-5
-------
APPENDIX H
• Masses of yellowish-green fatty deposits may be removed from above
the forelimbs and from above and in front of the hindlimbs. Fatty
deposits can also be found at the base of the neck near the point
where the neck enters the body cavity.
• The centrally located heart is positioned anterior to the liver.
• The large brownish liver is the predominant tissue in the body cavity
and is an edible tissue eaten by some populations. Note: The small
greenish-colored gall bladder lies on the dorsal side of the right lobe of
the liver (not visible unless the liver is lined upward and turned over).
The gall bladder is usually removed and discarded by consumers
because of its acrid taste (Liner, 1978).
• If the turtle specimen is a female, ovaries containing bright yellow-
colored spherical eggs of varying sizes are located posterior to the liver
and lie against the dorsal body wall.
Note: The fatty deposits, liver tissue, and eggs are highly lipophiiic tissues and
have been shown to accumulate chemical contaminants at concentrations 10 to
more than 100 times the concentrations reported from muscle tissue (Bryan et
al., 1987; Hebert et al., 1993; Olafsson et al., 1983, 1987; Ryan et al., 1986;
Stone et al., 1980). States may wish to resect the fatty tissues, liver, heart, and
eggs for inclusion in the turtle muscle tissue sample to obtain a conservative
estimate of the concentration to which the turtle-consuming public would be
exposed. Alternatively, States may want to retain these tissues for individual
analysis. Some States already advise their residents who consume turtles to
remove all fatty tissues (Minnesota Department of Health, 1994; New York State
Department of Health, 1994) and not to consume the liver and eggs (New York
State Department of Health, 1994). These cleaning procedures are
recommended as a risk-reducing strategy.
REFERENCES
Ashley, L.M. 1962. Laboratory Anatomy of the Turtle. W.C. Brown Company
Publishers, Dubuque, IA.
Bryan, A.M., P.G. Olafsson, and W.B. Stone. 1987. Disposition of low and high
environmental concentrations of PCBs in snapping turtle tissues. Bull.
Environ. Contam. Toxicol. 38:1000-1005.
Frye, F.L. 1994. Reptile Clinician's Handbook: A Compact Clinical and
Surgical Reference. Krieger Publishing Company, Malabar, FL.
H-6
-------
APPENDIX H
Hebert, C.E., V. Glooschenko, G.D. Haffner, and R. Lazar. 1993. Organic
contaminants in snapping turtle (Chelydra serpentina) populations from
Southern Ontario, Canada. Arch. Environ. Contam. Toxicol. 24:35-43.
Liner, E.A. 1978. A Herpetological Cookbook: How to Cook Amphibians and
Reptiles. Privately printed, Houma, LA.
Minnesota Department of Health. 1994. Minnesota Fish Consumption Advisory.
Minneapolis, MN.
New York State Department of Health. 1994. Health Advisory-Chemicals in
Sportfish and Game 1994-1995. #40820042. Division of Environmental
Health Assessment, Albany, NY.
Olafsson, P.G., A.M. Bryan, B. Bush, and W. Stone. 1983. Snapping turtles—A
biological screen for PCBs. Chemosphere 12 (11/12):1525-1532.
Ryan, J.J., P.Y. Lau, and J.A. Hardy. 1986. 2,3,7,8, Tetrachlorodibenzo-p-
dioxin and related dioxans and furans in snapping turtle (Chelydra
serpentina) tissues from the upper St. Lawrence River. Chemosphere 15
(5):537-548.
Stone, W.B., E. Kiviat, and S.A. Butkas. 1980. Toxicants in snapping turtles.
New York Fish and Game Journal 27 (1):39-50.
H-7
-------
-------
APPENDIX I
GENERAL PROCEDURES FOR REMOVING
EDIBLE TISSUES FROM SHELLFISH
-------
-------
Heading, peeling and deveining shrimp
To head a shrimp, hold it in
one hand. With your thumb
behind shrimp head, push head
off. Be sure to push just the
head off so that you do not lose
any meat.
If using a deveiner, insert it
at head end, just above the
vein.
Push through shrimp to the tail
and split and remove shell.
This removes vein at the same
time.
If you prefer to use a paring
knife, shell shrimp with your
fingers or knife. Then use
knife to gently remove vein.
Source: UNC Sea Grant Publication UNC-SG-88-02
1-3
-------
Cleaning soft-shell crabs
Hold crab in one hand and cut
across body just behind eyes to
remove eyes and mouth.
Turn crab on its back. Lift
and remove apron and vein
attached to it.
Turn crab over and lift one
side of top shell.
With a small knife, scrape
off grayish-feathery gills.
Repeat procedure on other
side.
Source: UNC Sea Grant Publication UNC-SG-88-02
1-4
-------
Cleaning hard-shell crabs
Hold crab in one hand. Turn
crab over and stab straight
down at point of apron with a
knife.
Make two cuts from this
point to form a V-pattern
that will remove mouth.
Do not remove knife after
making second cut. Firmly
press crab shell to cutting
surface without breaking back
shell. With other hand, grasp
crab by legs and claws on the
side where you are holding
knife, and pull up. This should
pull crab body free from back
shell.
1-5
-------
Remove gray, feathery gills,
which are attached just above
legs. Cut and scrape upward to
remove gills.
Remove all loose
material—viscera and
eggs—from body cavity.
If apron did not come loose
with shell, remove it.
Source: UNC Sea Grant Publication UNC-SG-88-02
1-6
-------
Shucking oysters
Oyster shells are especially
sharp; be sure to wear gloves
to protect your hands. Chip off
a small piece of shell from the
thin Up of the oyster until
there is a small opening.
Insert knife blade into the
opening and cut muscle free
from top and bottom shells.
Remove oyster meat from the
shell.
Source: UNC Sea Grant Publication UNC-SG-88-02
1-7
-------
Shucking clams
In the back of clam near the
hinge is a black ligament
Toward the front where
ligament ends is a weak spot.
Insert your knife at this spot.
Inside are two muscles.
Run the knife around the
shell to sever both
muscles.
Now insert the knife blade
into the front of the shell
and separate the two
shells.
Scrape the meat free
from the top and bottom
shell.
Source: UNO Sea Grant Publication UNC-SG-88-02
1-8
-------
APPENDIX J
COMPARISON OF TARGET ANALYTE SCREENING
VALUES (SVs) WITH DETECTION AND QUANTITATION LIMITS
OF CURRENT ANALYTICAL METHODS
-------
-------
APPENDIX J
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-------
APPENDIX J
iii
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T T T T 55
J-4
-------
APPENDIX M
Sokal, R.R., and F.J. Rohlf. 1981. Biometry. The Principles and Practice of
Statistics in Biological Research. Second Edition. W.H. Freeman and
Company, New York, NY. 859 pp.
Winer, BJ. 1962. Statistical Principles in Experimental Design. McGraw-Hill,
New York, NY.
M-10
-------
APPENDIX M
including those in Figure M-1, require uncorrelated data. Gilbert (1987)
discusses several methods for performing the required analyses in these cases.
Temporal trends in contaminant concentrations may be detected by regression
analyses, whereby the hypothesis is tested that concentrations are not changing
in a predictable fashion (usually linear) over time. If the hypothesis is rejected,
a trend may be inferred. States interested in performing regression analyses
should consult statistics textbooks such as Gilbert (1987) or Snedecor and
Cochran (1980).
M.3 REFERENCES
Gilbert, R.O. 1987. Statistical Methods for Environmental Pollution Monitoring.
Van Nostrand Reinhold Company, New York, NY. 320 pp.
Hays, W.L 1988. Statistics. Fourth Edition. CBS College Publishing, New
York, NY.
Hebert, C.E., and K.A. Keenleyside. 1995. To normalize or not to normalize?
Fat is the question. Environmental Toxicology and Chemistry 14(5):801-
807.
Hirsch, R.M., J.R. Slack, and R.A. Smith. 1982. Techniques of trend analysis
for monthly water quality data. Water Resources Research 18:107-121.
Lilliefors, H.W. 1967. The Kolmogorov-Smirnov test for normality with mean
and variance unknown. J. Amer. Stat. Assoc. 62:399-402.
Massey, F.J., Jr. 1951. The Kolmogorov:Smirnov test for goodness of fit. J.
Amer. Stat. Assoc. 46:68-78.
Milliken, G.A., and D.E. Johnson. 1984. Analysis of Messy Data: Volume 1.
Designed Experiments. Van Nostrand Reinhold Company, New York, NY.
