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
                   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
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                   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
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

-------
                                                                  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

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                                                                   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

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                                                                                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

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                                                                    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

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               3. TARGET SPECIES


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                                                    3. TARGET SPECIES
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-------
                                                                       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




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Esocidae

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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
7
9
10
8
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

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                                                    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

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                                                              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

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                                                          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

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                                                             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

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                                                           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

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                                                          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

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                                                                   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

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                                                                  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

-------
                                                                                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

-------
                                                   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

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                                                   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

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                                                                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

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                                                   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

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4. TARGET ANALYTES
              in
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                4-11

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                                                                  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
                                                                                   4-12

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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
                                                                                  4-13

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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).
                                                                                   4-14

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4. TARGET ANALYTES
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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
                                                                                 4-21

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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.
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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
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                                                                  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).
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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

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                  w
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                  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

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                                                                 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

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                                        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

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                                        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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Q-5 £
^i1
•|-° « co
Hl^'l
t *^~ c
K-'&S-c
S * «.e
»— 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
._
m
S

k_
•S
"n
c
o
.0
fc_
CD
CD
CO
£
Q.
£
o
a*
is intended
o
CO
CM
1
S
<
"o
LI-
CO
CD
H
§
CM
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
fl|iP
CO ^'x *" 00
"B "c -2 o i^
C CD (8 •£ C0_
i— -° .E •§ "S
33 _o *" co
*la*l
LU • ca o,g
cq 1 -1 'I 1
= 8 g 3. 2=
o^JS-
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

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                                             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

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                                                                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

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                                                                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

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                                                   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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                                                  6.  FIELD PROCEDURES
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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                                                                 6. FIELD PROCEDURES
                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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                                                                 6. FIELD PROCEDURES
               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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                                                                  6. FIELD PROCEDURES
                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.
                                                                                   6-14

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                                                                6. FIELD PROCEDURES
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
                                                                                 6-15

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                                                  6. FIELD PROCEDURES
 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

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                                                  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

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                                                  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

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                                                                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.  FIELD PROCEDURES
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                                             6.  FIELD PROCEDURES
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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

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                                                                 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

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                                                  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

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                                                                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

-------
                                                                      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

-------
                                                         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

-------
                                                      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

-------
                                   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

-------
                                   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

-------
                                        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

-------
                                  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

-------
                                    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

-------
                                      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.
                                                                                           7-13

-------
                                   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

-------
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

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                                    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

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                                      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

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                                     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

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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)".
                                                                                8-37

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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.
                                                                                 8-38

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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).
                                                                                8-39

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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)
                                                                                   8-40

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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.
                                                                                  8-41

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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.
                                                                  8-43

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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
                                                                                  8-44

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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.
                                                                   8-45

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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).
                                                                                  8-46

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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
                                                                                 8-48

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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:                                   '<
                                                                                 8-49

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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.
                                                               8-50

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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

LITERATURE CITED
              Abbott, R.T. 1974. American Seashells—The Marine Molluscs of the Atlantic
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              Agocs, M.M., R.A.  Etzel, R.G. Parrish, D.C. Paschal, P.R. Campagna, D S
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              Anderson, R.O., and SJ. Gutreuter.  1983.  Length, weight, and associated
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              Armbruster, G., K. Gerow, W. Gutenmann, C. Littmann, and D. Usk.  1987. The
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              Armbruster, G., K.L Gall, W.H. Gutenmann, and D.J. Lisk. 1989.  Effects  of
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              Ashley, .LM.  1962.  Laboratory Anatomy of the Turtle. W.C. Brown Company
                 Dubuque, IA.                                                 K   y'

