ECOLOGICAL RISK ASSESSMENT
       GUIDANCE FOR SUPERFUND:

PROCESS FOR DESIGNING AND CONDUCTING
     ECOLOGICAL RISK ASSESSMENTS
             INTERIM FINAL
       U.S. Environmental Protection Agency
          Environmental Response Team
                Edison, NJ
               June 5,1997

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                    UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                                 WASHINGTON, D,C. 20460'
                                      JUN -2  1397
                                                                         OFFICE OF
                                                             SOLID WASTE AND EMERGENCY RESPONSE
MEMORANDUM
SUBJECT:
FROM:
TO:
              Ecological Risk Assessment Guidance for Superfund, Process for Designing and
              Conducting Ecological Risk Assessments (EPA 540-R-97-006)
              Stephen D. Luftig, Director^
              Office of Emergency and Remedial Response
              Director, Office of Site Remediation and Restoration
                    Region I
              Director, Emergency and Remedial Response Division
                    Region n
              Director, Hazardous Waste Management Division
                    Regions HI, EX
              Director, Waste Management Division
                    Region IV
              Director, Superfund Division
                    Regions V, VI, VTI
              Assistant Regional Administrator, Ecosystem Protection and Remediation
                    Region VTU
              Director, Environmental Cleanup Office
                    Region X

       This memorandum transmits the interim final Ecological Risk Assessment Guidance for
Superfund, Process for Designing and Conducting Ecological Risk Assessments. This guidance
was prepared to address the questions posed by Remedial Project Managers and On-Scene
Coordinators related to  conducting ecological risk assessments. This guidance builds on
documents in preparation by the Office of Research and Development.

       Ecological risk assessment is an important part of both the removal and remedial work
conducted at Superfund sites.  The Ecological Risk Assessment Guidance for Superfund, Process
for Designing and Conducting Ecological Risk Assessments is an important step in making risk
assessments more consistent across the Regions. The guidance supersedes the Risk Assessment
Guidance for Superfund: Volume H - Environmental Evaluation Manual, 1989 (EPA/540/1-89-
                                                                           Printed on Recycled Paoer

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 001).  The new guidance will assist the Environmental Protection Agency (EPA) in continuing to
 meeting the requirements of sections 121(b)(l) and (d) of the Comprehensive Environmental
 Response, Compensation, and Liability Act (CERCLA), as amended by The Superfund
 Amendments and Reauthorization Act (SARA). Sections 121 (b)(0 and (d) require that remedial
 actions be protective of the environment. This guidance will also assist the EPA in complying with
 Section 121(c) which requires future reviews to ensure that the environment is protected at sites
 where contaminants remain after remedial actions were completed.

        The guidance has been extensively coordinated with the National Oceanic and
 Atmospheric Administration; Department of Interior, specifically the Fish and Wildlife Service;
 Department of Defense; and the Department of Energy which should.  Staffs in several of the
 Regions are already utilizing a draft of the guidance which should improve coordination with
 other Federal Agencies and Departments and lead to more consistent and transparent risk
 assessments. This guidance should be used for all ecological risk assessments conducted under
 CERCLA

       Scientific/Management Decision Points (SMDP) instituted in the document will bring risk
 managers and Natural Resource Trustees into the risk assessment process  earlier and streamline
 the process. Section 104(b) (2) of CERCLA requires that the EPA promptly notify Trustees of
 potential natural resource injuries and that the EPA seek to coordinate the assessments
 investigations, and planning of response activities with them.  As a matter of policy, the EPA
 should not only comply with the statutory directives, but should also make every effort to ensure
 Trustee participation at all stages of response.  The SMDP are an early opportunity to coordinate
 and engage the Trustees in the ecological risk assessment. These SMDP are also an opportunity
 for the EPA to comply with the National Contingency Plan (40 CFR Part 300);  Section 300.430
 (b) (7) requires that the "EPA seek to' coordinate necessary assessments, evaluations,
 investigations and planning with ...Trustees".

       If you have any questions regarding the Ecological Risk Assessment Guidance  for
 Superfund, please contact David W. Charters, Ph.D. at (732) 906-6825  or Mark D. Sprenger,
Ph.D. at (732) 906-6826.

Attachment

cc:     Department of Defense
             Department of the Army
             Department of the Navy
             Department of the Air Force
       Department of Interior
       Department of Commerce, NOAA
       Department of Energy

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                                   DISCLAIMER
The policies and procedures set forth here are intended as guidance to Agency and other
government employees. They do not constitute rulemaking by the Agency, and may not be
relied on to create a substantive or procedural right enforceable by any other person. The
Government may take action that is at variance with the policies and procedures in this
manual.

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II

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                           ACKNOWLEDGEMENTS
 The authors wish to acknowledge all the reviewers that have assisted the authors with
 insightful comments and assistance. We also wish to acknowledge the assistance of the
 Response Engineering and Analytic Contract Task Leader, Mark Huston and the editorial
 assistance of the ICF Consulting Group, primary editor Dr. Margaret McVey and Charles
 Chappell and Kimberly Osbom.

  Mark D.  Sprenger, Ph.D.                   David W. Charters, Ph.D.
  Environmental Response Team Center        Environmental Response Team Center
  Office of Emergency & Remedial Response   Office of Emergency & Remedial Response
Primary Reviewers:
Region I


Region II


Region HI
Susan Svirsky
Patti Tyler

Shari Stevens
Barbara O Kom
Robert Davis
Region IV    Lynn Wellman

Region V     Brenda Jones
             James Chapman, Ph.D.
Region VI   Susan Roddy
            Jon Rauscher, Ph.D.

Region VII   Steve Wharton
            Robert Koke

Region VIII  Gerry Henningsen, Ph.D., D.V.M.
            Dale Hoff, Ph.D.
            Mark Wickstrom, D.V.M.

Region DC   Clarence Callahan, Ph.D.
            Ned Black, Ph.D.

Region X    P. Bruce Duncan, Ph.D.
            Julius Nwosu
            Joe Goulet, Ph.D.
Headquarters:       Steve Ells

State of Texas:      Larry Champagne

U.S. Fish & Wildlife Service: Nancy Finley

Peer Review Committee:

      David Anderson, Ecology & Environment, Taylor, MI
      John Bascietto, DOE
      Tom Campbell, Woodward Clyde, Denver, CO
      Cherri Bassinger-Daniel, State of MO, Department of Health
                                        HI

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       Tom Dillon, U.S. Corps of Engineers
       Alyce Fritz, NOAA
       Duncan Gilroy, State of Oregon DEQ, Portland, OR
       Joe Greene, U.S. EPA
       Mark Harkins, Science & Space Technical Committee, Washington, DC
       Chris Ingersoll, U.S. DOI/NBS, Columbia, MO
       Mark Johnson, U.S. Army, Aberdeen, MD
       Lawrence Kapustka, EPT, Seattle, WA
       Alan Mclntosh, University of Vermont
       Gary Mangels, American Cyanamid
       Mary Malta, NOAA
       Jennifer Roberts, DOEC, State of Alaska, Department of Environmental Conservation
       Glen W. Suter, n, Martin Marietta  Energy Systems, Inc., Oak Ridge National
       Laboratory
       Randy Wentsel, U.S. Army
       Janet Whaley, U.S. Army, Aberdeen, MD

Stakeholder Meeting Attendees:

       Jeff Foran, Meeting Facilitator
       Judith Bland, Merck
       Jim Clark, Exxon Biomedical Sciences
       David Cragin, Elf Atochem
       Steve Geiger, Remediation Technology
       Simeon Hahn, U.S.  Navy
       David Hohreiter, Blasland, Bouck,  and Lee
       Kenneth Jenkins, consultant (Jenkins, Sanders, & Associates) representing General
       Electric
       Lorraine Keller, Rohm and Haas
       Bryce Landenberger, Dow Chemical
       Dale  Marino, Eastman Kodak
       Ellen Mihaich, Rhone-Poulenc
       Ron Porter, U.S. Air Force
       Mark Powell, Center for Risk Management at Resources for the Future
       Lee Salamone, Chemical Manufacturers Association
       Anne Sergeant, U.S. EPA
       Jean Snider, NOAA
       Ralph Stahl, DuPont
       Randy Wentsel, U.S. Army

Observers at the Stakeholder Meeting:

       Adam Ayers, Geraghty and  Miller
       Steve Ells, U.S. EPA
                                         IV

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Paul Hirsh, Chemical Manufacturers Association
Teresa Larson, National Association of Manufacturers
Reo Menning, American Industrial Health Council
Kevin Reinert, Rohm and Haas
Phil Sandine, Environmental Liability Management
Wendy Sherman, Chemical Manufacturers Association
Todd Slater, Elf Atochem

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VI

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                             CONTENTS


DISCLAIMER  	i

ACKNOWLEDGEMENTS . . .	iii

LISTS OF EXHIBITS, EXAMPLES, AND HIGHLIGHTS	xi

LIST OF ACRONYMS AND ABBREVIATIONS . .	 xiii

PREFACE	,	  xv

INTRODUCTION: ECOLOGICAL RISK ASSESSMENT FOR SUPERFUND	 1-1
      PURPOSE  	 1-1
      SCOPE	1-1
      BACKGROUND	 1-1
      DEFINITION OF ECOLOGICAL RISK ASSESSMENT	 1-3
      THE ECOLOGICAL RISK ASSESSMENT PROCESS	 1-3

STEP 1:  SCREENING-LEVEL PROBLEM FORMULATION AND ECOLOGICAL
      EFFECTS EVALUATION	1-1
      1.1   INTRODUCTION	 1,1
      1.2   SCREENING-LEVEL PROBLEM FORMULATION	 1-1
           1.2.1  Environmental Setting and Contaminants at the Site  	1-2
           1.2.2  Contaminant Fate and Transport	1-4
           1.2.3  Epotoxicity and Potential Receptors	1-4
           1.2.4  Complete Exposure Pathways	 1-5
           1.2.5  Assessment and Measurement Endpoints	1-7
      1.3   SCREENING-LEVEL ECOLOGICAL EFFECTS EVALUATION  	1-8
           1.3.1  Preferred Toxicity Data	 1-9
           1.3.2  Dose Conversions	 1-12
           1.3.3  Uncertainty Assessment	 1-12
      1.4   SUMMARY	 1-12

STEP 2:  SCREENING-LEVEL EXPOSURE ESTIMATE
      AND RISK CALCULATION	2-1
      2.1   INTRODUCTION	2-1
      2.2   SCREENING-LEVEL EXPOSURE ESTIMATES	2-1
           2.2.1  Exposure Parameters	 2-2
           2.2.2  Uncertainty Assessment	2-3
      2.3   SCREENING-LEVEL RISK CALCULATION	 2-4
      2.4   SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)	2-5
      2.5   SUMMARY	2-6
                                  VII

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STEP 3: BASELINE RISK ASSESSMENT PROBLEM FORMULATION	3-1
      3.1   THE PROBLEM-FORMULATION PROCESS	3-1
      3.2   REFINEMENT OF PRELIMINARY CONTAMINANTS OF
           CONCERN	3-3
      3.3   LITERATURE SEARCH ON KNOWN ECOLOGICAL EFFECTS  ..... 3-4
      3.4   CONTAMINANT FATE AND TRANSPORT, ECOSYSTEMS
           POTENTIALLY AT RISK, AND COMPLETE EXPOSURE
           PATHWAYS	3-4
           3.4.1  Contaminant Fate and Transport	3-5
           3.4.2  Ecosystems Potentially at Risk  	3-6
           3.4.3  Complete Exposure Pathways	3-7
      3.5   SELECTION OF ASSESSMENT ENDPOINTS	 . 3-8
      3.6   THE CONCEPTUAL MODEL AND RISK QUESTIONS	3-10
           3.6.1  Conceptual Model	3-10
           3.6.2  Risk Questions	3-14
      3.7   SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP) 	3-15
      3.8   SUMMARY	3-15

STEP 4: STUDY DESIGN AND DATA QUALITY OBJECTIVE PROCESS	4-1
      4.1   ESTABLISHING MEASUREMENT ENDPOINTS	4-2
           4.1.1  Species/Community/Habitat Considerations 	4-5
           4.1.2  Relationship of the Measurement Endpoints to the
                 Contaminant of Concern	 4-5
           4.1.3  Mechanisms of Ecoxicity	4-7
      4.2   STUDY DESIGN  	.4-7
           4.2.1  Bioaccumulation and Field Tissue Residue Studies	4-8
           4.2.2  Population/Community Evaluations	 4-12
           4.2.3  Toxicity Testing	4-13
      4.3   DATA QUALITY OBJECTIVES AND STATISTICAL
           CONSIDERATIONS	4-14
           4.3.1  Data Quality Objectives	4-14
           4.3.2  Statistical Considerations	4-15
      4.4   CONTENTS OF WORK PLAN AND  SAMPLING AND ANALYSIS
           PLAN 			 ,	4-15
           4.4.1  Work Plan			4-16
           4.4.2  Sampling and Analysis Plan . . ;	• • •  • 4-16
           4.4.3  Field Verification of Sampling Plan and Contingency Plans	4-18
      4.5   SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)	 4-18
      4.6   SUMMARY	4-18

STEP 5: FIELD VERIFICATION OF SAMPLING DESIGN	5-1
      5.1   PURPOSE  	5-1
      5.2   DETERMINING SAMPLING FEASIBILITY	5-2
      5.3   SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)	 . 5-3
                                   Vlll

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      5.4   SUMMARY	; .		5-4

 STEP 6:  SITE INVESTIGATION		. .  6-1
      6.1   INTRODUCTION	6-1
      6.2   SITE INVESTIGATION  	6-1
            6.2.1   Changing Field Conditions	6-2
            6.2.2   Unexpected Nature or Extent of Contamination  	6-2
      6.3   ANALYSIS OF ECOLOGICAL EXPOSURES AND EFFECTS	6-3
            6.3.1   Characterizing Exposures	6-3
            6.3.2   Characterizing Ecological Effects  	6-5
      6.4   SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)  ..........  6-6
      6.5   SUMMARY	6-7

 STEP 7:  RISK CHARACTERIZATION  	'.'	7-1
      7.1   INTRODUCTION .	7-1
      7.2   RISK ESTIMATION	7-1
      7.3   RISK DESCRIPTION			7-4
            7.3.1   Threshold for Effects on Assessment Endpoints  	7-4
            7.3.2   Likelihood of Risk  . . .".	 . .	7-5
            7.3.3   Additional Risk Information	:	7-5
      7.4   UNCERTAINTY ANALYSIS	-	7-5
            7.4.1   Categories of Uncertainty	7-6
            7.4.2   Tracking Uncertainties	7-7
      7.5   SUMMARY .	7-7

STEP 8: RISK MANAGEMENT	 . .	8-1
      8.1    INTRODUCTION	8-1
      8.2   ECOLOGICAL RISK MANAGEMENT IN SUPERFUND	8-1
            8.2.1   Other Risk Management Considerations	8-2
            8.2.2   Ecological Impacts of Remedial Options	8-3
            8.2.3   Monitoring	8-3
      8,3    SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)  	  8-4
      8.4    SUMMARY		,8-4

BIBLIOGRAPHY	Bibliography-1

GLOSSARY	 Glossary-1

APPENDIX A: EXAMPLE ECOLOGICAL RISK ASSESSMENTS FOR
      HYPOTHETICAL. SITES

      Example 1:  Copper Site	A-l
      Example 2:  Stream DDT Site	A-8
      Example 3:  PCB Site	 . A-14
                                    IX

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APPENDIX B: REPRESENTATIVE SAMPLING GUIDANCE DOCUMENT,
           VOLUMES:  ECOLOGICAL, DRAFT

     U.S. Environmental Protection Agency (U.S. EPA).  1997.  Representative Sampling
     Guidance Document,  Volume 3: Ecological, Draft.  Edison, NJ: Environmental
     Response Team, Office of Emergency and Remedial Response.

APPENDIX C: SUPPLEMENTAL GUIDANCE ON LITERATURE SEARCH

APPENDIX D: STATISTICAL CONSIDERATIONS

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          LISTS OF EXHIBITS, EXAMPLES, AND HIGHLIGHTS
List of Exhibits
      EXHIBIT 1-1: Ecological Risk Assessment Framework . . . . ,	  1-5
      EXHIBIT 1-2: Eight-step Ecological Risk Assessment
            Process for Superfund	f ......  1-9
      EXHIBIT 1-3: Steps in the Ecological Risk Assessment
            Process and Corresponding Decision Points in the
            Superfund Process	  1-10
      EXHIBIT 1-4: Ecological Risk Assessment Deliverables
            for the Risk Manager	  Ml
      EXHIBIT 1-5: Ecological Risk Assessment in the Remedial
            Investigation/Feasibility Study (RI/FS) Process	,  1-13
      EXHIBIT 1-1: List of Sensitive Environments
            in the Hazard Ranking System	 .  1-6
      EXHIBIT 6-1: Analysis Phase			6-4
      EXHIBIT 7-1: Risk Characterization	7-2
      EXHIBIT A-l:" Conceptual Model for the Copper Site  	A-5
      EXHIBIT A-2:  Conceptual Model for the Stream DDT Site  	A-l 1
      EXHIBIT A-3:  Conceptual Model for the Terrestrial PCB Site   	A-17
List of Examples
      EXAMPLE 1-1: Ecotoxicity-PCB Site	.		1-5
      EXAMPLE 1-2: Complete Exposure Pathways for Mammals-PCB Site	1-8
      EXAMPLE 3-1: Exposure Pathway Model-DDT Site	-...-.	3-7
      EXAMPLE 3-2: Potential for Food Chain Transfer-Copper
            and DDT Sites 	3-8
      EXAMPLE 3-3: Assessment Endpoint Selection-DDT,
            Copper, and PCB Sites	3-11
      EXAMPLE 3-4: Description of the Conceptual Model-DDT Site	3-12
      EXAMPLE 3-5: Conceptual Model Diagram-DDT Site  	3-13
      EXAMPLE 4-1: Lines of Evidence-Copper Site	  4-4
      EXAMPLE 4-2: Selecting Measurement Endpoints-DDT Site	4-6
      EXAMPLE 4-3: Tissue Residue Studies-DDT Site	4-9
      EXAMPLE 5-1: Field Verification of Sampling Design-Copper Site 	5-4
      EXAMPLE 5-2: Field Verification of Sampling Design-DDT Site	5-5
      EXAMPLE 6-1: Fish Sampling Contingency Plan-DDT Site	  6-2
                                       XI

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List of Highlights

      HIGHLIGHT 1-1:  The RI/FS Process	  1-2
      HIGHLIGHT 1-2:  Example Assessment Endpoints	  1-6

      HIGHLIGHT 1-3:  Example Measurement Endpoints .	 .  1-6
      HIGHLIGHT 1-4:  Ecological Impact and Risk Assessment	  1-8
      HIGHLIGHT 1-1:  Screening-level Risk Assessments 	1-2
      HIGHLIGHT 1-2:  Industrial or Urban Settings  	1-4
      HIGHLIGHT 1-3:  Exposure Pathway and Exposure Route 	1-7
      HIGHLIGHT 1-4:  Non-Chemical Stressors	1-9
      HIGHLIGHT 1-5:  Data Hierarchy for Deriving Screening
            Ecotoxiciry Values  	 1-10
                      NOAEL Preferred to LOAEL	 1-11
                      Area Use Factor	2-2
                      Hazard Index (HI) Calculation	2-5
                      Tiering an Ecological Risk Assessment	 .	3-3
                      Environmental Fate and Exposure 	3-5
                      Definitions:  Null and Test Hypotheses	3-14'
                      Importance of Distinguishing Measurement
            from Assessment Endpoints .	4-3
      HIGHLIGHT 4-2: Terminology and Definitions	4-6
      HIGHLIGHT 4-3: Elements of a QAPP		4-17
      HIGHLIGHT 6-1: Uncertainty in Exposure Models 	6-5
HIGHLIGHT 1-6:
HIGHLIGHT 2-1:
HIGHLIGHT 2-2:
HIGHLIGHT 3-1:
HIGHLIGHT 3-2:
HIGHLIGHT 3-3:
HIGHLIGHT 4-1:
                                      xu

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               LIST OF ACRONYMS AND ABBREVIATIONS
AQUIRE:    U.S. EPA's AQUatic Information REtrieval database
ARAR:      Applicable or Relevant and Appropriate Requirements
ASTM:      American Society of Testing and Materials
BAF:        Bioaccumulation Factor
BCF:   .     Bioconcentration Factor
BIOSIS:      Biosciences Information Services
BTAG:      Biological Technical Assistance Group
CERCLA:    Comprehensive Environmental Response, Compensation, and Liability Act
CLP:        Contract Laboratory Program
DDT:        Dichlorodiphenyltrichloroethane
DQO:        Data Quality Objective
EC50:        Effective Concentration for producing a specified effect in 50 percent of the
             test organisms
EEC:        Estimated Environmental Concentration
EPA:        Environmental Protection Agency
FS:          Feasibility Study
FSP:         Field Sampling Plan
FWS:        Fish and Wildlife Service
HEAST:      National Center for Environmental Assessment's Health Effects Assessment
             Summary Tables
HI:          Hazard Index
HQ:         Hazard Quotient
HSDB:       National Library of Medicine's Hazardous Substances Data Bank
IRIS:        EPA's Integrated Risk Information System
LC50:        Concentration Lethal to 50 percent of the test organisms
Li   "       Liter
LOAEL:      Lowest-Observed-Adverse-Effect Level
NCP:        National Oil and Hazardous Substances Pollution Contingency Plan
NOAA:      National Oceanic and Atmospheric Administration
NOAEL:      No-Observed-Adverse-Effect Level
NRC:        National Research Council
NRDA:      Natural Resource Damage Assessment
OERR:       U.S. EPA Office of Emergency and Remedial Response
OSC:        On-Scene Coordinator
OSWER:     U.S. EPA Office of Solid  Waste and Emergency Response
PA          Preliminary Assessment
PAH:        Polycyclic Aromatic Hydrocarbons
PC^:        Polychlorinated Biphenyl compound
PRP:         Potentially Responsible Party
QAPP:       Quality Assurance Project Plan
QA/QC:      Quality Assurance and Quality Control
                                        XIII

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RBP:        Rapid Bioassessment Protocol
RI:          Remedial Investigation
ROD:        Record of Decision
RPM:        Remedial Project Manager
SAP:        Sampling and Analysis Plan
SARA:       Superfund Amendments and Reauthorization Act of 1986
SI:          Site Investigation
SMDP:       Scientific/Management Decision Point
TOC:        Total Organic Carbon
WP:         Work Plan
                                        XIV

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                                      PREFACE
       This document provides guidance on the process of designing and conducting
 technically defensible ecological risk assessments for the Superfund Program. It is intended
 to promote consistency and a science-based approach within the Program and is based on the
 Proposed Guidelines for Ecological Risk Assessment (1996a) and the Framework for
 Ecological Risk Assessment (1992a) developed by the Risk Assessment Forum of the U.S.
 Environmental Protection Agency. When the Agency publishes its final Guidelines for
 Ecological Risk Assessment, ibis guidance will be reviewed and revised if necessary to ensure
 consistency with the Agency guidelines.

       This document is directed to the site managers (i.e., On-Scene Coordinators [OSCs]
 and Remedial Project Managers  [RPMs]) who are legally responsible for the management of a
 site.  However, it is anticipated that ecological risk assessors, as well as other individuals with
 input to the ecological risk assessment, will use this document.  -

       Ecological risk assessment is an integral pan of the Remedial  Investigation and
 Feasibility Study (RI/FS) process, which is designed to support risk management decision-
 making for Superfund  sites. The RI component of the process characterizes the nature and
 extent of contamination at a hazardous waste  site and estimates risks to human health and the
 environment posed  by  contaminants at the site.  The FS component of the process develops
 and evaluates  remedial options.  Thus, ecological risk assessment is fundamental to the RI
 and ecological considerations are also part of the FS process.

       This document  is intended to facilitate defensible site-specific  ecological risk
 assessments.   It is not  intended to determine the appropriate scale or complexity of an
 ecological risk assessment or to direct the user in the selection of specific protocols or
 investigation methods.  Professional judgment is essential in  designing and determining the
 data needs for any ecological risk assessment. However, when the process outlined in this
 document is followed,  a technically defensible and appropriately scaled site-specific
 ecological risk assessment should result.

       Ecological risk  assessment is an interdisciplinary field drawing upon environmental
 toxicology, ecology, and environmental chemistry, as well as other areas of science and
 mathematics.  It is important that users of this document understand that ecological risk
 assessment is  a complex, non-linear process, with many parallel activities. The user should
 have a basic understanding of ecotoxicology and ecological risk assessment and read through
 this document in its entirety prior to engaging in the ecological risk assessment process.
Without the basic understanding of the field and of this guidance,  the reader might not
recognize  the  relationships among different components of the risk assessment process.

       To assist the user in interpreting this guidance document, three illustrations of
planning an ecological risk assessment for a hazardous  waste site are provided in

                                           XV

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Appendix A. These are simplified, hypothetical examples that demonstrate and highlight
specific points in the ecological risk assessment process. These  examples are incomplete and
not intended to present a thorough discussion of the ecological or ecotoxicological issues that
would exist at an actual site.  Instead, they are intended to illustrate the first five steps of the
process, which precede a full ecological field investigation.  Excerpts from the three examples
are included in the guidance document as "Example" boxes to illustrate specific points.  The
user is encouraged to read the three examples in Appendix A in  addition to the Example
boxes within the guidance document itself.

       Ecological risk assessment is a dynamic field, and this document represents a process
framework into which changes in ecological  risk assessment approaches can readily be
incorporated. Four appendices are included with this document; additional appendices may be
developed to address specific issues.

       This document supersedes the U.S. EPA's (1989b) Risk Assessment Guidance for
Superfund, Volume 2: Environmental Evaluation Manual as guidance on how to design and
conduct an ecological risk assessment for the Superfund Program.  The Environmental
Evaluation Manual contains useful information on the statutory and regulatory basis of
ecological assessment, basic ecological concepts, and other background information that is not
repeated in this document.
                                           XVI

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                                INTRODUCTION:
          ECOLOGICAL RISK ASSESSMENT FOR SUPERFUND
 PURPOSE

       This document provides guidance on how to design and conduct consistent and
 technically defensible ecological risk assessments for the Superfund Program.  It is based on
 the Proposed Guidelines for Ecological Risk Assessment (1996a) and the Framework for
 Ecological Risk Assessment (1992a) developed by the Risk Assessment Forum of the U.S.
 Environmental Protection Agency (U.S. EPA or the Agency).  When the Agency finalizes its
 (1996a) Proposed Guidelines for Ecological Risk Assessment, this guidance will be reviewed
 and revised if necessary to ensure consistency with the  Agency guidelines.

       This document is directed to the site managers (i.e., On-Scene Coordinators [OSCs]
 and Remedial Project Managers [RPMsJ) who are legally responsible for managing site
 activities.  However, it is anticipated that the ecological risk assessors, as  well as all other
 individuals involved with ecological risk assessments, will use this document.
SCOPE

      This document is intended to facilitate defensible and appropriately-scaled site-specific
ecological risk assessments.  It is not intended to dictate the scale, complexity, protocols, data
needs, or investigation methods for such assessments.  Professional judgment is required to
apply the process outlined in this document to ecological risk assessments at specific sites.
BACKGROUND

Superfund Program

      The Comprehensive Environmental Response, Compensation, and Liability Act of
1980 (CERCLA or Superfund), as amended by the Superfund Amendments and
Reauthorization Act of 1986 (SARA), authorizes the U.S. EPA to protect public health and
welfare and the environment from the release or potential release of any hazardous substance,
pollutant, or contaminant.  U.S. EPA's Superfund Program carries out the Agency's mandate
under CERCLA/SARA.

      The primary regulation issued by U.S. EPA's Superfund Program is the National  Oil
and Hazardous Substances Pollution Contingency Plan (NCP).  The NCP calls for the
identification and mitigation  of environmental impacts (such as toxicity, bioaccumulation,
death, reproductive impairment, growth impairment, and loss of critical habitat) at hazardous
waste sites, and for the selection of remedial actions to protect the environment.  In addition,

                                        1-1

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numerous other federal and state laws and regulations concerning environmental protection
can be designated under Superfund as "applicable" or "relevant and appropriate" requirements
(ARARs) for particular sites. Compliance with these other laws and regulations generally
requires an evaluation of site-related ecological effects and the measures necessary to mitigate
those effects.
Risk Assessment in Superfund

       An important part of the NCP is the
requirement for a Remedial Investigation
and Feasibility Study (RI/FS) (see Highlight
1-1).  The RI^S is an analytical process
designed to  support risk management
decision-making for Superfund sites.  The
RI component of the process characterizes
the nature and extent of contamination at a
hazardous waste site and estimates risks to
human health and the environment posed by
contaminants at the site.  The FS component
of the process develops and  evaluates
remedial options.
           HIGHLIGHT 1-1
         The RI/FS Process

       Risk assessment is an integral part of
the RI/FS.  The three pans of the RI are: (!)
characterization of the nature and extent of
contamination; (2) ecological risk
assessment; and (3) human health risk
assessment. The investigation of the nature
and extent  of contamination determines the
chemicals present on site as well as their
distribution and concentrations.  The
ecological risk and human health risk
assessments determine the potential for
adverse effects to the environment and
human health, respectively.
       Although U.S. EPA has established
detailed guidelines for human health risk
assessment in the Superfund program (U.S.
EPA, 1989a, 1991a,b), similarly detailed guidelines for site-specific ecological risk assessment
do not exist for the Superfund program. Risk Assessment Guidance for Superfund, Volume 2:
Environmental Evaluation Manual (U.S. EPA, 1989b) provides conceptual guidance in
planning studies to evaluate a hazardous waste site's "environmental resources" (as used in
the manual, the phrase "environmental resources" is largely synonymous with "ecological
resources").  U.S. EPA also is publishing supplemental information on specific ecological risk
assessment topics for Superfund in the ECO Update series (U.S. EPA, 1995b, I994b,c,d,e,
1992b,c,d,  1991c,d).  However, those documents do not describe an overall, step-by-step
process by which an ecological risk assessment is designed and executed.  The Agency's
Framework for Ecological Risk Assessment (U.S. EPA, 1992a) provides a basic structure and
a consistent approach for conducting ecological risk assessments, but is not intended to
provide program-specific guidance. The Guidelines for Ecological Risk Assessment, currently
being developed by the Agency's Risk  Assessment Forum (1996a), will expand on the
Framework, but again, will not provide program-specific guidance.

       This document outlines a step-by-step ecological risk assessment process that is both
specific to  the Superfund Program and  consistent with the more general U.S. EPA Framework
and guidelines under development.  While the Agency's Framework and future Agency-wide
ecological risk assessment guidelines are not enforceable regulations, the concepts in those
                                           1-2

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documents are appropriate to Superfund. The concepts in the published Framework have
been incorporated into this document with minimal modification.  The definitions of terms
used in this ecological risk assessment guidance for Superfund (and listed in the Glossary) are
consistent with the definitions in the U.S. EPA Framework document unless noted otherwise.
DEFINITION OF ECOLOGICAL RISK ASSESSMENT

U.S. EPA "Framework" Document

       Ecological risk assessment is defined in the Framework as a process that evaluates the
likelihood that adverse ecological effects are occurring or may occur as a result of exposure
to one or more stressors (U.S. EPA, 1992a). The Framework defines a stressor as any
physical, chemical, or biological entity that can induce an adverse ecological response.
Adverse responses can range from Sublethal chronic effects in individual organisms to a loss
of ecosystem function. Although stressors can be biological (e.g., introduced species), only
chemical or physical stressors will be addressed in this document, because these are the
stressors subject to risk management decisions at Superfund sites.

Superfund Program

       The phrase "ecological risk assessment," as used specifically for the Superfund
Program in this document, refers to a qualitative  and/or quantitative appraisal of the actual  or
potential impacts of contaminants from a hazardous waste site on plants and animals other
than humans and domesticated species. A risk does not exist unless:  (1) the stressor has the
ability  to cause one or more adverse effects, and (2) it co-occurs with or contacts  an
ecological component long enough and at a sufficient intensity to elicit the identified adverse
effect.
THE ECOLOGICAL RISK ASSESSMENT PROCESS

U.S. EPA "Framework" Document

       The Framework describes the basic elements of a process for scientifically evaluating
the adverse effects of stressors on ecosystems and components of ecosystems.  The document
describes the basic process and principles to be used in ecological risk assessments conducted
for the U.S. EPA, provides operational definitions for terms used in ecological risk
assessments, and outlines basic principles around which program-specific guidelines for
ecological risk assessment should be organized.

       The Framework is similar to the National Research Council's (NRC) paradigm for
human health risk assessments (NRC, 1983) and the more recent NRC ecological risk
paradigm (NRC, 1993). The  1983 NRC paradigm consists of four fundamental phases:

                                         1-3

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hazard identification, dose-response assessment, exposure assessment, and risk
characterization.  The Framework differs from the 1983 NRC paradigm in a few ways:

       •      Problem formulation is incorporated into the beginning of the process to   .
              determine the focus and scope of the assessment;

       •      Hazard identification and dose-response assessment are combined in an  .
              ecological effects assessment phase; and

       •      The phrase "dose-response" is replaced by "stressor-response" to emphasize the
              possibility that physical changes (which are not measured in "doses") as well as
              chemical contamination can stress ecosystems.

Moreover, the Framework emphasizes the parallel nature of the ecological effects and
exposure assessments by joining the two assessments  in an .analysis phase between problem
formulation and risk characterization, as shown in Exhibit 1-1.

       During problem formulation, the risk assessor  establishes the goals, breadth, and focus
of the assessment (U.S. EPA,  1992a).  As indicated in the Framework, problem formulation is
a systematic planning step that identifies the major factors to be  considered and is linked to
the regulatory and policy contexts of the assessment.  Problem formulation includes  .
discussions between the risk assessor and risk manager, and other involved parties, to identify
the stressor characteristics, ecosystems potentially at risk, and ecological effects to be
evaluated.  During problem formulation,  assessment and measurement endpoints for the
ecological  risk assessment are identified, as described below.

       The Agency defines assessment endpoints as explicit expressions of the actual
environmental values (e.g., ecological resources) that  are to be protected (U.S. EPA, 1992a).
Valuable ecological resources include those without which ecosystem function would be
significantly impaired, those providing critical resources (e.g., habitat, fisheries), and those
perceived as valuable by humans (e.g., endangered species and other issues addressed by
legislation). Because assessment endpoints focus the  risk assessment design and analysis,
appropriate selection and definition of these endpoints are critical to the utility of a risk
assessment.

       Assessment endpoints should relate to statutory mandates (e.g., protection of the
environment), but must be specific enough to guide the development of the risk assessment
study design at a particular site.  Useful assessment endpoints define both the valued
ecological  entity at the site (e.g., a species, ecological resource, or habitat type) and a
characteristic(s) of the entity to protect (e.g., reproductive success, production per unit area,
areal extent).  Highlight 1-2 provides some examples  of specific  assessment endpoints related
to the general goal of protecting aquatic  ecosystems.
                                           1-4

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                     EXHIBIT 1-1
Ecological Risk Assessment Framework (U.S. EPA, 1992a)


Discussion
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Risk Assessor
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(Planning)


















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                       Risk Management
                        1-5

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                                             biological response to a stressor that can be
                                             assessment endpoint (U.S. EPA, 1992a;
       A measurement endpoint is a measurable
related to the valued characteristic chosen as the
although this definition may change—see
U.S. EPA,  1996a).  Sometimes, the
assessment endpoint can be measured
directly; usually, however, an assessment
endpoint encompasses too many species  or
species  that are difficult to evaluate (e.g.,
top-level predators).  In these cases, the
measurement endpoints are different from
the assessment endpoint, but can be used to
make inferences about risks to the
assessment endpoints. For example,
measures of responses in particularly
sensitive species and life stages might be
used to  infer responses in the remaining
species  and life stages in a specific
community. Such inferences must be
clearly described to demonstrate the link
between measurement and assessment
endpoints.  Highlight 1-3 provides examples
of measurement endpoints.

      Measures of exposure also can be used to make inferences about risks to assessment
endpoints at Superfund sites.  For example, measures of water concentrations of a
contaminant can be compared with concentrations known from the literature to be lethal to
sensitive aquatic organisms to infer something about risks to aquatic community structure.  As
a consequence, for purposes of this guidance, measurement endpoints include both measures
of effect and measures of exposure.
                                                         HIGHLIGHT 1-2
                                                Example Assessment Endpoints

                                                •   Sustained aquatic community
                                                    structure, including species
                                                    composition and relative abundance
                                                    and trophic structure.

                                                •   Sufficient rates of survival, growth,
                                                    and reproduction to sustain
                                                    populations of carnivores typical for
                                                    the area.

                                                •   Sustained fishery diversity and
                                                    abundance.
       A product of problem formulation is
a conceptual model for the ecological risk
assessment that describes how a given
stressor might affect ecological components
of the environment. The conceptual model
also describes questions about how stressors
affect the assessment endpoints, the
relationships among the assessment and
measurement endpoints, the data required to
answer the questions, and the methods that
will be used to analyze the data (U.S. EPA,
1992a).
                                                         HIGHLIGHT 1-3
                                               Example Measurement Endpoints

                                                •    Community analysis of benthic
                                                    macroin vertebrates.

                                                •    Survival and growth of fish fry in
                                                    response to exposure to copper.

                                                •    Community structure of fishery in
                                                    proximity to the site.
                                          1-6

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Superfund Program

       The goal of the ecological risk assessment process in the Superfund Program is to
provide the risk information necessary to assist risk managers at Superfund sites (OSCs and
RPMs) in making informed decisions regarding substances designated as hazardous under
CERCLA (see 40 CFR 302.4).  The specific objectives of the process, as stated in OSWER
Directive 9285.7-17, are:  (1) to identify and characterize the current and potential threats to
the environment from a hazardous substance release; and (2) to identify cleanup levels that
would protect those natural resources from risk.  Threats to the environment include existing
adverse ecological impacts and the risk of such impacts in the future. Highlight 1-4 provides
an overview of ecological risk assessment in the Superfund Program.

       Problem formulation is the most critical  step of an ecological risk assessment and must
precede any attempt to design a site investigation and analysis plan.  To ensure that the risk
manager can use the results of an ecological risk assessment to inform risk management
decisions for a  Superfund site, it is important that all involved parties contribute to the
problem  formulation phase and that the risk manager is clearly identified to all parties. These
parties include the remedial project manager (RPM), who is the risk manager with ultimate
responsibility for the site, the ecological risk assessment team, the Regional Superfund
Biological Technical Assistance Group (STAG), potentially responsible parties (PRPs),
Natural Resource Trustees, and stakeholders in the  natural resources at issue (e.g., local
communities, state agencies) (U.S. EPA, 1994a, 1995b). The U.S. EPA's (1994a) Edgewater
Consensus on an EPA Strategy for Ecosystem Protection in particular calls for the Agency to
develop a "place-driven" orientation,  that is, to focus on the environmental  needs of specific
communities and ecosystems, rather than on piecemeal program mandates.  Participation in
problem  formulation by all involved parties helps to achieve the place-driven focus.

       Issues such as restoration, mitigation, and replacement are important to the Superfund
Program; but are reserved for investigations that might or might not be included in the RI
phase. During  the risk management process of selecting the preferred remedial option leading
to the Record of Decision (ROD), issues of mitigation and restoration should be addressed.
In selecting a remedy, the risk manager must also consider the degree to which the remedial
alternatives reduce risk and thereby also reduce the need for restoration or mitigation.