Royston, J.P. 1982. An extension of Shapiro and Wilk's W test for normality to
large samples. Applied Statistics 31:115-124.
Sen, P.K. 1968. On a class of aligned rank order tests in two-way layouts.
Annals of Mathematical Statistics 39:1115-1124.
Shapiro, S.S., M.B. Wilk, and H.J. Chen. 1968. A comparative study of various
tests of normality. J. Amer. Stat. Assoc. 63:1343-1372.
Snedecor, G.W., and W.G. Cochran. 1980. Statistical Methods. 7th edition.
Iowa State University Press, Ames, Iowa.
M-9
-------
APPENDIX M
tion from any single station would not truly represent the potential contaminant
exposure to fish consumers in the waterbody of concern.
M.2 TEMPORAL COMPARISON OF STATIONS
Both screening and intensive studies are often repeated over time to ensure that
public health is adequately protected. By examining monitoring data from
several time periods from a single site, it may be possible to detect trends in
contaminant concentrations in fish tissues. Trend analysis data should never be
used to conduct risk assessments. Procedures for conducting risk assessments
are adequately covered elsewhere in this document (see Section 6.1.2.7). Trend
analysis may, however, be useful for monitoring the effects of various environ-
mental changes or policies on the contaminant concentrations in the target
species. For example, a State may have issued a fish advisory for a contami-
nant for which the source is known or suspected. Source control for this
contaminant is the obvious solution to the environmental problem. An evaluation
of the effectiveness of the source control may be made easier by trend analysis.
The State would still need to perform statistical calculations comparing data from
each sampling site to the SV, but trend analysis could yield valuable information
about the success of remediation efforts even if the fish advisory remained in
place because of SV exceedances.
Trend analysis can be performed using the statistical framework outlined in
Figure M-1, but complexities in pollution data collected over time may make this
approach unsuitable in some instances. The types of complexities for which
other statistical approaches might be warranted can be divided into four groups:
(1) changes in sampling and/or analysis procedures, (2) seasonality, and (3)
correlated data (Gilbert, 1987). Each of these subjects is discussed briefly here.
Changes in the designation of an analytical laboratory to perform analyses or
changes in sampling and/or analytical procedures are not uncommon in long-
term monitoring programs. These changes may result in shifts in the mean or
variance of the measured values, which could be incorrectly attributed to natural
or manmade changes in the processes generating the pollution (Gilbert, 1987).
Ideally, when changes occur in the methods used by the monitoring program,
comparative studies should be performed to estimate the magnitude of these
changes.
Seasonality may introduce variability that masks any underlying long-term trend.
Statistically, this problem can be alleviated by removing the cycle before applying
tests or by using tests unaffected by cycles (Gilbert, 1987). Such tests will not
be discussed here. States interested in performing temporal analyses with data
for which a seasonal effect is hypothesized should consult the nonparametric
test developed by Sen (1968) or the seasonal Kendall test (Hirsch et al., 1982).
Measurements of contaminant concentrations taken over relatively short periods
of time are likely to be positively correlated. Most statistical tests, however,
M-8
-------
APPENDIX M
A general statistical flowchart for comparing contaminant concentration data from
several stations to each other is presented in Figure M-1. The cadmium data in
Table M-1 may be additionally analyzed using the tests in Figure M-1. All of the
statistical tests in Figure M-1 can be performed using commercial statistical
software packages. By performing a spatial analysis of the data, the details of
the risk assessment might be further refined. For example, one component of
a fish advisory is often the establishment of risk-based consumption limits (see
Volume II of this series). In order to calculate these limits, an estimate of the
contaminant concentration in the target species must be available. In the
example shown in Table M-1, there are three estimates of cadmium
concentration. A spatial analysis of these data can help to identify which of the
concentrations (if any) to use in establishing risk-based consumption limits.
The initial steps in the flowchart on Figure M-1 are to determine whether
parametric or nonparametric statistical tests should be used. The first step is to
test whether each of the three groups of data are from populations that are
normally distributed. Three tests that may be used for this purpose are the
Kolmogorov-Smirnov test for normality (Massey, 1951), Shapiro and Wilk's W
test (Shapiro et al., 1968; Royston, 1982), and Lilliefors' test (Ulliefors, 1967).
The results for the W test on each of the three groups of data indicate that each
group was sampled from populations that are normally distributed (Table M-1).
The next step is to test for homogeneity of variances between the three groups.
Three tests that may be used for this purpose are Levene's test (Milliken and
Johnson, 1984), the Hartley F-max test (Sokal and Rohlf, 1981), and the
Cochran C test (Winer, 1962). The result of Levene's test indicates that the
variances of the three groups of data are not significantly different from each
other (Table M-1). These test results mean that parametric statistics (the left
side of Figure M-1) are appropriate for this dataset.
An appropriate parametric test to perform to determine whether the three mean
cadmium concentrations are significantly different from each other is a 1 -way
ANOVA. The result of this test indicates that the three means are significantly
different (Table M-1). What this result does not show, however, is whether each
mean concentration is significantly different from both of the other mean
concentrations. For this answer, multiple comparison tests can be used to
perform all possible pairwise comparisons between each mean.
Three tests that can be used to perform a multiple comparison are the Newrnan-
Keul test (Sokal and Rohlf, 1981), Duncan's Multiple Range test (Hays, 1988;
Milliken and Johnson, 1984), and the Tukey Honest Significant Difference test
(Hays, 1988; Milliken and Johnson, 1984). Three pairwise comparisons are
possible between three means (1 vs. 2, 1 vs. 3, and 2 vs. 3). The results of
Duncan's Multiple Range test indicate that the mean concentration at station 1
(21.5 ppm) is significantly lower than the mean concentrations at both station 2
(29.4 ppm) and station 3 (31.3 ppm), which in turn are not significantly different
from each other. Therefore, to be most conservative (i.e., protective), the State
could use the mean of the 16 replicate samples from stations 2 and 3 to
calculate risk-based consumption limits. In this example, use of the concentra-
M-7
-------
APPENDIX M
each location and the statistical comparisons between the three groups are
presented in Table M-1.
The mean cadmium concentration at each of three locations was more than
twice the SV of 10 ppm (Table M-1). The most important statistical test, as
indicated in Section 6.1.2.7, is a comparison of the mean target analyte concen-
tration for each location with the appropriate SV for that target analyte using a
f-test These tests must be performed before any analysis of spatial trends is
performed. The results of the f-tests indicate that each of the three mean tissue
concentrations is significantly greater than the SV (Table M-1). By itself, these
results indicate that a risk assessment is warranted.
Table M-1. Hypothetical Cadmium Concentrations (ppm) in Target Species A at
Three River Locations
Replicate samples
1
2
3
4
5
6
7
8
Mean
Standard deviation
p-Value for Mest with SV
p-Value for W test
p-Value for Levene's test
p-Value for ANOVA
p-Value for Duncan's-1 vs. 2
p-Value for Duncan's-1 vs. 3
p-Value for Duncan's-2 vs. 3
Station 1
20
18
25
22
21
22
23
21
21.5
2.07
<0.001
0.97
Station 2
28
27
34
28
30
29
30
29
29.4
2.13
<0.001
0.83
0.52
<0.0001
<0.0001
>0.0001
0.17
Station 3
33
30
30
28
20
39
31
30
31.3
3.45
<0.001
0.78
M-6
-------
APPENDIX M
difference in mean concentrations between two group means can be further
investigated using a multiple comparison test (Figure M-1). These tests indicate
which specific means are significantly different from each other, rather than just
indicating that one or more means are different, as the ANOVA does.
If the underlying assumptions for parametric testing are not met, nonparametric
tests of significance can be employed. Nonparametric tests of significant differ-
ences in central tendencies are often performed on transformed data, that is, the
ranks. Multiple comparison tests comparable to those used for parametric data
sets are not available for nonparametric data sets. For data sets including three
or more groups, a series of two-sample tests can be performed that can yield
similar information to-that derived from multiple comparison tests.
Because the concentrations of contaminants, particularly nonpolar organics, are
often correlated with the percentage of lipid in a tissue sample (see Section
8.1.2), contaminant data are often normalized to the lipid concentration before
statistical analyses are performed. This procedure can, in some instances,
improve the power of the statistical tests. States wishing to examine the
relationship between contaminant concentrations and percentage of lipid should
refer to Hebert and Keenleyside (1995) for a discussion of the possible statistical
approaches.