              ASTM (American Society for Testing and Materials).  1976.  ASTM Manual on
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             ASTM (American Society for Testing and Materials).  1983.  Standard Practice
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             ATSDR (Agency for Toxic Substances and Disease  Registry).  1987a. Draft
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                 Washington, DC.
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                                                 10.  LITERATURE CITED
ATSDR (Agency for Toxic Substances and Disease Registry).  1987b.  Draft
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ATSDR  (Agency for Toxic Substances and  Disease  Registry).   1987c.
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ATSDR  (Agency for Toxic Substances and  Disease  Registry).   1987d.
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ATSDR  (Agency for Toxic Substances and  Disease  Registry).   1989a.
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    Systems, Inc., for ATSDR.  U.S. Public Health Service in collaboration with
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ATSDR  (Agency for Toxic Substances and  Disease  Registry).   1989b.
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ATSDR  (Agency  for  Toxic  Substances and  Disease  Registry).    1990.
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ATSDR  (Agency for  Toxic  Substances and  Disease  Registry).    1993.
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 Bache, C.A., W.H. Gutenmann, and D.J. Lisk. 1971. Residues of total mercury
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 Ballschmitter, K., and  M. Zell.   1980.  Analysis  of polychlorinated biphenyls
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 Barnes, D.G., and J.S. Bellin.  1989.  Interim Procedures for Estimating Risks
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Battelle Memorial Institute.  1989.  Work/Quality Assurance Project Plan for the
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Beauchemin, D., K.W.M.  Siu,  J.W.  McLaren,  and S.S. Berman.   1989.
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    Valley in  1989.  Draft. Chattanooga, TN.

 U.S. DHHS  (U.S.  Department of Health  and  Human  Services).   1990.
    Toxicological Profile for  Polycyclic Aromatic Hydrocarbons.   TP-90-20.
    Agency for Toxic Substances and Disease Registry, Public Health Service
    Atlanta, GA.

 U.S.  EPA  (U.S.   Environmental  Protection  Agency).    1978.     Metal
    Bioaccumulation in Fish  and  Aquatic Invertebrates.  EPA-600/3-78-103.
    Environmental Research Laboratory, Office of Research and Development
    Springfield, VA.

U.S. EPA (U.S. Environmental Protection Agency).  1979a. Health Assessment
    Document for Cadmium. EPA-600/8-79-003. Environmental Standards and
    Criteria, Office of Research and Development, Research Triangle Park, NC.

U.S. EPA (U.S. Environmental Protection Agency).  1979b.  Methods for the
    Chemical Analysis of Water and Wastes. EPA-600/4-79-020.  Environmental
    Monitoring and Support Laboratory, Cincinnati, OH.
                                                                10-25

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                                                10. LITERATURE CITED
U.S. EPA (U.S. Environmental Protection Agency).  1980a.  Ambient Water
    Quality Criteria for Endrin. EPA-440/5-80-047. Office of Water Regulations
    and Standards, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1980b. Interim Guidelines
    and  Specifications  for  Preparing  Quality Assurance  Project  Plans.
    QAMS-005/80.  Quality Assurance Management Staff, Washington, DC,

U.S. EPA (U.S. Environmental Protection Agency).   1981 a.  An Exposure and
    Risk  Assessment for  Mercury.   EPA-440/4-85-011.  Office  of Water
    Regulations and Standards, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1981b. Interim Methods for
    the Sampling and Analysis of Priority Pollutants in Sediments and Fish
    Tissue.   EPA-600/4-81-055.    Environmental  Monitoring and Support
    Laboratory, Cincinnati, OH.

U.S. EPA (U.S. Environmental Protection Agency).  1982a.  Methods for the
    Chemical   Analysis  of  Municipal   and   Industrial   Wastewater.
    EPA-600/4-82-057.   Environmental  Monitoring  and Support Laboratory,
    Cincinnati, OH.

U.S. EPA (U.S.  Environmental Protection Agency).   1982b.  Arsenic.  In:
    Intermedia Priority Pollutant Guidance Documents. Office of Pesticides and
    Toxic Substances, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1983a.  Analyses of the
    Risks and Benefits of Seven Chemicals Used for Subterranean Termite
    Control.  EPA-540/9-83-005. Office of Pesticide Programs, Washington, DC.

U.S. EPA (U.S.  Environmental Protection Agency). 1983b.   Pesticide Fact
    Sheet—Dicofol.  Office of Pesticide Programs, Washington, DC.

U.S.  EPA  (U.S. Environmental  Protection  Agency).    1984a.    Internal
    memorandum from G. LaRoccato B. Burnam et al., August 16,1984. Office
    of Pesticide Programs, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1984b. Policy and Program
    Requirements to Implement the Quality Assurance Program.   EPA Order
    5360.1.  Quality Assurance Management Staff, Washington, DC.