       A natural resource damage assessment (NRDA) may be conducted at a Superfund site
at the discretion of Natural Resource Trustees for specific resources associated with a site.
An  ecological risk assessment is a necessary step for an NRDA, because it establishes the
causal link between site contaminants and specific adverse ecological effects.  The risk
assessment also  can provide information on what residual risks are likely for different
remediation options. However, the ecological risk  assessment does not constitute an NRDA.
The NRDA is the sole  responsibility  of the Natural Resource Trustees, not  of the U.S. EPA;
therefore, NRDAs will not be addressed in this guidance. For additional information on the
role of Natural  Resource Trustees in  the Superfund process, see ECO Update  Volume 1,
Number 3 (U.S. EPA, 1992c).

                                           1-7

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                                        HIGHLIGHT 1-4
                          Ecological Impact and Risk Assessment

             Ecological risk assessment within the Superfund Program can be a risk evaluation
      (potentially predictive), impact evaluation, or a combination of those approaches.  The
      functions of the ecological risk assessment are to:

             (1)     Document whether actual or potential ecological risks exist at a site;

             (2)     Identify which contaminants present at a site pose an ecological risk; and

             (3)     Generate data to be used in evaluating cleanup options.

      Ecological risk assessments can have  their greatest influence on risk management at a site in
      the evaluation and selection of site remedies. The ecological risk assessment should identify
      contamination levels that bound a threshold for adverse effects on the assessment endpoint.
      The threshold values provide a yardstick for evaluating the effectiveness of remedial options
      and can be used to set cleanup goals if appropriate.

             To justify a site action based upon ecological concerns, the ecological risk assessment
      must establish that an actual or potential ecological threat exists at a site. The potential for
      (i.e., risk of)  impacts can be the threat of impacts from a future release or redistribution of
      contaminants, which could be avoided by taking actions on "hot spots" or source areas. Risk
      also can be viewed as the likelihood that current impacts are occurring (e.g., diminished
      population size), although this can be difficult to demonstrate. For example, it may not be
      practical or technically possible to document existing ecological impacts, either due to limited
      technique resolution, the localized nature of the actual impact, or limitations resulting from
      the biological or ecological constraints of the field measurements (e.g., measurement
      endpoints, exposure point evaluation).  Actually demonstrating existing impacts confirms that
      a "risk" exists. Evaluating a gradient of existing impacts along a gradient of contamination
      can provide an stressor-response assessment that helps to identify cleanup levels.

             As noted above, the ecological risk assessment should provide the information needed
      to make risk  management decisions (e.g., to select the appropriate site remedy).  A
      management  option should not be selected first, and then the risk assessment tailored to
     justify the option.
This Guidance Document

       This ecological risk assessment guidance for Superfund is composed of eight steps
(see Exhibit 1-2) and several scientific/management decision points (SMDPs) (see Exhibit
1-3).  An SMDP requires a meeting between the risk manager and risk assessment team to
evaluate and approve or redirect the work up to that point. (Consultation with the Regional
BTAG is recommended for SMDPs  (a) through (d) in Exhibit 1-3.)  The group decides

                                              1-8

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                              EXHIBIT 1-2
     Eight-step Ecological Risk Assessment Process for Superfund
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STEP1: SCREENING-LEVEL
  •  Site Visit
  •  Problem Formulation
  •  Toxicity Evaluation
STEP 2: SCREENING-LEVEL:
  •  Exposure Estimate
  •  Risk Calculation
                  STEP 3: PROBLEM FORMULATION
                          Toxicity Evaluation
                Assessment
                Endpoints
                        I
                  Conceptual Model
                  Exposure Pathways
                        Questions/Hypotheses
                  STEP 5: VERIFICATION OF FIELD
                         SAMPLING DESIGN
                  STEP 6: SITE INVESTIGATION AND
                          DATA ANALYSIS
                 STEP 7: RISK CHARACTERIZATION
  Risk Assessor
and Risk Manager
   Agreement
     i
                                                                SMDP
STEP 4: STUDY DESIGN AND DQO PROCESS
• Lines of Evidence
• Measurement Endpoints
Work Plan and Sampling and Analysis Plan



SMDP
STEP 8:
RISK MANAGEMENT


SMDP ,
                                 1-9

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                                    EXHIBIT 1-3
               Steps in the Ecological Risk Assessment Process
        and Corresponding Decision Points in the Superfund Process

Steps and Scientific/Management Decision Points (SMDPs):

       1.      Screening-Level Problem Formulation and Ecological
              Effects Evaluation

       2.      Screening-Level Preliminary Exposure Estimate and
              Risk Calculation                                        SMDP (a)

       3.      Baseline Risk Assessment Problem Formulation              SMDP (b)

       4.      Study Design and Data Quality Objectives                   SMDP (c)

       5.      Field Verification of Sampling Design                      SMDP (d)

       6.      Site Investigation and Analysis of Exposure
              and Effects                                    ,         [SMDP]

       7.      Risk Characterization

       8.      Risk Management                                       SMDP (e)

Corresponding Decision Points in the Superfund Process:

       (a)     Decision about whether a full ecological risk assessment
              is necessary.

       (b)     Agreement among the risk assessors, risk manager, and
              other involved panics on the conceptual model,
              including assessment endpoints, exposure pathways, and
              .questions or risk hypotheses.

       (c)     Agreement among the risk assessors and risk manager on the
              measurement endpoints, study design, and data interpretation
              and analysis.

       (d)     Signing approval of the work plan and sampling and analysis
              plan for the ecological risk assessment.

       (e)     Signing the Record of Decision.

       [SMDP] only if change  to the sampling and analysis plan is necessary.
                                        1-10

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whether or not the risk assessment is proceeding in a direction that is acceptable to the risk
assessors and manager. The SMDPs include a discussion of the uncertainty associated with
the risk assessment, that might be reduced, if necessary, with increased effort. SMDPs are
significant communication points which should be passed with the consensus of all involved
parties.  The risk manager should expect deliverables that document specific SMDPs as
outlined in Exhibit 1-4. This approach is intended to minimize both the cost of and time
required for the Superfund risk assessment process.

       This guidance provides a technically valid approach for ecological risk assessments at
hazardous waste sites, although other approaches also can be valid.  The discipline of
ecological risk assessment is dynamic and continually evolving; the assessments rely on data
that are complex and  sometimes ambiguous.  Thus, if an approach other than the one
described in this guidance document is used, there must be clear documentation of the
process,  including process design and interpretation of the results, to  ensure a technically
defensible assessment. Clear documentation, consistency, and objectivity in the assessment
process are necessary for  the Superfund Program.

       An interdisciplinary team  including, but not limited to, biologists, ecologists, and
environmental lexicologists, is needed to design and implement a successful risk assessment
and to evaluate the weight of the evidence obtained to reach conclusions about ecological
risks.  Some of the many  points at which the Superfund ecological risk assessment process
requires professional judgment include:
                                      EXHIBIT 1-4
                      Ecological Risk Assessment Deliverables
                                 for the Risk Manager

  If the process stops at the end of Step 2:

         (1)    Full documentation of the screening-level assessment and SMDP not to continue
                the assessment.

  If the process continues to Step 3:

         (1)    Documentation of the conceptual model, including assessment endpoints,
                exposure pathways, risk hypotheses, and SMDP at the end of Step 3.

         (2)    The approved  and signed work plan and sampling and analysis plan,
                documenting the SMDPs at the end of Steps 4 and 5.

         (3)    The baseline risk assessment documentation (including documentation of the
                screening-level assessment used in the baseline assessment) developed  in Step 7.
                                          1-11

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        •      Determining the level of effort needed to assess ecological risk at a particular
              site;

        •      Determining the relevance of available data to the risk assessment;

        •      Designing a conceptual model of the ecological threats at a site and measures
              to assess those threats;

        •      Selecting methods and .models to be used in the various  components of the risk
              assessment;

        •      Developing assumptions to fill data gaps for toxicity and exposure assessments
              based on logic and scientific principles;  and

        •      Interpreting the ecological significance of observed or predicted effects.

The lead risk assessor should coordinate with appropriate professionals to  make many of these
decisions.. Specialists are needed for the more  technical questions  concerning the risk
assessment (e.g., which model, which assumptions).

       This guidance document focuses on the risk assessment process in  Superfund and does
not address all of the issues that a risk manager will need to consider.  After the risk
assessment is complete, the risk manager might require additional professional assistance in
interpreting the implications of the baseline  ecological  risk assessment  and selecting a
remedial option.

       The risk assessment process must be structured to ensure that site management
decisions can be made without the need for repeated studies or delays.  The first two steps in
the assessment process are a streamlined version of the complete Framework process and are
intended to allow a rapid  determination by the  risk assessment team and risk manager that the
site poses no or negligible 'ecological risk, or to identify which contaminants and  exposure
pathways require further evaluation.  Steps 3 through 7 are a more detailed version of the
complete Framework process.

       The ecological risk assessment process should be coordinated with the overall RI/FS
process to the extent possible.  Overall site-assessment costs are minimized when the needs of
the ecological and human health risk assessments are incorporated  into the chemical sampling
program to determine the  nature and extent  of contamination during the RI.   For sites at
which an RI  has not yet been planned or conducted, Exhibit 1-5 illustrates the relationship
between the eight ecological risk assessment steps and the overall  Superfund process and
decision points.  For older sites at which an RI was conducted before an ecological risk
assessment was considered, the ecological risk  assessment process  should  build on the
information already developed for the site.
                                           I-I2

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                                               EXHIBIT 1-5
                         Ecological Risk Assessment in the RI/FS Process
FROM:
• Preliminary Assessment
• Site Inspection
• NPL Listing
                                 Remedial Investigation
                                WP
                                and
                                SAP
                    Site
                 Investigation
                                        Feasibility Study
    Establish
    Remedial
   Objectives
    SREENINQ
ECOLOGICAL RISK
  ASSESSMENT

  (STEPS 1 & 2)
    FIELD
VERIFICATION

  (STEP 5)
    PROBLEM
FORMULATION AND
  STUDY DESIGN

   (STEPS 3 & 4)
Refine remedial
goals based on
risk assessment
                        ANALYSIS OF
                   EXPOSURE AND EFFECTS
                   RISK CHARACTERIZATION

                        (STEPS 6 » 7)
Development
and Analysis
of Alternatives
                                                                   TO:
                                                                   •  Remedy Selection
                                                                   •  Record of Decision
                                                                   •  Remedial Design
                                                                   •  Remedial Action
    Conduct risk
    evaluation of
     remedial
    alternatives
Ecological
Monitoring

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       It is important to realize that this eight-step approach is not a simple linear or
sequential process.  The order of actions taken will depend upon the stage of the RI/FS
atwhich the site is currently, the amount and types of site information available, as well as
other factors.  The process can be iterative, and in some iterations, certain individual steps
might not be needed. In many cases, it might be appropriate and desirable to conduct several
steps concurrently.

       Tasks that should be accomplished in each of the eight steps in Exhibits 1-2 and 1-3
are described in the eight following sections.  The eight sections include example boxes based
on the three hypothetical Superfund sites in Appendix A as well as exhibits and highlight
boxes.
                                           1-14

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        STEP 1: SCREENING-LEVEL PROBLEM  FORMULATION
                AND ECOLOGICAL EFFECTS EVALUATION
                                    OVERVIEW

          The screening-level problem formulation and ecological effects evaluation is
   pan of the initial ecological risk screening assessment.  For this initial step, it is likely
   that site-specific information for determining the nature and extent of contamination
   and for characterizing ecological receptors at the site is limited. This step includes all
   the functions of problem formulation (more fully described in Steps 3 and 4) and
   ecological effects analysis, but on a screening level.  The results of this step will be
   used in conjunction with exposure estimates in  the preliminary risk calculation in
   Step 2.
1.1    INTRODUCTION

       Step 1 is the screening-level problem formulation process and ecological effects
evaluation (Highlight  1-1 defines screening-level risk assessments).  Consultation with the
STAG is recommended at this stage.  How to brief the BTAG on the setting, history, and
ecology of a site is described in ECO Update  Volume 1, Number 5 (U.S. EPA, 1992d).
Section 1.2 describes the screening-level problem formulation, and Section 1.3 describes the
screening-level ecological effects evaluation. Section  1.4 summarizes this step.
1.2    SCREENING-LEVEL PROBLEM FORMULATION
                   /

       For the screening-level problem formulation, the risk assessor develops a conceptual
model for the site that addresses five issues:

     (1)  Environmental setting and contaminants known or suspected to exist at the site
          (Section 1.2.1);

     (2)  Contaminant fate and transport mechanisms that might exist at the site (Section
          1.2.2);

     (3)  The mechanisms of ecotoxicity associated with contaminants and likely categories
          of receptors that could be affected (Section 1.2.3);
                                        1-1

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       (4)  What complete exposure
           pathways might exist at the site
           (a complete exposure pathway is
           one in which the chemical can
           be traced or expected to travel
           from the source to a receptor
           that can be affected by the
           chemical) (Section 1.2.4); and

       (5)  Selection of endpoints to screen
           for ecological risk (Section
           1.2.5).

1.2.1  Environmental Setting  and
       Contaminants at the Site
            HIGHLIGHT 1-1
 Screening-level Risk Assessments

       Screening-level risk assessments are
simplified risk assessments -that can be
conducted with limited data by assuming
values for parameters for which data are
lacking. At the screening level, it is
important to minimize the chances of
concluding that there is no risk when in fact
a risk exists.  Thus, for exposure and toxicity
parameters for which site-specific information
is lacking, assumed values should
consistently be biased in the direction of
overestimating risk. This ensures that sites
that might pose an ecological risk are studied
further.  Without this bias, a screening
evaluation could not provide a defensible
conclusion that negligible ecological risk
exists or that certain contaminants and
exposure pathways can be eliminated from
consideration.
       To begin the screening-level
problem formulation, there must be at least
a rudimentary knowledge of the potential
environmental setting and chemical
contamination at the site. The first step is
to compile information from the site history
and from reports related to the site,
including the Preliminary Assessment  (PA)
or Site Investigation (SI). The second step is to use the environmental checklist presented in
Representative Sampling Guidance Document. Volume 3: Ecological (U.S. EPA, 1997; see
Appendix B) to begin characterizing the site for problem, formulation.  Key questions
addressed by the checklist include:

       •      What are the on- and off-site land uses (e.g., industrial, residential, or
              undeveloped; current and  future)?

       •      What type of facility existed or exists at  the site?

       •      What are the suspected  contaminants at the site?

       •      What is the environmental setting, including natural areas (e.g., upland forest,
              on-site stream, nearby wildlife refuge) as well as disturbed/man-made areas
              (e.g., waste lagoons)?

       •      Which habitats present on site are potentially contaminated or otherwise
              disturbed?
                                            1-2

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        •      Has contamination migrated from source areas and resulted in "off-site"
              impacts or the threat of impacts in addition to on-site threats or impacts?

        These questions should be answered using the site reports, maps (e.g. U.S. Geological
 Survey, National Wetlands Inventory), available aerial photographs, communication with
 appropriate agencies (e.g., U.S. Fish and Wildlife Service, National Oceanic and Atmospheric
 Administration, State Natural Heritage Programs), and a site visit.  Activities that should be
 conducted during  the site visit include:

        •      Note the layout and topography of the site;

        •      Note and describe  any water bodies and wetlands;

        •      Identify and map evidence indicating contamination or potential contamination
              (e.g., areas of no vegetation, runoff gullies to surface waters);

        •      Describe existing aquatic, terrestrial, and wetland ecological  habitat types (e.g.,
              forest, old field), and estimate the area covered by those habitats;

        •      Note any potentially sensitive environments (see Section 1.2.3 for examples of
              sensitive environments);

        •      Describe and, if possible, map soil and water types, land uses, and the
              dominant vegetation species present; and

       •      Record any observations of animal species or sign of a species.

       Mapping can be useful in establishing  a "picture" of the site to assist in problem
formulation. The  completed checklist  (U.S. EPA, 1997) will provide information regarding
habitats and species potentially or actually present on site, potential contaminant migration
pathways, exposure pathways, and the potential for non-chemical stresses at the site.

       After finishing the checklist, it might be possible to determine that present or future
ecological impacts are negligible  because complete exposure pathways do not exist and could
not exist in the future.  Many Superfund sites are located in highly  industrialized areas where
there could be few if any ecological receptors or where site-related  impacts might be
indistinguishable from non-site-related impacts (see Highlight 1-2).  For such sites,
remediation to reduce ecological risks might not be needed. However, all sites should be
evaluated by qualified personnel to determine whether this conclusion is appropriate:

       Other Superfund sites are  located in less disturbed areas with protected or sensitive
environments that  could be at risk of adverse  effects from contaminants from the site.  State
and federal laws (e:g., the Clean Water Act, the Endangered Species Act) designate certain
types of environments as  requiring protection.  Other types of habitats unique to certain areas

                                            1-3

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                HIGHLIGHT 1-2
         Industrial or Urban Settings

             Many hazardous waste sites exist
       in currently or historically industrialized
       or urbanized areas.  In these instances, it
       can be difficult to distinguish between
       impacts related to contaminants from a
       particular site and impacts related to
       non-contaminant stressors or to
       contaminants from other sites. However,
       even in these cases, it could be
       appropriate to take some remedial
       actions based on ecological risks. These
       actions might be limited to source
       removal or might be more extensive.
       An ecological risk assessment can assist
       the risk manager in determining what
       action, if any, is appropriate.
also could need special consideration in the risk
assessment (see Section 1.2.3).

1.2.2  Contaminant Fate and Transport

       During problem formulation, pathways
for migration of a contaminant (e.g., windblown
dust, surface water runoff, erosion) should be
identified. These pathways can exhibit a
decreasing gradient of contamination with
increasing distance from a site. There are
exceptions, however, because physical and
chemical  characteristics of the media also
influence contaminant distribution (e.g., the
pattern of sediment deposition in  streams varies
depending on stream flow and bottom
characteristics).  For the screening-level risk
assessment, the  highest contaminant
concentrations measured on the site should be
documented for each medium.

1.2.3  Ecotoxicity and Potential Receptors

       Understanding the toxic mechanism of a contaminant helps to evaluate the importance
of potential exposure pathways (see  Section 1.2.4) and to focus the selection of assessment
endpoints (see Section 1.2.5).  Some contaminants, for example, affect primarily vertebrate
animals by interfering  with organ systems not found in invertebrates or plants (e.g., distal
tubules of vertebrate kidneys, vertebrate hormone systems).  Other substances might affect
primarily  certain insect groups (e.g., by interfering with hormones needed for metamorphosis),
plants  (e.g., herbicides), or other groups of organisms.  For substances that  affect, for
example,  reproduction of mammals at much lower environmental exposure  levels than they
affect other groups of organisms, the screening-level risk assessment can initially focus on
exposure pathways and risks to mammals.  Example 1-1 illustrates this point using the PCB
site example provided  in Appendix A. A review of some of the more recent ecological risk
and toxicity assessment literature can help identify likely effects of the more common
contaminants at  Superfund sites.

       An experienced biologist or ecologist can determine what plants, animals, and habitats
exist or can be expected to exist in the area of the Superfund site. Exhibit 1-1, adapted from
the Superfund Hazard Ranking System, is a partial list of types of sensitive environments that
could require protection or special consideration. Information obtained for the environmental
checklist (Section  1.2.1), existing information  and maps, and aerial photographs  should be
used to identify  the presence of sensitive environments on or near a site that  might be
threatened by contaminants from  the site.
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                                     EXAMPLE 1-1
                                 Ecotoxicity-PCB Site

          Some PCBs are reproductive toxins in mammals (Ringer et al., 1972; Aulerich et al.,
    1985; Wren et al., 1991; Kamrin and Ringer, 1996). When ingested, they induce (i.e., increase
   concentrations and activity of) enzymes in the liver, which might affect the metabolism of some
   steroid hormones (Rice and O'Keefe,  1995). Whatever the mechanism of action, several
   physiological functions that are controlled by steroid hormones can be altered by the exposure
   of mammals to certain PCBs, and reproduction appears to be the most sensitive endpoint for
   PCB toxicity in mammals (Rice and O'Keefe, 1995).  Given this information, the screening
   ecological risk assessment should include potential exposure pathways for mammals to PCBs
   that are reproductive toxins (see Example 1-2).
1.2.4  Complete Exposure Pathways

       Evaluating potential exposure pathways is one of the primary tasks of the screening-
level ecological characterization of the site.  For an exposure pathway to be complete, a
contaminant must be able to travel from the source to ecological receptors and to be taken up
by the receptors via one or more exposure routes.  (Highlight 1-3 defines exposure pathway
and exposure route.)  Identifying complete exposure pathways prior to a quantitative
evaluation of toxicity allows the assessment to focus on only those contaminants that can
reach ecological receptors.

       Different exposure routes are important for different groups of organisms.  For
terrestrial animals, three basic exposure routes need to be evaluated:  inhalation, ingestion,
and dermal absorption.  For terrestrial plants, root absorption of contaminants in soils and leaf
absorption of contaminants evaporating from the soil or deposited on the leaves are of
concern at Superfund sites.  For aquatic animals, direct contact  (of water or sediment with the
gills or integument) and ingestion of food (and sometimes sediments) should be considered.
For aquatic plants, direct contact with water, and  sometimes with air or sediments, is of
primary concern.

       The most likely  exposure pathways and exposure routes also are related to the physical
and chemical properties of the contaminant (e.g., whether or not the contaminant is bound to
a matrix, such as organic carbon). Of the basic exposure routes identified above, more
information generally is available to quantify.exposure levels for ingestion by terrestrial
animals and for direct contact with water or sediments by aquatic organisms than for other
exposure routes and receptors.  Although other exposure routes can be  important, more
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                                       EXHIBIT 1-1
        List of Sensitive Environments in the Hazard  Ranking System8
Critical habitat for Federal designated endangered or threatened species
Marine Sanctuary
National Park
Designated Federal Wilderness Area
Areas identified under the Coastal Zone Management Act  .   •
Sensitive areas identified under the National Estuary Program or Near Coastal Waters Program
Critical areas identified under the Clean Lakes Program
National Monument
National Seashore Recreational Area
National Lakeshore Recreational Area
Habitat known to be used by Federal designated or proposed endangered or threatened species
National Preserve
National or State Wildlife Refuge
Unit of Coastal Barrier Resources System
Coastal Barrier (undeveloped)
Federal land designated for protection of natural ecosystems
Administratively Proposed Federal Wilderness Area
Spawning areas critical for the maintenance of fish/shellfish species within river, lake, or
        coastal tidal waters
Migratory pathways and feeding areas critical for maintenance of anadromous fish species within river
        reaches or areas in lakes or coastal tidal waters in which the fish spend extended periods of time
Terrestrial areas utilized for breeding by large or dense aggregations of animals
National river reach designated as Recreational
Habitat known to be used by state designated  endangered or threatened species
Habitat known to be used by species under review as to its Federal endangered or threatened status
Coastal Barrier (partially developed)
Federally-designated Scenic or Wild River
State land designated for wildlife or game  management
State-designated Scenic or Wild River                            -
State-designated Natural Areas
Particular areas, relatively small in size, important to maintenance of unique biotic communities
State-designated areas for protection or maintenance of aquatic life
Wetlands6

        a The categories are listed in groups  from  those assigned higher factor values to those assigned
lower factor values in the Hazard Ranking System (HRS) for listing hazardous waste sites on the National
Priorities List (U.S.  EPA,  1990b).  See Federal Register, Vol. 55. pp. 51624 and 51648 for additional
information regarding definitions.
        b Under the HRS. wetlands are rated on the basis of size.  See Federal Register, Vol. 55, pp.
51625 and 51662 for additional information.
                                            1-6

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               HIGHLIGHT 1-3
          Exposure Pathway and
              Exposure Route

    Exposure Pathway:  The pathway by
    which a contaminant travels from a source
    (e.g., drums, contaminated soils) to
    receptors.  A pathway can involve multiple
    media (e.g., soil runoff to surface waters and
    sedimentation, or volatilization to the
    atmosphere).

    Exposure Route: A  point of contact/entry
    of a contaminant from the environment into
    an organism (e.g., inhalation, ingestion,
    dermal absorption).
 assumptions are needed to estimate exposure
 levels for those routes, and the results are
 less certain.  Professional judgment is
 needed to determine if evaluating those
 routes sufficiently improves a risk
 assessment to warrant the effort.

       If an exposure pathway is not
 complete for a specific contaminant (i.e.,
 ecological receptors cannot be exposed to
 the contaminant), that exposure pathway
 does not need to be evaluated further. For
 example, suppose a contaminant that impairs
 reproduction in mammals occurs only in
 soils that are well below the root zone of
 plants that occur or are expected to occur on
 a site. Herbivorous mammals would not be
 exposed to the contaminant through their
 diets because plants would not be
 contaminated.  Assuming that most soil macroinvertebrates available for ingestion live in the
 root zone, insectivorous mammals also would be unlikely to be exposed.  In this case, a
 complete exposure pathway for this contaminant for ground-dwelling mammals would not
 exist, and the contaminant would not pose a significant risk to this group of organisms.
 Secondary questions might include whether the contaminant is leaching from the soil to
 ground water that discharges  to surface water, thereby posing a risk to the aquatic
 environment or to terrestrial mammals that drink the water or consume aquatic prey.
 Example 1-2 illustrates the process of identifying complete exposure pathways based  on the
 hypothetical PCB site described in Appendix A.

 1.2.5  Assessment and Measurement Endpoints

       For the  screening-level ecological risk assessment, assessment endpoints are any
adverse effects on ecological  receptors, where receptors are plant and animal populations and
communities, habitats, and sensitive environments.  Adverse effects on populations can be
inferred from measures related to impaired reproduction, growth, and survival.  Adverse
effects on communities can be inferred from changes in community structure or function.
 Adverse  effects on habitats can be inferred from changes in composition and characteristics
that reduce the habitats1 ability to support plant  and animal populations and communities.

       Many of the screening ecotoxicity values now available or likely to be available in the
future for the Superfund program (see Section 1.3)  are based on generic assessment endpoints
(e.g., protection of aquatic communities from changes in structure or function) and are
assumed to be widely applicable to sites around the United States.
1-7

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                                      EXAMPLE 1-2
                Complete Exposure Pathways for Mammals-PCB Site

          Three possible exposure pathways for mammals were evaluated at the PCB Site:
   inhalation, ingestion through the food chain, and incidental soil/sediment ingestion.

          Inhalation. PCBs are not highly volatile, so the inhalation of PCB vapors by
   mammals would be an essentially incomplete exposure pathway. Inhalation of PCBs adsorbed
   to soil panicles might need consideration in areas with exposed soils, but this site is well
   vegetated.

          Ingestion through the food chain. PCBs tend to bioaccumulate and biomagnify in
   food chains. PCBs in soils are not taken up by most plants, but are accumulated by soil
   macroinvertebrates. Thus, in areas without significant soil deposition on the surfaces of plants,
   mammalian herbivores would not be exposed to PCBs in most of their diet. In contrast,
   mammalian insectivores, such as shrews, could be exposed to PCBs in most of their diet.  For
   PCBs, the ingestion route for mammals would be essentially incomplete for herbivores but
   complete for insectivores.  For the PCB site, therefore, the ingestion exposure route for a
   mammalian insectivore (e.g., shrew) would be a complete exposure pathway that should be
   evaluated.

          Incidental soil/sediment ingestion. Mammals can ingest some quantity of soils or
   sediments incidentally, as they groom their fur or consume plants or animals from the soil.
   Burrowing mammals are likely to ingest greater quantities of soils during grooming than non-
   burrowing mammals, and mammals that consume plant roots or soil-dwelling macroinvertebrates
   are likely to ingest greater quantities of soils attached to the surface of their foods than
   mammals that consume other foods. The intake of PCBs from incidental ingestion of PCB-
   contaminated soils is difficult to estimate, but for insectivores that forage at ground level, it is
   likely to be far less than the intake of PCBs in the diet. For herbivores, the incidental  intake of
   PCBs in soils might be higher than the intake of PCBs in their diet, but still less than the intake
   of PCBs by mammals feeding on soil macroinvertebrates.  Thus, the exposure pathway for
   ground-dwelling mammalian insectivores remains the exposure pathway that should be
   evaluated.
1.3    SCREENING-LEVEL ECOLOGICAL EFFECTS EVALUATION

       The next step in the screening-level risk assessment is the preliminary ecological
effects evaluation and the establishment of contaminant exposure levels that represent
conservative thresholds for adverse ecological effects. In this guidance, those conservative
thresholds are called screening ecotoxicity values.  Physical stresses unrelated to contaminants
at the site are not the focus of the risk assessment (see Highlight 1-4),  although they can be
considered later when evaluating effects of remedial alternatives.
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        A literature search for studies that
 quantify toxicity (i.e., exposure-response) is
 necessary to evaluate the likelihood of toxic
 effects in different groups of organisms.
 Appendix C provides a basic introduction to
 conducting a literature search, but an expert
 should be consulted to minimize time and
 costs.  The toxicity profile should describe
 the toxic mechanisms of action for the
 exposure routes being evaluated and the
 dose or environmental concentration that
 causes a specified adverse effect.

        For each complete  exposure pathway,
 route, and contaminant, a screening
 ecotoxicity value should be developed.1
 The U.S. EPA  Office of Emergency and
 Remedial Response has developed screening
 ecotoxicity values [called ecotox threshold
 values (U.S.  EPA,  1996c)].  The values are
 for surface waters and sediments,  and are
 based on direct exposures  routes only;
 bioaccumulation and biomagnification in
 food chains  have not been accounted for.
 The following subsections describe preferred
 data (Section 1.3.1), dose conversions
 (Section 1.3.2), and analyzing uncertainty in
 the values (Section 1.3.3).

 1.3.1  Preferred Toxicity  Data

       Screening ecotoxicity values should represent a no-observed-adverse-effect-level
 (NOAEL) for long-term (chronic) exposures to a contaminant.  Ecological effects of most
 concern  are those that can  impact  populations (or higher levels of biological organization).
Those include adverse  effects on development, reproduction, and survivorship.  Community-
 level effects also can be  of concern, but toxicity data on community-level endpoints are
 limited and might be difficult to extrapolate from one community to another.
            HIGHLIGHT 1-4
       Non-Chemical Stressors

       Ecosystems can be stressed by
physical, as well as by chemical, alterations
of their environment. For this reason,
EPA's (1992a) Framework for Ecological
Risk Assessment addresses "stressor-
response" evaluation to include all types of
stress instead of "dose-response" or
"exposure-response" evaluation, which
implies that the stressor must be a toxic
substance.

       For Superfund sites, however, the
baseline risk assessment addresses risks from
hazardous substances released to the
environment, not risks from  physical
alterations of the environment, unless caused
indirectly by a hazardous substances (e.g.,
loss of vegetation from a chemical release
leading to serious erosion).   This guidance
document, therefore, focuses on exposure-
response evaluations for toxic substances.
Physical destruction of habitat that might be
associated with a particular remedy is
considered in the Feasibility  Study.
    '  It is possible to conduct a screening risk assessment with limited information and conservative
assumptions.  If site-specific information is too limited, however, the risk assessment is almost certain to move
into Steps 3 through 7. which require field-collected data.  The more complete the initial information, the better
the decision that can be made at this preliminary stage.
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       When reviewing the literature, one
 should be aware of the limitations of
 published information in characterizing
 actual or probable hazards at a specific site.
 U.S. EPA discourages reliance on secondary
 references because study details relevant for
 determining the applicability of findings to a
 given site usually are not reported in
 secondary sources. Only primary literature
 that has  been carefully reviewed by an
 ecotoxicologist should be used to support a
 decision. Several considerations and data
 preferences are summarized ,in Highlight 1-5
 and described more fully below.

       NOAELS and  LOAELS. For each
 contaminant for which  a complete exposure
 pathway/route exists, the literature should be
 reviewed for the lowest exposure level (e.g.,
 concentration in water or in the diet, ingested
 dose) shown to produce adverse effects (e.g.,
 reduced  growth, impaired reproduction,
 increased mortality) in a potential receptor
 species.  This value is called a lowest-
 observed-adverse-effect-level or LOAEL.
 For those contaminants with documented
 adverse effects, one also should identify the
 highest exposure level that is a NOAEL. A
 NOAEL is more appropriate than a LOAEL
 to use as an screening ecotoxicity value to
 ensure that risk is not underestimated (see Highlight 1-6). However, NOAELs currently are
 not available for many groups of organisms and many chemicals. When a LOAEL value, but
 not a NOAEL value, is available from the literature, a standard practice is to multiply the
 LOAEL  by  0.1 and to use the product as the screening ecotoxicity value.  Support for this
 practice comes from a data review indicating that 96 percent of chemicals included in the
 review had LOAEL/NOAEL ratios of five or less, and that all were ten or less (Dourson and
 Stara,  1983).

       Exposure duration.  Data from studies of chronic exposure are preferable to data
 from medium-term (subchronic), short-term (acute), or single-exposure studies because
exposures at Superfund remedial sites usually are long-term.  Literature reviews by
McNamara (1976) and Weil and McCollister (1963) indicate that chronic NOAELs can be
           HIGHLIGHT 1-5
    Data Hierarchy for Deriving
   Screening Ecotoxicity Values

       To develop a chronic NOAEL for a
screening ecotoxicity value from existing
literature, the following data hierarchy
minimizes extrapolations and uncertainties
in the value:

  •     A NOAEL is preferred to a
       LOAEL, which is preferred to an
       LC50 or an EC50.

  •     Long-term (chronic) studies are
       preferred to medium-term
       (subchronic) studies, which are
       preferred to short-term (acute)
       studies.

  •    If exposure at the site is by
       ingestion, dietary studies are
       preferred to gavage studies, which
       are preferred to non-ingestion routes
       of exposure.  Similarly, if exposure
       at the site is  dermal, dermal studies
       are preferred to studies using other
       exposure routes.
                                           MO

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 lower than subchronic (90-day duration for
 rats) NOAELs by up to a factor of ten.2

    N  . Exposure route.  The exposure
 route and medium used in the toxicity study
 should be comparable to the exposure route
 in the risk assessment.  For example, data
 from studies where exposure  is by gavage
 generally are not preferred for estimating
 dietary concentrations that could produce
 adverse effects, because the rate at which
 the substance is absorbed from the
 gastrointestinal tract usually is greater
 following gavage than following dietary
 administration.  Similarly,  intravenous
 injection of a substance results in
 "instantaneous absorption" and does not
 allow the substance to first pass through the
 liver, as it would following dietary
 exposure.  If it is necessary to attempt to
 extrapolate toxicity test results from one
 route of exposure  to another,  the
 extrapolation should be performed or
 reviewed by a toxicologist experienced in
 route-to-route extrapolations for the class of
                 HIGHLIGHT 1-6
          NOAEL Preferred to LOAEL

            Because the NOAEL and LOAEL
     are estimated by hypothesis testing (i.e., by
     comparing the response level of a test group
     to the response level of a control group for a
     statistically significant difference), the actual
     proportion of the test animals showing the
     adverse response at an identified LOAEL
     depends on sample size, variability of the
     response, and the dose interval. LOAELs,
     and even NOAELs, can represent a
     30 percent or higher effect  level for the
     minimum sample sizes recommended for
     standard test protocols.  For this reason, U.S.
     EPA recommends that the more conservative
     NOAELs, instead of LOAELs, are used to
     determine a .screening exposure level that is.
     unlikely to adversely impact populations.  If
     dose-response data are available, a site-
     specific low-effect level may be determined.
animals at issue.
       Field versus laboratory.  Most toxicity studies evaluate effects of a single
contaminant on a single species under controlled laboratory conditions.  Results from these
studies might not be directly applicable to the field, where organisms typically are exposed to
more than one contaminant in environmental situations that are not comparable to a  laboratory
setting and where genetic composition of the population can be more heterogeneous  than that
of organisms bred for laboratory use.  In addition, the bioavailability of a contaminant might
be different at a site than in a laboratory toxicity test.  In a field  situation, organisms also will
be subject to other environmental variables, such as unusual weather conditions, infectious
diseases, and food shortages.  These variables can have either positive or negative effects on
   2 The literature reviews of McNamara (1976) and Weil and McCollister (1963) included both rodent and
non-rodent species. The duration of the subchronic exposure usually was 90 days, but ranged from 30 to 210
days. A wide variety of endpoints and criteria for adverse effects were included in these reviews. Despite this
variation in the original studies, their findings provide a general indication of the ratio between subchronic to
chronic NOAELs for effects other than cancer and reproductive effects.  For some chemicals, chronic dosing
resulted in increased chemical tolerance. For over 50 percent of the compounds tested, the chronic NOAEL was
less than the 90-day NOAEL by a factor of 2 or less. However, in a few cases, the chronic NOAEL was up to  a
factor of 10 less than the subchronic NOAEL (U.S. EPA, 1993e).
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the organism's response to a toxic contaminant that only a site-specific field study would be
able to evaluate.  Moreover, single-species toxicity tests seldom provide information regarding
toxicant-related changes in community interactions (e.g., behavioral changes in prey species
that make them more susceptible to predation).

1.3.2  Dose Conversions

       For some data reported in the literature, conversions are necessary to allow the data to
be used for species other than those tested or for measures of exposure other than those
reported.  Many doses in laboratory studies are reported in terms of concentration in the diet
(e.g., mg contaminant/kg diet or ppm in the diet).  Dietary concentrations can be converted to
dose (e.g., mg contaminant/kg body weight/day) for comparison with estimated contaminant
intake  levels in the receptor species.

       When converting doses, it is important to identify whether weights are measured as
wet or dry weights.  Usually, body weights are reported on a wet-weight, not dry-weight
basis.  Concentration of the contaminant in the diet might be reported on a  wet- or dry-weight
basis.

       Ingestion rates and body weights for a test species often are reported in a toxicity
study or can  be obtained from other literature sources (e.g., U.S.  EPA, 1993a,b).  For
extrapolations between animal species with different metabolic rates as well as dietary
composition, consult U.S. EPA 1992e and 1996b.

1.3.3  Uncertainty Assessment

       Professional judgment is needed to determine the uncertainty associated with
information taken from the literature and any extrapolations used in developing a screening
ecotoxicity value. The risk assessor should be consistently conservative in selecting literature
values  and describe the limitations of using those values in the context of a particular site.
Consideration of the study design, endpoints, and other factors are important in determining
the utility of toxicity data in the screening-level risk assessment.   All of those factors should
be addressed in a brief evaluation of uncertainties prior to the screening-level risk calculation.
1.4    SUMMARY

       At the conclusion of the screening-level problem formulation and ecological effects
evaluation, the following information should have been compiled:

       •     Environmental setting and contaminants known or suspected to exist at the site
             and the maximum concentrations present (for each medium);

       •     Contaminant fate and transport mechanisms that might exist at the site;

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       •      The mechanisms of ecotoxicity associated with contaminants and likely
              categories of receptors that could be affected;

       •      The complete exposure pathways that might exist at the site from contaminant
              sources to receptors that could be affected; and

       •      Screening ecotoxicity  values equivalent to chronic NOAELs based on
              conservative assumptions.