Intensive studies may include the collection offish contaminant data from several
locations within a region of interest or for multiple time periods (e.g., seasons or
years) from a single location, or a combination of both. Data from intensive
studies such as these may be used to perform spatial (i.e., between stations) or
temporal (i.e., over time) analyses. It should be noted that these types of
analyses, if performed, are performed in addition to the statistical comparisons
of mean target analyte concentrations with SVs described in Section 6.1.2.7. It
is only the latter type of comparison that should be used to make decisions
regarding the necessity of performing risk assessments and the issuance of fish
consumption advisories. Spatial and temporal comparisons of contaminant data,
however, may yield important information about the variability of target analyte
concentrations in specific populations of a particular target species.
M.1 SPATIAL COMPARISON OF STATIONS
Intensive studies also may involve the collection of contaminant data from
multiple stations within a waterbody of interest. The stations could be located
in different lakes within a single drainage basin, upstream and downstream of a
point source of concern along a single river, or randomly located within a single
waterbody if an estimate of random spatial variability is desired. The use of an
example will serve to illustrate how a spatial analysis of contaminant data might
be performed. In this example, a State has determined from a screening study
on a river that cadmium is present in a target species at 20 ppm, which is two
times the SV of 10 ppm (see Table 5-2). An intensive survey was undertaken
in which eight samples were collected from three locations on the river of
potential concern and analyzed for cadmium. The results of the analyses for
_ — —
-------
APPENDIX M
Test for Normality
Kolmogorov-Smimov test
Wtest
LJIIiefors test
Transform
Data
Distribution
Normal
(p=0.05)
Test for Homogeneity of
Variance
Test for Normality
Kolmogorov-Smimov test
Wtest
LJIIiefors test
Levene's test
Hartley F-max
Cochran C test
variances
are Equal
(p=0.05)
Distribution
Normal
(p=0.05)
Test of Significant
Differences Between
Groups
1-wayANOVA (n>2)
t-test(n=2)
Test of Significant Differences Between
Groups
Kruskal-Wallis ANOVA by Ranks (n>2)
Kolmogorov-Smimov test (n=2)
Groups
are Equal
(p=0.05)
Groups
are Equal
(p=0.05)
Multiple Comparison Test
Newman-Keul
Duncan's Multiple Range test
Tukey Honest Significant Difference test
Report
Results
Report
Results
Report
Results
Report
Results
Figure M-1. Statistical approach to testing for significant differences
between different groups of contaminant monitoring data.
M-4
-------
APPENDIX M
APPENDIX M
STATISTICAL METHODS FOR COMPARING SAMPLES:
SPATIAL AND TEMPORAL CONSIDERATIONS
The primary objective of Tier 2 intensive studies is to assess the magnitude and
geographic extent of contamination in selected target species by determining
whether the mean contaminant concentration exceeds the screening value (SV)
for any target analyte. Secondary objectives of intensive studies may include
defining the geographical region where fish contaminant concentrations exceed
screening values (SVs), identifying geographic distribution of contaminant
concentrations, and, in conjunction with historical or future data collection,
assessing changes in fish contaminant concentrations over time. This appendix
discusses some of the statistical methods that may be used to compare fish
contaminant levels measured at different locations or over time.
The recommended statistical approach for comparing replicated contaminant
measurements between two or more groups is outlined below and in Figure M-1.
For each type of test, several options are provided, each of which may be
appropriate in specific cases. State staff should consult a statistician as to the
specific statistical tests to use for a particular data set.
Statistical tests of significant differences between means (or other measures of
central tendency) can be divided into parametric and nonparametric types.
Parametric tests assume that the contaminant concentrations in the population
being sampled are normally distributed and that the population variances in the
groups being tested are not significantly different from each other (Gilbert, 1987).
If either of these assumptions is violated, a nonparametric test may be more
appropriate. However, nonparametric tests should be used only when necessary
because the power of parametric tests generally is greater than the power of
nonparametric tests when the assumptions of the parametric test have been met
(Sokal and Rohlf, 1981).
Because the populations of many environmental measurements are not normally
distributed, logarithmic transformation is often performed on the sampled data
(Gilbert, 1987). However, transformation may not be appropriate in all cases.
If the data are sampled from a population that is normally distributed, then there
is no need for transformation (Figure M-1).
If the assumptions of normality and equality of variance are met, parametric tests
of significant differences between means, such as the one-way Analysis of
Variance (ANOVA) and the f-test, should be performed. If three or more groups
are compared using the ANOVA that results in a significant difference, the
-------
-------
APPENDIX M
STATISTICAL METHODS FOR COMPARING SAMPLES:
SPATIAL AND TEMPORAL CONSIDERATIONS
-------
APPENDIX L
RECOMMENDED PUBLICATIONS ON CERTIFIED STANDARDS
AND REFERENCE MATERIALS
Standard and Reference Materials for Marine Science (NOAA, 1992).
Available from
Dr. Adrianna Cantillo
National Ocean Service
National Oceanic and Atmospheric Administration
U.S. Department of Commerce
6001 Executive Blvd., Room 323
Rockville, MD 20852
This catalog lists approximately 2,000 reference materials from 16
producers and includes information on their use, sources, matrix type,
analyte concentrations, proper use, availability, and costs. Reference
materials are categorized as follows: ashes, gases, instrumental
performance, oils, physical properties, rocks, sediments, sludges, tissues,
and waters. This catalog has been published independently by both NOAA
and IOC/UNEP and is available in electronic form from the Office of Ocean
Resources, Conservation, and Assessment, NOAA/NOS.
Biological and Environmental Reference Materials for Trace Elements,
Nuclldes and Organic Microcontamlnants (Toro et al., 1990). Available
from
Dr. R.M. Parr
Section of Nutritional and Health-Related Environmental Studies
International Atomic Energy Agency
P.O. Box 100
A-1400 Vienna, Austria
This report contains approximately 2,700 analyte values for 117 analytes
in 116 biological and 77 nonbiological environmental reference materials
from more than 20 sources. Additional information on cost, sample size
available, and minimum amount of material recommended for analysis is
also provided.
REFERENCES
NOAA (National Oceanic and Atmospheric Administration). 1992. Standard and
Reference Materials for Marine Science. Third Edition. U.S. Department
of Commerce, Rockville, Maryland.
Toro, E. Cortes, R. M. Parr, and S. A. Clements. 1990. Biological and
Environmental Reference Materials for Trace Elements, Nuclides and
Organic Microcontaminants: A Survey. IAEA/RL/128(Rev. 1). International
Atomic Energy Agency, Vienna.
L-8
-------
APPENDIX L
RETAILERS OF ERA-CERTIFIED NEAT ORGANIC STANDARDS
(Including the Former EPA Pesticide Repository Standards)
Absolute Standards
498 Russel Street
New Haven, CT 06513
Tel: 800-368-1131
FAX: 203-468-7407
Contact: JackCiscio
Accustandard
25 Science Park Road
New Haven, CT 06511
Tel: 203-786-5290
FAX: 203-786-5287
Contact: Mike Bolgar
Alltech Associates
2051 Waukegan Road
Deerfield, IL 60015
Tel: 708-948-8600
FAX: 708-948-1078
Contact: Tom Rendl
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
Tel: 401-294-9400
FAX: 401-295-2330
Contact: Dr. Bill Russo
RETAILERS OF ERA-CERTIFIED INORGANIC QUALITY CONTROL SAMPLES
SPEX Industries, Inc.
3880 Park Avenue
Edison, NJ 08820
Tel: 1-201-549-7144 or 1-800-GET-SPEX
FAX: 1-201-549-5125
RETAILERS OF ERA-CERTIFIED SOLID MATRIX QUALITY CONTROL SAMPLES
Fisher Scientific
711 Forbes Avenue
Pittsburgh, PA 15219
L-7
-------
APPENDIX L
RETAILERS OF ERA-CERTIFIED ORGANIC SOLUTION STANDARDS
(Formerly the EPA Toxic and Hazardous Materials Repository)
Absolute Standards
498 Russel Street
New Haven, CT 06513
Tel: 800-368-1131
FAX: 203-468-7407
Contact: Jack Ciscio
Accustandard
25 Science Park Road
New Haven, CT 06511
Tel: 203-786-5290
FAX: 203-786-5287
Contact: Mike Bolgar
Alltech Associates
2051 Waukegan Road
Deerfield, IL 60015
Tel: 708-948-8600
FAX: 708-948-1078
Contact: Tom Rendl
Alarheda Chemical and Scientific
922 East Southern Pacific Drive
Phoenix, AZ 85034
Tel: 602-256-7044
FAX: 602-256-6566
Bodman Chemicals
P.O. Box 2221
Aston, PA 19014
Tel: 215-459-5600
FAX: 215-459-8036
Contact: Kirk Lind
Cambridge Isotope Laboratories
20 Commerce Way
Woburn, MA 01801-9894
Tel: 800-322-1174 or 617-938-0067
FAX: 617-932-9721
NSI Environmental Solutions, Inc.