U.S. EPA (U.S.  Environmental Protection Agency).  1984c.   Pesticide Fact
    Sheet—Chlorpyrifos. Office of Pesticides and Toxic Substances, Office of
    Pesticide Programs, Washington, DC.

U.S. EPA (U.S.  Environmental Protection Agency).  1984d.   Pesticide Fact
    Sheet—Disulfoton.  Office of  Pesticides and Toxic Substances, Office of
    Pesticide Programs, Washington, DC.

                                                                 1CJI26

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                                                 10. LITERATURE CITED
U.S. EPA (U.S. Environmental Protection Agency).  1985a.  Bioaccumulation
    Monitoring Guidance: 3. Recommended Analytical Detection Limits. EPA-
    503/6-90-001. Office of Marine and Estuarine Protection, Washington, DC.

U.S. EPA (U.S. Environmental Protection Ageficy).  1985b.  Development of
    Statistical Distribution for Ranges of Standard Factors Used in Exposure
    Assessment.  EPA-600/8-85-010.  Office  of Health and  Environmental
    Assessment, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1985c.  Guidance for the
    Registration  of  Pesticide  Products Containing Lindane  as the Active
    Ingredient. EPA-540/RS-86-121. Office of Pesticide Programs, Washington,
    DC.

U.S. EPA (U.S.  Environmental Protection Agency).  1985d.   Pesticide Fact
    Sheet—Terbufos.  Office of Pesticides  and Toxic Substances, Office'of
    Pesticide Programs, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1986a.  Ambient  Water
    Quality Criteria  for Chlorpyrifos.   EPA-440/5-86-005.   Office of Water
    Regulations and Standards, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1986b.  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). 1986c. Health Assessment
    Document for Polychlorinated Dibenzofurans. Draft.  EPA 600/8-86-018A.
    Environmental Criteria and Assessment Office, Cincinnati, OH.

U.S. EPA (U.S. Environmental  Protection Agency).  1986d.   Pesticide Fact
    Sheet—Diazinon.  Office of  Pesticides  and Toxic Substances, Office of
    Pesticide Programs, Washington, DC.

U.S. EPA (U.S. Environmental  Protection Agency).  1986e.   Research and
    Development  Methodology for Evaluating  Potential Carcinogenicity in
    Support of Reportable Quality Adjustments to CERCLA Section 102. OHEA-
    C-073 Draft.  Carcinogen Assessment Group Office of  Environmental
    Assessment, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1986f. Test Methods for the
    Evaluation of Solid Waste, Physical/Chemical Methods. SW-846; 3rd Edition
    (with 1990 updates).  Office of Solid Waste and Emergency Response,
    Washington, DC.
                                                                10-27

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                                                 10. LITERATURE CITED
U.S. EPA (U.S. Environmental Protection Agency).  1987a.  Bioaccumulation
    Monitoring Guidance:  2.  Selection of Target Species and Review of
    Available Data.   EPA-430/9-86-005.   Office of Marine  and Estuarine
    Protection, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1987b.  Bioaccumulation
    Monitoring Guidance: 5,  Strategies for Sample Replication and Composit-
    ing.   EPA-430/9-87-003. Office  of  Marine  and Estuarine  Protection,
    Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1987c.  Cadmium Health
    Advisory Draft. Office of Drinking Water, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).   1987d. National Dioxin
    Study.   Tiers 3,  5,  6,  and 7.  EPA-440/4-87-003.   Office of Water
    Regulations and Standards, Washington, DC.

U.S.  EPA  (U.S.   Environmental  Protection  Agency).    1987e.    Quality
    Assurance/Quality Control (QA/QC) for 301(h) Monitoring Programs:
    Guidance on Field and Laboratory Methods.  EPA-430/9-86-004.  Office of
    Marine and Estuarine Protection, Washington, DC.
U.S.  EPA  (U.S.  Environmental  Protection Agency).
    Assessment Guidelines of 1986.  EPA/600/8-87/045.
    Environmental Assessment, Washington, DC.
 1987f.   The  Risk
Office of Health and
U.S. EPA (U.S. Environmental Protection Agency).  1988a.  Drinking Water
    Criteria Document for Polychlorinated Biphenyls (PCBs). ECAO-CIN-414.
    Prepared by Environmental Criteria and Assessment Office for Office of
    Drinking Water, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1988b. Public Health Risk
    Evaluation Database.  Office  of  Emergency  and Remedial  Response,
    Washington, DC.