       For the screening-level ecological risk assessment, assessment endpoints will include
any likely adverse ecological effects on receptors for which exposure pathways are complete,
as determined from the  information listed above.  Measurement  endpoints will be based on
the available literature regarding mechanisms of toxicity and  will be used to establish the
screening ecotoxicity values.  Those values will be used with estimated exposure levels to
screen for ecological risks, as described in Step 2.
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          STEP 2:  SCREENING-LEVEL EXPOSURE ESTIMATE
                          AND RISK CALCULATION
                                    OVERVIEW

         The screening-level exposure estimate and risk calculation comprise the second
   step in the ecological risk screening for a site.  Risk is estimated by comparing
   maximum documented exposure concentrations with the ecotoxicity screening values
   from Step 1. At the conclusion of Step 2, the risk manager and risk assessment team
   will decide that either the screening-level ecological risk assessment is adequate to
   determine that ecological threats are negligible, or the process should continue to a
   more detailed ecological risk assessment (Steps 3 through 7).  If the process continues,
   the screening-level assessment serves to identify exposure pathways and preliminary
   contaminants of concern for the baseline risk assessment by eliminating those
   contaminants and exposure pathways that pose negligible risks.
2.1    INTRODUCTION

       This step includes estimating exposure levels and screening for ecological risks as the
last two phases of the screening-level ecological risk assessment. The process concludes with
a SMDP at which it is determined that:  (1) ecological threats are negligible; (2) the
ecological risk assessment should continue to.determine whether a risk exists; or (3) there is a
potential for  adverse ecological effects, and a more detailed ecological risk assessment,
incorporating more site-specific information, is needed.

       Section 2.2 describes the screening-level exposure assessment, focusing on the
complete exposure pathways identified in Step 1.  Section 2.3 describes the risk calculation
process, including estimating a hazard quotient, documenting the uncertainties in the quotient,
and summarizing the overall confidence in the screening-level ecological risk assessment.
Section 2.4 describes the SMDP that concludes Step 2.
2.2   SCREENING-LEVEL EXPOSURE ESTIMATES

      To estimate exposures for the screening-level ecological risk calculation, on-site
contaminant levels and general information on the types of biological receptors that might be
exposed should be known from Step 1.  Only complete exposure pathways should be
evaluated. For these, the highest measured or estimated on-site contaminant concentration for
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each environmental medium should be used to estimate exposures.  This should ensure that
potential ecological threats are not missed.

2.2.1  Exposure Parameters

       For parameters needed to estimate exposures for which sound site-specific information
is lacking or difficult to develop, conservative assumptions should be used at this screening
level.  Examples of conservative assumptions are listed below and described in the following
paragraphs:
   •    Area-use factor - 100 percent (factor
       related to home range and population
       density; see Highlight 2-1);

   •    Unavailability - 100 percent;

   •    Life stage - most sensitive life stage;

   •    Body weight and food ingestion rate
       - minimum body weight to
       maximum ingestion rate; and

   •    Dietary composition - 100 percent of
       diet consists of the most
       contaminated dietary component.

       Area-use factor.  For the
screening-level exposure estimate for	
terrestrial animals, assume  that  the home
range of one or more animals is entirely within the contaminated area, and thus the animals
are exposed 100 percent of the  time.  This is a conservative assumption and, as an
assumption, is only applicable to the screening-level phase of the risk assessment.  Species-
and site-specific home range information would be needed later, in Step 6, to estimate more
accurately the percentage of time an animal would use a contaminated area.  Also evaluate
the possibility that some species might actually focus their activities in contaminated areas of
the site.  For example, if contamination has reduced emergent vegetation in a pond, the pond
might be more heavily used for feeding by waterfowl than uncontaminated ponds with little
open water.

       Bioavailability. For the screening-level exposure estimate, in the absence of site-
specific information, assume that the bioavailability of contaminants at the site is 100 percent.
For example, at the screening-level, lead would be assumed to be 100 percent bioavailable to
mammals. While some literature indicates that mammals absorb approximately 10 percent of
ingested lead, absorption efficiency can be higher, up to about 60 percent, because dietary
           HIGHLIGHT 2-1
           Area-use Factor

       An animal's area-use factor can be
defined as the ratio of the area of
contamination (or the site area under
investigation) to the area' used by the animal,
e.g., its home range, breeding range, or
feeding/foraging range.  To ensure that
ecological risks are not underestimated, the
highest density and smallest area used by
each animal should be assumed.  This allows
the maximum number of animals to be
exposed to site contaminants and makes it
more likely that "hot spots" (i.e., areas of
unusually high contamination levels) will be
significant proportions of an individual
animal's home range.
                                           2-2

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 factors such as fasting, and calcium and phosphate content of the diet, can affect the
 absorption rate (Kenzaburo, 1986).  Because few species have been tested for bioavailability,
 and because Steps 3 through 6 provide an opportunity for this issue to be addressed
 specifically, the most conservative assumption is appropriate for this step.

       Life stage.  For the screening-level assessment, assume that the most sensitive life
 stages are present.  If an early life stage is the most sensitive, the population should be
 assumed to include or to be in that life stage.  For vertebrate populations, it is likely that most
 of the population is not in the most sensitive life stage most of the time.  However, for many
 invertebrate species, the entire population  can be at an early stage of development during
 certain seasons.

       Body weight and food ingestion rates.  Estimates of body weight and food
 ingestion  rates  of the receptor animals also should  be made conservatively to maximize the
 dose (intake of contaminants) on a body-weight basis and to avoid understating risk, although
 uncertainties in these factors  are far less than the uncertainties associated with the
 environmental contaminant concentrations. U.S. EPA's Wildlife Exposure Factors Handbook
 (U.S. EPA, 1993a,b) is a good source or reference to sources  of this information.

       Bioaccumulation.  Bioaccumulation values obtained from a literature search  can  be
 used to estimate contaminant accumulation and food-chain transfer at a Superfund site at the
 screening stage. Because many  environmental factors influence the degree of
 bioaccumulation, sometimes by several orders of magnitude, the most conservative (i.e.,
 highest) bioaccumulation factor (BAF) reported in  the literature should be used in the absence
 of site-specific  information.

       Dietary composition.  For species that feed on more than one type of food, the
 screening-level assumption should be that the diet is composed  entirely of whichever type  of
 food is most contaminated.  For example, if some foods (e.g., insects) are likely to be  more
 contaminated than other foods (e.g., seeds and fruits) typical in the diet of a receptor species,
 assume that the receptor species feeds exclusively on the more contaminated type of food.
 Again, EPA's Wildlife Exposure Factors Handbook (U.S. EPA, 1993a,b) is a good source  or
reference  to sources of this information.

2.2.2  Uncertainty Assessment

       Professional judgment is  needed to determine the uncertainty associated with
 information taken from the literature and any extrapolations used in developing a parameter to
estimate exposures.  All assumptions used to estimate exposures should be stated, including
some description of the degree of bias possible in each.  Where literature values are used,  an
 indication of the range of values that could be considered appropriate also should be
 indicated.
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 2.3   SCREENING-LEVEL RISK CALCULATION

       A quantitative screening-level risk can be estimated using the exposure estimates
 developed according to Section 2.2 and the screening ecotoxicity values developed according
 to Section 1.3. For the screening-level risk calculation, the hazard quotient approach, which
 compares point estimates of screening ecotoxicity values and exposure values, is adequate to
 estimate risk.  As described in Section 1.3, a screening ecotoxicity value should be equivalent
 to a documented  and/or best conservatively estimated chronic NOAEL.  Thus, for each
 contaminant and  environmental medium, the hazard quotient  can be expressed as the ratio of
 a potential exposure level to the NOAEL:
                           BQ .           or  HQ
                                  NOAEL             NOAEL

where:                           .

       HQ =         hazard quotient;

       Dose =       estimated contaminant intake at the site (e.g., mg contaminant/kg body
                     weight per day);

       EEC =        estimated environmental concentration at the site (e.g., mg
                     contarninant/L water, mg contaminant/kg soil, mg contaminant/kg food);
                     and

       NOAEL =    no-observed-adverse-effects-level (in units that match the dose or EEC).

An HQ less than one (unity) indicates that the contaminant alone is unlikely to cause adverse
ecological effects. If multiple contaminants of potential ecological concern exist at the site, it
might be appropriate to sum the HQs for receptors that could be simultaneously exposed to
the contaminants that produce effects by the same toxic mechanism (U.S. EPA, 1986a). The
sum of the HQs is called a hazard index (HI); (see Highlight 2-2). An HI less than one
indicates that the group of contaminants is unlikely to cause adverse ecological effects.  An
HQ or HI less than one does  not indicate the absence of ecological risk; rather, it should be
interpreted based on the severity of the effect reported and the magnitude of the calculated
quotient.  As certainty in the  exposure concentrations and the NOAEL increase, there is
greater confidence in  the predictive value of the hazard quotient model, and unity (HQ = 1)
becomes a more certain pass/fail decision point.

       The screening-level risk calculation is a conservative estimate to ensure that potential
ecological threats are not overlooked.  The calculation is used to document a decision about
whether or not there is a negligible potential for ecological impacts,  based on the information
available at this stage. If the potential for ecological impacts exists, this calculation can be

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 used to eliminate the negligible-risk
 combinations of contaminants and exposure
 pathways from further consideration.

       If the screening-level risk assessment
 indicates that adverse ecological effects are
 possible at environmental concentrations
 below standard quantitation limits, a "non
 detect" based on those limits cannot be used
 to support a "no risk" decision.  Instead, the
 risk assessment team and risk manager
 should request appropriate  detection limits
 or agree to continue to Steps 3 through 7,
 where exposure concentrations will be
 estimated from other information (e.g., fate-
 and-transport modeling, assumed or
 estimated values for non-detects).
2.4    SCIENTIFIC/MANAGEMENT
       DECISION POINT (SMDP)
           HIGHLIGHT 2-2
    Hazard Index (HI) Calculation

    For contaminants that produce adverse
effects by the same toxic mechanism:
Hazard Index =
EEC,/NOAEL,
EEC2/NOAEL2
EECj/NOAELj
 where:
          estimated environmental
          concentration for the 1th
          contaminant; and
NOAELi =
          NOAEL for the i* contaminant
          (expressed either as a dose or
          environmental concentration).

The EEC and the NOAEL are expressed in
the same units and represent the same
exposure period (e.g., chronic).  Dose could
be substimted for EEC throughout provided
the NOAEL is expressed as a dose.
       At the end of Step 2, the lead risk
assessor communicates the results of the
preliminary ecological risk assessment to the
risk manager. The risk manager needs to
decide whether the information available is
adequate to make a risk management
decision and  might require technical advice from the ecological risk assessment team to reach
a decision. There are only three possible decisions at this point:

       (1)    There  is adequate information to conclude that ecological risks are negligible
             and therefore no need for remediation on the basis of ecological risk;

       (2)    The information is not adequate to make a decision at this point, and the
             ecological  risk assessment process will continue to Step 3; or

       (3)    The information indicates a  potential for adverse ecological effects, and a more
             thorough assessment is warranted.

       Note that the  SMDP made at the end of the screening-level risk calculation will not
set a preliminary  cleanup goal. Screening ecotoxicity values are  derived to avoid
underestimating risk.  Requiring a cleanup based solely on those  values would not be
technically defensible.
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       The risk manager should document both the decision and the basis for it. If the risk
 characterization supports the first decision (i.e., negligible risk), the ecological risk assessment
 process ends here with appropriate documentation to support the decision. The  documentation
 should include all analyses and references used in the assessment, including a discussion of
 the uncertainties associated with the HQ and HI estimates.

       For assessments that proceed to Step 3, the screening-level analysis in Step 2 can
 indicate and justify which contaminants and exposure pathways can be eliminated from
 further assessment because they are unlikely to pose a substantive risk,  (If new contaminants
 are discovered or contaminants are found at higher concentrations later in the site
 investigation, those contaminants might need to be added to the ecological risk assessment at
 that time.)

       U.S. EPA must be confident that the SMDP made after completion of this calculation
 will protect the ecological components of the environment.  The decision  to continue beyond
 the screening-level risk calculation does  not indicate whether remediation  is necessary  at the
 site.  That decision will be made in Step 8 of the process.
2.5    SUMMARY

       At the conclusion of the exposure estimate and screening-level risk calculation step,
the following information should have been compiled:

       (1)    Exposure estimates based on conservative assumptions and maximum
              concentrations present; and

       (2)    Hazard quotients (or hazard  indices) indicating which, if any, contaminants and
              exposure pathways might pose ecological threats.

       Based on the results of the screening-level ecological risk calculation, the risk manager
and lead risk assessor will determine whether or not contaminants from the  site pose an
ecological threat.  If there  are sufficient data to determine that ecological threats are
negligible, the ecological risk assessment will be complete at this step with  a finding of
negligible ecological risk.  If the data indicate that there is (or might be) a risk of adverse
ecological effects, the ecological risk assessment process will continue.
                                                (
       Conservative assumptions have been used for each step of the screening-level
ecological risk assessment.  Therefore, requiring a cleanup based solely on this information
would not be  technically defensible.  To end the assessment at this stage, the conclusion of
negligible ecological risk must be adequately documented and technically defensible. A lack
of information on  the toxicity of a contaminant or on complete exposure pathways will result
in a decision to  continue with the ecological risk assessment process (Steps 3 through 7)—not
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a decision to delay the ecological risk assessment until a later date, when more information
might be available.
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  STEP 3:  BASELINE RISK ASSESSMENT PROBLEM  FORMULATION
                                     OVERVIEW

          Step 3 of the eight-step process initiates the problem-formulation phase of the
    baseline ecological risk assessment.  Step 3 refines the screening-level problem
    formulation and, with input from stakeholders and other involved parties, expands on
    the ecological issues that are of concern at the particular site.  In the screening-level
    assessment, conservative assumptions were used where site-specific information was
    lacking.  In Step 3,  the results of the screening assessment and additional site-specific
    information are used to determine the scope and goals of the baseline ecological risk
    assessment. Steps 3 through 7 are required only for sites for which the screening-level
    assessment indicated a need for further ecological  risk evaluation.

          Problem formulation at Step 3 includes several activities:

      •    Refining preliminary contaminants of ecological concern;
      •    Further characterizing ecological effects of contaminants;
      •    Reviewing and refining information on contaminant fate and transport, complete
          exposure pathways, and ecosystems potentially at risk;
      •    Selecting assessment endpoints; and
      •    Developing a conceptual model with working hypotheses or questions that the
          site investigation will address.

    At the conclusion of Step 3, there is a SMDP, which consists of agreement on four
    items: the assessment endpoints, the exposure pathways, the risk questions, and
    conceptual model integrating these components. The products of Step 3 are used to
    select measurement  endpoints and to develop the ecological  risk assessment work plan
    (WP) and sampling  and analysis plan (SAP) for the site in Step 4.  Steps 3 and 4 are,
    effectively, the data quality objective (DQO) process for the baseline ecological risk
    assessment.
3.1    THE PROBLEM-FORMULATION  PROCESS

       In Step 3, problem formulation establishes the goals, breadth, and focus of the baseline
ecological risk assessment.  It also establishes the assessment endpoints, or specific ecological
values to be protected (U.S. EPA, 1992a). Through Step 3, the questions and issues that need
to be addressed in the baseline ecological risk assessment are defined based on potentially
complete exposure pathways and ecological  effects.  A conceptual model of the site is

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developed that includes questions about the assessment endpoints and the relationship between
exposure and effects. Step 3 culminates in an SMDP, which is agreement between the risk
manager and risk assessor on the assessment endpoints, exposure pathways, and questions as
portrayed in the conceptual model of the site.

       The conceptual model, which is completed in Step 4, also will describe the approach,
types of data, and analytical tools to be used for the analysis phase of the ecological risk
assessment (Step 6). Those components of the conceptual model are formally described in
the ecological risk WP and SAP in Step 4 of this eight-step process.  If there is not
agreement among the risk manager, lead risk assessor, and the other professionals involved
with the ecological risk assessment on the initial conceptual model developed in Step 3, the
final conceptual model and field study  design developed in Step 4 might not resolve the
issues that must be considered to manage risks effectively.

       The complexity of questions developed during problem formulation does not depend
on the size of a site or the magnitude of its contamination.  Large areas of contamination can
provoke simple questions and, conversely, small sites with numerous contaminants can require
a complex series of questions and assessment endpoints.  There is no rule that can be applied
to gauge the effort needed for an ecological risk assessment based on site size or number of
contaminants; each site should be evaluated individually.

       At the beginning of Step 3, some basic information should exist for the site.  At a
minimum, information should be available from the site history, PA, SI, and Steps 1 and 2 of
this eight-step process.  For large or complex sites, information might be available from
earlier site investigations.

       It is important to be as complete as possible early in the process so that Steps 3
through 8 need not be repeated.  Repeating the selection of assessment  endpoints and/or the
questions and hypotheses concerning those endpoints is appropriate only if new information
indicating new threats becomes available.  The SMDP process should prevent having to return
to the problem formulation step because of changing opinions on the questions being asked.
Repetition of Step 3 should not be confused with the intentional tiering (or phasing) of
ecological site investigations at large or complex sites (see Highlight 3-1). The process of
problem formulation at complex sites is the same as at more simple sites, but the number,
complexity, and/or level of resolution of the questions and hypotheses can be greater at
complex sites.

       While problem formulation is conceptually simple, in practice it can be a complex and
interactive process. Defining the ecological problems to be addressed during the baseline risk
assessment involves identifying toxic mechanisms of the contaminants,  characterizing
potential receptors, and estimating exposure and potential ecological effects. Problem
formulation also constitutes the DQO process for the baseline ecological risk assessment (U.S.
EPA, 1993c,d).
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       The remainder of this section
 describes six activities to be conducted
 prior to the SMDP for this step:
 refining preliminary contaminants of
 ecological concern (Section 3.2); a
 literature search on the potential
 ecological effects of the contaminants
 (Section 3.3); qualitative evaluation of
 complete exposure pathways and
 ecosystems potentially at risk (Section
 3.4); selecting assessment endpoints
 (Section 3.5); and developing the
 conceptual model and establishing risk
 questions (Section 3.6).
             HIGHLIGHT 3-1
       Tiering an Ecological Risk
               Assessment

       Most ecological risk assessments at
Superfund sites are at least a two-tier process.
Steps 1 and 2 of this guidance serve as a first,
or screening, tier prior to expending a larger
effort for a detailed, site-specific ecological risk
assessment. The baseline risk assessment may
serve as the second tier. Additional tiers could
be needed in the baseline risk assessment for
large or complex sites where there is a need to
sequentially test interdependent hypotheses
developed during problem formulation (i.e.,
evaluating the results of one field assessment
before designing a subsequent field study).

       While tiering can be an effective way to
manage site investigations, multiple sampling
phases typically require some resampling of
matrices sampled during earlier tiers and
increased field-mobilization costs. Thus, in
some cases, a multi-tiered ecological risk
assessment might cost more than a two-tiered
assessment. The benefits of tiering should be
weighed against the costs.
3.2   REFINEMENT OF
       PRELIMINARY
       CONTAMINANTS OF
       CONCERN

       The results of the screening-level
risk assessment (Steps 1 and 2) should
have indicated which contaminants
found at the site can be eliminated from
further consideration and which should
be evaluated further. It is important to
realize that contaminants that might pose
an ecological  risk can  be different from
those that might pose a human health  risk because of differing exposure pathways,
sensitivities, and responses to contaminants.

       The initial list of contaminants investigated in Steps 1 and 2 included all contaminants
identified or suspected to be at the site.  During Steps 1 and 2, it is likely that several of the
contaminants found at the site were eliminated from further assessment because the risk
screen indicated that they posed a negligible ecological risk.  Because of the conservative
assumptions used during the risk screen, some of the contaminants retained for Step 3 might
also pose negligible risk.  At this stage, the risk assessor should review the assumptions used
(e.g.,  100 percent bioavailability) against  values reported in the literature (e.g., only up to 60
percent for a particular contaminant), and consider how the HQs would change if more
realistic conservative assumptions were used instead (see Section 3.4.1).  For those
contaminants for which the HQs drop to near or below unity, the lead risk assessor and risk
manager should discuss and  agree on which  can  be eliminated from further consideration at
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this time.  The reasons for dropping any contaminants from consideration at this step must be
documented in the baseline risk assessment.

       Sometimes, new information becomes available that indicates the initial assumptions
that screened some contaminants out in Step 2 are no longer valid (e.g., site contaminant
levels are higher than originally reported).  In this case, contaminants can be placed back on
the list of contaminants to be investigated with that justification.

        Note that a contaminant should not be eliminated from the list of contaminants to be
investigated only because toxicity information is lacking;  instead, limited or missing toxicity
information must be addressed using best professional judgment and discussed as an
uncertainty.
3.3    LITERATURE SEARCH ON KNOWN ECOLOGICAL EFFECTS

       The literature search conducted in Step 1 for the screening-level risk assessment might
need to be expanded to obtain the information needed for the more detailed problem
formulation phase of the baseline ecological risk assessment. The literature search should
identify NOAELs, LOAELs, exposure-response functions, and the mechanisms of toxic
responses for contaminants for which those data were not collected in Step 1.  Appendix C
presents a discussion of some  of the factors important in conducting a literature search.
Several U.S. EPA publications (e.g., U.S. EPA, 1995a,e,g,h) provide a window to original
toxicity literature for contaminants often found at Superfund sites. For all retained
contaminants, it is important to obtain and review the primary literature.
3.4    CONTAMINANT FATE AND TRANSPORT, ECOSYSTEMS POTENTIALLY AT
       RISK, AND COMPLETE EXPOSURE PATHWAYS

       A preliminary identification of contaminant fate and transport, ecosystems potentially
at risk, and complete exposure pathways was conducted in the screening ecological risk
assessment.  In Step 3, the exposure pathways and the ecosystems associated with the
assessment endpoints that were retained by the screening risk assessment are evaluated in
more detail. This effort typically involves compiling additional information on:

       (1)    The environmental fate and transport of the contaminants;

       (2)    The ecological setting and general flora and fauna of the site (including habitat,
             potential receptors, etc.); and

       (3)    The magnitude and extent of contamination, including its spatial and temporal
             variability relative to the assessment endpoints.
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        For individual contaminants, it is frequently possible to reduce the number of exposure
 pathways that need to be evaluated to one or a few "critical exposure pathways" which (1)
 reflect maximum exposures of receptors within the ecosystem, or (2) constitute exposure
 pathways to ecological receptors sensitive to the contaminant. The critical exposure pathways
 influence the selection of assessment endpoints for a particular site.  If multiple critical
 exposure pathways exist, they each should be evaluated, because it is often difficult to predict
 which pathways could be responsible for the greatest ecological risk.

 3.4.1   Contaminant Fate and Transport

        Information on how the contaminants will or could be transported or transformed in
 the environment physically, chemically, and biologically is used to identify the exposure
 pathways that might lead to significant ecological effects (see  Highlight 3-2).  Chemically,
 contaminants can undergo several processes in the environment:
       •      Degradation,3
       •      Complexation,
       •      lonization,
       •      Precipitation, and/or
       •      Adsorption.

Physically, contaminants might move
through the environment by one or more
means:

       •      Volatilization,
       •      Erosion,
       •      Deposition (contaminant
              sinks),
       •      Weathering of parent material
              with subsequent transport,
              and/or
       •      Water transport:
                  in solution,
                  as suspended material in the water, and
                  bulk transport of solid material.

Several biological processes  also affect contaminant fate and transport in the environment:

       •      Bioaccumulation,
       •      Biodegradation,
           HIGHLIGHT 3-2
 Environmental Fate and Exposure

       If a contaminant in an aquatic
ecosystem is highly lipophilic (i.e.,
essentially insoluble in water), it is likely to
partition primarily into sediments and not
into the water column.  Factors such as
sediment particle size and organic carbon
influence contaminant partitioning; therefore,
these attributes should be characterized when
sampling sediments. Similar considerations
regarding partitioning should be applied to
contaminants in soils.
     The product might be more or less toxic than the parent compound.
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       •      Biological transformation,4
       •      Food chain transfers, and/or
       •      Excretion.

       Additional information should be gathered on past as well as current mechanisms of
 contaminant release from source areas at the site.  The mechanisms of release along with the
 chemical and physical form of a contaminant can affect its fate, transport, and potential for
 reaching ecological receptors.

       A contaminant flow diagram (or exposure pathway diagram) comprises a large part of
 the conceptual  model, as illustrated in Section 3.6.  A contaminant flow diagram originates at
 the primary contaminant source(s) and identifies primary release mechanisms and contaminant
 transport pathways.  The release and movement of the contaminants can create secondary
 sources (e.g., contaminated sediments in a river; see Example 3-1), and even tertiary sources.

       The above information is used to evaluate where the contaminants are likely to
 partition in the environment, and the bioavailability of the contaminant (historically, currently,
 or in the future).  As indicated in Section 3.2, it might be possible for the risk assessment
 team and the risk manager to use this information to replace some of the conservative
 assumptions used in the screening-level risk assessment and to eliminate additional chemicals
 from further evaluation at this point.  Any such negotiations must be documented in the
 baseline risk assessment.,

 3.4.2  Ecosystems Potentially at Risk

       The ecosystems or habitats potentially at risk depend oh the ecological setting of a
 site.  An initial source of information on the ecological setting of a site is the data collected
 during the  preliminary site visit and characterization (Step 1), including the site ecological
 checklist (Appendix B).  The site description should provide answers to several questions
 including:

       •      What habitats  (e.g., maple-beech hardwood forest, early-successional fields) are
             present?
       •      What types of water bodies are present, if any?
       •      Do any other habitats listed in Exhibit 1-1 exist on or adjacent to the site?
       While adequately documented information should be used, it is not critical that
complete site setting information be collected during this phase of the risk assessment.
However, it is important that habitats at the site are not overlooked; hence, a site visit might
be needed to supplement the one conducted during the screening risk assessment.  If a habitat
     The product might be more or less toxic than the parent compound.


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                                     EXAMPLE 3-1
                          Exposure Pathway Model-DDT Site

          An abandoned pesticide production facility had released DDT to soils through poor
   handling practices during its operation. Due to erosion of contaminated soils, DDT migrated to
   stream sediments.  The contaminated sediments represent a secondary source that might affect
   benthic organisms through direct contact or ingestion. Benthic organisms that have accumulated
   DDT can be consumed by fish, and fish that have accumulated DDT can be consumed by
   piscivorous birds, which are considered a valuable component of the local ecosystem. This
   example illustrates how contaminant transport is traced from a primary source to a secondary
   source and from there through a food chain to an  exposure point that can affect an assessment
   endpoint.
actually present on the site is omitted during the problem formulation phase, this step might
need to be repeated later when the habitat is found, resulting in delays and additional costs
for the risk assessment.

        Available information on ecological effects of contaminants (see Section 3.3) can help
focus the assessment on specific ecological resources that should be evaluated more
thoroughly, because some groups of organisms can be more sensitive than others to a
particular contaminant.  For example, a species  or group of species could be physiologically
sensitive to a particular contaminant (e.g., the contaminant might interfere with its vascular
system); or, the species might not be able to metabolize and detoxify the  particular
contaminant(s) (e.g., honey bees and grass shrimp cannot effectively biodegrade PAHs,
whereas fish generally can).  Alternatively, an already-stressed population (e.g.,  due to habitat
degradation)  could be particularly sensitive to any added stresses.

       Variation in sensitivity should not be confused with variation in exposure, which can
result from behavioral and dietary differences among species.  For example, predators can be
exposed to higher levels of contaminants that biomagnify in food chains than herbivores.  A
specialist predator could feed primarily on one prey type that is a primary receptor of the
contaminant.  Some species might preferentially feed in a habitat where the contaminant tends
to accumulate.  On the  other hand, a species might change its behavior to avoid contaminated
areas.  Both sensitivity  to toxic  effects of a contaminant and behaviors that affect exposure
levels can influence risks for particular groups of organisms.

3.4.3  Complete Exposure Pathways

       The potentially complete exposure pathways identified in Steps  1  and 2 are described
in more detail in Step 3 on the basis of the refined contaminant fate and  transport evaluations
(Section  3.4.1) and evaluation of potential ecological receptors (Section 3.4.2).'
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       Some of the potentially complete exposure pathways identified in Steps 1 and 2 might
be ruled out from further consideration at this time.  Sometimes, additional exposure
pathways might be identified, particularly those originating from secondary sources. Any data
gaps that result in questions about whether an exposure pathway is complete should be
identified, and the type of data needed to answer those questions should be described to assist
in developing the WP and SAP in Step 4.                                   .

       During Step 3, the potential for food-chain exposures deserves particular attention.
Some contaminants are effectively transferred through food chains, while others are not. To
illustrate this point, copper and DDT are compared in Example 3-2.
                                     EXAMPLE 3-2
             Potential for Food Chain Transfer-Copper and DDT Sites

          Copper can be toxic in aquatic ecosystems and to terrestrial plants.  However, it is an
   essential nutrient for both plants and animals, and organisms can regulate internal copper
   concentrations within limits. For this reason, copper tends not to accumulate in most organisms
   or to biomagnify in food chains, and thus tends not to reach levels high enough to cause
   adverse responses through food chain transfer to upper-trophic-level organisms.  (Copper is
   known to accumulate by several orders of magnitude in phytoplankton and in filter-feeding
   mollusks, however, and thus can pose a threat to organisms that feed on those components of
   aquatic ecosystems; U.S. EPA, 1985a.) In contrast, DDT, a contaminant that accumulates  in
   fatty tissues, can biomagnify in many different types of food chains. Upper-trophic-level
   species (such as predatory birds), therefore, are likely to be exposed to higher levels of DDT
   through their prey than are lower-trophic-level species in the ecosystem.
3.5    SELECTION OF ASSESSMENT ENDPOINTS

       As noted in the introduction to this guidance, an assessment endpoint is "an explicit
expression of the environmental value that is to be protected" (U.S. EPA, 1992a). In human
health risk assessment, only one species is evaluated, and cancer and noncancer effects are the
usual assessment endpoints.  Ecological risk assessment, on the other hand, involves multiple
species that  are likely to be exposed to differing degrees and to respond differently to the
same contaminant.  Nonetheless, it is not practical or possible to directly evaluate risks to  all
of the individual  components of the ecosystem at a site.  Instead, assessment endpoints focus
the risk assessment on particular components of the ecosystem that could be adversely
affected by contaminants from the site.

       The selection of assessment endpoints includes discussion between the  lead risk
assessor and the risk manager concerning management policy goals and ecological values.
The lead risk assessor and risk manager should seek input from the regional BTAG, PRPs,
and other stakeholders associated with a site when identifying assessment endpoints for a site.

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Stakeholder input at this stage will help ensure that the risk manager can readily defend the
assessment endpoints when making decisions for the site.  ECO Update Volume 3, Number 1,
briefly summarizes the process of selecting assessment endpoints (U.S. EPA, 1995b).

       Individual assessment endpoints usually encompass  a group of species or populations
with some common characteristics, such as a specific exposure route or contaminant
sensitivity.  Sometimes, individual assessment endpoints are limited to one species (e.g., a
species known to be particularly sensitive to a site contaminant).  Assessment endpoints can
also encompass the typical structure and function of biological communities  or ecosystems
associated with a site.

       Assessment endpoints for the baseline ecological risk assessment must be selected
based on the ecosystems, communities, and/or species potentially present at the site. The
selection of assessment endpoints depends on:

       (1)    The contaminants present and their concentrations;
                                                           /
       (2)    Mechanisms of toxicity of the contaminants to different groups of organisms;

       (3)    Ecologically relevant receptor groups that are potentially sensitive or highly
              exposed to the contaminant and attributes of their natural history; and

       (4)    Potentially complete exposure pathways.

Thus, the  process of selecting assessment endpoints can be intertwined with  other phases of
problem formulation.

       The risk assessment team must  think through the contaminant mechanism(s) of
ecotoxicity to determine  what receptors will or could be at risk. This understanding must
include how the adverse  effects of the  contaminants might be expressed (e.g., eggshell
thinning in birds), as  well as how the chemical and physical form of the contaminants
influence bioavailability  and the type and magnitude of adverse response (e.g., inorganic
versus organic mercury).

       The risk assessment team also should determine if the contaminants can adversely
affect organisms in direct contact with  the contaminated media (e.g., direct exposure to water,
sediment,  soil) or if the contaminants accumulate in food chains, resulting in adverse effects
in organisms that are not directly exposed or are minimally exposed to the original
contaminated media (indirect exposure).  The team should decide  if the risk  assessment
should focus on toxicity  resulting from direct or indirect exposures, or if both must be
evaluated.

       Broad assessment endpoints (e.g., protecting aquatic communities) are generally of less
value in problem formulation than specific assessment endpoints (e.g., maintaining aquatic

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community composition and structure downstream of a site similar to that upstream of the
site). Specific assessment endpoints define the ecological value in sufficient detail to identify
the measures needed to answer specific questions or to test specific hypotheses. Example 3-3
provides three examples of assessment endpoint selection based on the hypothetical sites in
Appendix A.

       The formal identification of assessment endpoints is part of the SMDP for this step.
Regardless of the level of effort to be expended on the subsequent phases of the risk
assessment, the assessment endpoints identified are critical elements in the design of the
ecological risk assessment and must be agreed upon as the focus of the risk assessment.
Once assessment endpoints have been selected, testable hypotheses and measurement
endpoints can be developed to determine whether or not a potential threat to the assessment
endpoints exists.  Testable hypotheses and measurement endpoints cannot be developed
without agreement on the assessment endpoints among the risk manager, risk assessors, and
other involved professionals.
3.6    THE CONCEPTUAL MODEL AND RISK QUESTIONS

       The site conceptual model establishes the complete exposure pathways that will be
evaluated in the ecological risk assessment and the relationship of the measurement endpoints
to the assessment endpoints.  In the conceptual model, the possible exposure pathways are
depicted in an exposure pathway diagram and must be linked directly to the assessment
endpoints identified in Section 3.5.  Developing the conceptual model and risk questions are
described in Sections 3.6.1 and 3.6:2, respectively. Selection of measurement endpoints,
completing the conceptual model, is described in Step 4.

3.6.1  Conceptual Model

       Based on the information obtained from Steps 1 and 2, knowledge of the contaminants
present, the exposure pathway diagram, and the assessment endpoints, an integrated
conceptual model is developed (see Example 3-4). The conceptual model includes a
contaminant fate-and-transport diagram that traces the contaminants' movement from sources
through the ecosystem to receptors that include the assessment endpoints (see Example 3-5).
Contaminant exposure pathways that do not lead to a species or group of species associated
with the proposed assessment endpoint indicate that either:

       (1)    There is an incomplete exposure pathway to the receptor(s) associated with the
             proposed assessment endpoint; or

       (2)    There are  missing components or data necessary to demonstrate a complete
             exposure pathway.
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                                    EXAMPLE 3-3
         Assessment Endpoint Selection-DDT, Copper, and PCB Sites

 DDT Site

        An assessment endpoint such as "protection of the ecosystem from the effects of DDT"
 would give little direction to the risk assessment. However, "protection of piscivorous birds
 from eggshell thinning due to DDT exposure" directs the risk assessment toward the food-chain
 transfer of DDT that results in eggshell thinning in a specific group of birds. This assessment
 endpoint provides the foundation for identifying appropriate measures of effect and exposure
 and ultimately the design of the site investigation.  It is not necessary that a specific species of
 bird be identified on site.  It is necessary that the exposure  pathway exists and that the presence
 of a piscivorous bird could be expected.

 Copper Site

        Copper can  be acutely or chronically toxic to organisms in  an aquatic community
 through direct exposure of the organisms to copper in the water and sediments. Threats of
 copper toxicity to higher-trophic-level organisms are unlikely to exceed threats to organisms at
 the base of the food chain, because copper is an essential nutrient which is effectively regulated
 by most organisms if the exposure  is below immediately toxic levels. Aquatic plants
 (particularly phytoplankton) and mollusks,  however, are poor at regulating copper and might be
 sensitive receptors or effective in transferring copper to the next trophic level.  In addition, fish
 fry  can  be very sensitive to copper in water.  Based on these receptors and the potential for both
 acute and chronic toxicity, an appropriate general assessment endpoint for the system could be
 the  maintenance of aquatic community composition.  An operational definition of the
 assessment endpoint for this site would be  pond fish and invertebrate community composition
 similar to that of other ponds of similar size and characteristics in the area.

 PCB Site

       The primary ecological threat of PCBs in ecosystems  is not through  direct exposure and
acute toxicity.  Instead, PCBs bioaccumulate in food chains and can diminish reproductive
success in some vertebrate species.  PCBs  have been implicated as  a cause of reduced
reproductive success of piscivorous birds (e.g., cormorants,  terns) in the Great Lakes (Kubiak  et
al.,  1989;  Fox et al., 1991) and of mink along several waterways (Aulerich and Ringer, 1977;
Foley et al., 1988).  Therefore, reduced reproductive success in high-trophic-level species
exposed via their diet is a more appropriate assessment endpoint than either  toxicity to
organisms via direct exposure to PCBs in water, sediments, or soils, or reproductive impairment
 in lower-trophic-level species.
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                                      EXAMPLE 3-4
                    Description of the Conceptual Model-DDT Site

          One of the assessment endpoints selected for the DDT site (Appendix A) is the
   protection of piscivorous birds.  The site 'conceptual model includes the release of DDT from
   the spill areas to the adjacent stream, followed by food chain accumulation of DDT from the
   sediments and water through the lower trophic levels to forage fish in the stream.  The forage
   fish are the exposure point for piscivorous birds.  Eggshell thinning was selected as the  measure
   of effect.  During the literature review of the ecological effects of DDT, toxicity studies were
   found that reported reduced reproductive success  (i.e., number of young fledged) in birds that
   experienced eggshell thinning of 20 percent or more (Anderson and Hickey,  1972; Dilwqrth et
   al., 1972).  Based  on those data, the lead risk assessor and risk manager agreed  that eggshell
   thinning of 20 percent or more would be considered an adverse effect for piscivorous birds.

          Chronic DDT exposure can also reduce some animals' ability to escape  predation.
   Thus, DDT can indirectly increase the mortality rate of these organisms by making them more
   susceptible to predators (Cooke, 1971; Krebs et al., 1974). That effect of DDT  oh prey also can
   have an indirect consequence for the predators. If predators are more likely  to capture the more
   contaminated prey, the predators could be exposed to DDT at levels higher than represented in
   the average prey population.
If case (1) is true, the proposed assessment endpoint should be reevaluated to determine if it
is an appropriate endpoint for the site. If case (2) is true, then additional field data could be
needed to evaluate contaminant fate and transport at the site.  Failure to identify a complete
exposure pathway that does exist at the site can  result in incorrect conclusions or in extra
time and effort being expended on a supplementary investigation.

       As indicated in Section 3.5, appropriate assessment endpoints differ from site to site,
and can  be at one or more  levels of biological organization.  At any particular site, the
appropriate assessment endpoints might involve  local populations of a particular species,
community-level integrity,  and/or habitat  preservation.  The site conceptual  model must
encompass the level of biological organization appropriate for the assessment endpoints for
the site.  The conceptual model can use assumptions that generally represent a group of
organisms or ecosystem components.

       The intent of the conceptual model is not to describe a particular species or site
exactly as much  as it is to  be systematic,  representative, and conservative where information
is lacking (with assumptions  biased to be  more likely to overestimate than to underestimate
risk). For example, it is not  necessary or even recommended to develop new test protocols to
use species that exist at a site to test the toxicity of site media (See Step 4).  Species used in
standardized laboratory toxicity tests (e.g., fathead minnows, Hyallela amphipods) usually are
adequate surrogates for species in their general taxa and habitat at the site.