P.O. Box 12313
2 Triangle Drive
Research Triangle Park, NC 27709
Tel: 800-234-7837 or 919-549-8980
FAX: 919-544-0334
Contact: Zora Bunn
Promochem
Postfach 1246
D 4230 Wesel
West Germany
Tel: 0281/530081
FAX: 0281/89991-93
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
Tel: 401-294-9400
FAX: 401-295-2330
Contact: Dr. Bill Russo
L-6
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APPENDIX L
RETAILERS OF EPA-CERTIFIED ORGANIC QUALITY CONTROL SAMPLES
Accurate Chemical and Scientific
300 Shamee Drive
Westbury, NY 11590
Tel: 516-443-4900
FAX: 516-997-4938
Contact: Rudy Rosenberg
Accustandard
25 Science Park Road
New Haven, CT 06511
Tel: 203-786-5290
FAX: 203-786-5287
Contact: Mike Bolgar
Aldrich Chemical Company, Inc.
940 West Saint Paul Avenue
Milwaukee, Wl 53233
Tel: 414-273-3850
FAX: 800-962-9591
Contact: Roy Pickering
Alltech Associates/Applied
Science/Wescan Instruments
2051 Waukegan Road
Deerfield, IL 60015
Tel: 708-948-8600
FAX: 708-948-1078
Contact: Tom Rendl
Analytical Products Group
2730 Washington Boulevard
Belpre, OH 45714
Tel: 614-423-4200
FAX: 614-423-5588
Contact: Tom Coyner
Bodman Chemicals
P. O. Box 2221
Aston, PA 19014
Tel: 215-459-5600
FAX: 215-459-8036
Contact: Kirk Lind
Chemical Research Supply
P. O. Box 888
Addison, IL 60101
Tel: 708-543-0290
FAX: 708-543-0294
Contact: Nelson Armstrong
Crescent Chemical Corporation
1324 Motor Parkway
Hauppauge, NY 11788
Tel: 516-348-0333
FAX: 516-348-0913
Contact: Eric Rudnick
Curtis Matheson Scientific
P. O. Box 1546
9999 Veterans Memorial Drive
Houston, TX 77251-1546
Tel: 713-820-9898
FAX: 713-878-2221
Contact: Mitchel Martin
Environmental Research Associates
5540 Marshall Street
Arvada, CO 80002
Tel: 303-431-8454
FAX: 303-421-0159
Contact: Mark Carter
Restek Corporation
110 Benner Circle
Bellefonte, PA 16823
Tel: 814-353-1300
FAX: 814-353-1309
Contact: Eric Steindle
Supelco
Supelco Park
Bellefonte, PA 16823-0048
Tel: 800-247-6628 or 814-359-3441
FAX: 814-359-3044
Contact: Linda Alexander
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
Tel: 401-294-9400
FAX: 401-295-2330
Contact: Dr. Bill Russo
L-5
-------
APPENDIX L
Ultra Scientific
250 Smith Street
North Kingston, Rl 02852
Tel: 1-401-294-9400
FAX: 1-401-295-2330
Contact: Dr. Bill Russo
EPA-certified inorganic quality control samples, including trace metals,
minerals, and nutrients, are produced by:
SPEX Industries, Inc.
3880 Park Avenue
Edison, NJ 08820
Tel: 1-201-549-7144 or 1-800-GET-SPEX
FAX: 1-201-549-5125
EPA-certified solid matrix quality control samples, including standards for
pesticides in fish tissue, are produced by:
Fisher Scientific
711 Forbes Avenue
Pittsburgh, PA 15219
The most recent information on EPA-certified materials is available on the EPA
Electronic Bulletin Board (Modum No. 513-569-7610). Names and addresses
of retailers of EPA-certified CRADA QA/QC samples or standards as of February
20, 1991, are given below. When ordering these materials, specify "EPA
Certified Materials."
L-4
-------
APPENDIX L
APPENDIX L
SOURCES OF RECOMMENDED REFERENCE MATERIALS
AND STANDARDS
SOURCES OF ERA-CERTIFIED REFERENCE MATERIALS
EPA-certified analytical reference materials for priority pollutants and related
compounds are currently produced under five Cooperative Research and
Development Agreements (CRADAs) for: organic quality control samples;
organic solution standards; organic neat standards; inorganic quality control
standards; and solid matrix quality control standards. The CRADA cooperators
are listed below.
EPA-certified organic quality control samples, including standards for
pesticides in fish tissue, are produced by:
Supelco, Inc.
Supelco Park
Bellefonte, PA 16823-0048
Tel: 1-800-247-6628 or 1-814-359-3441
FAX: 1-814-359-3044
Contact: Linda Alexander
EPA-certified organic solution standards for toxic and hazardous materials
(formerly the EPA Toxic and Hazardous Materials Repository) are produced
by:
NSI Environmental Solutions, Inc.
P. O. Box 12313
2 Triangle Drive
Research Triangle Park, NC 27709
Tel: 1 -800-234-7837 or 1-919-549-8980
FAX: 1-919-544-0334
EPA-certified neat organic standards, including neat pesticide standards
(formerly the EPA Pesticide Repository), are produced by:
L-3
-------
-------
APPENDIX L
SOURCES OF RECOMMENDED
REFERENCE MATERIALS AND STANDARDS
-------
REFERENCES
1.
2.
3.
4.
Braman, R. S. , D. L. Johnson, C. C. Foreback, J. M. Ammons and J. L. Bricker.
Separation and determination of nanogram amounts of inorganic arsenic and
methyl arsenic compounds. Analytical Chemistry Vol. 49 No. 4 (1977) 621-625.
Andreae, M. 0. Determination of arsenic species in natural waters.
Analytical Chemistry Vol. 49, p. 820. May 1977.
Andreae, M. 0. Methods of Seawater Analysis. Arsenic (by hydride
generation/AAS), pp. 168-173 (1983) Verlag Chemie (Florida).
Maher, W. A. Determination of inorganic and methylated arsenic species in
marine organisms and sediments. Analytica Chemica Acta 126 (1981) 157-165.
2-28
-------
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2-27
-------
Precision for Sediments and Water
The precision or reproducibility for replicate analyses of arsenic species in
field samples is shown in Table 2-11. Collection of these field samples is
described in Section 3 of this report. The sediment was analyzed for Teachable
As (III) and As (V). Interstitial water and water from Hyco Reservoir were also
analyzed for As (III) and (V). The results indicate that the relative standard
deviations (RSD) for arsenic (III) and (V) in sediment are approximately 20% while
the RSD for these species in interstitial water and in the water column are
approximately 15% and 7%.
CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER WORK
Arsenic speciation of a variety of materials in the limnological environment is
simply and reproducibly achieved using selective hydride
generation/low-temperature trapping techniques in conjunction with atomic
absorption detection. The most difficult problem is the unambiguous determination
of total arsenic in solids by this technique. Other related techniques which
might be investigated include dry ashing, lithium metaborate fusion, and graphite
furnace atomic absorption. An alternate method is to analyze select samples by
X-ray fluorescence spectrometry.
2-26
-------
Interlaboratory Comparison
An Inter!aboratory comparison exercise was conducted between Battelle-Northwest
(BNW) and Dr. M. 0. Andreae of Florida State University (FSU) to demonstrate the
effectiveness of the sample storage and shipping procedure and varify the accuracy
of the anlaytical technique for determination of arsenic species in fresh water.
Three samples were prepared as follows: (1) Oungeness River water (DRW) was
filtered, (2) filtered DRW was spiked with nominally 0.45 pg I-1 of As (V) and
2 jjg L-1 each of DMA and MMA, and (3) coal fly ash, standard reference material
NBS-1633, was leached with DRW then filtered. All solutions were frozen
immediately after preparation in liquid nitrogen then transferred and stored at
-80°C. Samples were shipped on dry ice. Samples were analyzed at BNW and FSU the
same week approximately two months after preparation. The results in Table 2-10
show good agreement between these two laboratories even for concentrations below
0.1 ug L-1. We believe this inter!aboratory exercise has demonstrated that these
storage and shipping procedures are appropriate for freshwater samples and the
analytical method used for arsenic speciation is sensitive and accurate for
concentrations of inorganic arsenic greater than approximately 0.05 and for
organic arsenic concentrations greater than 0.2 ug L-1.