U.S. EPA (U.S. Environmental Protection  Agency).   1988c.  Pesticide  Fact
    Sheet: Tributyltin (Antifouling Paints). Number  143. September 23, 1988.
    Office of Pesticides and Toxic Substances, Office of Pesticide Programs,
    Washington, DC.

U.S. EPA  (U.S.  Environmental  Protection Agency).   1989a.   Analytical
    Procedures and Quality Assurance Plan for the Determination of Mercury in
    Fish. Draft.  Environmental Research Laboratory, Duluth MN.

U.S. EPA  (U.S.  Environmental  Protection Agency).  -1989b.   Analytical
    Procedures  and  Quality Assurance   Plan  for  the  Determination  of
    PCDD/PCDF  in  Fish.   EPA-600/3-90-022.    Environmental  Research
    Laboratory, Duluth, MN.
                                                                10-28

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                                                 10. LITERATURE CITED
 U.S. EPA  (U.S.  Environmental  Protection  Agency).    1989c.    Analytical
     Procedures and Quality Assurance Plan for the Determination ofXenobiotic
     Chemical  Contaminants  in  Fish.  EPA-600/3-90-023.   Environmental
     Research Laboratory, Duluth, NM.

 U.S. EPA (U.S. Environmental Protection Agency).  1989d. Assessing Human
     Health Risks from Chemically Contaminated Fish and Shellfish:  A Guidance
     Manual.  EPA-503/8-89-002. Office  of Water Regulations and Standards,
     Office of Marine and Estuarine Protection, Washington, DC.

 U.S. EPA  (U.S. Environmental  Protection Agency).  1989e.  Pesticide Fact
     Sheet—Ethion.  , Office of Pesticides and  Toxic Substances,  Office of
     Pesticide Programs, Washington, DC.

 U.S. EPA (U.S. Environmental Protection Agency).  1990a. Exposure Factors
     Handbook.  EPA-600/8-89/043.   Office of Health  and  Environmental
    Assessment, Washington,  DC.

 U.S. EPA (U.S. Environmental Protection Agency).  1990b.  Test Methods for
     Evaluating Solid Waste, Physical/Chemical Methods. SW-846,  3rd edition,
    proposed  Update II.  Office of Solid Waste and Emergency Response,
    Washington, DC.

 U.S. EPA (U.S. Environmental Protection  Agency). 1990c. Tetrachlorodibenzo-
    p-Dioxins and-Dibenzofurans in Edible Fish  Tissue at Selected Sites in
    Arkansas and Texas. Water Quality Management Branch and Surveillance
    Branch, Region 6, Dallas, TX.

 U.S. EPA (U.S. Environmental Protection Agency).  1990d.  Work Plan for FY
    91 Regional Ambient Fish Tissue Monitoring Program Activity No. ELR 80.
    Environmental Monitoring and Compliance Branch, Region 7, Kansas City,
    KS..

 U.S. EPA (U.S. Environmental Protection Agency).  1991a. Assessment and
    Control of Bioconcentratable Contaminants in Surface Waters. Draft. Office
    of Research and Development,  Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1991 b.  Contract Laboratory
    Program  Statement  of  Work  for  Inorganic Analysis,  Multi-Media,
    Multi-Concentration. SOW 788, July. Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1991c.  Contract Laboratory
    Program Statement of Work for Organic Analysis. Washington, DC.
                                                                10-29

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                                                10.  LITERATURE CITED
U.S. EPA (U.S. Environmental Protection Agency).  1991d.   Environmental
    Monitoring and Assessment  Program  (EMAP)  Near  Coastal  Program
    Laboratory Methods for Filleting and Compositing Fish for Organic and
    Inorganic Contaminant Analyses.  Draft.  U.S. EPA Office of Research and
    Development, Environmental Research Laboratory Narragansett, Rl.