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                                                  EXAMPLE 3-5
                                       Conceptual Model Diagram-DDT Site
                                                             SECONDARY
                                                              RECEPTOR
                                                                (Fish)
u*
              PRIMARY SOURCE
                  (Plant site)
 SECONDARY
   SOURCE
(Surface drainage)
 TERTIARY SOURCE
   (Stream sediments,
exposure point for fish and
   macroinvertebrates)
                                                 ASSESSMENT
                                                  ENDPOINT
                                             TERTIARY RECEPTOR
                                                (Piscivorous bird)
PRIMARY RECEPTOR
      (Benthic
  macroinvertebrates,
exposure point for fish)

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 3.6.2 Risk Questions

       Ecological risk questions for the
 baseline risk assessment at Superfund .sites
 are basically questions about the
 relationships among assessment endpoints
 and their predicted responses when exposed
 to contaminants. The risk questions should
 be based on the assessment endpoints and
 provide a basis for developing the study
 design (Step 4) and for evaluating the
 results of the site investigation in the
 analysis phase (Step 6) and during risk
 characterization (Step 7).

       The most basic question applicable
 to virtually all Superfund sites is whether
 site-related contaminants are causing or have
 the potential to cause adverse effects on the
 assessment endpoint(s).  To use the baseline
 ecological risk assessment in the FS to
 evaluate remedial alternatives,  it is helpful if
 the specific contaminant(s) responsible can
 be identified.  Thus refined, the question
 becomes  "does (or  could) chemical X cause adverse effects on the assessment endpoint?"  In
 general,  there are four lines of evidence that can be used to answer this question:

    (1)     Comparing estimated or measured exposure levels to chemical X with levels that
           are known from the literature to be toxic to receptors associated with the
           assessment endpoints;

    (2)     Comparing laboratory bioassays with media from the site and bioassays with media
           from a reference site;

    (3)     Comparing in situ toxicity tests at the site with in situ toxicity tests in a reference
           body of water; and

    (4)     Comparing observed effects in the receptors associated with the site with similar
           receptors at a reference site.

These lines of evidence are considered further in Step 4, as measurement endpoints are
selected  to complete the conceptual model and the site-specific study is designed.
           HIGHLIGHT 3-3
             Definitions:
      Null and Test Hypotheses

Null hypothesis:  Usually a hypothesis of
no differences between two populations
formulated for the express purpose of being
rejected.

Test (or alternative) hypothesis: An
operational statement of the investigator's
research hypothesis.

      "When appropriate, formal hypothesis
testing is preferred to make explicit what
error rates are acceptable and what
magnitude of effect is considered
biologically important. However, it might
not be practical for many assessment
endpoints or be the only acceptable way to
state questions about those  endpoints. See
Example 4-1 in the next chapter.
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3.7    SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)

       At the conclusion of Step 3, there is .a SMDP. The SMDP consists of agreement on
four items: contaminants of concern, assessment endpoints, exposure pathways, and risk
questions. Those items can be summarized with the assistance of the diagram of the
conceptual model.  Without agreement between the risk manager, risk assessors, and other
involved professionals on the conceptual model to this point, measurement endpoints cannot
be selected, and a site study cannot be developed effectively.  Example 3-5 shows the
conceptual model for the DDT site example in Appendix A.
3.8    SUMMARY

       By combining information on:  (1) the potential contaminants present; (2) the
ecotoxicity of the contaminants; (3) environmental fate and transport; (4) the ecological
setting; and (5) complete exposure pathways, an evaluation is made of what aspects of the
ecosystem at the site could be at risk and what the adverse ecological response could be.
"Critical exposure pathways" are based on:  (1) exposure pathways to sensitive species'
populations or communities; and (2) exposure levels associated with predominant fate and
transport mechanisms at a site.

       Based on that information, the risk assessors and risk manager agree on assessment
endpoints and specific questions or testable hypotheses that, together with the rest of the
conceptual  model, form the basis for the site investigation.  At this stage, site-specific
information on exposure pathways and/or the presence of specific species is likely to be
incomplete. By using  the conceptual model developed thus far, measurement endpoints can
be selected, and a plan for filling information gaps can be developed and written into the
ecological WP and SAP as described in Step 4.
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               STEP 4:  STUDY DESIGN AND DATA QUALITY
                              OBJECTIVE PROCESS
                                     OVERVIEW

          The site conceptual model begun in Step 3, which includes assessment
   endpoints, exposure pathways, and risk questions or hypotheses, is completed in Step 4
   with the development of measurement endpoints.  The conceptual model then is used
   to develop the study design and data quality objectives.  The products of Step 4 are the
   ecological risk assessment WP and SAP, which describe the details of the site
   investigation as well as the data analysis methods and data quality objectives (DQOs).
   As part of the DQO process, the SAP specifies acceptable levels of decision errors that
   will be used as the basis for establishing the quantity and quality of data needed to
   support ecological risk management decisions.

          The lead risk assessor and the risk manager should agree that the WP and SAP
   describe a study that will provide the risk manager with the information needed to
   fulfill the requirements of the baseline risk assessment and to incorporate ecological
   considerations into the site remedial process.  Once this step is completed, most of the
   professional judgment needed for the ecological risk assessment will have been
   incorporated into  the design and details of the WP and SAP.  This does not limit the
   need for qualified professionals in the implementation of the investigation, data
   acquisition, or data  interpretation.  However, there should be no fundamental changes
   in goals or approach to the ecological risk assessment once the WP and SAP are
   finalized.

          It is important to coordinate this  step with the WP and SAP for the site
   investigation, which is used to document the nature and extent of contamination and to
   evaluate human health risks.
       Step 4 of the ecological risk assessment establishes the measurement endpoints
(Section 4.1), completing the conceptual model begun in Step 3. Step 4 also establishes the
study design (Section 4.2) and data quality objectives based on statistical considerations
(Section 4.3) for the site assessment that will accompany site-specific studies for the remedial
investigation! The site conceptual model is used to identify which points or assumptions in
the risk assessment include the greatest degree of conservatism or uncertainty.  The field
sampling then can be designed to address  the risk model parameters that have important
effects on the risk estimates (e.g., bioavailability and toxicity of contaminants in the field,
contaminant concentrations at exposure points).

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       The products of Step 4 are the WP and SAP for the ecological component of the field
investigations (Section 4.4).  Involvement of the BTAG in the preparation, review, and
approval of WPs and SAPs can help ensure that the ecological risk assessment is well
focused, performed efficiently, and technically correct.

       The WP and SAP should specify the site conceptual model developed in Step  3, and
the measurement endpoints developed in  the beginning of Step 4. The WP describes:

       •     Assessment endpoints;
       •     Exposure pathways;
       •     Questions and testable hypotheses;
       •     Measurement endpoints and their relation to assessment endpoints; and
       •     Uncertainties and assumptions.

The SAP should describe:

       •     Data needs;
       •     Scientifically valid and sufficient study design and data analysis  procedures;
       •     Study methodology and protocols, including sampling techniques;
       •     Data reduction and interpretation techniques, including statistical analyses; and
       •     Quality assurance procedures and quality control techniques.

The SAP must include the data reduction and interpretation techniques, because it is necessary
to known how the  data will be interpreted to specify the number of samples needed.

       Prior to formal agreement on the WP and SAP, the proposed field sampling plan is
verified in Step 5.
4.1    ESTABLISHING MEASUREMENT ENDPOINTS

       As indicated in the Introduction, a measurement endpoint is defined as "a measurable
ecological characteristic that is related to the valued characteristic chosen as the assessment
endpoint" and is a measure of biological effects (e.g., mortality, reproduction, growth) (U.S.
EPA, 1992a; although this definition may change—see U.S. EPA 1996a).  Measurement
endpoints are frequently numerical expressions of observations (e.g., toxicity test results,
community diversity measures) that can be compared statistically to a control or reference site
to detect adverse responses to a site contaminant.  As used in this guidance, measurement
endpoints can include measures of exposure (e.g., contaminant concentrations in water) as
well as measures of effect.  The relationship between measurement and assessment endpoints
must be clearly described within the conceptual model and must be based on scientific
evidence. This is critical because the assessment and measurement endpoints usually are
different endpoints (see the Introduction and Highlight 4-1).
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       Typically, the number of
 measurement endpoints that are potentially
 appropriate for any given assessment
 endpoint and circumstance is  limited.  The
 most appropriate measurement endpoints for
 an assessment endpoint depend on several
 considerations, a primary one being how
 many and which lines of evidence are
 needed to support risk-management
 decisions at the site (see Section 3.6.2).
 Given the potential ramifications of site
 actions, the site risk manager  might want to
 use more than one line of evidence to
 identify site-specific thresholds for effects.
 The risk manager and risk assessors must
 consider the utility of each type of data
 given the cost of collecting those  data and
 the likely sensitivity of the risk estimates to
 the data.
           HIGHLIGHT 4-1
    Importance of Distinguishing
  Measurement from Assessment
              Endpoints

       If a measurement endpoint is
mistaken for an assessment endpoint, the
misperceptionvcan arise that Superfund is
basing a remediation  on an arbitrary or
esoteric justification.  For example,
protection of a few invertebrate and  algal
species could be mistaken as the basis for a
remedial decision, when the actual basis for
the decision is the protection of the aquatic
community as a whole (including higher-
trophic-level game fish  that depend on lower
trophic levels in the community), as
indicated by a few sensitive invertebrate and
algal species.
       There are some situations in which it
might only be necessary or possible to compare estimated or measured contaminant exposure
levels at a site to ecotoxicity values derived from the literature.  For example, for
contaminants in surface waters for which there are state water-quality standards, exceedance
of the standards indicates that remediation to reduce contaminant concentrations in surface
waters to below these levels could be  needed whether impacts are occurring or not.  For
assessment endpoints for which impacts are difficult to demonstrate in the field (e.g., because
of high natural variability), and toxicity tests are not possible (e.g., food-chain accumulation is
involved), comparing environmental concentrations with  a well-supported ecotoxicity value
might have to suffice.

       A bioassay using contaminated media from the site can suffice if the risk  manager and
risk assessor agree that laboratory tests with surrogate species will be taken as  indicative of
likely effects on the assessment endpoint.  For sites with complex mixtures of contaminants
without robust ecotoxicity  values and high natural variability in potential measures for the
assessment endpoint, either laboratory or in situ toxicity  testing might be the best technique
for evaluating risks to the assessment endpoint. For inorganic substances in soils or
sediments, bioassays often are needed to determine the degree to which a contaminant is
bioavailable at a particular site. Laboratory toxicity tests can indicate the potential for
adverse impacts in the field, while in situ toxicity testing with resident organisms can provide
evidence of actual impacts occurring in the field.

       Sometimes more  than one  line  of evidence is needed to reasonably demonstrate that
contaminants from a site are likely to  cause adverse effects on the assessment endpoint.  For
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example, total recoverable copper in a surface water body to which a water quality standard
did not apply could exceed aquatic ecotoxicity values, but not cause adverse effects because
the copper is only partially bioavailable or because the ecotoxicity value is too conservative
for the particular ecosystem.  Additional evidence from bioassays or community surveys
could help resolve whether the copper is actually causing adverse effects (See Example 4-1).
Alternatively, if stream community surveys indicate impairment of community structure
downstream of a site, comparing contaminant concentrations with aquatic toxicity values can
help identify which contaminants are most likely to be causing the effect.  When some lines
of evidence  conflict with others, professional judgment is needed to determine which data
should be considered more reliable or relevant to the questions.
                                    EXAMPLE 4-1
                           Lines of Evidence-Copper Site

   Primary question: Are ambient copper levels in sediments causing adverse effects in
         benthic organisms in the pond?

   Possible lines of evidence phrased as test hypotheses:
                      x
         (1)    Mortality in early life stages of benthic aquatic insects in contact  with
                sediments from the site significantly exceeds mortality in the same kinds
                of organisms in contact with sediments from a reference site (e.g.,
         (2)    Mortality in in situ toxicity tests in sediments at the pond significantly
                exceeds mortality in in situ toxicity tests in sediments at a reference pond
                (e.g., p< 0.1).

         (3)    There are significantly fewer numbers of benthic aquatic insect species
                present per m2 of sediment at the pond near the seep than at the opposite
                side of the pond (e.g., p < 0.1).

   Statistical and biological significance:  Differences in the incidence of adverse
   effects between groups of organisms exposed to contaminants from the site and groups
   not exposed might be statistically significant, but not biologically important, depending
   on the endpoint and the power of the statistical test. Natural systems can sustain some
   level of perturbation without changing in structure or function.  The risk assessor needs
   to evaluate what level of effect will be considered biologically important.  Given the
   limited power of small sample sizes to detect an effect, the risk assessor might decide
   that any difference that is statistically detectable at  a p level of 0.1 or lessjs important
   biologically.
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       Once there is agreement on which lines of evidence are required to answer questions
 concerning the assessment endpoint, the measurement endpoints by which the questions or
 test hypotheses will be examined can be selected.

       Each measurement endpoint should represent the same exposure pathway and toxic
 mechanism  of action as the assessment endpoint it represents; otherwise, irrelevant exposure
 pathways or toxic mechanisms might be evaluated.  For example, if a contaminant primarily
 causes damage to vertebrate kidneys, the use of daphnids (which do not have kidneys) would
 be inappropriate.                                                '   .          ,

       Potential measurement endpoints in toxicity tests or in field studies should be
 evaluated according to how well they can answer questions about the assessment endpoint or
 support or refute  the hypotheses developed for the conceptual model. Statistical
 considerations, including sample size and statistical  power described in Section 4.3, also must
 be considered in selecting the measurement endpoints.  The following subsections describe
 additional considerations for selecting measurement endpoints, including
 species/community/habitat (Section 4.1.1), relationship to the contaminant(s) of concern
 (Section 4.1.2), and mechanisms of ecotoxicity (Section 4.1.3).
                                            i
 4.1.1  Species/Community/Habitat Considerations

       The function of a measurement endpoint is to represent an assessment endpoint for the
 site. The measurement endpoint must allow clear inferences about potential changes in the
 assessment endpoint.  Whenever assessment and measurement endpoints are not the same
 (which usually  is  the case), measurement  endpoints  should be selected to be inclusive of risks
 to all of the species, populations, or groups included in the assessment endpoint that are not
 directly measured. In other words, the measurement endpoint should be representative of the
 assessment endpoint for the site and not lead to an underestimate of risk to the assessment
 endpoint. Example 4-2 illustrates this point for the  DDT site in Appendix A.

       In selecting a measurement endpoint, the species and life stage, population, or
community chosen should be the one(s) most susceptible to the contaminant for the
assessment endpoint in question. For species and populations, this selection is based on a
review of the species:  (1) life history; (2) habitat utilization; (3) behavioral characteristics;
and (4) physiological parameters.  Selection of measurement endpoints also should be based
 on which routes of exposure are likely. For communities, careful evaluation of the
contaminant fate and transport in the environment is essential.

4.1.2  Relationship of the Measurement Endpoints to the Contaminant of
       Concern

       Additional criteria to consider when selecting measurement endpoints are inherent
properties (such as the physiology or behavioral characteristics of the species) or life history
parameters that make a species useful in evaluating the effects of site-specific contaminants.

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                                                     For example, Chironomus tentans (a
                                                     species of midge that is used as a
                                                     standard sediment toxicity testing
                                                     species in the larval stage) is
                                                     considered more tolerant of metals
                                                     contamination than is C. riparius, a
                                                     similar species (Klemm et al., 1990;
                                                     Nebeker et al., 1984;  Pascoe et al.,
                                                     1989). To assess the effects of
                                                     exposure of benthic communities to
                                                     metal-contaminated sediment,  C.
                                                     riparius might be the better species to
                                                     use as a test organism for many aquatic
                                                     systems to ensure that risks are not
                                                     underestimated.  In general, the most
                                                     sensitive of the measurement endpoints
                                                     appropriate for inferring risks  to the
                                                     assessment endpoint should be used.  If
all else is equal, however, species that are commonly used in the laboratory  are preferred over
non-standard laboratory species to improve test precision.

       Some species have been identified as being particularly sensitive to certain
contaminants.  For example, numerous studies have demonstrated that mink  are among the
most sensitive  of the tested mammalian species to the toxic effects of PCBs  (U.S. EPA,
1995a).  Species that rely on quick reactions or behavioral responses to avoid predators can
be particularly  sensitive to contaminants affecting the central nervous system, such as
mercury.  Thus, the sensitivity of the measurement endpoint relative to the assessment
endpoint should be considered for each contaminant of concern.
              HIGHLIGHT 4-2
      Terminology and Definitions

       In the field of ecotoxicology, there
historically have been multiple definitions for
some terms, including definitions for direct
effects, indirect effects, acute effects, chronic
effects, acute tests, and chronic tests.  This
multiplicity of definitions has resulted in
misunderstandings and inaccurate communication
of study designs. Definitions of these and other
terms, as they are used in this document, are
provided in the glossary.  When consulting other
reference materials, the user should evaluate  how
the authors defined terms.
                                      EXAMPLE 4-2
                     Selecting Measurement Endpoints-DDT Site

          As described in Example 3-1, one of the assessment endpoints selected for the DDT site
   is the protection of piscivorous birds from egg-shell thinning due to DDT exposure.  The belted
   kingfisher was selected as a piscivorous bird with the smallest home range that could utilize the
   area of the site, thereby maximizing the calculated dose to a receptor. In this illustration, the
   kingfishers are used as the most highly exposed of the piscivorous birds potentially present.
   Thus, one can conclude that, if the risk assessment shows no threat of eggshell thinning to the
   kingfisher, there should be minimal or no threat to other piscivorous birds that might utilize the
   site.  Thus, eggshell thinning in belted kingfishers is an appropriate measurement endpoint for
   this site.
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4.1.3  Mechanisms of Ecoxicity

       A contaminant can exert adverse ecological effects in many ways.  First, a
contaminant might affect an organism after exposure for a short period of time (acute) or after
exposure over an extended period of time (chronic). Second, the effect of a contaminant
could be lethal (killing the organism) or sublethal (causing adverse effects other than death,
such as reduced growth, behavioral changes, etc.).  Sublethal effects can reduce an organism's
lifespan or reproductive success. For example, if a contaminant reduces the reaction speed of
a prey species, the prey can become more susceptible to predation. Third, a contaminant
might act directly or indirectly on an organism.  Direct effects include lethal or sublethal
effects of the chemical on the organism. Indirect effects occur when the contaminant
damages the food, habitat, predator-prey relationships, or competition of the organism in its
community.

       Mechanisms of ecotoxicity and exposure pathways have already been considered
during problem formulation and identification of the assessment endpoints. However, toxicity
issues are revisited when selecting  appropriate measurement endpoints to ensure that they
measure  the assessment endpoint's toxic response of concern.
4.2    STUDY DESIGN

       In Section 4.1, one or more lines of evidence that could be used to answer questions
or to test hypotheses concerning the assessment endpoint(s) were identified.  This section
provides recommendations on how to design a field study for:  bioaccumulation and field ^
tissue residue studies (Section 4.2.1); population/community evaluations (Section 4.2.2); and
toxicity testing (Section 4.2.3).  A thorough understanding  of the strengths and limitations of
these types of field studies is necessary to properly design  any investigation.

       Typically, no one line of evidence can stand on its own.  Analytic chemistry on co-
located samples and other lines of evidence are needed to support a conclusion.  When
population/community evaluations are coupled with toxicity testing and media chemistry, the
procedure often is referred to as a triad approach (Chapman et al., 1992; Long and Chapman,
1985).  This method has proven effective in defining the area affected by contaminants in
sediments of several large bays and estuaries.

       The development of exposure-response relationships is critical for evaluating risk
'management options; thus, for all three types  of studies, sampling is applied to a
contamination gradient when possible as well as compared to reference data.  Reference data
are baseline values or characteristics  that should represent the site in the absence of
contaminants released from the site.  Reference data might be data collected from the site
before  contamination occurred or new data collected from a reference site.  The reference site
can be the least impacted (or unimpacted) area of the Superfund site or a. nearby site that is

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 ecologically similar, but not affected by the site's contaminants.  For additional information
 on selecting and using reference information in Superfund ecological risk assessments, see
 ECO Update Volume 2, Number 1 (U.S. EPA, 1994e).

       The following subsections present a starting point for selecting an appropriate study
 design for the different types of biological sampling that might apply to the site investigation.

 4.2.1  Bioaccumulation and Field Tissue Residue Studies

       Bioaccumulation and field tissue residue studies typically are conducted at sites where
 contaminants are likely to accumulate in food chains;  The studies help to evaluate
 contaminant exposure levels associated with measures of effect for assessment endpoint
 species.

       The degree to which a contaminant is transferred through a food chain can be
 evaluated in several ways.  The most common type of study reported in the literature is a
 contaminant bioaccumulation (uptake) study. As indicated in Section 2.2.1, the most
 conservative BAF values identified in the literature generally are used to estimate
 bioaccumulation in Step 2 of the screening-level risk assessment.  Where the potential for
 overestimating bioaccumulation by using conservative literature values to represent the site is
 substantial, additional evaluation of the literature for values more likely to apply to the site or
 a site-specific tissue residue study might be  advisable.

       A tissue  residue study generally is conducted on  organisms that are in the exposure
pathway (i.e., food chain) associated with the assessment endpoint. Data seldom are available
to link tissue residue levels in the sampled organisms  to adverse effects in those organisms.
Literature toxiciry studies usually associate effects  with an administered dose (or data that can
be converted to  an administered dose), not a tissue residue level.  Thus,  the purpose of a field
tissue residue study usually is to measure contaminant concentrations in foods consumed by
the species associated with the assessment endpoint.  This measurement minimizes the
uncertainty associated with estimating a dose (or intake) to that species, particularly in
situations in which several media and trophic levels are  in the  exposure pathway.

       The concentration of a contaminant in the primary  prey/food also should be linked to
an exposure concentration from a contaminated medium (e.g.,  soil, sediment, water), because
it is the medium, not the food chain, that will be remediated.  Thus, contaminant
concentrations must be measured in environmental media at the same locations at which the
organisms  are collected along contaminant gradients and at reference locations. Co-located
samples of the contaminated medium and organisms are needed to establish a correlation
between  the tissue residue levels and contamination levels in the medium under evaluation;
these studies are most effective if conducted over a gradient of contaminant concentrations.
In addition, tissue residues from sessile organisms  (e.g., rooted plants, clams) are easier to
attribute  to specific contaminated areas than are tissue residues from mobile organisms (e.g..
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 large fish).  Example 4-3 illustrates these concepts using the DDT site example in
 Appendix A.
                                     EXAMPLE 4-3
                          Tissue Residue Studies-DDT Site

          In the DDT site example, a forage fish (e.g., creek chub) will be collected at
   several locations with known DDT concentrations in sediments.  The forage fish will be
   analyzed for body burdens of DDT, and the relationship between the DDT levels in the
   sediments and the levels in the forage fish will be established. The forage fish DDT
   concentrations can be used to evaluate the DDT threat to piscivorous birds feeding on
   the forage fish at each location.  Using the DDT concentrations measured in fish that
   correspond to a LOAEL and NOAEL for adverse effects in birds and the relationship
   between  the DDT levels in  the sediments and in the forage fish, the corresponding
   sediment contamination levels can be estimated. Those sediment DDT concentrations
   can then  be used  to estimate a cleanup level that would reduce threats of eggshell
   thinning  to piscivorous birds.
       Although it might seem obvious, it is important to confirm that the organisms
examined for tissue residue levels are in the exposure pathways of concern established by the
conceptual model. Food items targeted for collection should be those that are likely to
constitute a large portion of the diet of the species of concern (e.g., new growth on maple
trees, rather than cattails, as a food source for deer) and/or represent pathways of maximum
exposure.  If not, erroneous conclusions or study delays and added costs can result.  Because
specific organisms often can only be captured in one season, the timing of the study can be
critical, and failure to plan accordingly can result in serious site management difficulties.

       There are numerous factors that must be considered when selecting a species in which
to measure contaminant residue levels. Several investigators have discussed the "ideal"
characteristics of the species to be collected and analyzed. The recommendations of Phillips
(1977, 1978) include that the species selected should be:

       (I)     Able to accumulate the chemical of concern without being adversely affected
              by the levels encountered at the site;

       (2)     Sedentary (small home range) in order to be representative of the area of
              collection;

       (3)     Abundant in the study area; and
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       (4)    Of reasonable size to give adequate tissue for analysis (e.g., 10 grams for
              organic analysis and 0.5 gram for metal analysis for many laboratories (Roy F.
              Weston, Inc., 1994)).

Additional considerations for some situations would be that the species is:

       (5)    Sufficiently long-lived to allow for sampling more than one age class; and

       (6)    Easy to sample and hardy enough to survive in the laboratory (allowing for the
              organisms to eliminate contaminants from their gastrointestinal tract prior to
              analysis, if desired, and allowing for laboratory studies on the uptake of the
              contaminant).

       It is usually not possible or necessary to find an organism that fulfills all of the above
requirements. The selection of an organism for tissue analysis should balance these
characteristics with the hypotheses being tested, knowledge of the contaminants' fate and
transport, and the practicality of using the particular species.  In the following sections,
several of the factors mentioned above are described in greater detail.

       Ability to accumulate the contaminant.  The objectives of a tissue residue study
are (1) to measure bioavailability directly; (2) to provide site-specific estimates of exposure to
higher-trophic-level organisms; and (3) to relate tissue residue levels to concentrations in
environmental media (e.g., in soil, sediment, or water). Sometimes these studies also can be
used to link tissue residue levels with observed effects in the organisms sampled. However,
in a "pure" accumulation  study, the species selected for collection and tissue analysis should
be ones that can accumulate a contaminant(s)  without being adversely affected by the levels
encountered in the environment.  While it is difficult  to evaluate whether or not a population
in the  field is affected by accumulation  of a contaminant, it is important,to try. Exposure that
results in  adverse responses might alter the animal's feeding rates or efficiency, diet, degree
of activity, or metabolic rate, and  thereby influence the animal's daily intake or accumulation
of the  contaminant and the estimated BAF.  For example, if the rate of bioaccumulation of a
contaminant in an organism decreases with increasing environmental concentrations (e.g., its
toxic effects reduce food  consumption rates), using a  BAF determined at low environmental
concentrations to estimate bioaccumulation at  high environmental concentrations would
overestimate  risk.  Conversely, if bioaccumulation increased with increasing environmental
concentrations (e.g., its toxic effects impair the organisms' ability to excrete the contaminant),
using a BAF determined at low environmental concentrations would underestimate risks at
higher environmental concentrations.

       Consideration of the physiology  and biochemistry of the species selected for residue
analysis also is important.  Some species can  metabolize certain organic contaminant(s) (e.g.,
fish can metabolize PAHs).  If several different types of prey are consumed by a species of
concern, it would be more appropriate to analyze prey species that do not metabolize the
contaminant.

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        Home range.  When selecting species for residue analyses, one should be confident
 that the contaminant levels found in the organism depend on the contaminant levels in the
 environmental media under evaluation. Otherwise,  valid conclusions cannot be drawn about
 ecological risks posed by contaminants at the site.  The home range, particularly the foraging
 areas within the home range, and movement patterns of a species are important in making this
 determination. Organisms do not utilize the environment uniformly.  For species that have
 large home ranges or are migratory, it can be difficult to evaluate potential exposure to
 contaminants at the site.  Attribution of contaminant levels in an organism to contaminant
 levels in the surrounding environment is easiest for animals  with small home and foraging
 ranges and limited movement patterns.  Examples of organisms with small home ranges
 include young-of-the-year fish, burrowing Crustacea (such as fiddler crabs or some crayfish),
 and small mammals.

       Species also should be selected for residue analysis to maximize the overlap between
 the area of contamination and the species' home range or feeding range. This provides a
 conservative evaluation of potential exposure levels. The possibility that a species' preferred
 foraging areas within a home range overlap the areas of maximum contamination also should
.be considered.

       Population size. A species selected for tissue residue analysis should be sufficiently
 abundant at the site that adequate numbers (and sizes) of individuals can be collected to
 support the tissue mass requirements for chemical analysis and to achieve the sample size
 needed for statistical comparisons.  The organisms actually collected should be not only of
 the same species, but also of similar age or size to reduce data variability when BAFs are
 being evaluated.  The practicality of using a particular species is evaluated in Step 5.

       Size/composites.  When selecting species in which to measure tissue residue levels,
 it  is best to have individual animals large enough for chemical analysis, without having to
 pool (combine) individuals prior to chemical analysis.  However, composite samples will be
 needed if individuals  from the species selected  cannot yield  sufficient tissue for the required
 analytical methods. Linking contaminant levels in organisms to concentrations in
 environmental media  is easier if composites are made up of  members of the same species,
 sex, size, and age, and therefore exhibit similar accumulation characteristics.  When deciding
 whether or not to pool samples, it is important  to consider what impact the loss of
 information on variability of contaminant levels along these  dimensions will  have on data
 interpretation.  The size, age, and sex of the species collected should be representative of the
 range of prey consumed by the species of concern.

       Summary. Although it can be difficult to meet all of the suggested criteria for
 selecting a species for tissue residue studies, an attempt should be made to meet as many
 criteria as possible. No formula is available for ranking the  factors in order of importance
 within a particular site investigation because the ranking depends on the study objectives.
 However, a key criterion is that the organism be sedentary or have a limited home range.  It
 is  difficult  to connect site contamination to organisms that migrate over great distances  or that

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have extremely large home ranges.  Further information on factors that can influence
bioaccumulation is available from the literature (e.g., Phillips. 1977, 1978; U.S. EPA, 1995d).

4.2.2  Population/Community Evaluations

       Population/community evaluations, or biological field surveys, are potentially useful
for both contaminants that are toxic to organisms through direct exposure to the contaminated
medium and contaminants that bioaccumulate in food chains.  In either case, careful
consideration must be given to the mechanism of contaminant effects.  Since
population/community evaluations are "impact" evaluations, they typically are not predictive.
The release of the contaminant must already have occurred and exerted an effect in order for
the population/community evaluation to be an effective tool for a risk assessment.

       Population and community surveys evaluate the current status of an ecosystem, often
using several measures of population or community structure (e.g., standing biomass, species
richness) or function (e.g., feeding group analysis).  The most commonly used measures
include number of species and abundance of organisms in an ecosystem, although some
species are difficult to evaluate.  It is difficult to detect changes in top predator populations
affected by bioaccumulation of substances in their food chain due to the  mobility of top
predators.  Some species, most notably insects, can develop a tolerance to contaminants
(particularly pesticides); in these cases, a population/community survey would be ineffective
for evaluating existing impacts.  While population/community evaluations can be useful, the
risk assessors should consider the level of effort required as well as the difficulty in
accounting for natural variability.

       A variety of population/community evaluations have been used at Superfund sites.
Benthic macroinvertebrate surveys are the most commonly conducted population/community
evaluations. There are methods  manuals (e.g., U.S. EPA 1989c,  1990a) and publications that
describe the technical procedures for conducting these studies.  In certain instances, fish
community evaluations have proven useful at Superfund sites.  However, these investigations
typically are more labor-intensive and costly than a comparable macroinvertebrate study. In
addition, fish generally are not sensitive measures of the effects of sediment contamination,
because they usually are  more mobile than benthic macroinvenebrates. Terrestrial plant
community evaluations have been used to a limited extent at Superfund sites.  For those
surveys, it is important to include information about historical land use and physical habitat
disruption in the uncertainty analysis.

       Additional information on designing field studies and on field study methods can be
found in ECO Update Volume 2. Number 3 (U.S. EPA, 1994d).

       Although population- and community-level  studies can be valuable, several factors can
confound the interpretation of the results. For example, many fish and small mammal
populations  normally cycle in relation to population density, food availability, and other
factors.  Vole populations have been known to reach thousands of individuals per acre and

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then to decline to as low as tens of individuals per acre the following years without an
identifiable external strcssor (Geller, 1979). It is important that the "noise of the system" be
evaluated so that the impacts attributed to chemical contamination at the site are not actually
the result of different, "natural" factors.  Populations located relatively close to each other can
be affected independently:  one might undergo a crash, while another is peaking.  Physical
characteristics of a site can isolate populations so that one population level is not a good
indicator of another; for example, a paved highway can be as effective a barrier as a river,
and populations on either side can fluctuate independently.  Failure to evaluate such issues
can result in erroneous conclusions.  The level of effort required to resolve some of these
issues can make population/community evaluations impractical in some circumstances.

4.2.3  Toxicity Testing

       The bioavailability  and toxicity of site contaminants can be tested directly with
toxicity tests. As with other methods, it is critical that the media tested are in exposure
pathways relevant to the assessment endpoint.  If the  site  conceptual model  involves exposure
of benthic invertebrates to contaminated sediments, then a solid-phase toxicity test using
contaminated sediments (as opposed to a water-column exposure test) and an infaunal species
would be appropriate.  As  indicated earlier, the species tested and the responses measured
must be  compatible with the mechanism of toxicity. Some common site contaminants are not
toxic to most organisms at the same environmental concentrations that threaten top predators
because the contaminant biomagnifies  in food chains (e.g., PCBs); toxicity tests using
contaminated media from the site would not be appropriate for evaluating this type of
ecological  threat.

       There are numerous U.S. EPA methods manuals and ASTM guides and procedures for
conducting toxicity tests (see references in  the Bibliography). While documented methods
exist for a wide variety of toxicity tests, particularly laboratory tests, the risk assessor must
evaluate what a particular toxicity test measures and, just  as importantly, what it does not
measure.  Questions to consider when  selecting an  appropriate toxicity test include:

       (1)     What is the  mechanism  of toxicity of the contaminant(s)?

       (2)     What contaminated media are being evaluated (water, soil, sediment)?

       (3)     What toxicity test species are available to test the media being evaluated?

       (4)     What life stage of the species should be tested?

       (5)     What should the duration of the toxicity test be?

       (6)     Should the test organisms be fed during the test?

       (7)     What endpoints should be measured?

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       There are a limited number of toxicity tests that are readily available for testing
environmental media. Many of the aquatic toxicity tests were developed for the regulation of
aqueous discharges to surface waters.  These tests are useful, but one must consider the
original purpose of the test

       New toxicity  tests are being developed continually  and can  be of value in designing a
Superfund site ecological risk assessment.  However, when non-standard tests are used,
complete documentation of the specific test procedures is necessary to support use of the data.

       In  situ toxicity tests involve placing organisms in locations  that might be affected by
site contaminants and in reference locations.  Non-native species should not be used, because
of the risk of their release into the environment in which they could adversely affect (e.g.,
prey on or outcompete) resident species. In situ tests might provide more realistic evidence
of existing adverse effects than laboratory toxicity tests; however, the investigator has little
control over many environmental parameters and the experimental  organisms can be lost to
adverse weather or other events (e.g., human interference) at the site or reference location.

       For additional information on  using toxicity tests in ecological risk assessments, see
ECO Update Volume 2, Numbers 1 and 2 (U.S. EPA, 1994b,c).

4.3    DATA QUALITY OBJECTIVES AND STATISTICAL CONSIDERATIONS

       The SAP indicates the number and  location of samples to be taken, the number of
replicates  for each sampling location, and the method for determining sampling locations. In
specifying those parameters, the investigator  needs to consider, among other things,  the DQOs
and statistical methods that will be used to analyze the data.

4.3.1  Data Quality Objectives

      The DQO process represents a series  of planning steps that can be employed
throughout the development of the WP and SAP to ensure that the type, quantity, and quality
of environmental data to be collected during  the ecological investigation are adequate to
support  the intended  application. Problem formulation in Steps 3 and 4 is essentially the
DQO process. By employing problem formulation and the DQO process, the investigator is
able to define data requirements and error levels that are acceptable for the  investigation prior
to the collection of data.  This approach helps ensure that  results are appropriate and
defensible for decision making. The  specific goals of the  general DQO process are  to:

       •     Clarify the study objective and define the most appropriate types  of data to
             collect;

       •     Determine the most appropriate field conditions under which to collect the data;
             and
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       •      Specify acceptable levels of decision errors that will be used as the basis for
              establishing the quantity and quality of data needed to support risk management
              decisions.

 As the discussion of Steps 3 and 4 indicates, those goals are subsumed in the problem
 formulation phase of an ecological risk assessment. Several U.S. EPA publications provide
 detailed descriptions of the DQO process (U.S. EPA,  1993c,d,f, 1994f). Because many of the
 steps of the DQO process are already covered during problem formulation, the DQO process
 should be reviewed by the investigator and applied as needed.

 4.3.2  Statistical Considerations

       Sampling locations can be selected "randomly" to characterize an area or non-
 randomly, as along a contaminant concentration gradient. The way in which sampling
 locations are selected determines which statistical tests, if any, are appropriate for evaluating
 test hypotheses.

       If a toxicity test is to be used to identify contaminant concentrations in the
 environment associated with a threshold for adverse effects, the statistical power of the test is
 important.  The threshold for effects is assumed to be between the NOAEL and LOAEL of a
 toxicity test (see Section 7.3.1).  For toxicity tests that use a small number of test and control
 organisms or for which the toxic response is highly variable, the increase in response rate of
 the test animals compared with controls often must be relatively high (e.g., 30 to 50 percent
 increase) for the response to be considered a LOAEL  (i.e., statistically increased level of an
 adverse response compared with control levels). If a NOAEL-to-LOAEL range that might
 represent a 20 to 50 percent increase'in adverse effect is unacceptable (e.g., a population is
 unlikely to sustain itself with an additional 40 percent mortality), then the power of the study
 design must be increased, usually by increasing sample size, but sometimes by  taking full
 advantage of all available information to improve the power of the design (e.g., stratified
 sampling, special tests for trends, etc.).  A limitation on the use of toxicity values from the
 literature is that often, the investigator does  not discuss the statistical power of the study
design, and hence does not indicate the minimum statistically detectable effect level.
Appendix D describes additional statistical considerations, including a description of Type I
and Type II error, statistical power, statistical models, and power efficiency.

       In evaluating the results of statistical analyses, one should remember that a statistically
significant difference relative to a control or reference population does not necessarily imply a
biologically important or ecologically significant difference (see Example 4-1).
4.4    CONTENTS OF WORK PLAN AND SAMPLING AND ANALYSIS PLAN

       The WP and SAP for the ecological investigation should be developed as pan of the
initial RI sampling  event if possible.  If not, the WP and SAP can be developed as an

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 additional phase of the site investigation. In either case, the format of the WP and SAP
 should be similar to that described by U.S. EPA (1988a, 1989b).  Accordingly, those
 documents should be consulted when developing the ecological investigation WP and SAP.

       The WP and SAP are typically written as separate documents.  In that case, the WP
 can be submitted for the risk manager's  review so that any concerns with the approach can be
 resolved prior to the development of the SAP.  For some smaller sites, it might be more
 practical to combine the two documents, in which  case, the investigators should discuss the
 overall objectives and approach with the risk manager to ensure that all parties agree.

       The WP and SAP are briefly described in Sections 4.4.1 and 4.4.2, respectively. A
 plan for testing the SAP before the site WP and SAP are signed and the investigation begins
 is described in Section 4.4.3.

 4.4.1  Work Plan

       The purpose of the WP is to document the  decisions and evaluations made during
 problem  formulation and to identify additional investigative tasks needed to complete the
 evaluation of risks to ecological resources. As presented in U.S. EPA (1988a), the WP
 generally includes the following:                             '

       •       A general overview and background of the site including the site's physical
              setting, ecology, and previous uses;

       •       A summary and analysis of previous site investigations and conclusions;

       •       A site conceptual model, including an  identification of the potential exposure
              pathways selected for analysis, the assessment endpoints and questions or
              testable hypotheses, and the measurement endpoints selected for analysis;

       •       The identification of additional site investigations needed to conduct the
              ecological risk assessment; and

       •       A description of assumptions used and the major sources of uncertainty in the
              site  conceptual model and existing information.

The general  scope of the additional sampling activities also is presented in the WP.  A
detailed description of the additional sampling activities is presented in the SAP along with an
anticipated schedule of the site activities.