Table 2-10
ARSENIC SPECIATION INTERCOMPARISON EXERCISE
ug &-1
As (III)
BNW
0.061
±0. 004
0.061
±0.005
0.052
±0.006
Andreae
0.067
0.066
0.031
As
BNW
0.042
±0.008
0.468
±0.028
12.9
±0.2
(V)
Andreae
0.023
0.421
12.0
MMA
BNW
<0.01
1.96
±0.11
<0.01
Andreae
0.002
1.67
ND
DMW
BNW
<0.01
1.92
±0.13
<0.01
Andreae
0.067
1.82
ND
SDRW
FA
Inter-comparison exercise results with Meinrat 0. Andreae for arsenic speciation
in limnological samples. DRW is filtered Dungeness River water; SDRW is Dungeness
River water spiked with nominally 0.45 ug-2-1 As (V), and 2 H9'*-1 each DMA and
MMA. FA is the filtrate of 1000 mg-Jfc-1 NBS coal fly ash leached with DRW.
BNW results are the mean of (3) determinations. ND means not detected. ± = one
standard deviation.
2-25
-------
CD
O
t—H
Z
LU
18
16
14
12
10
8
6
4
2
-€> flS(III)
TOTRL INORGBNIC flS
INTERSTITIflL VOTER
CONDITIONS
ROOM TEMPERRTURE
flTMOSPHERIC CONTflCT
10 15 20
TIME (HOURS)
25
Figure 2-7. Plot of the concentration of AsIII and total inorganic
arsenic versus storage time Tn interstitial water.
2-24
-------
Interstitial Water. Interstitial water is collected from mud by pressure
filtration under nitrogen. An aliquot (~100 g) of mud is placed into a plastic
pressure filtration vessel with 1.0 u acid-cleaned filter, and tapped down to
remove air bubbles. The system is pressurized to 75 psi, and after discarding the
first 1 to 2 ml of filtrate, the interstitial water is collected into a 30-ml
polyethylene bottle under nitrogen. The As(III) stability curve in Figure 2-7 was
generated on a sample in contact with air. Within 5 minutes, the sample had
changed from colorless to brown, indicating that Fe(II) had oxidized to Fe(III),
and precipitated as colloidal Fe(OH)3. If an aliquot of sediment is filtered
under nitrogen and then frozen at -196°C, as for water samples, within 5 to
10 minutes, minimal changes in the As(III)/As(V) ratio should have taken place.
Using the above technique, a sample of spiked, Lake Washington sediment was
analyzed for interstitial water arsenic speciation 30 days after spiking with
arsenic. This data is presented in Table 2-9 and shows that the distribution
coefficients (K.) of the various species between the solid and aqueous phases
increase in the following order: DMA«MMA10,000
371
364
23
2-23
-------
mud (LWM) and spiked LWM were placed into polyethylene bottles and frozen at
-18°C, while three aliquots were kept refrigerated at 0 to 4°C. After 30 days
these samples were analyzed for arsenic species, the results of which are shown in
Table 2-8. These data indicate that small changes in the concentrations of the
various species may be occurring, with significant decreases (20-30%) in the
organic species being seen. These changes are small enough, however, that if the
samples were analyzed as soon as possible after collection, they should not be of
great importance.
Table 2-8
THIRTY-DAY STORAGE RESULTS FOR ARSENIC SPECIATION IN SEDIMENTS
Lake Washington mud
Arsenic
species
pg-g-1Arsenic, dry weight basis
Initial
concentration
Concentrations after 30-day aging
Refrigerated, 0-4°C Frozen, -18°C
As(III)
As(V)
MMA
DMA
2.2 ± 0.3
4.4 ± 0.3
<0.8
<0.8
2.2 ± 0.4
5.2 ± 0.4
<0.8
<0.8
2.3 ± 0.3
5.4 ± 0.4
<0.8
<0.8
Spiked Lake Washington mud
Arsenic
species
As(III)
As(V)
MMA
DMA
Initial
concentrati on
8.2 ± 1.4
13.5 ± 1.7
51.3 ±6.0
47.0 ±4.2
jjg-g-1ArsenicJ^ dry weight
basis
Concentrations after 30-day aging
Refrigerated, 0-4°C
7.1 ± 2.7
13.8 ± 1.0
39.9 ± 1.6
46.5 ±3.2
Frozen, -18°C
9.9 ± 1.3
16.0 ± 0.5
46.2 ±3.5
40.0 ±2.4
2-22
-------
5.0
to
a
7«
3.0
2.0
1.O
-I 1 1-
-4 1 1-
\Aillll)
-I 1 1 t—l 1-
1234567 8 9 10 11 12
4O
^30
a
20
10
H 1 1 1-
«•— spiked concentration —.>
-1 1
5 ' 6 7 8 9 10 11 12
pH
Figure 2-6. Arsenic species released from sediments as a function
of solution pH. Plot of arsenic in sediment leached, pg g-1 dry
weight basis (DWB), versus pH of leachate.
2-21
-------
Arsenic Speclation of Sediments. Maher (4) has shown that various arsenic species
that may be removed from solids at different pH values. This approach was tested
on a sample of spiked Lake Washington mud, over a wide range of pH using phosphate
buffers. The results of these experiments, shown as arsenic recovered versus pH
for all four species, are illustrated in Figure 2-6. Notice that the maximum
recovery of As(III) occurs at about pH = 2.8 and that the maximum for As(V), MMA
and DMA occur at pH >12. From these data, the two convenient buffers of 0.1 M
H3P04 (pH = 1.5) and Na3P04 (pH = 12) were chosen to selectively extract the
arsenic species from sediments. Samples extracted with H3P04 (final pH = 2.3) are
analyzed only for As(III) whereas those extracted with Na3P04 (final pH = 11.9)
are analyzed only for total As, which gives As(V), MMA and DMA, as As(III) is not
extracted at this pH. On untested sediment types it would be wise to test this
relationship to be sure it holds true before instituting an analytical regime.
Recovery of arsenic species from spiked Lake Washington mud is illustrated in
Table 2-7. The calculated spike was added to the mud, which was then aged 14 days
at 4°C before analysis. All analysis were carried out in quintuplicate. The
yields are good and within the day-to-day variability for the respective species.
Table 2-7
RECOVERY OF ARSENIC SPECIES FROM SPIKED LAKE WASHINGTON
MUD BY SELECTIVE LEACHING
Arsenic
species
As(III)
As(V)
MMA
DMA
ug-g-1
Lake Washington
mud
2.2 ± 0.3
4.4 ± 0.3
<0.8
<0.8
Arsenic, dry
Spike
added
5.8
9.5
58.0
54.0
weight basis
Total
recovered
8.2 ± 14
13.5 ± 17
51.3 ±6.0
47.0 ± 4.2
Percent
recovery
103%
96%
88%
87%
The values of the above analysis were then taken as the time zero values, and the
mud divided and stored in one of two ways. Three aliquots each of Lake Washington
2-20
-------
TIME, MINUTES
Figure 2-5. Chromatogra'm of digested (HN03/H2S04) spiked
Lake Washington mud. Vertical axis absorbance, horizontal
axis time. Note absence of DMA peak and presence of
unidentified higher boiling compound.
2-19
-------
Table 2-6
COMPARISON OF X-RAY FLUORESCENCE SPECTROSCOPY AND HYDRIDE
GENERATION AA IN THE DETERMINATION OF TOTAL ARSENIC
ENVIRONMENTAL SEDIMENTS. ALL REPRESENT TOTAL INORGANIC
ARSENIC BY HOT ACID DIGESTION EXCEPT (*) SLWM,
WHICH IS THE SUM OF SPECIES BY LEACHING
Type of Sediment
Total Arsenic, ug-g-1 dry weight basis
XRF Hydride AA
Lake Washington (silt)
Spiked Lake Washington (silt)
BCSS-1, clean estuarine (mud)
Contaminated Puget Sound (sandy)
Duwamish River (sand)
14.
124.
11.
108.
8.
6
1
7
0
0
±
±
±
±
0.
3.
0.
24.
1
4
7
0
n=3
n=3
n=3
n=3
n=l
14.
120.
9.
93.
2.
5
0
9
0
6
± 1.
± 7.
± 1.