U.S. EPA (U.S. Environmental Protection Agency).  1991e.   Environmental
    Monitoring and Assessment  Program  (EMAP)  Near  Coastal  Virginian
    Province Quality Assurance Project Plan.  Draft.  Office of Research and
    Development, Environmental Research Laboratory, Narragansett, Rl.

U.S. EPA (U.S. Environmental Protection  Agency).   1991f.   Environmental
    Monitoring Methods Index, Version 1.0 Software, User's Manual, EMMIUser
    Support. Office of Water, Sample Control Center,  Alexandria, VA.

U.S. EPA (U.S. Environmental Protection Agency).   1991g.  Methods for the
    Determination of Metals in Environmental Samples.  EPA-600/4-91/010.
    Environmental  Monitoring  Systems Laboratory,  Office  of  Research and
    Development, Cincinnati, OH.

U.S.  EPA (U.S.   Environmental  Protection  Agency).    1991h.   National
    Bioaccumulation Study. Draft. Office of Water Regulations and Standards,
    Washington, DC.

U.S. EPA (U.S. Environmental  Protection Agency).  1991i. Proposed Water
    Quality Guidance for the  Great Lakes  System.   Office of Science and
    Technology, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1991J. Workshop Report on
    Toxicity Equivalency Factors  for  Polychlorinated Biphenyl Congeners.
    EPA/625/3-91/020. Risk Assessment Forum, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1992a.  Classification List
    of Chemicals Evaluated for Carcinogenicity Potential.  Office of Pesticide
    Programs, Washington, DC.

U.S.  EPA  (U.S.  Environmental Protection  Agency).   1992b.   Consumption
    Surveys for Fish and Shellfish:  A Review and Analysis of Survey Methods.
    EPA-822/R-92-001. Office of Water, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1992c.  National Study of
    Chemical Residues in Fish.  Volume I. EPA-823/R-92-008a.   Office  of
    Science and Technology, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1992d.  National Study of
    Chemical Residues in Fish. Volume II.  EPA-823/R-92-008b.   Office  of
    Science and Technology, Washington, DC.
                                                                10-30

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                                                10.  LITERATURE CITED
U.S. EPA (U.S. Environmental Protection Agency). 1992e. 304(a) Criteria and
    Related Information for Toxic Pollutants.   Spreadsheet.  Water Quality
    Standards Unit, Water Management Division, Region 4, Atlanta, GA.

U.S. EPA  (U.S. Environmental Protection Agency).  1993a.  Fate One  Uner
    Database.  Office of Pesticide Programs, Washington, DC.

U.S. EPA  (U.S. Environmental Protection  Agency).  1993b.  Reference  Dose
    Tracking Report.  Office of Pesticide Programs, Health  Effects  Division,
    Washington, DC.

U.S. EPA (U.S. Environmental  Protection Agency).   1993c.   Provisional
    Guidance  for  Quantitative  Risk  Assessment of  Pgtycyclic  Aromatic
    Hydrocarbons. EPA/600/R-93/089. Environmental Criteria and Assessment
    Office, Office of Health and Environmental Assessment, Cincinnati, OH.

U.S. EPA (U.S. Environmental Protection Agency).  1993d.  Workshop Report
    on Developmental Neurotoxic Effects Associated with Exposure to PCBs.
    September 14-15, 1992, Research Triangle Park,  NC.  Risk Assessment
    Forum, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency).  1993e. Proceedings from
    National Workshop on PCBs in Fish Tissue. May 11-12,1993, Washington,
    DC. EPA 823-R-93-003. Office of Water, Washington, DC.

U.S. EPA  (U.S. Environmental Protection  Agency).  1994a.  Endangered and
    Threatened Wildlife and Plants. 50 CFR 17.11 and 17.12 June 30, 1994.

U.S. EPA (U.S.  Environmental Protection Agency).  1994b.   Guidance for
    Assessing Chemical Contaminant Data for Use in Fish Advisories—Volume
    II Risk Assessment and Fish Consumption Limits.  EPA 823-B-94-004.
    Office  of Water.

U.S. EPA (U.S. Environmental Protection Agency). 1995a. 304a Criteria Toxic
    Substance Spreadsheet.  EPA Region IV.  Water Management  Division.
    Atlanta, GA.