4.4.2  Sampling and Analysis Plan

       The SAP typically  consists of two components:  a field sampling plan (FSP) and a
quality assurance project plan (QAPP).  The FSP provides guidance for all field work by

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 providing a detailed description of the sampling and data-gathering procedures to be used for
 the project. The QAPP provides a description of the steps required to achieve the objectives
 dictated by the intended use of the data.

        Field sampling plan. The FSP provides a detailed description of the samples
 needed to meet the objectives and scope of the investigation outlined in the WP. The FSP for
 the ecological assessment should be detailed enough that a sampling team unfamiliar with the
 site would be able to gather all the samples and/or required field data based on the guidelines
 presented in the document.  The FSP for the ecological investigation should include a
 description of the following elements:

       •     Sampling type and objectives;
       •     Sampling location, timing, and frequency;
       •     Sample designation;
       •     Sampling equipment and procedures; and
       •     Sample handling and analysis.
A detailed description of those elements for chemical analyses is provided in Appendix B of
U.S. EPA (1988a).  Similar specifications should be developed for the biological sampling.

       Quality assurance project plan.  The objective of the QAPP is to provide a
description of the policy, organization, functional activities, and quality control protocols
necessary for achieving the study objectives. Highlight 4-3 presents the elements typically
contained in a QAPP.
       U.S. EPA has prepared guidance on
the contents of a QAPP (U.S. EPA, 1987a,
1988a, 1989a).  Formal quality assurance
and quality control (QA/QC) procedures
exist for some types of ecological
assessments, for example, for laboratory
toxicity tests on aquatic species.  For
standardized laboratory tests, there are
formal QA/QC procedures that specify  (1)
sampling and handling of hazardous wastes;
(2) sources and culturing of test organisms;
(3) use of reference toxicants, controls, and
exposure replicates; (4) instrument
calibration; (5) record keeping; and (6) data
evaluation.  For other types of ecological
assessments, however,  QA/QC procedures
are less well defined (e.g., for  biosurveys of
vegetation, terrestrial vertebrates).  BTAG
         HIGHLIGHT 4-3
       Elements of a QAPP

 (1)  Project description
 (2)  Designation of QA/QC
       responsibilities
 (3)  Statistical tests and data quality
       objectives
 (4)  Sample collection and chain of
       custody
 (5)  Sample analysis
 (6)  System controls and preventive
       maintenance
 (7)  Record keeping
 (8)  Audits
 (9)  Corrective actions
(10)  Quality control reports
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members can provide input on appropriate QA/QC procedures based on their experience with
Superfund sites.

4.4.3  Field Verification of Sampling Plan and Contingency Plans

       For biological sampling, uncontrolled variables can influence the availability of species
to be sampled, the efficiency of different types of sampling techniques, and the level of effort
required to achieve the sample sizes specified in the SAP.  As a consequence,  the risk
assessor should develop a plan to test the sampling design before the WP and SAP are signed
and the •site investigation begins.  Otherwise, field sampling during the site investigation could
fail to meet the DQOs specified in the SAP, and the study could fail to meet its objectives.
Step 5 provides a description of the field verification of the sampling design.

       To the extent that potential field problems can be anticipated, contingency plans also
should be specified in the SAP. An example of a contingency plan is provided in Steps 5 and
6 (Examples 5-2 and 6-1).
4.5    SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)

       The completion of the ecological risk assessment WP and SAP should coincide with
an SMDP. Within this SMDP, the ecological risk assessor and the ecological risk manager
agree on:  (1) selection of measurement endpoints; (2) selection of the site investigation
methods; and (3) selection of data reduction and interpretation techniques. The WP or SAP
also should specify how inferences will  be drawn from the measurement to the assessment
endpoints.
4.6    SUMMARY

       At the conclusion of Step 4, there will be an agreement on the contents of the WP and
SAP. As noted earlier, these plans can be parts of a larger WP and SAP that are developed
to meet other remedial investigation needs, or they can be separate documents.  When
possible, any field sampling efforts for the ecological risk assessment should overlap with
other site data collection efforts to reduce sampling costs and to prevent redundant sampling.

       The WP and/or the SAP should specify the methods by which the collected data will
be analyzed.  The plan(s) should include all food-chain-exposure-model parameters,  data
reduction techniques, data  interpretation methods, and statistical analyses that will be used.
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          STEP 5:  FIELD VERIFICATION OF SAMPLING DESIGN
                                      OVERVIEW
                                            4
          Before the WP and SAP are signed, it is important to verify that the field
   sampling plan they specify is appropriate and implementable at the site. If this has not
   already been done, it should be done now.  During field verification of the sampling
   design, the testable hypotheses, exposure pathway models, and measurement endpoints
   are evaluated for their appropriateness and implementability. The  assessment
   endpoint(s), however, should not be under evaluation in this step; the appropriateness
   of the assessment endpoint should have been resolved in Step 3. If an assessment
   endpoint is changed at this step, the risk assessor must return to Step 3, because the
   enure process leading to the actual site investigation in Step 6 assumes the selection of
   appropriate assessment endpoints.
5.1    PURPOSE

       The primary purpose of field verification of the sampling plan is to ensure that the
samples specified by the SAP actually can be collected.  A species that will be associated
with a measurement endpoint and/or exposure point concentration should have been observed
at the preliminary site characterization or noted during previous site visits. During this step,
previously obtained information should be verified and the feasibility of sampling will need to
be checked by a site visit.  Preliminary sampling will determine if the targeted species is
present and—equally important—collectable in sufficient numbers or total biomass to meet
data quality  objectives.  This preliminary field assessment also allows for final confirmation
of the habitats that exist on or near the site.  Habitat maps are verified a final time, and
interpretations of aerial  photographs can be checked.

       Final decisions on reference areas also should be made in this step.  The reference
areas should be chosen  to be as similar as possible to the site in all aspects except
contamination.  Parameters to be evaluated for similarity include, but are not limited to:
slope, habitat, species potentially present, soil and sediment characteristics, and for surface
waters, flow rates, substrate type, water depth, temperature, turbidity, oxygen levels, water
hardness, pH, and other standard water quality parameters. If several on-site habitats or
habitat variables are being investigated, then several reference areas could be required.
Reference areas should  be as free of site-related contaminants above background levels as
practical.
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 5.2   DETERMINING SAMPLING FEASIBILITY

       When sampling biota, it is difficult to predict what level of effort will be necessary to
 obtain an adequate number of individuals of the required size.  Some preliminary field
 measurements often can help determine adequate sampling efforts to attain the sample sizes
 specified in the SAP for statistical analyses. The WP and SAP should be signed and the site
 investigation should be implemented immediately after verification of the sampling design to
 limit effects of uncontrolled field variables.  For example, evaluation of current small
 mammal population density might indicate to the investigator that 400 trap-nights instead of
 50 are necessary to collect the required number of small mammals.  If there is a time lag
 between the field sampling verification and the actual site investigation, it could be necessary
 to reverify the field sampling to determine if conditions have changed.

       Sampling methods for abiotic media also should be tested. There is a wide variety of
 sampling devices and methods, and it is important to use the most appropriate, as the
 following examples illustrate:

       •     When sampling a stream's surface water, if the stream is only three inches
             deep, collecting the water directly into 32-ounce bottles would not be practical.

       •     Sampling the substrate in a stream might be desirable, but if the substrate is
             bedrock, it might not be feasible or the intent of the sampling design.

       An  exposure-response relationship between contamination and biological effects is a
 key component of establishing causality during the analysis phase of the  baseline risk
 assessment (Step 6).  If extent-of-contamination sampling is conducted in phases, abiotic
 exposure media and biotic samples must be collected simultaneously because the interactions
 (both temporal and spatial) between the matrix to be remediated and the biota are crucial to
 the development of a field exposure-response relationship. Failure to collect one sample
 properly or to coordinate samples temporally can significantly impact the  interpretation of the
 data.

       Sampling locations need to be checked to make sure that they are appropriately
 described and placed within the context of the sampling  plan.  Directions for a sediment
 sample "to be taken 5 feet from the north side of stream A," could cause confusion if the
 stream is only 4 feet wide, or if the sampler doesn't know if the sample should be taken in
 the stream, or 5 feet  away from the edge of the stream.  All samples should be checked
 against the intended use of the data to be obtained.

       All  pathways for the migration of contaminants off site should be evaluated, such as
windblown dust, surface water runoff, and erosion.  Along these pathways, a gradient of
decreasing  contamination with increasing distance from the site might exist.  Site-specific
ecological evaluations and risk assessments can be more useful to risk managers if gradients
of contamination can be located and evaluated.

                                           5-2

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       Contaminant migration pathways might have changed, either due to natural causes
 (e.g., storms) or site remediation activities (e.g., erosion channels might have been filled or
 dug up to prevent further migration of contaminants).  Channels of small or large streams,
 brooks, or rivers might have moved; sites might have been flooded. All of the assumptions
 of the migration and exposure pathways need to be verified prior to the full site investigation.
 If a contaminant gradient is necessary for the sampling plan, it is important to verify that the
 gradient exists and  that the range of contaminant concentrations is appropriate. A gradient of
 contamination that causes no  impacts at the highest concentration measured has as little value
 as a gradient that kills everything at the lowest concentration measured; in either case, the
 gradient  would not  provide useful exposure-response information.  A gradient verification
 requires  chemical sampling, but field screening-level analyses might be effective.

       These and other problems associated with the practical implementation of sampling
 should be resolved prior to finalizing the SAP to the extent practicable.  Assessing the
 feasibility of the sampling plan before the site investigation begins saves costs in the long
 term because it minimizes the chances of failing to meet DQOs during the site investigation.

       Examples 5-1 and 5-2 describe the field verification of the sampling plan for the
 hypothetical copper and DDT sites illustrated in Appendix A. Note that the scope of the field
 verification differs for the copper and DDT sites.  For the  DDT site, a modification to  the
 study design was necessary.  For both sites, the issues were resolved and a sign-off was
 obtained at the  SMDP for this step.

       Any change  in measurement endpoints will require that exposure pathways  to the new
 measurement endpoint be checked. The new measurement endpoint must fit into the
 established conceptual model. Changes to measurement endpoints might require revision of
 the conceptual model  and agreement to the changes at the  SMDP. It is highly desirable that
 the agreed-upon conceptual model should be modified and approved by the same basic group
 of individuals who developed it.                             .
5.3    SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)

       The SMDP for the field verification of the sampling design is the signing of the
finalized WP and SAP.  Any changes to the investigation proposed in Step 4 must be made
with agreement from the risk manager and risk assessment team.  The risk manager must
understand what changes have been made and why, and must ensure that the risk management
decisions can be made from the information that the new study design can provide. The risk
assessors must be involved to ensure that the assessment endpoints and testable hypotheses
are still being addressed.

       In the worst cases, changes in the measurement endpoints could be necessary,  with
corresponding changes to the risk hypotheses and sampling design.  Any new measurement
endpoints must  be evaluated according to their utility for inferring changes in the assessment

                                          5-3

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                                     EXAMPLE 5-1
                 Field Verification of Sampling Design-Copper Site

          Copper was released from a seep area of a landfill adjacent to a small pond; the release
   and resulting elevated copper levels in the pond are of concern. The problem formulation and
   conceptual model stated that the assessment endpoint was the maintenance of a typical pond
   community for the area, including the benthic invertebrates and fish. Toxicity testing was
   selected to evaluate the potential toxicity of copper to aquatic organisms. Three toxicity tests
   were selected: a 10-day solid-phase sediment toxicity test (with the amphipod Hyalella azteca),
   and two water column tests (i.e., the 7-day growth test with the green alga Selenastrum
   capricornutum and the fathead minnow, Pimephales promelas, 1-day larval growth test).  The
   study design specified that sediment and water for the toxicity tests would be collected at the
   leachate seeps known to be at the pond edge, and at three additional equidistant locations
   transecting the pond (including  the point of maximum pond depth). The pond contains water
   year-round; however, the seep flow depends on rainfall.  Therefore, it is only necessary to verify
   that the leachate seep is active at the time of sampling.
endpoints and their compatibility with the site conceptual model (from Steps 3 and 4).  Loss
of the relationship between measurement endpoints and the assessment endpoints, the risk
questions or testable hypothesis, and the site conceptual model will result in a failure to meet
study objectives.

       Despite one's best efforts to conduct a sound site assessment, unexpected
circumstances might still make it necessary for the sampling plan  to be changed in the field.
Any changes should be agreed to and  documented by the lead risk assessor in consultation
with the risk manager.

       Once the finalized WP and SAP  are approved and signed,  Step 6 should begin.
5.4    SUMMARY

       In summary, field verification of the sampling plan is very important to ensuring that
the DQOs of the site investigation can be met.  This step verifies that the selected assessment
endpoints, testable hypotheses, exposure pathway model, measurement endpoints, and study
design from Steps 3 and 4 are appropriate and implementable at the site. By verifying the
field sampling plan prior to conducting the full site investigation, well-considered alterations
can be made to  the study design and/or implementation if necessary.  These changes will
ensure that the ecological risk assessment meets the study objectives.
                                            5-4

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       If changing conditions force changes to the sampling plan in the field (e.g., selection
of a different reference site), the changes should be agreed to and documented by the lead
risk assessor in consultation with the risk manager.
                                       EXAMPLE 5-2
                    Field Verification of Sampling Design-DDT Site

          For the stream DDT site, the assessment endpoint was protection of piscivorous birds
   from adverse reproductive effects.  The conceptual model included the exposure pathway of
   sediment to forage fish to the kingfisher. The measurement endpoint selected was tissue residue
  ' levels in creek chub (Semotilus atromaculatus), which could be associated with contaminant
   levels in sediments.  Existing information on the stream contamination indicates that a gradient
   of contamination exists and that five specific sampling locations should be sufficient to
   characterize the  gradient to the  point where concentrations are unlikely to have  adverse effects.
   The study design specified that 10 creek chub of the same size and sex be collected at each
   location.  Each chub should be  approximately 20 grams, so that minimum sample mass
   requirements could be met without using composite samples for analysis.  In addition, QA/QC
   protocol requires that 10 more fish be collected at one of the  locations.

          In this example, a site assessment was necessary to verify that a sufficient number of
   creek chub of the specified size would be present to meet the sampling  requirements.  Stream
   conditions were  evaluated to determine what fish sampling technique would work at the targeted
   locations. A field assessment was conducted, and several fish collection techniques were used
   to determine which was the most effective for the site. Collected creek chub and other fish
   were examined to determine the size range available and whether the sex of the individuals
   could be determined.                                          .        .    .  .

          The site  assessment indicated that the creek chub might not be present in sufficient
   numbers to provide the necessary  biomass for chemical analyses.  Based upon those findings, a
   contingency plan was agreed to, which stated that both the creek chub and the longnosed dace
   (Rhinichthys cataractae) would be collected. If the creek chub were collected at all locations in
   sufficient numbers, then those samples would be analyzed and the dace would be released.  If
   sufficient creek chub could not  be collected but sufficient longnosed dace could, the longnosed
   dace would  be analyzed and the creek chub released.  If neither species could be collected at all
   locations in  sufficient numbers, then a mix of the two species would be used; however, for any
   given sampling location only one species would be used to make the sample. In addition, at
   one location, which preferably had high DDT levels in the sediment, sufficient numbers (20
   grams) of both species would be collected to allow comparison (and calibration) of the
   accumulation between the two species.
                                             5-5

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         STEP 6:  SITE INVESTIGATION AND ANALYSIS PHASE
                                     OVERVIEW

          Information collected during the site investigation is used to characterize
   exposures and ecological effects. The site investigation  includes all of the field
   sampling and surveys that are conducted as part of the ecological risk assessment.  The
   site investigation and analysis of exposure and effects should be straightforward,
   following the WP and SAP developed in Step 4 and tested in Step 5.

          Exposure characterization relies heavily on data from the site investigation and
   can involve fate-and-transport modeling. Much of the information for characterizing
   potential ecological effects was gathered from the literature review during problem
   formulation, but the site investigation might provide evidence of existing ecological
   impacts and additional exposure-response information.
6.1    INTRODUCTION

       The site investigation (Section 6.2) and analysis phase (Section 6.3) of the ecological
risk assessment should be straightforward.  In Step 4, all issues related to the study design,
sample collection, DQOs, and procedures for data reduction and interpretation should have
been identified and resolved.  However, as described in Step 5, there are circumstances that
can arise during a site investigation that could require modifications to the original study
design.  If any unforeseen events do require a change to the WP or SAP, all changes must be
agreed upon at the SMDP (Section 6.4).  The results of Step 6 are used to characterize
ecological risks in Step 7.
6.2    SITE INVESTIGATION

       The WP for the site investigation is based on the site conceptual model and should
specify the assessment endpoints, risk questions, and testable hypotheses.  The SAP for the
site investigation should specify the relationship between measurement and assessment
endpoints, the  necessary number, volume, and types of samples to be collected, and the
sampling techniques to be used. The SAP also should specify the data reduction and
interpretation techniques and the DQOs. The feasibility of the sampling design was tested in
Step 5. Therefore, the site investigation should be a direct implementation of the previously
designed study.


                                          6-1

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       During the site investigation, it is important to adhere to the DQOs and to any
requirements for co-located sampling.  Failure to collect one sample properly or to coordinate
samples temporally can significantly affect interpretation of ihe data.  Changing field
conditions (Section 6.2.1) and new information on the nature and extent of contamination
(Section 6.2.2) can require a change in the SAP.

6.2.1  Changing Field Conditions

       In instances where unexpected conditions arise in the field that make the collection of
specified samples impractical or not ideal, the ecological risk assessor should reevaluate the
feasibility of the sampling design as described in Step 5.  Field efforts should not necessarily
be halted, but decisions to change sampling procedures or design must be agreed to by the
risk manager and lead risk assessor or project-delegated equivalents.

       Field modifications to study designs are not uncommon during field investigations.
When the WP and SAP provide a precise conceptual model and study design- with specified
data analyses, informed modifications to the SAP can be made to comply with the objectives
of the study.  As indicated in Step 4, contingency plans can be included in the original SAP
in anticipation of situations that might arise during the site investigation (see  Example 6-1).
Any modifications, and the reasons for the modifications, must be documented in the baseline
risk assessment.
                                    EXAMPLE 6-1
                    Fish Sampling Contingency Plan-DDT Site

          At the DDT site where creek chub are to be collected for DDT tissue residue analyses,
   a contingency plan for the site investigation was developed. An alternate species, the longnosed
   dace, was specified  with the expectation that, at one or all locations, the creek chub might be
   absent at the time of the site investigation. Such contingency plans are prudent even when the
   verification  of the field  sampling design  described in Step 5 indicates  that the samples are
   obtainable.
6.2.2  Unexpected Nature or Extent of Contamination
                                                        *.
       It is not uncommon for an initial sampling phase of the RI to reveal that
contamination at levels of concern extend beyond areas initially established for characterizing
contamination and ecological effects at the site or that contaminant gradients are much steeper
than anticipated.  If this contingency changes the opportunity for evaluating  biological effects
along a contamination gradient, the ecological risk assessors and risk manager need to
determine whether additional sampling (e.g., further downstream from the site) is needed.
                                           6-2

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 Thus, it is important for the ecological risk assessors to track information on the nature and
 extent of contamination as RI sampling is conducted.

        On occasion, new contaminants are identified during an RI.  In this case, the risk
 assessors and site manager will need to return to Step 1 to screen the new contaminants for
 ecological risk.

        Immediate analysis of the data for each type of sampling and communication between
 the risk assessors and risk managers can help ensure that the site investigation is adequate to
 achieve the study goals and objectives when field modifications are necessary. If a change to
 the WP or SAP is needed, the lead risk assessor and risk manager must agree on all changes
 (the SMDP in Section 6.4).
 6.3   ANALYSIS OF ECOLOGICAL EXPOSURES AND EFFECTS

       The analysis phase of the ecological risk assessment consists of the technical
 evaluation of data on existing and potential exposures (Section 6.3.1) and ecological effects
 (Section 6.3.2) at the site. The analysis is  based on the information collected during Steps 1
'through 5 and often includes additional assumptions or models to interpret the data in the
 context of the site conceptual  model.  As illustrated in Exhibit 6-1, analysis of exposure and
 effects is performed interactively, with the  analysis of one informing the analysis of the other.
 This step follows the data interpretation and analysis methods specified in the WP and SAP,
 and therefore should be a straightforward process.

       In the analysis phase, the site-specific data obtained during the site investigation
 replace many of the assumptions that were made for the screening-level analysis in Steps  1
 and 2.  For the exposure and ecological effects characterizations, the uncertainties associated
 with the field measurements and with assumptions where site-specific data are not available
 must be documented.

 6.3.1  Characterizing Exposures

       Exposure can be expressed as  the co-occurrence  or contact of the stressor with the
 ecological  components, both in time and space (U.S. EPA, 1992a).  Thus, both the stressor
 and the ecosystem must be characterized on similar temporal and spatial scales.  The result of
 the exposure analysis is an exposure profile that quantifies the magnitude and spatial and
 temporal patterns of exposure as they relate to the assessment endpoints and risk questions
 developed  during problem formulation. The exposure profile and a description of associated
 uncertainties and assumptions serve as input to the risk  characterization in Step 7.

       Stressor characterization  involves determining the stressor's distribution and pattern of
 change.  The analytic approach for characterizing ecological exposures should have been
 established in the WP and SAP  on the basis of the site conceptual model.  For chemical

                                           6-3

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           EXHIBIT 6-1
  Analysis Phase (U.S. EPA, 1992a)
                    PROBLEM FORMULATION


                    ANALYSIS


                  v RISK CHARACTERIZATION
                      N
PROBLEM FORMULATION
Characterization of Exposure

•

Stressor x Ecosy

Pattern of Change Abie
\\ //
Y Exposure [/
N Analysis
1

Exposure
Profile
IHLV
Characterization of



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\\
Ecological Effects


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Evaluation
of Relevant
Effects Data
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\ Ecological
Response
Analysis
1



Stressor-Response
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RISK CHARACTERIZATION
               6-4

-------
stressors at Superfund sites, usually a combination of fate-and-transport modeling and
sampling data from the site are used to predict the current and likely future nature and extent
of contamination at a site.
                                                          HIGHLIGHT 6-1
                                                 Uncertainty in Exposure Models

                                                     The accuracy of an exposure model
                                               depends on the accuracy of the input
                                               parameter values and the validity of the
                                               model's structure (i.e., the degree to which it
                                               represents the actual relationships among
                                               parameters at the site). Field measurements
                                               can be used to calibrate model outputs or
                                               intermediate calculations.  Such field
                                               measurements should be specified in the WP
                                               and SAP.  For example, studies of tissue
                                               residue levels often are used to calibrate
                                               exposure and food-chain models.
        When characterizing exposures, the
 ecological context of the site  established
 during problem formulation is analyzed
 further, both to understand potential effects
 of the ecosystem on fate and  transport of
 chemicals in the environment and to
 evaluate site-specific characteristics of
 species or communities of concern. Any
 site-specific information that can be used to
 replace assumptions based on information
 from the literature or from other sites is
 incorporated into the description of the
 ecological components of the site.
 Remaining assumptions  and uncertainties in
 the exposure model  (Highlight 6-1) should
 be documented.

 6.3.2   Characterizing Ecological Effects

       At this point, all  evidence for existing and potential adverse effects on the assessment
 endpoints is analyzed. The information from the literature review on ecological effects is
 integrated with any evidence of existing impacts based on the site investigation (e.g., toxicity
 testing). The methods for analyzing site-specific data should have been specified in the WP
 and SAP, and thus should be  straightforward.  Both exposure-response  information and
 evidence that site contaminants are causing or can cause adverse effects are evaluated.

       Exposure-response analysis.  The exposure-response analysis for a Superfund site
 describes the relationship between the magnitude, frequency, or duration of a contaminant
stressor in an experimental or observational setting and the magnitude of response. In this
phase of the analysis,  measurement endpoints are related  to the assessment endpoints using
the logical structure  provided by the conceptual model. Any extrapolations that are required
to relate measurement to assessment endpoints (e.g., between species, between response
 levels, from laboratory to field) are explained. Finally, an exposure-response relationship is
described to the extent possible (e.g., by a regression equation), including the confidence
limits (quantitative or  qualitative) associated with the relationship.

       Under some circumstances,  site-specific exposure-response information  can be
obtained by evaluating existing ecological impacts along a contamination gradient at  the site.
Statistical techniques to identify or describe the relationship between exposure and response
from the field data should have been specified in the WP and SAP.  The potential for
                                           6-5

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 confounding stressors that might correlate with the contamination gradient should be
 documented (e.g., decreasing water temperature downstream of a site; reduced soil erosion
 further from a site).

       An exposure-response analysis is of particular importance to risk managers who must
 balance human health and ecological concerns against the feasibility and effectiveness of
 remedial options. An exposure-response  function can help a risk manager to specify the
 trade-off between the degree of cleanup and likely benefits of the cleanup and to balance
 ecological and financial costs and benefits of different remedial options, as discussed in
 Step 8.

       When exposure-response data are  not available or cannot be developed, a threshold for
 adverse effects can be developed instead, as in Step 2. For the baseline risk assessment,
 however,  site-specific information should be used instead of conservative assumptions
 whenever possible.

       Evidence of causality.  At Superfund sites, evidence of causality is key to the risk
 assessment. Thus, it is important to evaluate the strength of the causal association between
 site-related contaminants and effects on the measurement and assessment endpoints.
 Demonstrating a correlation between a contaminant gradient and ecological impacts  at a site
 is a key component of establishing causality, but other evidence can be used in the absence of
 such a demonstration. Moreover, an exposure-response correlation at a site is not sufficient to
 demonstrate causality, but requires one or more types of supporting evidence and  analysis of
 potential confounding factors.  Hill's (1965) criteria for evaluating causal associations are
 outlined in the Framework (U.S. EPA, 1992a).
6.4    SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)

       An SMDP during the site investigation and analysis phase is needed only if alterations
to the WP or SAP become necessary. In the worst case, changes in measurement endpoints
could be required, with corresponding changes to the testable hypotheses and sampling
design.  Any new measurement endpoints must be evaluated according to their utility for
inferring changes in the assessment endpoints and their compatibility with the site conceptual
model; otherwise, the study could fail to meet its objectives.

       Proposed changes to the SAP must be made in consultation with the risk manager and
the risk assessors. The risk manager must understand what  changes have been made and
why, and must ensure that the risk management decisions can be made from the information
that the new study design can  provide. The risk assessors must be involved to ensure that the
assessment endpoints and study questions or testable hypotheses are still being addressed.
                                           6-6

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6.5    SUMMARY

       The site investigation step of the ecological risk assessment should be a
straightforward implementation of the study designed in Step 4 and verified in Step 5.  In
instances where unexpected conditions arise in the field that indicate a need to change the
study design, the ecological risk assessors should reevaluate the feasibility or adequacy of the
sampling design.  Any proposed changes to the WP or SAP must be agreed upon by both the
risk assessment team and the risk manager and must be documented in the baseline risk
assessment.

       The analysis phase of the ecological risk  assessment consists  of the technical
evaluation of data on existing and potential exposures and ecological effects and is based on
the information collected during Steps  1 through 5 and the site investigation in Step 6.
Analyses of exposure and effects are performed interactively, and follow the data
interpretation and analysis methods specified in the WP and SAP.  Site-specific data obtained
during Step  6 replace many of the assumptions that were made for the screening-level
analysis in Steps  1  and 2.  Evidence of an exposure-response relationship between
contamination and ecological responses at a site  helps to establish causality. The results of
Step 6 are used to characterize ecological risks in Step 7.
                                           6-7

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                     STEP 7:  RISK CHARACTERIZATION
                                      OVERVIEW

          In risk characterization, data on exposure and effects are integrated into a
    statement about risk to the assessment endpoints established during problem
    formulation.  A weight-of-evidence approach is used to interpret the implications of
    different studies or tests for the assessment endpoints.  In a well-designed study, risk'
    characterization should be straightforward, because the procedures were established in
    the WP and SAP.  The risk characterization section of the baseline ecological risk
    assessment should include a qualitative and quantitative presentation of the risk results
    and associated uncertainties.
7.1    INTRODUCTION

       Risk characterization is the final phase of the risk assessment process and includes two
major components:  risk estimation and risk description (U.S. EPA, 1992a; Exhibit 7-1).  Risk
estimation (Section 7.2) consists of integrating the exposure profiles with the exposure-effects
information  and summarizing the associated uncertainties. The risk description (Section 7.3)
provides information important for interpreting the risk results and, in the Superfund Program,
identifies a threshold for adverse effects on the assessment endpoints (Section 7.4).

       It is U.S. EPA policy that risk characterization should be consistent with the values of
"transparency, clarity, consistency, and reasonableness" (U.S. EPA, 1995f).  "Well-balanced
risk characterizations present risk conclusions and information regarding the strengths and
limitations of the assessment for other risk assessors, EPA decision-makers, and the public"
(U.S. EPA, 1995f).  Thus, when preparing the risk characterization, the risk assessment team
should make sure that the documentation of risks is easy  to follow and understand, with all
assumptions, defaults, uncertainties, professional judgments, and any other inputs to the risk
estimate  clearly identified and easy to find.
7.2    RISK ESTIMATION

       Documentation of the risk estimates should describe how inferences are made from the
measurement endpoints to the assessment endpoints established in problem formulation. As
stated earlier, it is not the purpose of this document to provide a detailed guidance on the
selection and utilization of risk models.  The risk assessment team should have developed and
the risk manager should have agreed  upon the conceptual model used to characterize risk, its

                                          7-1

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              EXHIBIT 7-1
Risk Characterization (U.S. EPA, 1992a)
                       PROBLEM FORMULATION


                       ANALYSIS


                     x  RISK CHARACTERIZATION
                         X
          ANALYSIS
1
Risk Estimation
Integration



Uncertainty
Analysis



             J
          Ecological
            Risk
          Summary
        Interpretation of
          Ecological
         Significance
             I
    Discussion Between the
 Risk Assessor and Risk Manager
           (Results)
                                                      s?
2.
5*


o

0

(O
       Risk Management
                   7-2

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 assumptions, uncertainties, and interpretation in Steps 3 through 5. This agreement is
 specified in the site WP and SAP and is the purpose of the SMDPs in Steps 3 through 5.

       Unless the site investigation during Step 6 discovers unexpected information, the risk
 assessment should move smoothly through the risk characterization phase, because the data
 interpretation procedures were specified in the WP and SAP.  While it might be informative
 to investigate a data set for trends, outliers, or other statistical indicators, these investigations
 should be secondary to the data interpretations specified in the SAP.  Analysis of the data
 beyond the purposes for which it was collected might be informative, but could lead to
 biased, conflicting, or superfluous conclusions.  Those outcomes can divert or confound the
 risk characterization process.

       For ecological  risk assessments that entail more than one type of study (or line of
 evidence), a strength-of-evidence approach is used to integrate different types of data to
 support a conclusion.  The data might include toxicity test results, assessments of existing
 impacts at a site, or risk calculations comparing exposures estimated for the site with toxicity
 values from the literature.  Balancing and interpreting the different types of data can be a
 major task and require professional judgment.  As indicated above, the strength of evidence
 provided  by different types of tests and the precedence that one type of study might have over
 another should already have been  established during Step 4.  Taking this approach will ensure
 that data interpretation is objective and not biased to support a preconceived answer.
 Additional strength-of-evidence  considerations at this stage include the degree to which DQOs
 were met and whether confounding factors became evident during the site  investigation and
 analysis phase.

       For some biological tests (e.g., toxicity tests, benthic macroinvertebrate studies), all or
 some of the data interpretation process is outlined in existing documents, such as in toxicity
 testing manuals.  However, in most cases, the SAP must provide the details on how the data
 are to be  interpreted for a site.  The data interpretation methods also should be presented in
 the risk characterization documentation.  For example, if the triad  approach was used to
evaluate contaminated sediments, the risk estimation  section should describe how the three
types of studies (i.e., toxicity  test, benthic invertebrate survey, and sediment chemistry) are
integrated to  draw conclusions about risk.

       Where exposure-response functions are not available or developed,  the quotient
method of comparing  an estimated exposure concentration to a threshold for response can be
used, as in Step 2.  Whenever possible, however, presentation of full exposure-response
functions  provides the risk manager with more information on which to base site decisions.
This guidance has recommended the use of on-site contamination  gradients to demonstrate on-
site exposure-response functions.  Where such data have been collected, they should be
presented along with the risk  estimates.  Hazard quotients, hazard indices (for contaminants
with the same mechanism of toxicity), the results of in situ toxicity testing, or community
survey data can be mapped along  with analytic chemistry data to provide a clear picture of
the relationship between areas of contamination and effects.

                                            7-3

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       In addition to developing point estimates of exposure concentrations, as for the hazard
quotient approach, it might be possible to develop a distribution of exposure levels based on
the potential variability in various exposure parameters (see Section 7.3.2). Probabilities of
exceeding a threshold for adverse effects might then be estimated. Again, the risk assessment
team and risk manager should have already agreed to what analyses will be used to
characterize risks.
7.3    RISK DESCRIPTION

       A key to risk description for Superfund sites is documentation of environmental
contamination levels that bound the threshold for adverse effects on the assessment endpoints
(Section 7.3.1).  The risk description can also provide information to help the risk manager
judge the likelihood and ecological significance of the estimated risks (Sections 7.3.2 and
7.3.3, respectively).

7.3.1  Threshold for Effects on Assessment Endpoints

       Key outputs of the risk characterization step are contaminant concentrations in each
environmental medium that bound the threshold for estimated adverse ecological effects given
the uncertainty inherent in the data and models used. The lower bound of the threshold
would be based on consistent conservative assumptions and NOAEL toxicity values. The
upper bound would be based on observed impacts or predictions that ecological impacts could
occur. This upper bound would be developed using consistent assumptions, site-specific data,
LOAEL toxicity values, or an impact evaluation.

       The approach to estimating environmental contaminant concentrations that represent
thresholds for adverse  ecological effects should have been specified in the study design  (Step
4); When higher-trophic-level organisms are associated with assessment endpoints, the study
design,should have described how monitoring data and contaminant-transfer models would be
used to back-calculate  an environmental concentration representing a threshold for effect.  If
the site investigation demonstrated a gradient of ecological effects along a contamination
gradient, the risk assessment team can identify and document the levels of contamination
below which no further improvements in the assessment endpoints are discernable or
expected.  If departures from the original analysis plan are necessary based on information
obtained during the site investigation or data analysis phase, the reasons for change should be
documented.

       When assessment endpoints include populations of animals that can travel moderate
distances, different ways of presenting a threshold for adverse effects are possible.  Various
combinations of level of contamination and area! extent of contamination relative to the
foraging range of the animals can result in similar contaminant intake levels by the animals.
In that case, a point of departure for identifying a threshold for effect would be to identify
that level of contamination, which if uniformly distributed both at the site and beyond, would

                                           7-4

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 not pose a threat. The assumption of uniform contamination has been used to back-calculate
 water-quality criteria to protect piscivorous wildlife in the Great Lakes (U.S. EPA, 1995a).
 Again, use of this approach should have been specified in the study design.

 7.3.2  Likelihood of Risk

        In addition to identifying one or more thresholds for effects, the risk assessment team
 might develop estimates of the probability that exposure levels would exceed the ecotoxicity
 thresholds given the distribution  of values likely for various exposure parameters (e.g., home
 range size, population density). A distributional analysis might be used to estimate the range
 of likely exposure levels associated with a given exposure model based on ranges for the
 input variables.

 7.3.3  Additional  Risk Information

        In addition to developing numerical estimates of existing impacts, risks, and thresholds
 for effect, the risk assessor should put the estimates in context with a description of their
 extent, magnitude, and potential ecological significance.  Additional ecological risk
 descriptors are listed below:

        •      The location and area] extent of existing contamination above a threshold for
              adverse effects;

        •      The degree  to which the threshold for contamination is exceeded or is likely to
              be exceeded in the future, particularly if exposure-response functions are
              available; and

        •      The expected half-life  (qualitative or quantitative) of contaminants in the
              environment (e.g., sediments, food chain) and the potential for natural recovery
              once  the sources of contamination are removed.

 To interpret the information in light of remedial options, the risk manager might need to
 solicit input from specific  experts.

        At this stage, it is important for the risk  assessors to consider carefully several
 principles of risk communication, as  described in U.S. EPA's  (1996a) Proposed Guidelines
for Ecological Risk  Assessment.
7.4    UNCERTAINTY ANALYSIS

       There are several sources of uncertainties associated with Superfund ecological risk
estimates.  One is the initial selection of substances of concern based on the sampling data  .
and available toxicity information.  Other sources of uncertainty include estimates of toxicity

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 to ecological receptors at the site based on limited data from the laboratory (usually on other
 species), from other ecosystems, or from the site over a limited period of time.  Additional
 uncertainties result from the exposure assessment, as a consequence of the uncertainty in
 chemical monitoring data and models used to estimate exposure concentrations or doses.
 Finally, further uncertainties are included in risk estimates when simultaneous exposures to
 multiple substances occur.

       Uncertainty should be distinguished from variability, which arises from true
 heterogeneity or variation in characteristics of the environment and receptors. Uncertainty, on
 the other hand, represents lack of knowledge about certain factors which can sometimes be
 reduced by additional study.

       This section briefly  notes  several categories of uncertainty (Section 7.4.1) and
 techniques for tracking uncertainty through a risk assessment (Section 7.4.2).  Additional
 guidance on discussing uncertainty and variability in risk characterization is provided in U.S.
 EPA's (19920 Guidance on Risk Characterization for Risk Managers and Risk Assessors.

 7A.I  Categories of Uncertainty

       There are three basic categories of uncertainties that apply to Superfund site risk
 assessments:  (1) conceptual model uncertainties; (2) natural variation and parameter error; and
 (3) model error.  Each of these is described below.

       There will be uncertainties associated with the conceptual model  used as the basis to
investigate the site.  The initial characterization of the ecological problems at a Superfund
site, likely exposure pathways, chemicals of concern, and exposed ecological components,
requires professional judgments and assumptions. To the extent possible, the risk assessment
 team should describe what judgments and assumptions were included in  the conceptual model
that formed the basis of the WP and SAP.

       Parameter values  (e.g., water concentrations, tissue residue levels, food ingestion rates)
usually can be characterized as a distribution of values, described by central tendencies,
ranges, and percentiles, among other descriptors.  When evaluating uncertainty in parameter
values, it is important to distinguish uncertainty from variability.  Ecosystems include highly
variable abiotic (e.g., weather, soils) and biotic  (e.g., population density) components. If all
instances of a parameter (e.g., all members of a population) could be sampled, the "true"
parameter value distribution could be described. In practical terms, however, only a fraction
of the instances (e.g., a few of the members of  the population) can be sampled, leaving
uncertainty concerning the true parameter value distribution. The risk assessor should provide
either quantitative or qualitative descriptions of uncertainties in parameter value distributions.

       Finally, there is uncertainty associated with how well a model (e.g., fate and transport
model) approximates true relationships between site-specific environmental conditions.
Models available at present tend to be fairly simple and at best, only partially validated with

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field tests. As a consequence, it is important to identify key model assumptions and their
potential impacts on the risk estimates.

7.4.2  Tracking Uncertainties

       In general, there are two approaches to tracking uncertainties through a risk
assessment: (1) using various point estimates of exposure and response to develop one or
more point estimates of risk; and (2) conducting a distributional analysis to predict a
distribution of risks based on a distribution of exposure levels and exposure-response
information.  Whether one or the other or both  approaches are taken should have been agreed
to during Step 4, and the specific type of analyses to be conducted should have been specified
in the SAP.