± 21
1
5
0
.0
n=6
n=5*
n=5
n=3
n=l
However, when Lake Washington sediment spiked with inorganic as well as organic
forms was analyzed by this method, the following was observed:
1.
2.
3.
All of the MMA was recovered as MMA.
All of the inorganic arsenic was recovered as inorganic arsenic.
None of the DMA was recovered, but an unidentified higher boiling
peak was generated.
This peak is clearly illustrated in Figure 2-5. Even after the above samples were
re-digested to near-dryness (white fumes) in HN03 plus HC104, the same results
were obtained. Therefore, at this point we recommend no hydride generation method
to determine total arsenic in sediments, though this may be achieved using either
neutron activation analysis or X-ray fluorescence spectroscopy. On the other
hand, since no organic forms have been detected in any natural sediment and since
both MMA and DMA give observable peaks if they are present, it is safe to assume
as a general guideline that if only.an inorganic arsenic peak is generated by a
given sample, then it probably represents close to the total arsenic content of
the sample.
2-18
-------
Determination of Arsenic Species in Sediments
Two procedures were investigated in the determination of arsenic in sediments.
One, a wet-acid digestion was used to determine total arsenic. The second was a
mild, pH-selective leach to remove, various arsenic species intact.
Total Arsenic. In applying the hot HN03/H2S04 digestion to standard sediments and
air particulate matter, good agreement was attained between the established values
and the measured values (Table 2-5). Also>, in the case of estuarine and riverine
sediments collected in the Puget Sound area, there was good agreement between
X-ray fluorescence spectroscopy and this method (Table 2-6). In either case, all
observed arsenic was in the inorganic form.
Table 2-5
TOTAL INORGANIC ARSENIC IN STANDARD SEDIMENTS BY
HN03/H2S04
Total (inorqanic) arsenic pq-q-1 dry weight basis
Replicate
1
2
3
4
5
N
X
s
RSD
Certified
+
MESS-1
Estuarine
sediment
8.9
8.8
8.8
9.6
10.1
5
9.2
0.6
6.5%
10.6
1.2
BCSS-1
Estuarine
sediment
10.9
8.5
9.4
9.8
10.7
5
9.9
1.0
10.12
11.1
1.4
NBS-1646
Estuarine
sediment
9.8
10.0
9.8
8.5
11.0
5
9.8
0.9
9.2%
11.6
1.3
NBS-1648
Air
parti cul ate
matter
123.0
136.0
115.0
-
3
125.0
11.0
8.8%
115.0
10.0
2-17
-------
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2-15
-------
concentration of these parameters was within ±20% of the initial in all cases.
The noise in the data is due mostly to the day-to-day analytical variability,
which has been observed to be about twice that of same-day replicate analysis. On
the other hand, these data also show that it is very difficult to preserve the
original As(III)/As(V) ratio in samples, even for a short time. Two major
observations are made: first, river water (Dungeness River water) tends to
spontaneously reduce As(V) to As(III), even though the water has been filtered to
0.4 u, thus removing most living creatures. This is also curious, as the natural
equilibrium As(III)/As(V) ratio is about 0.2 in Dungeness River water. It is
surmised that dissolved organic materials in the water are responsible for its
reducing properties, a conclusion that is supported by work involving the
reduction of Hg(II) to Hg(0) by humic acids (Bloom, unpublished work). The second
observation is that the freezing of water inexplicably, but reproducibly
causes the oxidation of As(III) to As(V) (Figure 2-4-g, i), except in the case of
very rapid freezing by immersion in LN2 (Figure 2-4-m, o).
In light of these observations, the following storage regimes are recommended for
arsenic in aqueous solution:
1. If only total inorganic arsenic plus MMA and DMA are to be
determined, the sample should be stored at 0 to 4°C in polyethylene
bottles until analysis. No chemical preservative is needed or
desired and the analysis should be carried out as soon as possible.
2. If the As(III)/As(V) ratio is to be maintained, the sample must be
quick-frozen to -196°C in liquid nitrogen, and then stored at at
least -80 C until analysis. Note that Figure 4-k shows that even
^o£e Case of rapid freez1r>9 to -196°C, followed by storage at
-18 C, a definite oxidation of As(III) to As(V) was observed.
A convenient and safe way to quick-freeze samples is to place 55 ml of sample into
a 60-ml narrow-mouth polyethylene bottle, screw on the cap (which has a 2 mm
diameter hole) tightly, and drop into a Dewar flask full of liquid nitrogen.
These bottles have been shown not to crack if less than 58 ml of water is placed
in them, and not to float in the LN2 if more than 50 ml is placed in them. After
returning to the laboratory, the bottles may be placed into a low temperature
freezer until analysis. Note of caution, if a small hole is not placed in the lid
of the bottles, which are frozen in liquid nitrogen, the bottles may explode when
removed from the liquid nitrogen.
2-14
-------
Table 2-4
PRECISION DATA FOR THREE ARSENIC SPECIES, ILLUSTRATING
THE DECREASE IN PRECISION WITH INCREASING BOILING
POINT OF SPECIES. THESE SAMPLES WERE SPIKED RIVER
WATER USED IN WATER STORAGE TESTS
Arsenic concentrations,
Replicate
N (8-24-83)
x
s
RSD
N (9-11-83)
x
s
RSD
Inorganic
arsenic
3
937
44
4.7%
3
800
24
3.0%
MMA
3
2483
79
3.2%
4
2342
'165
7.0%
nq-1-1
DMA
3
2173
181
8.3%
4
2393
260
10.9%
The detection limit of this technique has not been explored to the extreme as the
usual environmental sample benefits from less, not more sensitivity. For a chart
recorder expansion of 600 mau full scale, and the parameters given in the text,
and for a 30-ml sample aliquot, the following approximate detection limits are
found: As(V), 0.006 pg-1-1 (twice the standard deviation of the blank); As(III)
0.003 jjg-1'1 (O-5 chart units); MMA, 0.010 ug--1 as As (0.5 chart units); DMA,
0.012 ug-1-1 as As (0.5 chart units). For As(III), MMA and DMA, no contribution
to the blank has been found due to reagents, except for the As(III) present in the
river water used as a dilutant. As for As(V) a small contribution is found,
mostly from the NaBH4, and to a smaller extent from H3P04. These may be minimized
by selecting reagent lots of reagents found to be low in arsenic.
Water Storage Experiments
From the many experiments undertaken to determine a storage regime for arsenic
species, the following general conclusion can be made: Almost any storage scheme
will preserve the total arsenic, MMA, and DMA concentrations of river water in the
ug-1-1 range. This is illustrated in the Figures 2-4a-p, where the final
2-13
-------
As arsenic response 'is quite sensitive to the H2/02 ratio in the flame, it is
necessary to restandardize the instrument whenever it is set up. Usually,
however, the response is quite constant and stable over the entire day.
Precision, Accuracy and Detection Limits
Precision and accuracy are the greatest and the detection limits the lowest for
inorganic arsenic. The precision and accuracy of the inorganic arsenic
determination is illustrated at two concentrations in Table 2-3. The standard
seawater, NASS-1 (National Research Council of Canada) was run in 5.0-ml aliquots
and the "standard river water" (National Bureau of Standards) was run in 100-ul
aliquots. In either case, both the precision (RSD) and accuracy were about 5%.
Precision begins to decrease, as the boiling point of the compound increases, as
is illustrated in Table 2-4, for spiked river water. No standard reference
material has been found for the organic species.
Table 2-3
REPLICATE DETERMINATIONS OF TOTAL INORGANIC
ARSENIC IN SOME STANDARD WATERS
Replicate
1
2
3
4
5
N
X
S
RSD
Certified
±
Total (inorganic)
NASS-1
Seawater
1.579
1. 556
1.591
1.493
1.529
5
1.550
0.040
2.6%
1.65
0.19
arsenic, gg-1-1
NBS
River water
81.5
74.5
71.8
79.0
79.3
5
77.2
4.0
5.2%
76.0
7.0
M - number of replicates.
X - mean
S - ± one standard deviation
RSD - relative standard deviation
2-12
-------
10
15 20 25 30 35
flRSENIC (NRNOGRRMS)
Figure 2-3. Standard curves, absorbance versus concentration for arsenic.hydride
species, atomic absorption detector.
2-11
-------
Figure 2-2. Typical chromatogram of arsenic hydride
species. Vertical axis absorbance, horizontal axis
time.
2-10
-------
Conditions of temperature ranging from 20°C to -196°C were assessed, as well as
preservation with HC1 and ascorbic acid. Storage tests were carried out over a
period of one month for water samples.