U.S. EPA (U.S.  Environmental Protection Agency).  1995b.   Guidance on
    Establishing Trace Metal Clean Rooms in Existing Facilities.  Draft.  EPA
    821-B-95-001.   Office  of Water,  Engineering  and  Analysis  Division,
    Washington, DC.

U.S. EPA  (U.S. Environmental Protection Agency).  1995c.  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.
                                                                10-31

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                                                 10.  LITERATURE CITED
 U.S.  EPA (U.S. Environmental  Protection Agency).  1995d.  Method 1631:
    Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic
    Fluorescence Spectrometry.  Draft.  EPA 821-R-95-027.  Office of Water,
    Engineering and Analysis Division, Washington, DC.

 U.S.  EPA (U.S. Environmental  Protection Agency).  1995e.  Method 1632.
    Determination of Inorganic Arsenic in Water by Hydride Generation Flame
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    and Analysis Division, Washington, DC.

 U.S.  EPA (U.S. Environmental  Protection Agency).  1995f.  Method 1637:
    Determination  of  Trace  Elements  in  Ambient  Waters by  Chelation
    Preconcentration with Graphite Furnace Atomic Absorption. EPA 821 -R-95-
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 U.S.  EPA  (U.S. Environmental  Protection Agency).   1995g.  Method 1638:
    Determination of Trace Elements in Ambient Waters by Inductively Coupled
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 U.S.  EPA  (U.S. Environmental  Protection Agency).   1995h.  Method 1639:
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    Temperature Graphite Furnace Atomic Absorption.   EPA 321-R-95-032.
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    Documentation and Evaluation of Trace  Metals Data Collected for Clean
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    Engineering and Analysis Division, Washington, DC.

 U.S. EPA (U.S. Environmental Protection Agency).   1995J.  Reference Dose
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 U.S. EPA (U.S. Environmental Protection Agency). 1995k.  QA/QC Guidance
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                                                                10-32

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                                                  10.  LITERATURE CITED
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                                                                 10-33

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    loggerhead sea turtles, Caretta caretta, by incremental growth marks in the
    skeleton. Smithson.  Contrib. Zoo/. 427:1-34.

Zweig, G., and  J. Sherma (eds.).  1980.  Updated General Techniques and
    Additional Pesticides.  Volume 11. In: Analytical Methods for Pesticides and
    Plant-Growth Regulators.  Academic Press, New York,  NY.
                                                                 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

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                                                            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

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                                  APPENDIX A
(u)
                                         A-8

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                                                                          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.
     A Biometrical Approach.
     New York, NY. 633 pp.
1980. Principles and Procedures of Statistics.
Second Edition.  McGraw-Hill Book Company.
U.S. EPA (U.S. Environmental Protection Agency).  1989.  Risk Assessment
     Guidance for Superfund:  Volume I, Human Health Evaluation Manual.
     Office  of  Emergency  and Remedial  Response,  Washington, DC.
     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

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-------
                          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

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                                                            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

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                                                            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

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                                                                        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
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                                                      APPENDIX D
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                      APPENDIX E
      TARGET ANALYTE DOSE-RESPONSE
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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

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                                                              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

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                                                            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

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                                                             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

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                         APPENDIX G
  RECOMMENDED PROCEDURES FOR PREPARING
WHOLE FISH COMPOSITE HOMOGENATE SAMPLES

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                                                                     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

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                                                                                             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

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                                                                         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

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                                                                         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

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                                                                 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

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                                                                           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

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                                                                          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

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                                                                     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

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                                                                         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

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                                                          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

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                       APPENDIX H
     GENERAL PROCEDURES FOR REMOVING
EDIBLE TISSUES FROM FRESHWATER TURTLES

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                                                                       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

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                                                                APPENDIX H
              External Anatomy
                                                        Carapace
                                                         Bony Bridge
                                                     connecting carapace
                                                         and plastron
                                                        Plastron
Source: Ashley, 1962.
                                 Figure H-1.
                                                                        H-4

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                                                                      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

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                                                                         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

-------
                                                                       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



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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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                                                       2-16

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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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  Synno.
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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

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                                      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

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                                                             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

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            APPENDIX K
A RECOMMENDED METHOD FOR
INORGANIC ARSENIC ANALYSIS

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