7.5    SUMMARY

       Risk characterization integrates the results of the exposure profile and exposure-
response analyses, and is the final  phase of the  risk assessment process. It consists of risk
estimation and risk description, which together  provide information to help judge the
ecological significance of risk estimates in the absence of remedial activities.  The risk
description also identifies a threshold for effects on the assessment endpoint as a range
between contamination levels identified as posing no ecological risk and the lowest
contamination levels identified as likely to produce adverse ecological effects.  To ensure that
the risk characterization is transparent, clear,  and reasonable, information regarding the
strengths and limitations of the assessment must be identified and described.
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                        STEPS:  RISK MANAGEMENT
                                     OVERVIEW

          Risk management at a Superfund site is ultimately the responsibility of the site
   risk manager, who must balance risk reductions associated with cleanup of
   contaminants with potential impacts of the remedial actions themselves. The risk
   manager considers inputs from the risk assessors, BTAGs, stakeholders, and other
   involved parties.  In Step 7, the risk assessment team identified a threshold for effects
   on the assessment endpoint as a range between contamination levels identified as
   posing no ecological risk and the lowest contamination levels identified as likely to
   produce adverse ecological effects.  In Step 8, the risk manager evaluates several
   factors in  deciding whether or not to clean up to within that range.
8.1    INTRODUCTION

       Risk management is a distinctly different process from risk assessment (NRC, 1983,
1994; U.S. EPA, 1984a, 1995f).  The risk assessment establishes whether a risk is present and
defines a range or magnitude of the risk.  In risk management, the results of the risk
assessment are integrated with other considerations to make and justify risk management
decisions.  Additional risk management considerations can include the implications of existing
background levels of contamination, available technologies, tradeoffs between human and
ecological concerns, costs of alternative actions, and  remedy selection.  For further
information on management of ecological risks Agency-wide,  see U.S. EPA 1994h.  Some
Superfund-specific considerations are described below.
8.2    ECOLOGICAL RISK MANAGEMENT IN SUPERFUND
                                                                        »

       According to section 300.40 of the NCP, the purpose of the remedy selection process
is to eliminate, reduce, or control risks to human health and the environment. The NCP
indicates further that the results of the baseline risk assessment will help to establish
acceptable exposure levels for use in developing remedial alternatives during the FS.  Based
on the criteria for selecting the preferred remedy and, using information from the human
health and ecological risk assessments and the evaluation of remedial options in the FS, the
risk .manager then selects a preferred remedy.

       The risk manager must consider several types of information  in addition to the
baseline ecological risk assessment when evaluating remedial options (Section 8.2.1). Of

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particular concern for ecological risk management at Superfund sites is the potential for
remedial actions themselves to cause adverse ecological impacts (Section 8.2.2). There also
exists the opportunity to monitor ecological components at the site to gauge the effectiveness
(or impacts) of the selected remedy (Section 8.2.3).

8.2.1  Other Risk Management Considerations

       The baseline ecological risk assessment is not the only set of information that the  risk
manager must consider when evaluating remedial options during the FS phase of the
Superfund process.  The NCP (40 CFR 300.430(f)(l)(i)) specifies  that each remedial
alternative should be evaluated according to nine criteria. Two are considered threshold
criteria, and take  precedence over the others:

       (1)    Overall protection of human health and the environment; and

       (2)    Compliance with applicable or relevant and appropriate requirements (ARARs)
              (unless waiver applicable).

As described in Section 8.2.2 below, a particularly important consideration for the  first
criterion are the ecological impacts of the remedial options.

       Five of the nine criteria are considered primary balancing criteria to be considered
after the threshold criteria:

       (3)    Long-term effectiveness and permanence;

       (4)    Reduction of toxicity, mobility, or volume of hazardous wastes through the use
              of treatment;

       (5)    Short-term effectiveness;

       (6)    Implementability; and

       (7)    Cost.

       Finally, two additional criteria are referred to as modifying criteria that must be
considered:

       (8)    State acceptance, and

    .   (9)    Community acceptance.

Effective risk communication is particularly important to help ensure that a remedial option
that best satisfies  the other criteria can be implemented at a site. U.S. EPA's (1996a)

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Proposed Guidelines for Ecological Risk Assessment provides an overview of this topic and
identifies some of the relevant literature.

       Additional factors that the site risk manager takes into consideration include existing
background levels (see U.S. EPA, 1994g); current and likely future land uses (see U.S. EPA,
1995c); current and likely future resource uses in the area; and local, regional, and national
ecological significance of the site. Consideration of the ecological impacts of remedial
options and residual risks associated with leaving contaminants in place are very important
considerations, as described in the next section.

8.2.2  Ecological Impacts of Remedial Options

       Management of ecological risks must take into account the potential for impacts to the
ecological assessment endpoints from implementation of various remedial options. The risk
manager must balance:  (1) residual risks posed by site contaminants before and after
implementation of the selected remedy with (2) the potential impacts of the selected remedy
on the environment independent of contaminant effects.  The selection of a remedial
alternative could  require  tradeoffs between long-term and short-term risk.

       The ecological risks posed by the "no action"  alternative are the risks estimated by the
baseline ecological risk assessment.  In addition, each remedial option is likely to have its
own ecological impact.  This impact could be anything from a short-term loss to complete
and permanent loss of the present habitat and ecological communities. In instances where
substantial ecological  impacts will result from the remedy (e.g., dredging a wetland), the  risk
manager will need to consider ways to mitigate the impacts of the remedy and compare the
mitigated impacts to the  threats posed by the site contamination.

       During the FS, the boundaries of potential risk under the no-action alternative (i.e.,
baseline conditions) can be compared with the evaluation of potential impacts of the remedial
options to help justify the preferred remedy. As indicated above, the preferred remedy should
minimize the risk of long-term impacts that could result from the remedy and any residual
contamination. When the selected remedial option leaves some site contaminants presumed to
pose an ecological risk in place, the justification for the selected remedy must be clearly
documented.

       In short, consideration of the environmental effects of the remedy itself might result in
a decision to allow contaminants to remain on site  at levels higher than the threshold for
effects on the assessment endpoint. Thus, selection of the most appropriate ecologically
based remedy can result  in residual contamination that presents some risk.

8.2.3  Monitoring

       Ecological risk assessment is a relatively new field with limited data available to
validate its predictions.  At sites where remedial actions are taken to reduce ecological

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 impacts and risks, the results of the remediation efforts should be compared with the
 predictions made during the ecological risk assessment.

       While it often is difficult to demonstrate the effectiveness of remedial actions in
 reducing human health risks, it often is possible to demonstrate the effectiveness of
 remediations to reduce ecological risks, particularly if a several-year monitoring program is
 established. The site conceptual model provides the conceptual basis for monitoring options,
 and the site investigation should have indicated which options might be most practical for the
 site. Monitoring also is important to assess the effectiveness of a no-action alternative. For
 example, monitoring sediment contamination and benthic communities at intervals following
 removal of a contaminant source allows one to test predictions of the potential for the
 ecosystem to recover naturally over time.
8.3    SCIENTIFIC/MANAGEMENT DECISION POINT (SMDP)

       The risk management decision is finalized in the Record of Decision (ROD).  The
decision should minimize the risk of long-term impacts that could result from the remedy and
any residual contamination.  When the selected remedy leaves residual contamination at levels
higher than the upper-bound estimate of the threshold for adverse effects on the assessment
endpoint, the risk manager should justify the decision (e.g., describe how a more complete
physical remedy could jeopardize an ecological community more than the residual
contamination).
8.4    SUMMARY

       Risk-management decisions are the responsibility of the risk manager (the site
manager), not the risk assessor. The risk manager should have been involved in planning the
risk assessment; knowing the options available for reducing risks, the risk manager can help
to frame questions during the problem-formulation phase of the risk assessment.

       The risk manager must understand the risk assessment, including its uncertainties,
assumptions, and level of resolution.  With an understanding of potential adverse effects
posed by residual levels of site contaminants and posed by the remedial actions themselves,
the risk manager can balance the ecological costs and benefits of the available remedial
options.  Understanding the uncertainties associated with the risk assessment also is critical to
evaluating the overall protectiveness of any  remedy.
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                                 BIBLIOGRAPHY
       This combined reference list and bibliography is intended to provide a broad, but not
 all inclusive, list of other materials that may provide useful information for ecological risk
 assessments at Superfund sites.  These documents include other Superfund Program guidance
 documents, standard guides for toxicity testing, other EPA program office references with
 potential applications at Superfund sites, and other ecological risk assessment reference
 materials. References cited in the text are marked with an asterisk (*).
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American Society for Testing and Materials (ASTM).  I994a.  Annual Book of ASTM
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                                                            /
American Society for Testing and Materials (ASTM).  1994b.  Standard guide for conducting
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American Society for Testing and Materials (ASTM).  1993a.  Standard terminology relating
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American Society for Testing and Materials (ASTM).  I993b.  Standard guide for designing
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American Society for Testing and Materials (ASTM).  1993. ASTM Standards of Aquatic
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American Society for Testing and Materials (ASTM).  1992.  Standard guide for conducting
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American Society for Testing and Materials (ASTM).  1992.  Standard guide for conducting
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       Standard E  1367-92.

American Society for Testing and Materials (ASTM).  1990.  Standard guide for collection,
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       ASTM Standard E 1391-90.

American Society for Testing and Materials (ASTM).  1988.  Standard  guide for conducting
       early life-stage toxicity tests with fishes:  ASTM Standard E 1241-88.
                                     Bibliography-1

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 American Society for Testing and Materials (ASTM).  1984. Standard Practice for
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 Ankley, G.T.; Thomas, N.A.; Di Toro, D.M.;  et al.  1994. Assessing potential bioavailability
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 *Aulerich, R.J.; Ringer, R.K.  1977.  Current  status of PCS  toxicity to mink and effect on
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 *Aulerich, R.J.; Bursian, S.J.; Breslin, WJ.; et al.  1985. Toxicological manifestations of
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       in mink. J. Toxicol. Environ. Health  15:  63-79.

Barnhouse, L.W.; Suter, G.W.; Bartell, S.M.; et al.  1986. User's Manual for Ecological Risk
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Bartell, S.M.; Gardner, R.H.; O'Neill, R.V.  1992.  Ecological Risk Estimation. New York,
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Baudo, R.; Giesy, J.P.; Muntau, H.  1990. Sediments: Chemistry and Toxicity ofln-place
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Cairns, J. Jr.; Niederlehner, B.R.  1995.  Ecological Toxicity Testing:  Scale, Complexity, and
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Calabrese, E.J.; Baldwin, L.A.  1993.  Performing Ecological Risk Assessments.  New York,
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                                     BibIiography-2

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Davis, W.S.; Simon, T.P.  1995.  Biological Assessment and Criteria: Tools for Water
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U.S. Environmental Protection Agency (U.S. EPA). 1989. Report to  the Sediment Criteria
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U.S. Environmental Protection Agency (U.S. EPA). 1989. Sediment Classification Methods
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U.S. Environmental Protection Agency (U.S. EPA). 1989. Water Quality Criteria to Protect
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U.S. Environmental Protection Agency (U.S. EPA). 1989. Ecological Assessment of
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U.S. Environmental Protection Agency (U.S. EPA).  1989.  Superfund Exposure Assessment
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U.S. Environmental Protection Agency (U.S. EPA).  1989.  Protocols for Short-Term Toxicity
       Screening of Hazardous Waste Sites.  Office of Research and Development,
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U.S. Environmental Protection Agency (U.S. EPA).  1989.  Scoping Study of the Effects of
       Soil Contamination on Terrestrial Biota. Washington, DC:  Office of Toxic
       Substances.

*U.S. Environmental Protection Agency (U.S. EPA).  1988a.  Guidance for Conducting
       Remedial Investigations and Feasibility Studies Under CERCLA.  Washington, DC:
       Office of Emergency and Remedial Response; OSWER Directive No. 9355.3-01.

U.S. Environmental Protection Agency (U.S. EPA).  1988.  Estimating Toxicity of Industrial
       Chemicals to Aquatic Organisms Using Structure Activity Relationships. Office of
       Toxic Substances, Washington, DC: EPA/560/6-88/001.

U.S. Environmental Protection Agency (U.S. EPA).  1988.  CERCLA  Compliance with Other
       Laws Manual, Part 1. Washington, DC: Office of Emergency and  Remedial
       Response; OSWER Directive 9234.1-01.

U.S. Environmental Protection Agency (U.S. EPA).  1988.  Short-term Methods for
       Estimating the Chronic Toxicity of Effluents in Receiving Waters to Marine and
       Estuarine Organisms. Cincinnati, OH:  Office of Research and Development, Office
       of Environmental Monitoring and Support Laboratory; EPA/600/4-87/0928.

U.S. Environmental Protection Agency (U.S. EPA).  1988.  Estimating Toxicity of Industrial
       Chemicals to Aquatic Organisms Using Structure Activity Relationships. Washington,
       DC:  Office of Toxic Substances; EPA/560/6-88/001.

U.S. Environmental Protection Agency (U.S. EPA).  1988.  Short-term Methods for
       Estimating the Chronic Toxicity of Effluents in Receiving Waters to Marine and
       Estuarine Organisms. Cincinnati, OH:  Office of Research and Development,
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U.S. Environmental Protection Agency (U.S. EPA).  1988.  Methods for Aquatic Toxicity
       Identification Evaluations:  Phase II,  Toxicity Identification Procedures. Duluth, MM:
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U.S. Environmental Protection Agency (U.S. EPA).  1988.  Methods for Aquatic Toxicity
       Identification Evaluations:  Phase III, Toxicity Confirmation Procedures.  Duluth, MN:
       Office of Research and Development, Environmental Research Laboratory; EPA/600/3-
       88/036.

U.S. Environmental Protection Agency (U.S. EPA).  1988.  Superfund Exposure Assessment
       Manual.  Washington, DC: Office of Solid Waste and Emergency Response Directive
       9285.5-1; EPA/540/1-88/001.

*U.S. Environmental Protection Agency  (U.S. EPA).  1987a. Data Quality Objectives for
       Remedial Response Activities:  Development Process. Washington, DC:  Office of
       Solid Waste and Emergency Response, Office of Emergency and Remedial Response
       and Office of Waste Programs Enforcement, OSWER Directive 9355.0-7B;
       EPA/540/G-87/003.

U.S. Environmental Protection Agency (U.S. EPA).  1987.  Data Quality Objectives for
       Remedial Response Activities:  Example Scenario: RI/FS Activities at a Site with
       Contaminated Soils and Ground Water. Washington, DC:  Office of Solid Waste and
       Emergency  Response, Office of Emergency and Remedial Response and Office of
       Waste Programs Enforcement, OSWER Directive 9355.0-7B; EPA/540/G-87/004.
                                                 /
U.S. Environmental Protection Agency (U.S. EPA).  1987.  Permit Writer's Guide to Water
       Quality-Based Permitting for Toxic Pollutants. Washington, DC:  Office of Water
       Regulations and Standards; EPA/440/4-87/005.

U.S. Environmental Protection Agency (U.S. EPA).  1987.  Guidelines for Deriving Ambient
       Aquatic Life Advisory Concentrations.  Washington,  DC: Office of Water Regulations
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U.S. Environmental Protection Agency (U.S. EPA).  1987.  A Compendium of Superfund
       Field Operations Methods.  Washington DC:  Office of Solid Waste and Emergency
       Response, Office of Environmental and Remedial Response; EPA/540/P-87/001.

U.S. Environmental Protection Agency (U.S. EPA).  1987.  Role of Acute Toxicity Bioassays
       in the Remedial Action Process at Hazardous Waste Sites.  Corvallis, OR:  Office of
       Research  and Development, Environmental Research Laboratory; EPA/600/8-87/044.

U.S. Environmental Protection Agency (U.S. EPA).  1987.  Ecological Risk Assessment  in the
       Office of Toxic Substances: Problems and Progress 1984-1987. Washington, DC:
       Office of Toxic Substances, Health and Environmental Review Division (Author:
       Rodier, D.)
                                    Bibliography-20

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 *U.S. Environmental Protection Agency (U.S. EPA).  1986a.  Guidelines for the Health Risk
       Assessment of Chemical Mixtures. Washington, DC: Office of Health and
       Environmental Assessment; EPA/600/8-87/045.

 U.S. Environmental  Protection Agency (U.S. EPA).  1986.  Engineering Support Branch,
       Standard Operating Procedures and Quality Assurance Manual.  Region IV,
       Environmental Services Division.

 U.S. Environmental  Protection Agency (U.S. EPA).  1986.  Guidelines for Deriving
       Numerical Criteria for the Protection of Aquatic Organisms and Their Uses.
       Washington,  DC:  Office of Water Regulations and Standards.

 U.S. Environmental  Protection Agency (U.S. EPA).  1986.  Quality Criteria for Water 1986.
       Washington,  DC:  Office of Water Regulations and Standards; EPA/440/5-86/001.

 *U.S. Environmental Protection Agency (U.S. EPA).  1985a.  Ambient Water Quality Criteria
      for Copper-1984.  Washington, DC:  Office of Water, Regulations and Standards,
       Criteria and Standards Division.  EPA/440/5-84-031.  PB85-227023.

 U.S. Environmental  Protection Agency (U.S. EPA).  1985.  Development of Statistical  ,
       Distributions of Ranges of Standard Factors Used in Exposure Assessments.
       Washington,  DC:  Office of Health and Environmental Assessment, OHEA-E-161;
       EPA/600/8-85/010.

U.S. Environmental  Protection Agency (U.S. EPA).  1985.  Guide for Identifying Cleanup
      Alternatives at Hazardous Waste Sites and Spills.  Washington, DC:  Office of Solid
       Waste and Emergency Response; EPA/600/3-83/063, NTIS PB86-144664.

U.S. Environmental  Protection Agency (U.S. EPA).  1985. Methods for Measuring the Acute
       Toxicity of Effluents to Freshwater and Marine Organisms.  Cincinnati, OH:  Office of
       Research  and Development, Environmental Monitoring and Support Laboratory;
       EPA/600/4-85/013.

U.S. Environmental  Protection Agency (U.S. EPA).  1985.  Short-term Methods for
       Estimating the Chronic Toxicity of Effluents in Receiving Waters to Freshwater
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       Monitoring and Support Laboratory;  EPA/600/4-85/014.

*U.S. Environmental Protection Agency (U.S. EPA).  1984a.  Risk Assessment and
      Management: Framework for Decision Making.  Washington, DC:  Office of Policy,
       Planning, and Evaluation; EPA/600/9-85/002.
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       Concentrations of Chemical Substances in the Environment. Washington, DC:  Office
       of Toxic Substances, Environmental Effects Branch.

 U.S. Environmental Protection Agency (U.S. EPA).  1984.  Technical Support Manual:
       Waterbody Surveys and Assessments for Conducting Use Attainability Analyses:
       Volume II:  Estuarine Systems.  Washington,  DC: Office of Water Regulations and
       Standards.

 U.S. Environmental Protection Agency (U.S. EPA).  1984.  Technical Support Manual:
       Waterbody Surveys and Assessments for Conducting Use Attainability Analyses:
       Volume III:  Lake Systems. Washington, DC: Office of Water Regulations and
       Standards.

 U.S. Environmental Protection Agency (U.S. EPA).  1983.  Technical Support Manual:
       Waterbody Surveys and Assessments for Conducting Use Attainability Analyses.
       Washington, DC:  Office of Water Regulations and Standards (November).

 U.S. Environmental Protection Agency (U.S. EPA).  1983.  Environmental Effects of
       Regulatory Concern Under TSCA:  A Position Paper.  Washington, DC: Office of
       Toxic Substances, Health and Environmental  Review Division (Author:  Clements,
       R.G.)

 U.S. Environmental Protection Agency (U.S. EPA) and Department of the Army, U.S. Army
       Corps of Engineers (USAGE).  1994. Evaluation of Dredged Material Proposed for
       Discharge in Waters of the U.S.—Testing Manual (Draft); Inland Testing Manual.
       Washington, DC: EPA Office of Water. EPA/823/B-94/002.

 Watras, CJ.; Huckabee, J.W. (eds.).  1995. Mercury Pollution: Integration and Synthesis.
       Boca Raton, FL: CRC Press, Inc., Lewis Publishers.
                                    i
 *Weil, C.S.; McCollister, D.D.  1963.  Relationship between short- and long-term feeding
       studies in designing an effective toxicity test.  Agr. Food Chem.  11:  486-491.

Wentsel, R.S.; LaPoint, T.W.; Simini, M.; Checkai, R.T.; Ludwig, D.; Brewer, L.  1994.
       Procedural Guidelines for Ecological Risk Assessments at U.S. Army Sites, Volume I.
       Aberdeen Proving Ground, MD: Edgewood Research, Development, and Engineering
       Center, U.S.  Army Chemical and Biological Defense Command.   Rept. No. ERDEC-
       TR-221.

 *Wren, C.D.  1991. Cause-effect linkages between chemicals and populations of mink
       (Mus tola vison)  and otter (Lutra canadensis) in the Great Lakes basin.  J. Toxicol.
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                                    Bibliography-22

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                                     GLOSSARY
       This glossary includes definitions from several sources.  A superscript number next to a
 word identifies the reference from which the definition was adapted (listed at the end of the
 Glossary).
Abiotic.1  Characterized by absence of life; abiotic materials include non-living environmental
media (e.g., water, soils, sediments); abiotic characteristics include such factors as light,
temperature, pH, humidity, and other physical and chemical influences.

Absorption Efficiency.  A measure of the proportion of a substance that a living organism
absorbs across exchange boundaries (e.g., gastrointestinal tract).

Absorbed Dose.2 The amount of a substance penetrating the exchange boundaries of an
organism after contact. Absorbed dose for the inhalation and ingestion routes of exposure is
calculated from the intake and the absorption efficiency.  Absorbed dose for dermal contact
depends on the surface area exposed and absorption efficiency.

Accuracy.4  The degree to which a measurement reflects the true value of a variable.

Acute.   Having a sudden onset or lasting a short time.  An acute stimulus is severe enough
to induce a response rapidly. The word acute can be used to define either the exposure or the
response to an exposure  (effect).  The duration of an acute aquatic toxicity test is generally 4
days of less and mortality is the response usually measured.

Acute Response.  The response of (effect on) an organisms which has a rapid onset.  A
commonly measured rapid-onset response in toxicity tests is mortality.
                                                         i
Acute Tests.  A toxicity test of short duration, typically 4 days or less (i.e., of short duration
relative to the lifespan of the test organism).

Administered Dose.2  The mass  of a substance  given to an organism and in contact with an
exchange boundary (i.e., gastrointestinal tract) per unit wet body weight (BW) per unit time
(e.g., mg/kgBW/day).

Adsorption.14 Surface retention of molecules, atoms,  or ions by a  solid or liquid, as opposed
to absorption, which is penetration of substances into the bulk of a  solid or liquid.

Area Use Factor. The ratio of an organism's home range, breeding range, or
feeding/foraging range to the area of contamination of the site under investigation.
                                        Glossary-1

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 Assessment Endpoint.   An explicit expression of the environmental value that is to be
 protected.

 Benthic Community.7  The community of organisms dwelling at the bottom of a pond, river,
 lake, or ocean.

 Bioaccurnulation.^ General term describing a process by which chemicals are taken up by an
 organism either directly from exposure to a contaminated medium or by consumption of food
 containing the chemical.

 Bioccumulation Factor (BAF).3  The ratio of the concentration of a contaminant in an
 organism to the concentration in the ambient environment at steady state, where the organism
 can take in the contaminant through ingestion with its food as well as through direct contact.

 Bipassay.  Test used to evaluate the relative potency of a chemical by comparing its effect
 on living organisms with the effect of a standard preparation on the same type of organism.
 Bioassay and toxicity tests are not the same—see toxicity test.  Bioassays often are run on a
 series of dilutions of whole effluents.

 Bioassessment. A general term referring to environmental evaluations involving living
 organisms; can include bioassays,  community analyses, etc.

 Bioavailability.4  The degree to which a material in environmental media can be assimilated
 by an organism.

 Bioconcentration.  A process by which there is a net accumulation of a chemical directly
 from  an exposure medium into an organism.

 Biodegrade.15  Decompose into more elementary compounds by the action of living
 organisms, usually referring to microorganisms  such as bacteria.

Biomagnification.5 Result of the process of bioaccumulation and biotransfer by which tissue
concentrations of chemicals in organisms at one trophic  level exceed tissue concentrations in
organisms at the next lower trophic level in a food chain.

Biomarker.21   Biochemical, physiological, and histological changes in organisms that can be
 used to estimate either exposure to chemicals or the effects of exposure to chemicals.

Biomonitoring.5  Use of living organisms as "sensors" in environmental quality surveillance
to detect changes in environmental conditions that might threaten living organisms in the
environment.

Body Burden.  The concentration or total amount of a substance in a living organism;
implies accumulation  of a substance above background levels in exposed organisms.

                                       Glossary-2

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Breeding Range. The area utilized by an organism during the reproductive phase of its life
cycle and during the time that young are reared.

Bulk Sediment.8  Field collected sediments used to conduct toxicity tests; can contain
multiple contaminants and/or unknown concentrations of contaminants.

Characterization of Ecological Effects.6 A portion of the analysis phase of ecological risk
assessment that evaluates the ability of a stressor to cause adverse effects under a particular
set of circumstances.

Characterization of Exposure.6 A portion of the analysis phase of ecological risk
assessment that evaluates the interaction of the stressor with one  or more ecological
components. Exposure can be expressed as co-occurrence, or contact depending on the
stressor and ecological component involved.

Chemicals of Potential Concern.2 Chemicals that are potentially site-related and whose data
are of sufficient quality for use in a quantitative risk assessment.

Chronic.5  Involving a stimulus that is lingering or continues for a long time; often signifies
periods from several weeks to years, depending on the reproductive life cycle of the species.
Can be used to define either the exposure or the response to an exposure (effect).  Chronic
exposures typically induce a biological response of relatively slow progress and long duration.

Chronic Response.  The response of (or effect on) an organism to a chemical that is not
immediately  or directly lethal to the organism.

Chronic Tests.9  A toxicity test used to study  the effects of continuous, long-term exposure
of a chemical or other potentially toxic material on an organism.

Community.6  An assemblage of populations of different species within a specified location
and time.

Complexation.14  Formation of a group of compounds in which  a part of the molecular
bonding between compounds is of the coordinate type.

Concentration.  The relative amount of a substance in an environmental medium, expressed
by relative mass (e.g., mg/kg), volume (ml/L), or number of units (e.g., pans per million).

Concentration-Response Curve.5 A curve describing the relationship between exposure
concentration and percent of the test population responding.

Conceptual Model.6  Describes a series of working hypotheses of how the stressor might
affect ecological components.  Describes ecosystem or ecosystem components potentially at
                                        Glossary-3

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risk, and the relationships between measurement and assessment endpoints and exposure
scenarios.

Contaminant of (Ecological) Concern. A substance detected at a hazardous waste site that
has the potential to affect ecological receptors adversely due to its concentration, distribution,
and mode of toxicity.

Control.5  A treatment in a toxicity test that duplicates all the conditions of the exposure
treatments but contains no test material. The control is used to determine the response rate
expected in the test organisms in the absence of the test material.

Coordinate Bond.14  A chemical bond between two atoms in which a shared pair of
electrons forms the bond and the pair of electrons has been supplied by one of the two atoms.
Also known as a coordinate valence.

Correlation.10 An estimate of the degree  to which two sets of variables vary together, with
no distinction between dependent and independent variables.

Critical Exposure Pathway. An exposure pathway which either provides the highest
exposure levels or is the primary pathway of exposure to an identified receptor of concern.

Degradation.14  Conversion of an organic  compound to one containing a smaller number of
carbon atoms.

Deposition.14  The lying, placing, or throwing down of any material.

Depuration.  A process that results in elimination of toxic substances from an organism.

Depuration Rate.  The rate at which a substance is depurated from an organism.

Dietary Accumulation.9 The net accumulation of a substance by an organism as a result of
ingestion in the diet.

Direct Effect (toxin).6 An effect where the stressor itself acts directly on the ecological
component of interest, not through other components of the ecosystem.

Dose.11 A measure of exposure.  Examples include (1) the amount of a chemical ingested,
(2) the amount of a chemical absorbed, and (3) the product of ambient exposure  concentration
and the duration of exposure.

Dose-Response Curve.5  Similar to concentration-response curve except that the dose (i.e. the
quantity) of the chemical administered to the organism is known. The curve is plotted as
Dose versus Response.
                                       Glossary-4

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Duplicate.8  A sample taken from and representative of the same population as another
sample.  Both samples are carried through the steps of sampling, storage, and analysis in an
identical manner.

Ecological Component.6  Any part of an ecosystem, including individuals, populations,
communities, and the ecosystem itself.

Ecological Risk Assessment.6 The process that evaluates the likelihood that adverse
ecological effects may occur or are occurring as a result of exposure to one or more stressors.

Ecosystem.  The biotic community and abiotic environment within a specified location and
time, including the chemical, physical, and biological relationships among the biotic and
abiotic components.

Ecotoxicity.11  The study of toxic effects on nonhuman organisms, populations, or
communities.

Estimated or Expected Environmental Concentration.5 The concentration of a material
estimated as  being likely to occur in environmental media to which organisms are exposed.

Exposure.    Co-occurrence of or contact between a stressor and an ecological component.
The  contact reaction between a chemical and a biological system, or organism.

Exposure Assessment.2 The determination or estimation (qualitative or quantitative) of the
magnitude, frequency, duration, and route of exposure.

Exposure Pathway.2 The course a chemical or physical agent takes from a source to an
exposed organism.  Each exposure pathway incudes a source or release from a source, an
exposure point, and an exposure route.  If the  exposure point differs from the source,
transport/exposure media (i.e.,  air, water)  also are included.

Exposure Pathway Model.  A model in which potential pathways of exposure are  identified
for the selected receptor species.

Exposure Point.2  A location of potential contact between an organism and a chemical or
physical agent.

Exposure Point Concentration.  The concentration  of a contaminant occurring at an
exposure point.

Exposure Profile.6 The product of characterizing exposure in the analysis phase of
ecological risk assessment.  The exposure profile summarizes the  magnitude and spatial and
temporal patterns of exposure for the scenarios described in the conceptual model.
                                       Glossary-5

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Exposure Route.2  The way a chemical or physical agent comes in contact with an organism
(i.e., by ingestion, inhalation, or dermal contact).

Exposure Scenario.6  A set of assumptions concerning how an exposure takes place,
including assumptions about the exposure setting, stressor characteristics, and activities of an
organism that can lead to exposure.

False Negative. The conclusion that an event (e.g., response to a chemical) is negative when
it is in fact positive (see Appendix D).

False Positive.  The conclusion that an event is positive when it is in fact negative (see
Appendix D).

Fate.  Disposition of a material in various environmental compartments (e.g. soil or
sediment, water, air, biota) as a result of transport, transformation, and degradation.

Food-Chain Transfer. A  process by which substances in the tissues of lower-trophic-level
organisms are transferred to the higher-trophic-level organisms that feed on them.

Forage (feeding) Area.  The area utilized by an organism for hunting or gathering food.

Habitat.1 Place where a plant or animal lives, often characterized by a dominant plant form
and physical characteristics.

Hazard.  The likelihood that a substance will cause an injury or adverse effect under
specified conditions.

Hazard Identification.2 The process of determining whether exposure to a stressor can
cause an increase in the incidence of a particular adverse effect, and whether an adverse
effect is likely to occur.

Hazard Index.3 The sum  of more than one hazard quotient for multiple substances and/or
multiple exposure pathways.  The HI is calculated separately for chronic, subchronic, and
shorter-duration exposures.
                   \
Hazard Quotient.2  The ratio of an exposure level to a substance to a toxicity value selected
for the risk assessment for  that substance (e.g., LOAEL or NOAEL).

Home Range.12 The area  to which an animal confines its activities.

Hydrophilic.22  Denoting the property of attracting or associating with water molecules;
characteristic of polar or charged molecules.
                                        Glossary-6

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 Hydrophobic.12 With regard to a molecule or side group, tending to dissolve readily in
 organic solvents, but not in water, resisting wetting, not containing polar groups or sub-
 groups.

 Hypothesis.12  A proposition set forth as an explanation for a specified phenomenon or group
 of phenomena.

 Indirect Effect.6  An effect where the stressor acts on supporting components of the
 ecosystem, which in turn have an effect on the ecological component of interest.

 Ingestion Rate.  The rate at which an organism consumes food, water, or other materials
 (e.g., soil, sediment). Ingestion  rate  usually is expressed in terms of unit of mass or volume
 per unit of time (e.g., kg/day, L/day).

 lonization.    The process by which a neutral atom loses or gains electrons, thereby acquiring
 a net charge and becoming an ion.

 Lethal.5 Causing death by direct action.

 Lipid.13 One of a variety  of organic substances that are insoluble in polar solvents, such as
 water, but that  dissolve readily in non-polar organic solvents.  Includes fats, oils, waxes,
 steroids, phospholipids, and carotenes.

 Lowest-Observable-Adverse-Effect Level (LOAEL). The lowest level of a stressor
 evaluated in a toxicity test  or biological field survey that has a statistically significant adverse
 effect on the exposed organisms compared with unexposed organisms in a control or
 reference site.

 Matrix.14  The substance in which an analyte is embedded or contained; the properties of a
 matrix depend  on its constituents and form.

Measurement  Endpoint.6   A measurable ecological characteristic that is related to the valued
characteristic chosen as the assessment endpoint.  Measurement endpoints often are expressed
as the statistical or arithmetic summaries of the observations that make up the measurement.
 As used in this guidance document, measurement endpoints can include measures of effect
 and measures of exposure,  which is a departure from U.S. EPA's (1992a) definition which
 includes only measures of effect.

Media.15  Specific environmental compartments—air, water, soil—which are the subject of
regulatory concern and activities.

Median Effective Concentration (EC50).5  The concentration of a substance to which test
organisms are exposed that is estimated to be effective in producing some sublethal  response
in 50 percent of the test population.  The EC50 usually is expressed as a time-dependent value

                                        Glossary-7

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 (e.g., 24-hour EC50).  The sublethal response elicited from the test organisms as a result of
 exposure must be clearly defined.

 Median Lethal  Concentration (LC50).5 A statistically or graphically estimated
 concentration that is expected to be lethal to 50 percent of a group of organisms under
 specified conditions.

 Metric.    Relating to measurement; a type of measurement—for example a measurement of
 one of various components of community structure (e.g., species richness, % similarity).

 Mortality.  Death rate or proportion of deaths  in a population.

 No-Observed-Adverse-Effect Level (NOAEL).5  The highest level  of a stressor evaluated in
 a toxicity test or biological field survey that causes no statistically significant difference in
 effect compared with  the controls or a reference site.

 Nonparametric.17  Statistical methods that make no assumptions regarding the distribution of
 the data.

 Parameter.18 Constants applied to a model that are obtained by theoretical calculation or
 measurements taken at another time and/or  place, and are assumed to be appropriate for the
 place and time being  studied.

 Parametric.   Statistical methods used  when the distribution of the data is known.

 Population.   An aggregate of individuals of a species within a specified location in space
 and time.

 Power.10  The power of a statistical test indicates the probability of  rejecting the null
 hypothesis when it should be rejected (i.e.,  the null hypothesis is false).  Can be considered
 the sensitivity of a statistical test. (See also Appendix D.)

Precipitation.14 In analytic chemistry, the process of producing a separable solid  phase
within a liquid medium.

Precision.19 A  measure of the  closeness of agreement among individual measurements.

Reference Site.11  A  relatively  uncontaminated site used for comparison to contaminated sites
in  environmental monitoring studies, often incorrectly referred to as  a control.

Regression Analysis.10  Analysis of the functional relationship between two variables; the
independent variable is described on the X  axis and the dependent variable is described on the
Y  axis (i.e, the change in Y is a function of a change in X).
                                        Glossary-8

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Replicate.  Duplicate analysis of an individual sample. Replicate analyses are used for
quality control.

Representative Samples.18 Serving as a typical or characteristic sample; should provide
analytical results that correspond with actual environmental quality or the condition
experienced by the contaminant  receptor.

Risk.   The expected frequency  or probability of undesirable effects resulting from exposure
to known or expected stressors.

Risk Characterization.6  A phase of ecological risk assessment that integrates the results of
the exposure and ecological effects  analyses to evaluate the likelihood of adverse ecological
effects associated with exposure  to the stressor.  The ecological significance of the adverse
effects is discussed, including consideration of the types and magnitudes of the effects, their
spatial and  temporal  patterns, and the likelihood of recovery.

Sample.14  Fraction  of a material tested or analyzed; a selection or collection from a larger
collection.

Scientific/Management Decision Point (SMDP).  A point during the risk assessment process
when the risk assessor communicates results of the assessment at that stage to a risk manager.
At this point the risk manager determines whether the  information is  sufficient to arrive at a
decision regarding risk management strategies and/or the need for additional information to
characterize risk.

Sediment.20 Paniculate material lying below  water.

Sensitivity.  In relation to toxic  substances, organisms that are more sensitive exhibit adverse
(toxic) effects at lower exposure levels than organisms that are less sensitive.

Sensitive Life  Stage. The life stage (i.e., juvenile, adult,  etc.)  that exhibits the highest degree
of sensitivity (i.e., effects are evident at a lower exposure concentration) to a contaminant  in
toxicity tests.

Species.13   A group  of organisms that actually or potentially interbreed and are reproductively
isolated from all  other such groups; a taxonomic grouping of morphologically similar
individuals; the category below genus.

Statistic.10  A  computed or estimated statistical quantity such as  the mean, the standard
deviation, or the correlation coefficient.

Stressor.6   Any physical, chemical, or biological entity that can induce an adverse response.
                                         Glossary-9

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 Sublethal.5  Below the concentration that directly causes death. Exposure to sublethal
 concentrations of a substance can produce less obvious effects on behavior, biochemical
 and/or physiological functions, and the structure of cells and tissues in organisms.

 Threshold Concentration.5 A concentration above  which some effect (or response) will be
 produced and below which it will not.

 Toxic Mechanism of Action.23 The mechanism by which chemicals produce their toxic
 effects, i.e., the mechanism  by which a chemical alters normal cellular biochemistry and
 physiology.  Mechanisms can include; interference with normal receptor-ligand interactions,
 interference with membranae functions, interference with cellular energy production, and
 binding to biomolecules.

 Toxicity Assessment. Review of literature, results in toxicity tests, and data from field
 surveys regarding the toxicity of any given material to an appropriate receptor.

 Toxicity Test.5 The means by which the toxicity of a chemical or other test material is
 determined.  A toxicity test  is used to measure the degree of response produced by exposure
 to a specific  level of stimulus (or concentration of chemical) compared with an unexposed
 control.

 Toxicity Value.2  A numerical expression of a substance's exposure-response relationship that
 is used in risk assessments.'

 Toxicant.  A poisonous substance.

 Trophic Level.6  A functional classification of taxa within a community that is based on
 feeding relationships (e.g., aquatic and terrestrial plants make up the first trophic level, and
 herbivores make up the second).

 Type I Error.10  Rejection  of a true null hypothesis (see also Appendix D).

 Type II Error.10 Acceptance of a false null hypothesis (see also Appendix D).

 Uptake.5  A process by which materials  are transferred into or onto an organism.

 Uncertainty.] ]  Imperfect knowledge concerning the present or future state of the  system
 under consideration; a component of risk resulting from imperfect knowledge of the degree of
 hazard or of its spatial and temporal distribution.