The stability of the As(III)/As(V) ratio in interstitial water at room
temperature, in the presence of air was carried out over a 24-hour period to
determine the feasibility of the field collection of interstitial water.
Because of the time-consuming nature of sediment analysis, a two-point storage
test was carried out with triplicate samples analyzed for two sediments at two
temperatures (0°C and -18°C). Mud samples were stored in polyethylene vials and
analyzed at time zero and one month.
RESULTS AND DISCUSSION
Data Output
Using the procedures outlined above, and a mixed standard containing As(V), MMA,
and DMA, standard curves were prepared for each of the arsines generated. A
typical chromatogram from this procedure is illustrated in Figure 2.2. Under
the conditions described in this paper, the elution times for the various arsines
are as follows: AsH3, 24 ± 2 s; CH3AsH2, 53 ± 2 s and (CH3)2AsH, 66 ± 2 s.
Notice that the peaks are broadened and that the sensitivity decreases as the
boiling point of the compound increases. The small amount of signal after the DMA
peak is probably a higher boiling impurity in the DMA, or some DMA that is lagging
in the system during elution. We had previously noted much larger, multiple peaks
in this region when water was allowed to condense between the trap and the
detector. Such peaks were effectively eliminated and the DMA peak sharpened with
the addition of the heating coil between the trap and the detector.
The typical standard curves in Figure 2.3 are prepared from the mean of two
determinations at each concentration. Arsenic peak-height response appears to be
linear to at least 600 mau (milliabsorbance units), which is the full scale
setting used on our chart recorder. Andreae (3) shows that arsenic response is
extremely non-linear above this for the peak height mode, and recommends the use
of peak area integration to increase the linear range. We have chosen to simply
use a small enough sample aliquot to remain within 600 mau.
2-9
-------
settle overnight. An appropriate-sized aliquot of the supernatant liquid
(25-100 ul) is added to 20 ml of deionized water and run as for total arsenic.
teachable Arsem'te
An aliquot (~l-2 g) of fresh or freshly thawed wet homogeneous sediment is weighed
to the nearest 10 mg directly into a 40-ml acid-cleaned Oak Ridge type centrifuge
tube. To this is added 25 ml of 0.10 M H3P04 solution and the tubes are agitated
with the lids on. Periodic agitation is maintained for 18 to 24 hours, at which
time the tubes are centrifuged for 30 minutes at 2500 RPM. Twenty milliliter
aliquots of the supernatant liquid are removed by pipetting into cleaned
polyethylene vials and saved in the refrigerator until analysis. Analysis should
be accomplished within the next couple days.
For analysis, an appropriate-sized aliquot (10-100 pi) is added to 20 ml of
well-character!zed filtered river water (or other nonoxidizing/nonreducing water).
Enough 1.0 M NaOH solution is added to approximately neturalize the H3P04 (1/3 the
volume of the sample aliquot), and then 1.0 ml of Tris buffer is added. The
sample is then analyzed as for As(III).
teachable Arsenate, HMA and DMA
An aliquot (~l-2 g) of wet sediment is weighed into a centrifuge tube, as above.
To this are added 25 ml of 0.1 M Na3P04 solution, and the tubes agitated
periodically for 18 to 24 hours. After centrifugation the supernatant liquid
(dark brown due to released humic materials) is analyzed as for total arsenic
using an appropriate-sized aliquot in 20 ml of deionized water. The total
inorganic arsenic in this case should be only As(V), as As(III) is observed to not
be released at this pH. No pre-neutralization of the sample is necessary as the
HC1 added is well in excess of the sample alkalinity.
Interstitial Water Analysis
Interstitial water samples may be treated just as ordinary water, except that as
they are quite high in arsenic, usually an aliquot of 100 to 1000 ul diluted in
deionized water or river water is appropriate in most cases.
Storage Experiments
Storage experiments designed to preserve the original arsenic speciation of
samples were carried out for a wide variety of conditions. For water samples,
30-ml and 60-ml polyethylene bottles precleaned in 1 M HC1 were used.
2-8
-------
Table 2-2
REDUCTION PRODUCTS AND THEIR BOILING POINTS OF VARIOUS
AQUEOUS ARSENIC SPECIES
Aqueous form
As(III), arsenous acid, HAs02
As(V), arsenic acid, H3As04
HHA, CH3AsO(OH)2
DMA, (CH3)2AsO(OH)
Reduction product
AsH3
AsH3
CH3AsH2
(CH3)2AsH
B.P. ,
-55
-55
2
35.
°C
6
Arsenic (III) Determination
The same procedure as above is used to determine arsenite, except that the initial
pH is buffered at about 5 to 7 rather than <1, so as to isolate the arsenous acid
by its pKa (1). This is accomplished by the addition of 1.0 ml of Tris buffer to
a 5- to 30-ml aliquot of unacidified sample. (If the sample is acidic or basic,
it must be neutralized first, or the buffer will be exhausted.) , For the As(III)
procedure, 1.0 ml of NaBH4 is added in a single short (~10 seconds) injection, as
the rapid evolution of H2 does not occur at this pH.
Small, irreproducible quantities of organic arsines may be released at this pH and
should be ignored. The separation of arsenite, however, is quite reproducible and
essentially 100% complete. As(V) is calculated by subtracting the As(III)
determined in this step from the total inorganic arsenic determined on an aliquot
of the same sample previously.
SEDIMENTS
Total Inorganic Arsenic
A 1.00-g aliquot of freeze-dried and homogenized sediment is placed into a 100-ml
snap-cap volumetric flask. Five milliliters of deionized water is added to form a
slurry and then 7 ml of the acid digestion mixture is added. After 5 minutes, the
caps are replaced and the flasks heated at 80 to 90°C for 2 hours. Upon cooling
the samples are diluted to the mark with deionized water, shaken, and allowed to
2-7
-------
Iris Buffer. 394 g of Tris-HCl (tris (hydroxymethyl) aminomethane hydrochloride)
and 2.5 g of reagent grade NaOH are dissolved in deionized water to make
1.0 liter. This solution is 2.5 M in tris and 2.475 M in HC1, giving a pH of
about 6.2 when diluted 50-fold with deionized water.
Sodium Borohydride Solution. Four grams of >98% NaBH4 (previously analyzed and
found to be low in arsenic) are dissolved in 100 ml of 0.02 M NaOH solution. This
solution is stable 8-10 hours when kept covered at room temperature. It is
prepared daily.
Phosphoric Acid Leaching Solution. To prepare 1.0 liter of 0.10 M phosphoric acid
solution, 6.8 ml of reagent grade 85% H3P04 are dissolved in deionized water.
Trisodium Phosphate Leaching Solution. To prepare 1.0 liter of 0.10 M trisodium
phosphate solution, 6.8 ml of 85% H3P04 and 12 g of reagent grade NaOH are
dissolved in deionized water.
Acid Digestion Mixture. With constant stirring, 200 ml of concentrated reagent
grade H2S04 are slowly added to 800 ml concentrated HN03.
METHODS
Total Arsenic Determination
An aqueous sample (5-30 ml) is placed into the reaction vessel and 1.0 ml of
6M HC1 is added. The 4-way valve is put in place and turned to begin purging the
vessel. The G.C. trap is lowered into a Dewar flask containing liquid nitrogen
(LN2) and the flask topped off with LN2 to a constant level. A 2.0-ml aliquot of
NaBH4 solution is then introduced through the silicone rubber septum with a
disposable 3-ml hypodermic syringe and the timer turned on. The NaBH4 is slowly
added over a period of about 1 minute, being careful that the H2 liberated by the
reduction of water does not overpressurize the system or foam the contents out of
the reaction vessel.
After purging the vessel for 8 minutes, the stopcock is turned to pass helium
directly to the G.C. trap. In rapid order, the LN2 flask is removed, the trap
heating coil is turned on, and the chart recorder is turned on. The arsines are
eluted in the order: AsH3, CH3AsH2, (CH3)2AsH according to their increasing
boiling points given in Table 2.2 (1).
2-6
-------
Detector. Any atomic absorption unit may serve as a detector, once
been built to hold the quartz cuvette burner in the wave path. This
done using a Perkin-Elmer Model 5000® spectrophotometer with
discharge arsenic lamp. An analytical wavelength of 197.3 nm and
0..7 nm (low) are used throughout. This wavelength has been shown to
linear range, though about half the sensitivity of the 193.7
Background correction is not used as it increases the system noise
been found necessary on the types of sample discussed in this paper.
a bracket has
work has been.
electrode!ess
slit width of
have a longer
nm 1i ne (2).
and has never
Standards and Reagents
Arsemte (Asflim Standards. A 1000 mg-1-1 stock solution is made up by the
dissolution of 1.73 grams of reagent grade NaAs02 in 1.0-liter deionized water
containing 0.1% ascorbic acid. This solution is kept refrigerated in an amber
bottle. A 1.0-mg-1-1- working stock solution is made by dilution with 0.3%
ascorbic acid solution and stored as above. Under these conditions this solution
has been found stable for at least one year.