Volatilization.14  The conversion of a chemical  substance from a liquid or solid state to a
                                                                                       i
 gaseous vapor state.
                                       Glossary-10

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 Xenobiotic.6 A chemical or other stressor that does not occur naturally in the environment.
 Xenobiotics occur as a result of anthropogenic activities such as the application of pesticides
 and the discharge of industrial chemicals to air, land, or water.
 ENDNOTES

 1 Krebs 1978, 2 U.S. EPA 1989, 3 Calow 1993, 4 Freedman  1989, 5 Rand and Petrocelli
 1985, 6 U.S. EPA 1992a, 7 Ricklefs 1990, 8 U.S. EPA 1992b, 9 ASTM 1993a, 10 Sokal and
 Rohlf 1981, n Suter 1993, 12 Wallace et al. 1981, 13 Curtis  1983, 14 Parker 1994, 15 Sullivan
 1993, 16 U.S. EPA 1990, 17 Zar 1984, 18 Keith 1988, 19 Gilbert 1987,  20 ASTM 1993b,
 21 Huggett et al. 1992, ^ Stedman 1995, 23 Amdur et al. 1991.
GLOSSARY REFERENCES

Amdur, M.O.; Doull J.; Klaassen, C.D.  1991.  Casarett and Doull's Toxicology. Fourth
       Edition.  New York, NY: McGraw-Hill.                     .

American Society for Testing and Materials (ASTM). 1993a. ASTM Standard E 943.
       Standard terminology relating to biological effects and environmental fate.

American Society for Testing and Materials (ASTM). 1993b. ASTM Standard E 1525.
       Standard guide for designing biological tests with sediments.

Calow, P. (ed.).  1993. Handbook of Ecotoxicology. Volume!.  Boston, MA: Blackwell
       Publishing.

Curtis, H.  1983.  Biology.  Fourth Edition. New York, NY:  Worth.

Freedman, B. 1989.  Environmental Ecology. The Impacts of Pollution and Other Stresses
       on Ecosystem Structure and Function.  New York, NY: Academic Press.

Gilbert, R.O.  1987. Statistical Methods for Environmental Pollution Monitoring. New York,
       NY:  Reinhold.

Keith, L.H. (ed.).  1988. Principles of Environmental Sampling.  American Chemical Society.

Krebs, C.J. 1978.  Ecology: The experimental analysis of distribution and abundance.
       Second edition.  New York, NY: Harper & Row.

Huggett, R.J.; Kimerle, R.A.; Nehrle, P.M. Jr.; Bergman, H.L. (eds.). 1992. Biomarkers:
       Biochemical, Physiological, and Histological Markers of Anthropogenic Stress.  A
       Special Publication of SET AC.  Chelsea, MI: Lewis Publishers.

                                      Glossary-11

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 Parker, S.P. (ed.).  1994.  Dictionary of Scientific and Technical Terms.  Fifth Edition.  New
       York, NY:  McGraw-Hill.

 Rand, G.M.; Petrocelli, S.R.  1985. Fundamentals of Aquatic Toxicology. Methods and
       Applications.  New York, NY: McGraw Hill.

 Ricklefs, R.E. 1990.  Ecology.  Second Edition.  New York, NY: W.H. Freeman.

 Sokal, R.R.; Rohlf, FJ.  1981.  Biometry.  Second Edition.  New York, NY:  W.H. Freeman.

 Stedman, T.L. 1995.  Stedman's Medical Dictionary. 26th Edition. Baltimore, MD:
       Williams  and Wilkins.

 Sullivan, T.F.P.  1993. Environmental Regulatory Glossary. Government Institutes, Inc.

 Suter, G.W. n.  1993. Ecological Risk Assessment.  Ann Arbor, ML Lewis.

 U. S. Environmental Protection Agency (U.S. EPA). 1989. Risk Assessment Guidance for
       Superfund:  Volume 1 - Human Health. Washington, DC:  Office of Emergency and
       Remedial Response; EPA/540/1-89/002.

 U. S. Environmental Protection Agency (U.S. EPA). 1990.  Macroinvertebrate Field and
       Laboratory Methods for Evaluating the Biological Integrity of Surface Waters.
       Washington, DC:  Office of Water; EPA/600/4-90/030.

 U. S. Environmental Protection Agency (U.S. EPA). 1992a. Framework for Ecological Risk
       Assessment. Washington, DC: Risk Assessment Forum; EPA/630/R-02/011.

 U.S. Environmental Protection Agency (U.S. EPA).  1992b. Sediment  Classification Methods
       Compendium.   Washington, DC:  Office of Water; EPA/823/R-092/006.

Wallace, R.A.; King, J.L.; Sanders, G.P.  1981.  Biology. The Science of Life. Second
       Edition. IL: Scott, Foresman & Co.

Zar, J.H.   1984.  Biostatistical Analysis.  Princeton, NJ:  Prentice-Hall.
                                      Glossary-12

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            APPENDIX A

EXAMPLE ECOLOGICAL RISK ASSESSMENTS
       FOR HYPOTHETICAL SITES

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                                  INTRODUCTION

       Appendix A provides examples of Steps  1 through 5 of the ecological risk assessment
process for three hypothetical sites:

       (1)     A former municipal landfill from  which copper is leaching into a large pond
              down-gradient of the site (the copper site);

       (2)     A former chemical production facility that spilled DDT, which has been
              transported into a nearby stream by surface water runoff (the DDT site); and

       (3)     A former waste-oil recycling facility that disposed of PCBs in a lagoon from
              which extensive soil contamination has resulted (the PCB  site).

These examples are intended to illustrate key points in Steps 1 through 5 of the ecological
risk assessment process. No actual site is the basis for the examples.

       The examples stop with Step 5 because the remaining steps (6 through 8) of the
ecological risk assessment process and the risk management decisions depend on site-specific
data collected during a site investigation.  We have not attempted to develop hypothetical data
for analysis or the  full range of information that a site risk manager would consider when
evaluating remedial options.

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                          EXAMPLE 1:  COPPER SITE
 STEP 1: SCREENING-LEVEL PROBLEM FORMULATION AND ECOLOGICAL
 EFFECTS EVALUATION

       Site history. This is a former municipal landfill located in an upland area of the
 mid-Atlantic plain.  Residential, commercial, and industrial refuse was disposed of at this site
 in the 1960s and 1970s. Large amounts of copper wire also were disposed at this site over
 several years.  Currently, minimal cover has been placed over the fill and planted with
 grasses.  Terrestrial ecosystems in the vicinity of the landfill include upland forest and
 successional fields.  Nearby land uses include agriculture and residential and commercial uses.
 The landfill cover has deteriorated in several locations.  Leachate seeps have been noted on
 the slope of the landfill, and several seeps discharge to a five-acre pond down-gradient of the
 site.

       Site visit.  A preliminary site visit was conducted and the ecological checklist was
 completed. The checklist indicated that the pond has an organic substrate; emergent
 vegetation, including cattail and rushes, occurs along the shore near the leachate seeps; and
 the pond reaches a depth of five feet toward the middle. Fathead minnows, carp, and several
 species of sunfish were observed, and the benthic macroinvertebrate community appeared to
 be diverse. The pond water was clear, indicating an absence of phytoplankton.  The pond
 appears to function as a valuable habitat for fish and other wildlife using this area.
 Preliminary sampling indicated elevated copper levels in the seep as well as elevated base
 cations, total organic carbon (TOC), and depressed pH levels (pH 5.7).

       Problem formulation.  Copper is leaching from the landfill into the pond from a
 seep area. EPA's ambient water quality criteria document for copper (U.S. EPA, 1985)
 indicates that it can cause toxic effects in aquatic plants, aquatic  invertebrates, and young fish
 at relatively low water concentrations.  Thus, the seep might threaten the ability of the pond
 to suppon macroinvertebrate and fish communities and the wildlife that feed on them.
Terrestrial ecosystems do not need to be evaluated because the overland flow of the seeps is
limited to short gullies, a few inches wide.  Thus, the area of concern has been identified as
the  five-acre pond and the  associated leachate seeps.  Copper in  surface water and sediments
of the pond might be of ecological concern.

       Ecological effects evaluation.  Copper is toxic to both aquatic plants and aquatic
animals.  Therefore, aquatic toxicity-based data will  be used to screen for ecological risk in
the  preliminary risk calculation. The screening ecotoxicity value selected for water-column
exposure is the U.S. EPA chronic ambient water quality criterion (12 ug/L at a water, hardness
of 100 mg/L as CaCO3). A screening ecotoxicity value for copper in sediments was
identified as 34 mg/kg (U.S. EPA, 1996).
                                         A-l

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STEP 2:  SCREENING-LEVEL EXPOSURE ESTIMATE AND RISK CALCULATION

       Exposure estimate.  Preliminary sampling data indicate that the leachate contains
53 ug/L copper as well as elevated base cations, elevated TOC, and depressed pH (pH 5.7). '
Sediment concentrations range from 300 mg/kg to below detection (2 mg/kg), decreasing with
distance from the leachate seeps.
          i
       Risk calculation. The copper concentration in the seep water (53 Mg/L) exceeds the
chronic water quality criterion for copper (12 Mg/L). The maximum sediment copper
concentration of 300 mg/kg exceeds the screening ecotoxicity value for copper in sediments
(34 mg/kg).  Therefore, the screening-level hazard quotients for both sediment and  water
exceed one.  The decision at the Scientific/Management Decision Point (SMDP) is  to continue
the ecological risk assessment

       Similar screening for the levels of base cations generated hazard quotients below one
in the seep water.  .Although TOC and pH are not regulated under CERCLA, the possibility
that those parameters might affect the biota of the pond should be kept in mind if surveys of
the pond biota are conducted.  Sediment concentrations of chemicals other than copper
generated hazard quotients (HQs) of less than one at the maximum concentrations found.
STEP 3:  BASELINE RISK ASSESSMENT PROBLEM FORMULATION

       Based on the screening-level risk assessment, copper is known to be the only
contaminant of ecological concern at the site.

       Ecotoxicity literature review.  A review of the literature on the ecotoxicity of
copper to aquatic biota was conducted and revealed several types of information. Young
aquatic organisms are more sensitive to copper than adults (Demayo et al., 1982; Kaplan and
Yoh, 1961; Hubschman,  1965). Fish  larvae usually are more sensitive than embryos (McKim
et al., 1978; Weis and Weis, 1991), and fish become less sensitive to copper as body weight
increases (Demayo et al., 1982). Although the exact mechanism of toxicity to fish is
unknown, a loss of osmotic control has been noted in some studies (Demayo et al.  1982;
Cheng and  Sullivan, 1977).

       Flowthrough  toxicity studies in which copper concentrations were measured revealed
LC50 values ranging from 75 to 790 ug/L for fathead minnows and 63 to 800 ug/L for
common carp (U.S. EPA, 1985). Coldwater fish species, such as rainbow trout, can be more
sensitive, and species like pumpkinseeds (a sunfish) and bluegills are less sensitive (U.S.
EPA,  1985).  Although fish fry usually are the most sensitive life stage, this is not always the
case; Pickering et  al. (1977) determined an LC50 of 460 ug/L to 6-month-old juveniles and an
LC50 of 490 pg/L to 6-week-old fry for fathead minnows.  A copper concentration in water
of 37  ug/1 has been shown to cause a significant  reduction in fish egg production (Pickering
et al., 1977).

                                        A-2

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       Elevated levels of copper in sediments have been associated with changes in benthic
 community structure, notably reduced numbers of species (Winner et al.,  1975; Kraft and
 Sypniewski, 1981).  Studies also have been conducted with adult Hyalella azteca (an
 amphipod) exposed to copper in sediments.  One of these studies indicated an LC50 of 1,078
 mg/kg in the sediment (Cairns et al., 1984); however, a no-observed-adverse-effect level
 (NOAEL) for copper in sediments was not identified for an early life stage of a benthic
 invertebrate.

       A literature review of the ecotoxicity of copper to aquatic plants, both algae and
 vascular plants, did not reveal information on the toxic mechanism by which copper affects
 plants.  The review did indicate that exposure of plants to high copper levels inhibits
 photosynthesis and growth (U.S. EPA, 1985), and cell separation after cell division (Hatch,
 1978).  Several studies conducted using Selenastrum  capricornutum indicated that
 concentrations at 300 ug/L kill algae after 7 days, and a value of 90 ug/1 causes complete
 growth inhibition after 7 days (Bartlett et al., 1974).

       The  literature indicates that copper does not biomagnify in food chains and does not
 bioaccumulate in most animals because it is a biologically regulated essential element.
 Accumulation in phytoplankton and filter-feeding mollusks, however, does occur.  The
 toxicity of copper in water is influenced by water hardness, alkalinity, and pH (U.S. EPA,
 1985).

       Exposure pathways. A flow diagram was developed to  depict the environmental
 pathways that could  result in impacts of copper to the pond's biota (see Exhibit A-l). Direct
 exposure to copper in the pond water and sediments could cause acute or chronic toxicity in
 early life stages of fish and/or benthic invertebrates, and in aquatic plants. Risks to filter-
 feeding mollusks and phytoplankton as well as animals that feed on them are not considered
 because the mollusks and phytoplankton are unlikely to occur in significant quantities in  the
 pond.  The exposure pathways that will be evaluated, therefore,  are direct contact with
 contaminated sediments and water.

       Assessment endpoints and conceptual model. Based on the screening-level
risk assessment, the ecotoxicity literature review, and the complete exposure pathways,
development of a conceptual model for the site is initiated.  Copper can be acutely or
 chronically toxic to organisms in an aquatic community through direct exposure of the
 organisms to copper in the water and sediments.  Threats of copper to higher trophic level
 organisms are unlikely to exceed threats to organisms at the base of the food chain, because
 copper is an essential nutrient which is effectively regulated by most organisms if the
exposure is  below toxic levels.  Fish fry in particular can be very sensitive to copper in water.

       Based on these receptors and the potential  for both acute and chronic toxicity, an
appropriate general assessment endpoint for the ecosystem would be the maintenance of  the
community composition of the pond.  A more operational definition of the assessment
endpoint would be the maintenance of pond community structure typical for the locality  and

                                           A-3

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 for the physical attributes of the pond, with no loss of species or community alteration due to
 copper toxicity.

       Risk questions. One question is whether the concentrations of copper present in the
 sediments and water over at least pan of the pond are toxic to aquatic plants or animals.  A
 further question is what concentration of copper in sediments represents a threshold for
 adverse effects.  That level could be used as a preliminary cleanup goal.
STEP 4:  MEASUREMENT ENDPOINTS AND STUDY DESIGN

       To answer the hypothesis identified in Step 3, three lines of evidence were considered
when selecting measurement endpoints:  (1) whether the ambient copper levels are higher
than levels known to be directly toxic to aquatic organisms likely or known to be present in
the pond; (2) whether water and sediments taken from the pond are more toxic to aquatic
organisms than water and sediments from a reference pond; and  (3) whether the aquatic
community structure in the site pond is simplified  relative to a reference pond.

       Measurement endpoints.  Since the identified assessment endpoint is maintaining
a typical pond community structure, the possibility of directly measuring the condition of the
plant, fish, and macroinvertebrate communities in the pond was considered.  Consultation with
experts on benthic macroinvertebrates suggested that standard measures of the pond benthic
invertebrate community probably would be insensitive measures  of existing effects at this
particular site because of the high spatial variation in benthic communities within and among
ponds of this size.  Measuring the fish community also would be unsuitable, due to the
limited size of the pond and low diversity of fish species anticipated.  Since copper is not
expected to bioaccumulate or biomagnify in this pond, direct toxicity testing was selected as
appropriate. Because early life stages tend to be more sensitive to the toxic effects of copper
than older life stages, chronic toxicity would be measured on early life stages.  For animals,
toxicity is defined as a statistically significant decrease in survival  or juvenile growth rates
(measurement endpoints) of a test group exposed to water or sediments from the site
compared with  a test group exposed to water or sediments from  a reference site. For plants,
toxicity is defined as a statistically significant decrease in growth rate (measurement endpoint)
with the same comparison.

       One toxicity test selected is a 10-day (i.e., chronic) solid-phase sediment toxicity test
using an early life stage of Hyalella azteca.  The measures of effects for the test are mortality
rates and growth rates (measured as length and weight increases).  Two water-column toxicity
tests will be used:  (1) a 7-day test using the alga Selenasirum capricornutum (growth test)
and (2) a 7-day larval fish test using Pimephales promelas (mortality and growth endpoints).
The H. azteca and P.  promelas toxicity tests will be used to determine the effects of copper
on early life stages of invertebrates and fish in sediment and the water column, respectively.
The test on S. capricornutum will be used to determine the phytotoxicity of copper in the
water column.

                                          A-4

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             EXHIBIT A-1
Conceptual Model for the Copper Site
                          MEASUREMENT ENDPOINT
                        (Sediment loxicity to Hyatella azieca)
PRIMARY SOURCE
(Landfill)
h

SECONDARY
SOURCE
(Groundwater seep)
                 TERTIARY SOURCE
                (Sediment, exposure point
                 for aquatic receptors)
                 TERTIARY SOURCE
                (Surface water, exposure
               point for aquatic receptors)
   ASSESSMENT
    ENDPOINT
AQUATIC RECEPTOR
   ASSESSMENT
     ENDPOINT
AQUATIC RECEPTOR
                           MEASUREMENT ENDPOINT
                        (Surface water toxicity to Selenastrum
                       capricornatum and Pimephalespromelas)

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       Study design.  To answer the questions stated in the problem formulation step, the
 study design specified in the following.  The water column tests will be run on 100 percent
 seep water, 100 percent  pond water near the seep, 100 percent reference-site water, and the
 laboratory  control. U.S. EPA test protocols will be followed. Five sediment samples will be
 collected from the pond bottom at intervals along the observed concentration gradient, from a
 copper concentration of 300 mg/kg at the leachate seeps down to approximately 5 mg/kg near
 the other end of the pond.  The sediment sampling locations will transect the pond at
 equidistant locations  and include the point of maximum pond depth.  All sediment samples
 will be split so that copper concentrations can be measured in sediments from each sampling
 location. A reference sediment will be coUected and a laboratory control will be  run. Test .
 organisms  will not be fed during the test; sediments will be sieved to remove native
 organisms  and debris. Laboratory procedures will follow established protocols and will be
 documented and reviewed prior to initiation of the test. For the water-column test, statistical
 comparisons will be made between responses to each of the two pond samples and the
 reference site, as well as the laboratory control.  Statistical comparisons also will  be made of
 responses to sediments taken from each sampling location and responses to the reference
 sediment sample.

       Because leachate seeps can be intermittent (depending on rainfall), the study design
 specifies that a  pre-sampling visit is required to confirm that the seep is flowing and can be
 sampled. The study  design also specifies that both sediments and water will be sampled at
 the same time at each sampling location.

       As the work plan (WP) and sampling and analysis plan (SAP) were finished, the
ecological risk assessor and the risk manager agreed on the site conceptual model, assessment
endpoints,  and study  design (SMDP).
STEPS: FIELD VERIFICATION OF STUDY DESIGN

       A site assessment was conducted two days prior to the scheduled initiation of the site
investigation to confirm that the seep was active. It was determined that the seep was active
and that the site investigation could be initiated.

REFERENCES

Bartlett, L.; Rabe, F.W.; Funk, W.H.  1974.  Effects of copper, zinc, and cadmium on
       Selenastrum capricornutum.  Water Res. 8: 179-185.

Cairns, M.A.; Nebeker, A.V.; Gakstatter, J.H.; Griffis, W.L.  1984. Toxicity of copper-spiked
       sediments  to freshwater invertebrates. Environ. Toxicol. Chem. 3:  345-445.

Cheng, T.C.; Sullivan, J.T.  1977. Alterations in the osmoregulation of the pulmonate
       gastropod Biomphalaria glabrata due to copper.  J. Invert. Path. 28:  101.


                                          A-6

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Demayo, A., et al.  1982.  Effects of copper on humans, laboratory and farm animals,
       terrestrial plants, and aquatic life. CRC Crit. Rev. Environ. Control.  12:  183.

Hatch, R.C.  1978.  Poisons causing respiratory insufficiency. In: L.M. Jones, N.H, Booth
       and L.E. McDonald (eds.), Veterinary Pharmacology and Therapeutics.  Iowa State
       University, IA:  Ames Press.

Hubschman, J.H.  1965.  Effects of copper on the crayfish Orconectes rusticus (Girard). I.
       Acute toxicity.  Crustaceana 12: 33-42.

Kaplan, H.M.; Yoh, L.  1961.  Toxicity of copper to frogs.  Herpetologia 17:  131-135.

Kraft, KJ.; Sypniewski, R.H.   1981. Effect of sediment copper on the distribution of benthic
       macroinvertebrates  in the Keweenaw Waterway.  J. Great Lakes Res. 7:  258-263.

McKim, J.M.; Eaton, J.G.; Holcombe, G.W.  1978. Metal toxicity to embryos and larvae of
       eight species of freshwater fish.  II. Copper. Bull. Environ. Contain. Toxicol. 19:
       608-616.

Pickering, Q.; Brungs, W.; Gast, M. 1977.  Effect of exposure time and copper concentration
       of fathead minnows, Pimephales promelas (Rafinesque).  Aquatic Toxicol. 12:  107.

U.S. Environmental Protection Agency (U.S. EPA). 1996.  Ecotox Thresholds.  ECO Update,
       Intermittent Bulletin, Volume 3, Number 2.  Washington, DC:  Office of Emergency
       and Remedial Response, Hazardous Site Evaluation Division; Publication 9345.0-
       12FSI; EPA/540/F-95/038; NTIS PB95-963324.

U.S. Environmental Protection Agency (U.S. EPA). 1985.  Ambient Water Quality Criteria
      for Copper.  Washington, DC:  Office of Water; EPA/440/5-84/031.

Weis, P.; Weis, J.S.  1991. The developmental toxicity of metals and metalloids in fish.  In:
       Newman, M.C.; Mclntosh, A.W. (eds.), Metal Ecotoxicology:  Concepts and
       Applications.  Boca Raton, FL:  CRC Press, Inc., Lewis Publishers.

Winner, R.W.; Kelling, T.; Yeager, R.; et al.   1975. Response of a macroinvertebrate fauna
       to a copper gradient in an experimentally-polluted stream. Verb. Int. Ver. Limnol. 19:
       2121-2127.                              ,
                                         A-7

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                           EXAMPLE 2:  DDT SITE
 STEP 1:  SCREENING-LEVEL PROBLEM FORMULATION AND ECOLOGICAL
 EFFECTS EVALUATION

       Site history. This is the site of a former chemical production facility located
 adjacent to a stream.  The facility manufactured and packaged dichlorodiphenyltrichloroethane
 (DDT). Due to poor storage practices, several DDT spills have occurred.

       Site visit A preliminary site visit was conducted and the ecological checklist was
 completed. Information gathered indicates that surface water drainage from the site flows
 through several drainage swales toward an unnamed creek. This creek is a second-order
 stream containing riffle-run areas and small pools.  The stream substrate is composed of sand
 and gravel in the pools with some depositional areas in the backwaters and primarily cobble
 in the riffles.

       Problem formulation. Previous sampling efforts indicated the presence of DDT
 and its metabolites in the stream's sediments over several miles  at concentrations up to
 230 mg/kg. A variety of wildlife, especially piscivorous birds, use this area for feeding.
 Many species of minnow have been noted in this stream.  DDT is well known for its
 tendency to bioaccumulate and biomagnify in food chains, and available evidence indicates
 that it can cause reproductive  failure in birds due to eggshell thinning.

       The risk assessment team and risk manager agreed that the assessment endpoint is
 adverse effects on reproduction of high-trophic-level wildlife, particularly piscivorous birds.

       Ecological effects evaluation.  Because DDT is well studied, a dietary
 concentration above which eggshell thinning might occur was identified in existing U.S. EPA
 documents on the ecotoxicity of DDT.  Moreover,  a no-observed-adverse-effect-level
 (NOAEL)  for the ingestion route for birds also was identified.

 STEP  2:  SCREENING-LEVEL EXPOSURE ESTIMATE AND RISK CALCULATION

       Exposure estimate. For the screening-level exposure estimate, maximum
 concentrations  of DDT identified in the sediments were used. To estimate the concentration
 of DDT in forage fish, the maximum concentration in sediments was multiplied by the
 highest DDT bioaccumulation factor relating forage fish tissue concentrations to sediment
concentrations  reported in the literature.  Moreover, it was assumed that the piscivorous birds
 obtain  100 percent of their diet from the contaminated area.

       Risk calculation.  The predicted concentrations of DDT in forage fish were
compared  with the dietary NOAEL for DDT in birds. This risk screen indicated that DDT
concentrations  measured at this site might be high  enough to cause adverse reproductive

                                       A-8

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effects in birds.  Thus, transfer of DDT from the sediments to the stream and biota are of
concern at this site.
 STEP 3: BASELINE RISK ASSESSMENT PROBLEM FORMULATION

       Based on the screening-level risk assessment, potential bioaccumulation of DDT in
 aquatic food chains and effects of DDT on reproduction in piscivorous birds are known
 concerns. During refinement of the problem, the potential for additional ecological effects of
 DDT was examined.

       Ecotoxicity literature review.  In  freshwater systems, DDT can have direct effects
 on animals, particularly aquatic insects.  A literature review of the aquatic toxicity of DDT
 was conducted, and a NOAEL and LOAEL identified for the toxicity of DDT to aquatic
 insects.  Aquatic plants are not affected by DDT. Additional quantitative information on
 effects of DDT on birds was reviewed, particularly to identify what level of eggshell thinning
 is likely  to reduce reproductive success. A number of studies have correlated DDT residues
 measured in eggs of birds to increased eggshell thinning arid egg loss due to breakage.
 Eggshell thinning of more than 20 percent appears to result in decreased hatching success due
 to eggshell breakage (Anderson and Hickey,  1972; Dilworth et al., 1972). Information was
 not available for any piscivorous species of bird.  Lincer (1975) conducted a laboratory
 feeding study using American kestrels. Females fed a  diet of 6 mg/kg DDE1
 (1.1  mg/kgBW-day) produced eggs with shells  which were 25.5 percent thinner than archived
 eggshells collected prior to widespread use of DDT.  Based on this information, a LOAEL of
 1.1  mg/kgBW-day was selected to evaluate the effects  of DDT on piscivorous birds.

       Exposure pathways, assessment endpoints, and conceptual model. Based
 on knowledge  of the fate and transport of DDT in aquatic  systems and the ecotoxicity of
 DDT to aquatic organisms and birds, a conceptual model was initiated.  DDT buried in the
 sediments can  be released to the water column  during resuspension and redistribution of the
 sediments. Some diffusion of DDT to the water column from the sediment surface also will
 occur. The benthic community would be an initial receptor for the DDT in sediments, which
could result in reduced benthic species abundance and  DDT accumulation in species that
remain.  Fish that feed on benthic organisms might be  exposed to DDT both in the water
 column and in their food. Piscivorous birds  would be  exposed to the DDT that has
 accumulated in the fish, and could be exposed  at levels sufficiently high to cause more than
 20 percent eggshell thinning.  Based on this  information, two assessment endpoints were
 identified: (1) maintaining stream community structure typical for the stream order and
 location,  and (2) protecting piscivorous birds from eggshell thinning that could result in
 reduced reproductive success.
   1  DDE is a degradation product of DDT; typically, field measures of DDT are reported as the sum of the
concentrations of DDT, DDE, and DDD (another degradation product).

                                         A-9

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       A flow diagram of the exposure pathways for DDT was added to the conceptual model
(Exhibit A-2). The diagram identifies the primary, secondary, and tertiary sources of DDT at
the site, as well as the primary, secondary, and tertiary types of receptors that could be  -
exposed.

       Risk questions. Two questions were developed:  (1) has the stream community
been affected by the DDT, and (2) have food-chain accumulation and transfer of DDT
occurred to the extent that 20 percent or more eggshell thinning would be expected hi
piscivorous birds that use the area.
STEP 4:  MEASUREMENT ENDPOINTS AND STUDY DESIGN

       Measurement endpoints.  For the assessment endpoint of protecting piscivorous
birds from eggshell thinning, the conceptual model indicated that DDT in sediments could
reach piscivorous birds through forage fish. Belted kingfishers are known to feed in the
stream. They also have the smallest home range of the piscivorous birds hi the area, which
means that more kingfishers can forage entirely from the contaminated stream area than can
other species of piscivorous birds.  Thus, one can conclude that, if the risk assessment shows
no threat of eggshell thinning to the kingfisher, there should be minimal  or no threat to other
piscivorous birds that might utilize the site. Eggshell thinning hi the belted kingfisher
therefore was selected as the measure of effect.

       Data  from the literature suggest that DDT can have a bioaccumulation factor in
surface water systems  as high as six orders of magnitude (106); however, in most aquatic
ecosystems, the actual bioaccumulation of DDT  from the environment is lower, often
substantially lower.  Many factors influence the  actual accumulation of DDT in the
environment. There is considerable debate over the parameters of any proposed theoretical
bioaccumulation model; therefore, it was decided to measure tissue residue levels in the
forage fish at the site instead of estimating the tissue residue levels in forage fish using a
bioaccumulation factor (BAF).

       Existing information on the distribution of DDT in  the stream indicates that a general
gradient of DDT concentrations exists in the sediments, and five locations could be identified
that corresponded to a range of DDT concentrations in sediments. Based on information
available on  fish communities in streams.similar to the one in the site area, creek chub
(Semotilus atromaculaius) were selected to measure exposure levels  for kingfishers. Creek
chub feed on benthic invertebrates, which are in direct contact with the contaminated
sediments. Adult creek chub average  10 inches  and about 20 grams, allowing for analysis of
individual fish.  Creek chub also have small home ranges during the spring and summer, and
thus  it should be possible to relate DDT levels in the chub to DDT levels in the sediments.
                                         A-10

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                                        EXHIBIT A-2
                         Conceptual Model for the Stream DDT Site
                                                     MEASUREMENT ENDPOINT
                                                       (DDT concentration in fish
                                                        tissue, exposure point for
                                                             kingfishers)
                                                 SECONDARY
                                                  RECEPTOR
                                                    (Fish)
PRIMARY SOURCE
    (Plant site)
 SECONDARY
   SOURCE
(Surface drainage)
                                                   ASSESSMENT
                                                    ENDPOINT
                                               TERTIARY RECEPTOR
                                                  (Piscivorous bird)
 TERTIARY SOURCE
   (Stream sediments,
exposure point for fish and
   macroinvertebrales)
PRIMARY RECEPTOR
      (Benthic
  macroinvertebrates,
exposure point for fish)
                                                            MEASUREMENT ENDPOINT
                                                              (Benthic macroinvertebrate
                                                                community structure)

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       For the assessment endpoint of maintaining stream community structure, the selected
 measurement endpoints were several metrics describing the abundance and trophic structure of
 the stream benthic macroinvertebrate community.

       Study design.  The study design specified that creek chub would be collected at
 several locations with known DDT concentrations in sediments. The fish would be analyzed
 for body burdens of DDT, and the relationship between DDT levels in the sediments and in
 the creek chub would be established.  The fish DDT concentrations would be used to evaluate
 the DDT threat to piscivorous birds feeding on the fish at each location.  Using the DDT
 concentrations measured in fish that correspond to a LOAEL and NOAEL for adverse effects
 in birds,  the corresponding  sediment'contamination levels would be determined. Those
 sediment DDT levels then could be used to derive a cleanup level that would reduce threats
 of eggshell thinning to piscivorous birds.

       The study design for measuring DDT residue levels in creek chub specified that
 10 creek chub of the same size and sex would be collected at each location and that each
 creek chub be at least 20 grams, so that individuals could be analyzed. In addition, at one
 location, QA/QC  requirements dictated that an additional 10 fish be collected.  In this
 example, it was necessary to verify in the field that sufficient numbers of creek chub of the  '
 specified size were  present  to meet the tissue sampling requirements.  In  addition, the stream
 conditions needed to be evaluated to determine what fish sampling techniques would work
 best at the targeted  locations.

       The study design and methods for benthic macroinvertebrate collection followed the
 Rapid Bioassessment Protocol (RBP) manual for level three evaluation (U.S.  EPA, 1989).
 Benthic macroinvertebrate samples were co-located with sampling for fish tissue residue
 levels so that one set of co-located water and sediment samples for analytic chemistry could
 serve for comparison with both tissue analyses.

       The study design also specified that the hazard quotient (HQ) method would be used
 to evaluate the effects of DDT on the kingfisher during risk characterization.  To determine
the HQ, the estimated daily dose of DDT consumed by the kingfishers is divided by a
LOAEL of 1.1 mg/kgBW-day for kestrels.  To estimate the DDT dose to the kingfisher, the
 DDT concentrations in the chub is multiplied by the fish ingestion rate for kingfishers and
 divided by the body weight of kingfishers.  This dose is adjusted by the area use factor. The
 area use factor corresponds to the proportion of the diet  of a kingfisher that would consist of
 fish from the contaminated  area.  The area use factor is a function of the home range size of
kingfishers relative to the area of contamination.  The adjusted dose is compared to the
LOAEL.   A HQ of greater than one implies that impaired reproductive success in kingfishers
due to site contamination is likely, and an HQ of less than one implies impacts due to site
contaminants are unlikely (see text Section 2.3 for a description of HQs).
                                         A-12

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 STEP 5:  FIELD VERIFICATION OF STUDY DESIGN

       A field assessment was conducted and several small fish collection techniques were
 used to determine which technique was the most effective for capturing creek chub at the site.
 Collected chub were examined to determine the size range available and to determine if
 individuals could be sexed.

       Seine netting the areas targeted indicated that the creek chub might not be present in
 sufficient numbers to provide the necessary biomass for chemical analyses.  Based on these
 findings, a contingency plan was agreed to (SMDP), which stated that both the creek chub
 and the longnosed dace (Rhinichthys cataractae) would be collected. If the creek chub were
 collected at all locations in sufficient numbers, those samples would be analyzed and the dace
 would be released.  If sufficient creek chub could not be collected but sufficient longnosed
 dace could, the longnosed dace would be analyzed and the creek chub released.  If neither
 species could be collected at all locations in sufficient numbers, then a mix of the two species
 would be used; however, for any given  site only one species would be analyzed.  In addition,
 at one location, preferably one with high DDT levels in the sediment, sufficient numbers of
 approximately 20 gram individuals of both species would be collected to allow comparison
 (and calibration) of the accumulation between the two species.  If necessary to meet the
 analytic chemistry needs, similarly-sized individuals of both sexes of creek chub would be
 pooled. Pooling two or more individuals would be necessary for the smaller dace.  The risk
 assessment team decided that the fish samples would be collected by electro-shocking. Field
 notes for all samples would document the number of fish per sample pool, sex, weight,
 length, presence of parasites or deformities, and other measures and  might help to explain any
 anomalous data.
REFERENCES

Anderson, D.W.; Hickey, JJ.  1972.  Eggshell changes in certain North American birds.  In:
       Voos, K.H. (ed.), Proceedings: XV International Ornithological Congress. The
       Hague, Netherlands; pp. 514-540.

Dilworth, T.G., Keith, J.A.; Pearce, P.A.;  Reynolds, L.M.  1972. DDE and eggshell thickness
       in New Brunswick woodcock.  J. Wildl. Manage. 36:  1186-1193.

Lincer, J.L.  1975.  DDE-induced eggshell thinning in the American kestrel; a comparison of
       the field situation and laboratory results.  J. App. Ecol. 12:  781-793.

U.S. Environmental Protection Agency (U.S. EPA).  1989.  Rapid Bioassessment Protocols
      for Use in Streams and Rivers: Benthic Macroinvertebrates and Fish. Washington,
       DC:  Office of Water (Plafkin, J.L., Barbour, M.T., Porter, K.D.,  Gross, S.K., and
       Hughes, R.M., authors); EPA/440/4-89/001.
                                         A-13

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                            EXAMPLE 3:  PCB SITE
STEP 1:  SCREENING-LEVEL PROBLEM FORMULATION AND ECOLOGICAL
EFFECTS EVALUATION

       Site history. This is a former waste-oil recycling facility located in a remote area.
Oils contaminated with polychlorinated biphenyl compounds (PCBs) were disposed of in a
lagoon. The lagoon was not lined, and the soil is composed mostly of sand. Oils
contaminated with PCBs migrated through the soil and contaminated a wide area adjacent to
the site.

       Site visit  During the preliminary site visit, the ecological checklist was completed.
Most of the habitat is upland forest, old field, and successional terrestrial areas. Biological
surveys at this site have noted  a variety of small mammal.signs.  In addition, red-tailed hawks
were observed.

       Problem formulation.  At least 10 acres surrounding the site are known to be
contaminated with PCBs.  Some PCBs are reproductive toxins in mammals (Ringer et al.,
1972; Aulerich et al., 1985; Wren, 1991; Kamrin and Ringer, 1996). When ingested, they
induce (i.e., increase concentrations and activity of) enzymes in the liver, which might affect
the metabolism of some steroid hormones (Rice and O'Keefe, 1995).  Whatever the
mechanism of action, several physiological functions that are controlled by steroid hormones
can be altered by exposure of mammals to PCBs, and reproduction appears to be the most
sensitive endpoint for PCB toxicity in mammals (Rice and O'Keefe, 1995). Given this
information, the screening ecological risk assessment should include potential exposure
pathways for mammals to PCBs.,

       Several possible exposure pathways were evaluated for mammals.  PCBs are not
highly  volatile, so inhalation of PCBs by animals would not be an important exposure
pathway. PCBs in soils generally are hot taken up by most plants, but are accumulated by
soil macroinvertebrates.  Thus, herbivores, such as voles and rabbits, would not be exposed to
PCBs in most of their diets; whereas insectivores, such as shrews, or omnivores, such as  deer
mice, could be exposed to accumulated PCBs in their diets.  PCBs also are known to
biomagnify in terrestrial food chains; therefore, the ingestion exposure route needs evaluation,
and shrews and/or deer mice would be appropriate mammalian receptors to evaluate in this
exposure pathway.

       Potential reproductive effects on predators that feed on shrews or mice also would be
important to evaluate. The literature indicated that exposure to PCBs through the food chain
could cause reproductive impairment in predatory birds through a similar mechanism  as in
mammals.  The prey of red-tail hawks include voles, deer mice, and various insects.  Thus,
this raptor could be at risk of adverse reproductive effects.
                                        A-14

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       Ecological effects evaluation. No-observed-adverse-effect levels (NOAELs) for
 the effects of PCBs and other contaminants at the site on mammals, birds, and other biota
 were identified in the literature.
 STEP 2: SCREENING-LEVEL EXPOSURE ESTIMATE AND RISK CALCULATION

       Exposure estimate.  For the screening-level risk calculation, the highest PCB and
 other contaminant levels measured  on site were used to estimate exposures.

       Risk calculation. The potential contaminants of concern were screened based on
 NOAELs for exposure routes appropriate to each contaminant.  Based on this screen, PCBs
 were confirmed to be the only contaminants of concern to small mammals, and possibly to
 birds, based on the levels measured at this site. Thus, at the SMDP, the risk manager and
 lead risk assessor decided to continue to Step 3 of the ecological risk assessment process.
STEP 3:  BASELINE RISK ASSESSMENT PROBLEM FORMULATION

       The screening-level ecological risk assessment confirmed that PCBs are of concern to
small mammals based on the levels measured at the site and suggested that predatory birds
might be at risk from PCBs that accumulate in some of their mammalian prey.