Further dilutions of As(III) for analysis, or of samples to be analyzed for
As(III), are made in filtered Dungeness River water. It has been observed both
here and elsewhere (Andreae 1983) that deionized water can have an oxidizing
potential that causes a diminished As(III) response at low levels (1 ug-1-1 and
less). Dilute As(III) standards are prepared daily.
Arsenate (AsOO) Standards. To prepare a 1000 mg-1-1 stock solution, 4.16 g of
reagent grade Na2HAS04-7H20 are dissolved in 1.0 liter of deionized water.
Working standards are prepared by serial dilution with deionized water and
prepared monthly.
Honomethylarsonate (MMA) Standards. To prepare a stock solution of 1000 mg-1-1,
3.90 g of CH3AsO(ONa)2-6H20 is dissolved in 1.0 liter of deionized water. Working
standards are prepared by serial dilution with deionized water. Dilute standards
are prepared weekly.
Dimethvlarsinate (DMA) Standards. To prepare a stock solution of lOOOmg-1-1,
2.86 g of reagent grade (CH3)2As02Na-3H20 (cacodylic acid, sodium salt) is
dissolved in 1.0 liter deionized water. Dilute standards are handled as for MMA.
6M Hydrochloric Acid. Equal volumes of reagent grade concentrated HC1
deionized water are combined to give a solution approximately 6M in HC1.
and
2-5
-------
A. SCHEMATIC
DIAGRAM
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Atomlzer. The eluted arsines are detected by flame atomic absorption, using a
special atomizer designed by Andreae (2). This consists of a quartz cross tube as
shown in Figure 2-1-c. Air is admitted into one of the 6-mm o.d. side tubes
(optimal flows are given in Table 2-1), while a mixture of hydrogen and the
carrier gas from the trap is admitted into the other. This configuration is
superior to that in which the carrier gas is mixed with the air (Andreae, personal
communication 1983) due to the reduction of flame noise and possible extinguishing
of the flame by microexplosions when H2 is generated in the reaction vessel. To
light the flame, all of the gases are turned on, and a flame brought to the ends
of the quartz cuvette. At this point a flame will be burning out of the ends of
the tube. After allowing the quartz tube to heat up (~5 minutes) a flat metal
spatula is put smoothly first over one end of the tube, and then the other. An
invisible air/hydrogen flame should now be burning in the center of the cuvette.
This may be checked by placing a mirror near the tube ends and checking for water
condensation. Note that the flame must be burning only inside the cuvette for
precise, noise-free operation of the detector.
Table 2-1
OPTIMAL FLOWS AND PRESSURES FOR GASES
IN THE HYDRIDE GENERATION SYSTEM
Gas
He
H2
Air
Flow rate
ml-min-1
150
350
180
Pressure
lb-in-2
10
20
20
Precision and sensitivity are affected by the gas flow rates and these must be
individually optimized for each system, using the figures in Table 2-1 as an
initial guide. We have observed that as the 02/H2 ratio goes up, the sensitivity
increases and the precision decreases. As this system is inherently very
sensitive, adjustments are made to maximize precision.
2-3
-------
septum (Ace Glass #9096-32) to allow the air-free injection of sodium borohydride.
The standard impinger assembly is replaced with a 4-way Teflon stopcock impinger
(Laboratory Data control #700542) to allow rapid and convenient switching of the
helium from the purge to the analysis mode of operation.
t
GC TraP- Tne 1«* temperature GC trap is constructed from a 6 mm o.d. borosilicate
glass U-tube about 30-cm long with a 2-cm radius of bend (or similar dimensions to
fit into a tall widemouth Dewar flask. Before packing the trap, it is silanized
to reduce the number of active adsorption sites on the glass. This is
accomplished using a standard glass silanizing compound such as Sylon-Ct® (Supelco
Inc.). The column is half-packed with 15% OV-3 on Chromasorb® WAW-DMCS (45-60
mesh). A finer mesh size should not be used, as the restriction of the gas flow
is sufficient to overpressurize the system. After packing, the ends of the trap
are plugged with silanized glass wool.
The entire trap assembly is then preconditioned as follows: The input side of the
trap (non-packed side) is connected via silicone rubber tubing to helium at a flow
rate of 40 ml-min-1 and the whole assembly is placed into an oven at 175°C for
2 hours. After this time, two 25-ul aliquots of GC column conditioner (Silyl-8®,
Supelco Inc.) are injected by syringe through the silicone tubing into the glass
tubing. The column is then left in the oven with helium flowing through it for
24 hours. This process, which further neutralizes active adsorption sites and
purges the system of foreign volatiles, may be repeated whenever analate peaks are
observed to show broadening.
Once the column is conditioned, it is evenly wrapped with about 1.8 m of nichrome
wire (22 gauge) the ends of which are affixed to crimp on electrical contacts.
The wire-wrapped column is then coated about 2-mm thick all over with silicone
rubber caulking compound and allowed to dry overnight. The silicone rubber
provides an insulating layer which enhances peak separation by providing a longer
temperature ramp time.
The unpacked side of the column is connected via silicone rubber tubing to the
output from the reaction vessel. The output side of the trap is connected by a
nichrome-wire wrapped piece of 6-mm diameter borosilicate tubing to the input of
the flame atomizer. It is very important that the system be heated everywhere
(~80°C) from the trap to the atomizer to avoid the condensation of water. Such
condensation can interfere with the determination of dimethylarsine. All
glass-to-glass connections in the system are made with silicone rubber sleeves.
2-2
-------
Section 2
DETERMINATION OF ARSENIC SPECIES IN LIMNOLOGICAL SAMPLES
BY HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROSCOPY
INTRODUCTION
This section describes the analytical methods used to determine the arsenic
species in waters and sediments. Also, sample storage tests were conducted to
select methods of storing and shipping environmental samples that would minimize
changes in speciation. Based on results of previous studies we selected hydride
generation coupled with atomic absorption spectroscopy as *the method of
quantification of arsenic. In this technique arsenate, arsenite, methylarsenic
acid, and dimethylarsinic acid are volatilized from solution at a specific pH
after reduction to the corresponding arsines with sodium borohydride (1). The
volatilized arsines are then swept onto a liquid nitrogen cooled chromatographic
trap, which upon warming, allows for a separation of species based on boiling
points. The released arsines are swept by helium carrier gas into a quartz
cuvette burner cell (2), where they are decomposed to atomic arsenic. Arsenic
concentrations are determined by atomic absorption spectroscopy. Strictly
speaking, this technique does not determine the species of inorganic arsenic but.
rather the valence states of arsenate (V) and arsenite (III). The actual species
of inorganic arsenic are assumed to be those predicted by the geochemical
equilibrium model described in Section 1 of this report.
EXPERIMENTAL SECTION
Apparatus
The apparatus needed for the volatilization, separation and quantisation of
arsenic species is shown schematically in Figure 2-1-a. Briefly, it consists of a
reaction vessel, in which arsenic compounds are reduced to volatile arsines, a
liquid nitrogen cooled gas chromatographic trap, and a H2 flame atomic absorption
detector.
Reaction Vessel. The reaction vessel is made by grafting a side-arm inlet onto a
30-ml "Midget Impinger" (Ace Glass #7532-20), as illustrated in Figure 2-1-b. The
8-mm diameter side arm may then be sealed with a silicone rubber-stopper type
2-1
-------
-------
APPENDIX K
APPENDIX K
A RECOMMENDED METHOD FOR
INORGANIC ARSENIC ANALYSIS
Extracted from:
Crecelius, E.A., N.S. Bloom, C.E. Cowan, and E.A. Jenne. 1986. Speciation of
Selenium and Arsenic in Natural Waters and Sediments. Volume 2:
Arsenic Speciation, Section 2, in EPRI report #EA-4641, Vol. 2, pp. 2-1
to 2-28.
K-3
-------
-------
APPENDIX K
A RECOMMENDED METHOD FOR
INORGANIC ARSENIC ANALYSIS
-------
-------
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APPENDIX J
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