       Ecotoxicity literature review. A literature review was conducted to evaluate
potential reproductive effects in birds.  PCBs have been implicated as a cause of reduced.
reproductive success of piscivorous birds (e.g., cormorants, terns) in the Great Lakes (Kubiak
et al., 1989; Fox et al.,  1991). Limited information was available on the effects of PCBs to
red-tailed hawks.  A study on American kestrel indicated that consumption Of 33 mg/kgBW-
day PCBs resulted hi a  significant decrease in sperm concentration  in male kestrels (Bird et
al.,  1983).  Implications of this  decrease for mating success in kestrels was not evaluated in
the study, but studies on other bird species indicate that it could increase the incidence of
infertile eggs and therefore reduce the number of young fledged per pair.  The Great Lakes
International Joint Commission  (IJC) recommends 0.1 mg/kg total PCBs as a prey tissue level
that will protect predatory birds and mammals (IJC, 1988).  (This number is used as an
illustration and not to suggest that this particular level is appropriate for a given site.)

       Exposure pathways.  The complete exposure pathways identified during Steps 1
were considered appropriate for the baseline ecological risk assessment as well.

       Assessment endpoints and conceptual model.  Based on the screening-level
risk assessment for small mammals and the results of the ecotoxicity literature search for
birds, a conceptual model  was initiated for the site, which included consideration of predatory
birds (e.g., red-tailed hawks) and their prey. The ecological risk assessor and the risk
manager agreed (SMDP) that assessment endpoints for the site would be the protection of

                                        A-15

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small mammals and predatory birds from reproductive impairment caused by PCBs that had
accumulated in their prey.

       An exposure pathway diagram was developed for the conceptual model to identify the
exposure pathways by which predatory birds could be exposed to PCBs originating in the soil
at the site (see Exhibit A-3). While voles may be prevalent at the site, they are not part of
the exposure pathway for predators because they are herbivorous and PCBs do not accumulate
in plants.  Deer mice (Peromyscus maniculatus), on the other hand, also are abundant at the
site and, being omnivorous, are likely to be exposed to PCBs that have accumulated in the
insect component of their diet.  Preliminary calculations indicated that environmental levels
likely to cause reproductive effects in predatory birds are lower than those likely to cause
reproductive effects in mice because mice feed lower in the food chain than do raptors.  The
assessment endpoint was therefore restricted to reproductive impairment in predatory birds.

       Risk questions. Based on the conceptual model, one question was whether
predatory  birds could consume a high enough dose of PCBs in their diet to impair their
reproduction. Given the presence of red-tailed hawks on site, the question was refined to ask
whether that species could consume sufficient quantities of PCBs in their diet to affect
reproduction.
STEP 4:  MEASUREMENT ENDPOINTS AND STUDY DESIGN

       Measurement endpoints.  To determine whether PCB levels in prey of the red-
tailed hawk exceed levels that might impair their reproduction, PCB levels would be
measured in deer mice taken from the site (of all of the species in the diet of the red-tailed
hawk, deer mice are assumed to accumulate the highest levels of PCBs). Based on estimated
prey ingestion rates for red-tailed hawks, a total PCB dose would be estimated from the
measured PCB concentrations in the  mice.

       Study design.  The available measures of PCB concentrations in soil at the site
indicated a gradient of decreasing PCB concentration with increasing distance from the
unlined lagoon.  Three locations along this gradient were selected to measure PCB
concentrations in deer mice.  The study design specified that eight deer mice of the same size
and sex would be collected at each location. Each mouse should be approximately  20 grams
so that contaminant levels can be measured in individual mice.  With concentrations measured
in eight individual mice, it is possible to estimate a mean concentration and an  upper
confidence limit of the mean concentration in deer mice for the location.  In addition, QA/QC
requirements dictate that an additional eight deer mice should be collected at one location.

       For this site, it was necessary to verify that sufficient numbers of deer mice of the
specified size would be present to meet the sampling requirements.  In addition, habitat
                                         A-16

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                EXHIBIT A-3
Conceptual Model for the Terrestrial PCB Site
                       MEASUREMENT ENDPOINT
                       (PCBs in mouse tissue, exposure
                         point for red-tailed hawks)
PRIMARY SOURCE
(Waste lagoons)
w

SECONDARY
SOURCE
(Site soils)
\
- h
w
PRIMARY RECEPTOR
(Deer mouse)
h

. ASSESSMENT
ENDPOINT-
SECONDARY RECEPTOR
(Predatory birds)

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 conditions needed to be evaluated to determine what trapping techniques would work at the
 targeted locations.

       The study design specified further that the hazard quotient (HQ) method would be
 used to estimate the risk of reproductive impairment in the red-tailed hawk from exposure to
 PCBs in their prey. To determine the HQ, the measured DDT concentrations in deer mice is
 divided by the LOAEL of 33 mg/kgBW-day for a decrease in sperm concentration in kestrels.
 To estimate the dose to  the red-tailed hawk, the PCB concentrations in deer mice is
 multiplied by the quantity of deer mice that could be ingested by a red-tailed hawk each day
 and divided by the body weight of the hawk.  This  dose is adjusted by a factor that
 corresponds to the proportion of the diet of a red-tailed hawk that would come from the
 contaminated area. This area use factor is a function of the home range size of the hawks
 relative to the area of contamination.  A HQ of greater than one implies that impacts due to
 site contamination are likely, and an HQ of less than one implies impacts due to site
 contaminants are unlikely.
STEP 5: FIELD VERIFICATION OF STUDY DESIGN

       A field assessment using several trapping techniques was conducted to determine (1)
which technique was most effective for capturing deer mice at the site and (2) whether the
technique would yield sufficient numbers of mice over 20 grams to meet the specified
sampling design.  On the first evening of the field assessment, two survey lines of 10 live
traps were set for deer mice in typical old-field habitat in the  area believed to contain the
desired DDT concentration gradient for the study design.  At the beginning of the second day,
the traps were retrieved.  Two deer mice over 20 grams  were  captured in each of the  survey
lines. These results indicated that collection of deer mice over a period of a week or  less
with this number and spacing of live traps should be adequate,to meet the study objectives.
REFERENCES

Aulerich, R.J.; Bursian, SJ.; Breslin, WJ.; et al.  1985. Toxicological manifestations of
       2,4,5-,2',4',5'-, 2,3,6,2',3',6'-, and 3,4,5,3',4',5'-hexachlorobiphenyl and Aroclor 1254
       in mink.  J. Toxicol. Environ. Health 15:  63-79.

Bird, D.M.; Tucker, P.H.; Fox, G.A.; Lague, P.C.  1983.  Synergistic effects of Aroclor 1254
       and mirex on the semen characteristics of American kestrels.  Arch. Environ. Contain.
       Toxicol.  12:  633-640.

Fox, G.A.; Collins, B.; Hayaskawa, E.; et al.   1991.  Reproductive outcomes in colonial fish-
       eating birds: a biomarker for developmental toxicants in Great Lakes food chains,  n.
       Spatial variation in the occurrence and prevalence of bill defects in young double-
       crested cormorants in the Great Lakes. J. Great Lakes Res. 17:158-167.

                                         A-18

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International Joint Commission (IJC) of United States and Canada.  1988.  Great Lakes Water
       Quality Agreement.  Amended by protocol. Signed 18 November 1987. Ottawa,
       Canada.

Kamrin, M.A.; Ringer, R.K.  1996. lexicological implications of PCB residues in mammals.
       In:  Beyer, W.N.; Heinz, G.H.; Redmon-Norwood, A.R. (eds.). Environmental
       Contaminants in Wildlife: Interpreting Tissue Concentrations. A Special Publication
       of the Society of Environmental Toxicology and Chemistry  (SETAC), La Point, T.W.
       (series ed.).  Boca Raton, FL:  CRC Press, Inc., Lewis Publishers, pp  153-164.

Kubiak, T.J.; Harris, H.J.; Smith, L.M.; et al.  1989. Microcontaminants and reproductive
       impairment of the Forster's tem on Green Bay, Lake Michigan—1983.  Arch.  Environ.
       Contam. Toxicol. 18: 706-727.

Rice, C.P; O'Keefe, P. 1995. Sources, pathways, and effects of PCBs, dioxins, and
       dibenzofurans.  In:  Hoffman, D.J.; Rattner, B.A.; Burton, G.A. Jr.; Cairns, J.,  Jr.
       (eds.). Handbook of Ecotoxicology.  Ann Arbor, MI:  CRC Press, Inc., Lewis
       Publishers.

Ringer, R.K.; Aulerich, R.J.; Zabik, M.  1972. Effect of dietary polychlorinated biphenyls on
       growth and reproduction  of mink.  Extended abstract.  ACS (American Chemical
       Society) 164th  Annu. Meet.  12:  149-154.

Wren, C.D.  1991.  Cause-effect linkages between chemicals  and populations of mink
       (M us tola vison) and otter (Lutra canadensis) in the Great Lakes basin.  J. Toxicol.
       Environ. Health 33:  549-585.
                                         A-19

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               APPENDIX B

REPRESENTATIVE SAMPLING GUIDANCE DOCUMENT,
          VOLUMES:  ECOLOGICAL

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                                       OSWER Directive XXXXJDC
                                            EPA 540/R/94/XXX
                                                 PBxx-xxxxxx
                                                   May 1997
                DRAFT
         SUPERFUND PROGRAM

REPRESENTATIVE SAMPLING GUIDANCE


          VOLUMES: BIOLOGICAL



              INTERIM FINAL
          Environmental Response Team Center

        Office of Emergency and Remedial Response
        Office of Solid Waste and Emergency Response

          U.S. Environmental Protection Agency
               Washington. DC 20460

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                                                Notice
The policies and procedures established in this document are intended solely for the guidance of government personnel, for
use in the Superfund Program. They are not intended, and cannot be relied upon, to create any rights, substantive or
procedural, enforceable by any party in litigation with the United States. The Agency reserves the right to act at variance
with these policies and procedures and to change them at any time without public notice.

For more information on Biological Sampling procedures, refer to the Compendium ofERT Toxicity Testing Procedures.
OSWER Directive 9360-44)8. EPA/540/P-91/009 (U.S. EPA 1991a). Topics covered in this compendium include: toxiciry
testing; and surface water and sediment sampling.

Please note mat the procedures in this document should only be used by individuals properly trained and certified under a
40 Hour Hazardous Waste Site Training Course that meets the requirements set forth in 29 CFR 1910.120(e)(3).  It should
not be used to replace or supersede any information obtained in a 40 Hour Hazardous Waste Site Training Course.

Questions, comments, and recommendations are welcomed regarding the Superfund Program Representative Sampling
Guidance, Volume 3 -- Biological. Send remarks to:


                               Mark Sprenger PhD. - Environmental Scientist
                               David Charters Ph.D. - Environmental Scientist
                              U.S. EPA • Environmental Response Center (ERC)
                                          Building 18, MS-101
                                        2890 Woodbridge Avenue
                                          Edison. NJ 08837-3679


For additional copies of the Superfund Program Representative Sampling Guidance, Volume 3 - Biological, contact*


                                  National Technical Information Services
                                          5285 Port Royal Road
                                          Springfield. VA 22161
                                          Phone (703) 487-4650


U.S.  EPA employees can order a copy by calling the ERC at (908) 321-4212

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                                            Disclaimer


This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

The following trade names are mentioned in this document:

Havahart® - Allcock Manufacturing Co.. Lititz, PA

Longworth - Longworth Scientific Instrument Company, Ltd.. England

Museum Special - Woodstream Corporation, Lititz, PA

Sherman - H.B. Sherman Traps, Tallahassee, FL
                                                  ui

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This page intentionally left blank.
               IV

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                                             CONTENTS

Notice	;	ii

Disclaimer	  iii

List of Figures  	  viii

List of Tables	:	vii

Preface	;.  ix

1.0     INTRODUCTION	;	1

        1.1     Objective and Scope	 1
        1.2     Risk Assessment Overview	 1
        1.3     Conceptual Site Model	2
        1.4     Data Quality Objectives	3
        1.5     Technical Assistance	4

2.0     BIOLOGICAL/ECOLOGICAL ASSESSMENT APPROACHES	6

        2.1     Introduction	...'... 6
        2.2     RISK EVALUATION	'.	6
                2.2.1    Literature Screening Values	6
                2.2.2    Risk Calculations  		6
                2.2.3    Standard Field Studies	6
                        2.2.3.1   Reference Area Selection  		6
                        2.232   Receptor Selection	7
                        2.23.3   Exposure-Response Relationships	•	8
                        2.23.4   Chemical Residue Studies	8
                        2.23.5   Population/Community Response Studies	9
                        2.2.3.6   Toxiciry Testing/Bioassays	9

3.0     BIOLOGICAL SAMPLING METHODS 	11

        3.1      Chemical Residue Studies	 11
                3.1.1    Collection Methods	11
                        3.1.1.1   Comparability Considerations	 12
                        3.1.12   Mammals	,	i	12
                        3.1.1.3   Fish	13
                        3.1.1.4   Vegetation  	13
                3.1.2    Sample Handling and Preparation  	'	14
                3.1.3    Analytical Methods	14

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        3.2     Population/community Response Studies	15
                3.2.1    Terrestrial Vertebrate Surveys	15
                3.2.2    Benthic Macroinvenebrate Surveys	15
                        3.2.2.1  Rapid Bioassessment Protocols for Benthic Communities	   16
                        3.2.2.2  General Benthological Surveys	16
                        3.2.23  Reference Stations	16
                        3.2.2.4  Equipment for Benthic Surveys	16
                3.2.3    Fish Biosurveys	17
                        3.23.1  Rapid Bioassessment Protocols for Fish Biosurveys	17
        3.3     Tpxicity Tests	17
                3.3.1    Examples of Acute Toxicity Tests	•... 17
                3.3.2    Examples of Chronic Toxicity Tests	18

4.0     QUALITY ASSURANCE/QUALITy CONTROL 	21

        4.1   .  Introduction	21
        4.2     Data Categories		21
        4.3     Sources of Error	21
                4.3.1    Sampling Design	21
                4.3.2    Sampling Methodology and Sample Handling		.. 22
                4.3.3    Sample Homogeneity	22
                4.3.4    Sample Analysis	22
        4.4     QA/QC Samples 	i	23
                4.4.1    Replicate Samples	23
                4.4.2    Collocated Samples 	24
                4.43    Reference Samples	25
                4.4.4    Rinsate Blank Samples	25
                4.4.5    FieldBlank Samples	.v	25
                4.4.6    Trip Blank Samples	25
                4.4.7    Performance Evaluation/Laboratory Control Samples	25
                4.4.8    Controls ....'.	25
                4.4.9    Matrix Spike/Matrix Spike Duplicate Samples	26
                4.4.10  Laboratory Duplicate Samples	,	26
        4.5     Data Evaluation	.26
                4.5.1    Evaluation of Analytical Error	 26
                4.5.2    Data Validation	26

5.0     DATA ANALYSIS AND INTERPRETATION	27

        5.1     Introduction  	27
        5.2     Data Presentation And Analysis 	27
                                                   VI

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              5.2.1   Data Presentation Techniques	27
              522   Descriptive Statistics 	27
              5.23   Hypothesis Testing			27
       5.3     Data Interpretation	;	;:	28
              5.3.1   Chemical Residue Studies	28
              5.3.2   Population/Community Studies	.28
              5.3.3   Toxicity Testing  	:	28
              5.3.4   Risk Calculation	28

APPENDIX A - CHECKLIST FOR ECOLOGICAL ASSESSMENT/SAMPLING	30

APPENDK B - EXAMPLE OF FLOW DIAGRAM FOR CONCEPTUAL SITE MODEL	47

APPENDDC C - EXAMPLE SITES	50

REFERENCES 	53
                                            VII

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                                         List of Figures






FIGURE 1 - Conceptual Site Model	5



FIGURE 2 - Common Mammal Traps	19



FIGURES - Illustrations of Sample Plots			29
                                         List of Tables



TABLE 1 • Reference List of Standard Operating Procedures - Ecological Sampling Methods	20
                                               vui

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                                                  Preface


 This document is third in a series of guidance documents designed to assist Supcrfund Program Site Managers such as On-
 Scene Coordinators (OSCs). Site Assessment Managers (SAMs). and other field staff in obtaining representative samples
 at Supcrfund sites. It is intended to assist Supcrfund Program personnel in evaluating and documenting environmental threat
 in support of management decisions, including whether or not to pursue a response action. This document provides general
 guidance for collecting representative biological samples (i.e., measurement endpoints) once it has been determined by the
 Site Manager that additional sampling will assist in evaluating the potential for ecological risk. In addition, this document
 will:

 •       Assist field personnel in representative biological  sampling within the objectives and scope of the Superfund
         Program

 •       Facilitate the use of ecological assessments as an integral part of the overall site evaluation process

 •       Assist the Site Manager in determining whether an environmental threat exists and what methods are available to
         assess that threat

This document is intended to be used in conjunction with other existing guidance documents, most notably. Ecological Risk -
Assessment Guidance for Superfund: Process for Designing and Conducting Ecological Risk Assessments, OSWER, EPA
540-R-97/006.

The objective  of representative sampling is to ensure that a sample or a group of samples accurately characterizes site
conditions.  Biological information collected in  this manner  complements existing ecological assessment methods.
Representative  sampling within the objectives of the Superfund Program is used to:

         promote awareness of biological and ecological issues
         define the parameters of concern and the data quality objectives (DQOs)
         develop a biological sampling plan
         define biological sampling methods and equipment
         identify and collect suitable quality assurance/quality control (QA/QQ samples
         interpret and present the analytical and biological data

The National Contingency Plan (NCP) requires that short-term response (removal) actions contribute to the efficient
performance of any long-term site remediation, to the extent applicable.  Use of this document will help determine if
biological sampling should be conducted at a site, and if so, what samples will assist program personnel in the collection
of information required to make such a determination.
                                    j             -                                       '
Identification and assessment of potential environmental threats are important elements for the Site Manager to understand.
These activities can be accomplished through ecological assessments such as biological sampling. This document focuses
on the performance of ecological assessment screening approaches, more detailed ecological assessment approaches, and
biological sampling methods.
                                                     IX

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                                       1.0 INTRODUCTION
 1.1     OBJECTIVE AND SCOPE

 This document is intended to assist Superfund Program
 personnel in evaluating and documenting environmental
 threat in support of management decisions.  It presents
 ecological assessment and sampling as tools in meeting
 the objectives of trie Superfund Program, which include:

 • °      Determine threat to public health, welfare, and
         the environment

 •       Determine the need for long-term action

 •       Develop containment and control strategies

 •       Determine appropriate treatment and disposal
         options

 •       Document attainment of clean-up goals

 This document is intended to assist Superfund Program
 personnel in obtaining scientifically valid and defensible
 environmental data  for  the overall decision-making
 process  of site  actions.  Both the Comprehensive
 Environmental Response. Compensation, and Liability
 Act  (CERCLA)  [§104(a)(l)]. as amended  by the
 Superfund   Amendments  and  Reauthorization  Act
 (SARA), and the NCP (§300.400(a)(2)]. require  that the
 United States Environmental Protection Agency (U.S.
 EPA) "protect human health and the environment."

Environmental threats may. be independent of human
health threats, whether they co-exist at a site or are the
result of the  same causative agents.  It is therefore
 important  to determine and document   potential.
 substantial, and/or imminent threats to the environment
 separately from threats to human health.

 Representative sampling ensures that a sample or  a group
of sample accurately characterizes site conditions.
Representative biological sampling  and ecological risk
assessment include, but are not limited to.  the collection
of sue information and the collection of samples for
chemical  or lexicological analyses. Biological sampling
is dependent upon  specific  site  requirements during
limited  response actions or  in  emergency response
situations.    Applying   the   methods  of  collecting
environmental information, as outlined in this document,
can facilitate the decision-making process (e.g., during
chemical spill incidents).
The collection of representative samples is critical to the
site evaluation  process  since all  data  interpretation
assumes proper sample collection.  Samples collected
which inadvertently or intentionally direct the generated
data toward a conclusion are biased and therefore not
representative.

This document provides Superfund Program personnel
with general guidance  for collecting  representative
biological samples (i.e.,  measurement endpoints, [see
Section 1.2 for the definition of measurement endpoint]).
Representative biological sampling is conducted once the
Site Manager has determined that additional sampling
may assist in evaluating the potential for ecological risk.
This determination should be made in consultation with
a trained ecologist or biologist The topics covered in
this document include sampling methods and equipment,
QA/QC, and data analysis and interpretation.

The appendices in this document provide several types of
assistance.  Appendix A provides a checklist for initial
ecological assessment and sampling.   Appendix  B
provides an example flow diagram for the development
of a conceptual site model.  Appendix C provides
examples   of how  the  checklist  for   ecological
assessment/sampling is used to formulate a conceptual
site model that leads up to the design  of  a site
investigation.

This document is intended  to be used in conjunction with
other  existing  guidance documents, most notably,
Ecological Risk Assessment Guidance for Superfund:
Process for Designing and Conducting Ecological Risk
Assessments, EPA 540-R-97/006 (U.S. EPA 1997).

1.2    RISK ASSESSMENT OVERVIEW

The term ecological risk  assessment (ERA), as used in
this document, and  as  defined in  Ecological  Risk
Assessment Guidance for  Superfund:  Process for
Designing    and   Conducting   Ecological   Risk
Assessments, OSWER, EPA 540-R-97/006 (U.S. EPA
1997) refers to:
        "... a qualitative and/or quantitative
        appraisal of the actual  or potential
        impacts of a hazardous waste site on
        plants and animals other than humans
        and domesticated species."

Risk assessments are an integral pan of the Superfund

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  process and are conducted as part of the baseline risk
  assessment for the remedial investigation and feasibility
  study (RI/FS). The RI is defined by a characterization of
  the nature and extent of contamination, and ecological
  and human health risk assessments.  The nature and
  extent of contamination determines the chemicals present
  on the site.   The ecological and human health  risk
  assessments determine if the concentrations threaten the
  environment and human health.

  An ecological risk assessment is a formal process that
  integrates   knowledge   about   an   environmental
  contaminant (i.e., exposure assessment) and its potential
  effects to ecological receptors (i.e., hazard assessment).
  The  process  evaluates the  likelihood  that  adverse
  ecological effects may occur or are occurring as a result
  of exposure  to a stressor. As defined  by U.S. EPA
  (1992), a stressor is any physical, chemical or biological
  entity that can induce an adverse  ecological response.
 Adverse responses can range from sublethal chronic
  effects in an individual organism to a loss of ecosystem
  function.

 Although stressors can be biological (e.g., introduced
 species),  in  the   Superfund  Program  substances
 designated as hazardous under CERCLA are usually the
 stressors of concern. A risk does not exist unless (1) the
 stressor has the ability to cause one or  more adverse
 effects, and (2) it co-occurs with or contacts an ecological
 component long enough and at sufficient intensity to elicit
 the identified adverse effect.

 The  risk  assessment  process   also   involves  the
 identification of assessment and measurement endpoints.
 Assessment endpoints are explicit expressions of the
 actual environmental  values (e.g., ecological resources)
 that are to be protected. A measurement endpoint is a
 measurable biological response to a stressor that can be
 related  to  the  valued characteristic  chosen as  the
 assessment endpoint (U.S. EPA  1997).  Biological
 samples  are  collected  from a site to represent  these
 measurement endpoints. See Section 2.2 for a detailed
 discussion of assessment and measurement endpoints.

 Except where required under  other regulations, issues
 such as restoration,  mitigation, and replacement  are;
i important  to  the  program  but  are  reserved  for:
; investigations that may or may not be included in the RI i
I phase.   During the management  decision process of:
i selecting the preferred remedial option leading to the
! Record of Decision (ROD), mitigation and restoration
i issues should be addressed.  Note that these  issues are not j
i necessarily  issues within  the  baseline ecological risk I
 assessment.
 Guidelines for human health risk assessment have been
 established;  however,   comparable   protocols  for
 ecological risk assessment  do not  currently exist.
 Ecological Risk Assessment Guidance for Superfund:
 Process for Designing and Conducting Ecological Risk
 Assessments."  (U.S. EPA  1997) provides conceptual
 guidance  and  explains how  to design and  conduct
 ecological risk assessments  for a CERCLA RI/FS. The
 Framework for Ecological Risk Assessment (U.S. EPA
 1992) provides an Agency-wide structure for conducting
 ecological risk assessments and describes the basic
 elements for evaluating site-specific adverse effects of
 stressors on the environment-  These documents should
 be referred to for specific information regarding the risk
 assessment process.

 While the  ecological risk assessment is a necessary first
 step in a "natural  resource  damage  assessment"  to
 provide a  causal link, it is not a  damage evaluation.  A
 natural resource damage assessment may be conducted at
 any  Superfund  site at the  discretion  of the  Natural
 Resource  Trustees.   The portion  of the  damage
 assessment  beyond   the   risk  assessment  is the
responsibility of the Natural Resource Trustees, not of the
U.S.  EPA.   Therefore,   natural  resource   damage
assessment is not addressed in this guidance.

1.3     CONCEPTUAL SITE MODEL

A  conceptual site model is an integral pan of a site
 investigation and/or ecological  risk assessment as  it
provides the framework from which the study design is
structured.   The  conceptual  site model  follows
contaminants from their sources, through transport and
fate pathways (air, soil, surface water, groundwater),  to
the ecological receptors.  The conceptual model is a
strong  tool  in  the development of a  representative
sampling plan and is a requirement when conducting an
ecological risk assessment. It assists the Site Manager in
evaluating the interaction of different site features (e.g.,
drainage systems and the surrounding  topography),
thereby ensuring that contaminant sources, pathways, and
ecological  or human receptors  throughout the site have
been considered before sampling locations, techniques,
and media are chosen.

Frequently, a conceptual model is created as a site map
(Figure 1) or flow diagram that describes the potential
movement  of contaminants  to site  receptors  (see
Appendix B). Important considerations when creating a
conceptual model are:

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 •       The  state(s)  (or chemical  form)  of each
         contaminant and its potential mobility through
         various media
 •       Site topographical features
 •       Meteorological   conditions   (e.g.,   climate,
         precipitation, humidity, wind direction/speed)
 •       Wildlife area utilization.

 Preliminary and historical site information may provide
 the identification of the contaminants) of concern and the
 tevel(s) of the contamination. A sampling plan should be
 developed from the conceptual  model based on  the
 selected assessment endpoints.

 The conceptual site model (Figure 1) is applied to this
 document, Representative Sampling Guidance  Volume
 3:    Biological.   Based  on  the  model, you can
 approximate:

 • Potential Sources
         hazardous waste site (waste pile,  lagoon,
         emissions), drum dump (runoff, leachate),
         agricultural (runoff, dust, and particulates)

 • Potential Exposure Pathways
         ingestion
           waste, contained -in  the pile   on  the
           hazardous waste site; soil panicles near
           the  waste pile; drum dump; or  area of
           agricultural activity
         inhalation
           dust and particulates from  waste pile,
           drum dump, or area of agricultural activity
         absorption/direct contact
           soil near waste pile, drum dump, or area of
           agricultural  activity and  surface water
           downstream of sources

• Potential Migration Pathways
         air (paniculates and gases) from drum dump
         and area of agricultural activity
         soil (runoff) from the hazardous waste site.
         drum dump, and agricultural runoff
         surface water (river & lake) from hazardous
         waste site and agricultural runoff
         groundwater  (aquifer) from  drum dump
         leachate.

• Potential Receptors of Concern (and associated
potential  routes)
         wetland vegetation/mammals/invertebrates if
         suspected  to be in  contact with potentially
         contaminated soil and surface water
        rivenne  vegetation/aquatic   organisms  if
        suspected to be in contact with potentially
        contaminated surface water and soil
        lake vegetatum/mammals/aquatic organisms if
        suspected to be in contact with potentially
        contaminated surface water and leachate.

1.4    DATA  QUALITY OBJECTIVES

Data  quality objectives (DQOs) state  the  level  of
uncertainty  that is  acceptable  from data collection
activities. DQOs also define the data quality necessary to
make a certain decision. Consider the following when
establishing DQOs for a particular project:

•       Decision(s) to be made or question(s) to be
        answered;

•       Why environmental data are needed and  how
        the results will be used;

•       Time  and  resource  constraints  on  data
        collection;

•       Descriptions of the environmental data to be
        collected;

•       Applicable model or data interpretation method
        used to arrive at a conclusion;

•       Detection limits for analytes of concern; and

•       Sampling and analytical error.

In addition to these considerations, the quality assurance
components of precision, accuracy (bias), completeness.
representativeness, and comparability should  also be
considered. Quality assurance components are defined as
follows:

•       Precision - measurement of variability in the
        data collection process.

•       Accuracy (bias) - measurement of bias in the
        analytical process. The term "bias" throughout
        this document refers to the QA/QC accuracy
        component.

•       Completeness  —  percentage  of sampling
        measurements which are judged to be valid.

•       Representativeness - degree to which sample
        data accurately  and precisely  represent the

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         characteristics of the site contaminants and their
  •  .     concentrations.

 •        Comparability — evaluation of the similarity of
         conditions   (e.g.,  sample  depth,   sample
         homogeneity) under which separate sets of data
         are produced.

 Many of the DQOs and quality assurance considerations
 for soil, sediment, and water sampling are also applicable
 to  biological  sampling.   However,  there  are also
 additional considerations that are specific to biological
 sampling.

 •        Is biological  data  needed  to answer  the
         question(s) and, if so, how will the data be used;

 •        Seasonal,  logistical,  resource,  and  legal
         constraints on biological specimen collection;

 •        What component of the biological system will
         be collected  or evaluated (i.e., tissue samples.
         whole organisms, population data, community
         data, habitat data);

 •        The specific model or interpretation scheme to
         be utilized on the data set;

 •        The temporal, spatial, and behavioral variability
         inherent in natural systems.

 Quality assurance/quality control (QA/QC) objectives are
 discussed further in Chapter 4.

 1.5      TECHNICAL ASSISTANCE

 In this document,  ii is assumed that technical specialists
are available  to  assist Site Managers and other site
 personnel hi determining the best approach to ecological
 assessment. This assistance ensures that all approaches
 are up-to-date and that  best professional judgment is
 exercised.  Refer to Appendix A for more information.

 Support   in   designing  and   evaluating  ecological
assessments is currently available from regional technical
assistance  groups such   as   Biological  Technical
 Assistance  Groups (BTAGs).  Support is also available
 from the Environmental Response Team Center (ERTC)
as well as from other sources within each region.

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FIGURE 1:
CONCEPTUAL
SITE MODEL

WIND ROSE
STATE GAME
  LANDS
syN. r  N5j*iL3|S*&
^\%&l
                                                  WATER PLANT
                                                     INTAKE

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         2.0  BIOLOGICAL/ECOLOGICAL ASSESSMENT APPROACHES
 2.1      INTRODUCTION

 Biological  assessments vary in their level of effort,
 components,  and  complexity, depending  upon  the
 objectives of the study and specific site conditions.  An
 assessment  may  consist  of  literature-based  risk
 evaluations   and/or   site-specific   studies   (e.g.,
 population/community studies, toxicity tests/bioassays,
 and tissue residue analyses).

 Superfund Program personnel (RPMs and OSCs) may be
 limited to completing the ecological checklist (Appendix
 A) during  the. Preliminary Site Evaluations and to
 consulting an ecological specialist if it is determined that
 additional field data are  required.   The checklist is
 designed to be completed by one person during an initial
-.site visit. The checklist provides baseline data, is useful
 in designing sampling objectives; and requires a few
 hours to complete in the field.

 When the Site Manager determines that additional data
 collection is needed at a response site, the personnel and
 other resources  required  depends  on  the selected
 approach and the site complexity.

 To determine which biological assessment approach or
 combination of approaches is appropriate for a given site
 or situation, several factors must be considered. These
 include what management decisions will ultimately need
 to be made based  on  the data;  what are the  study
 objectives: and what should be the appropriate level of
 effort to obtain knowledge of contaminant fate/ transport
 and ecotoxicity.

 2.2     RISK EVALUATION

 Three common approaches to evaluating environmental
 risk to ecological receptors are (1) the use of literature
 screening values (e.g.. literature toxicity  values)  for
 comparison to  site-specific contaminant levels.  (2) a
 "desk-top" risk assessment which can model existing site-
 specific  contaminant data  to ecological receptors  for
 subsequent comparison to literature toxicity values, and
 (3) field investigation/laboratory analysis that involves a
 site investigation (which may utilize existing contaminant
 data for support) and laboratory analysis of contaminant
 levels in media and/or experimentation using bioassay
 procedures.  These three approaches are described in
 further detail next.
2.2.1  Literature Screening Values

To determine the environmental effects of contaminants
at a hazardous waste site, the levels of contaminants
found may be compared to literature toxicity screening
values or established screening criteria. These  values
should be derived from studies that involve testing of the
same matrix and a similar organism of concern.  Most
simply stated, if the contaminant levels on the site are
above the established criteria, further evaluation of the
site may be necessary to determine the presence of risk.
Site contaminant levels that are lower than established
criteria may indicate  that  no further evaluation  is
necessary at the site for that contaminant

2.2.2  Risk Calculations

The "desk-top" risk calculation approach compares site
contaminants to  information from  studies  found in
technical literature. This type of evaluation can serve as
a screening assessment or as a tier in a more complex
evaluation. Since many assumptions must be made due
to limited site-specific information, risk calculations are
necessarily conservative. The collection and inclusion of
site-specific field data can reduce the number and/or the
magnitude of these "conservative" assumptions, thereby
generating a  more realistic calculation of potential risk.
(See Chapter 5.0 for a complete discussion on risk
calculations.)

2.2.3  Standard Field Studies

Two important aspects of conducting a field study that
warrant discussion are the selection of a reference area
and the selection of the receptors of concern. These are
important to establish prior to conducting a field study.

2.2.3.1   Reference Area Selection

A reference area is defined in this document as an area
that is outside the chemical influence of the site but
possesses similar characteristics (e.g., habitat, substrate
type) that allows for the comparison of data between the
impacted area (Le., the site) and the unimpacted area (i.e.,
the reference  area).   Reference areas  can provide
information regarding naturally occurring compounds and
the existence of any regional contamination independent
of the site.  They can help determine if contaminants are
ubiquitous in the area and can separate site-related issues

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  from non-she related issues.

  The reference area must be of similar habitat type and
  support a species composition similar to the study area.
  The collection and analysis of samples from a reference
  area can support site-specific decisions regarding uptake,
  body burden, and accumulation of chemicals and toxicity.

  The reference area should be outside the area of influence
  of  the  site and  if possible, in an area  of minimal
  contamination or disturbance.  Location of reference
  areas in urban or industrial areas is frequently difficult,
  but an acceptable reference area is usually critical to the
  successful use of ecological assessment methods.

  2.2.3.2   Receptor Selection

  The selection of a receptor is dependent  upon the
  objectives of the study and the contaminants present The
.  first step is to determine the toxicity characteristics of the
  contaminants (i.e., acute, chronic, bioaccumulative, or
  non-persistent).   The next  step  is to determine the
  exposure route of the chemical (i.e., dermal, ingestion.
  inhalation).

  Selection of the  receptor or group of receptors is a
  component of establishing the measurement endpoint in
  the study design. When discussing the term measurement
  endpoint, it is useful to first define a related concept, the
  assessment endpoint. An assessment endpoint is defined
  as "an explicit expression of the environmental value that
  is to be protected." For example, "maintaining aquatic
  community composition and structure downstream of a
  site similar  to that upstream of the site" is an explicit
  assessment endpoint. Inherent in this assessment endpoint
  is the process of receptor selection  that  would  most
 appropriately answer the question that the endpoint
  raises.   Related  to this  assessment  endpoint is the
  measurement endpoint which is defined as "a measurable
  ecological characteristic that is related to the valued
  characteristic chosen as the assessment endpoint." For
 'example,  measurements of  biological effects such as
  mortality, reproduction, or growth of an  invertebrate
 community  are measurement endpoints.  Establishing
  these endpoints will ensure (1) that the proper receptor
  will be selected to best answer the questions raised by the
 assessment and measurement endpoints, and (2) that the
  focus of the study remains on  the component of the
 environment that may be used as the basis for decision.

 There are a number of factors that must be considered
 when selecting a target species. The behavioral habits
 and lifestyle of the species must be consistent with the
environmental fate and transport of the contaminants of
interest as well as pathways of exposure to receptor
species. For example, if the contaminants of concern at
the site are PCBs that are bioaccumulative, a mammal
such as a mink could be selected for the study since this
species  is  documented  to  be  sensitive   to  the
bioaccumulation of PCBs.  The r"'n^ in this case has
been selected to be used far establishing the measurement
endpoint that is representative of piscivorous mammals.
However, h may not be feasible to collect mink for study
due to their low availability in a given area. Therefore.
the food items of the mink (e.g., small tnammnic aquatic
vertebrates and invertebrates) may be collected and
analyzed for PCBs as an alternative means of evaluating
the risk to mink.  The resulting residue data may be
utilized to produce a dose model.  From this model, a
reference dose value may be determined from which the
probable effects to mink calculated.

The movement patterns of a measurement endpoint are
also important during the  receptor selection  process.
Species that are  migratory or that have large feeding
ranges are more difficult to link to site exposure than
those which  are  sessile, territorial, or have limited
movement patterns.
Ecological field studies offer direct or corroborative
evidence of a link between contamination and ecological
effects. Such evidence includes:

•       Reduction in population sizes of species that
        can not be otherwise explained  by naturally
        occurring population cycles
•       Absence of species-normally occurring in the
        habitat and geographical distribution
•       Dominance of species associated primarily with
        stressed habitat
•       Changes in community diversity or trophic
        structure relative to a reference location
•       High incidence of lesions, tumors,  or other
        pathologies
•       Development    of    exposure    response
        relationships.

Ecologists usually compare data of observed  adverse
effects to information obtained from a reference area not
affected by site contamination.  To accomplish this.
chemical  and biological  data should  be collected
simultaneously and then compared to determine if a
correlation exists  between contaminant concentrations
and ecological  effects (U.S.  EPA  1991b).   The
simultaneous  collection of the data is  important in
reducing the effect of temporal variability as a factor in
the correlation analysis.

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 The  type of field study selected  is directed  by the
 contaminants present linked to the assessment endpoint.
 Prior to  choosing a specific  study approach, the site
 contaminant must be determined using information about
 known or suspected site contaminants and how the nature
 of these  contaminants may be modified  by  several
 environmental and ecotoxicological factors. In addition,
 evaluation of chemical fate and transport information is
 necessary to determine  the  appropriate matrix  and
 technique.

 Contaminants can be a food chain threat, a lethal threat,
 a  direct non-lethal toxicant, indirect toxicant, or some
 combination of the four.  Chemical residue studies are
 appropriate if the contaminant of concern (COC) will
 bioaccumulate. Ecotoxicological information can provide
 insight about contaminants  that  are  expected  to
 accumulate in organisms. It can also provide information
 about which organisms provide the best data for the study
 objectives.     For   example,  the  species-specific
 bioaccumulation  rate must be considered along  with
 analytical detection  limits; the bioaccumulated levels
 need  to be above the analytical  detection limits.  In
 contrast, population/ community studies or toxicity testing
 may be more appropriate if the contaminants cause direct
 lethality.

 2.2.3.3  Exposure - Response Relationships

 The relationship between the exposure (or dose) of a
 contaminant and  the response  that  it elicits  is a
 fundamental concept in toxicology (Timbrell 1989). The
 simplest response to observe is death. Some examples of
 other responses that vary in terms of ease of measurement
 include pathological  lesions, cell necrosis, biochemical
changes, and behavioral changes. It is this foundation of
 exposure-response relationships upon which the concept
 of  chemical  residue  studies, population/community
 studies, and toxicity lesting/bioassays are built upon.

 2.2.3.4    Chemical Residue Studies

 Residue studies are  appropriate to use when  there is
 concern about the accumulation of contaminants in the
 tissues  of indigenous species.   Residue studies are
conducted by collecting organisms of one or more species
and comparing the contaminant bioaccumulation data to
 those  organisms collected from a reference area.

 Chemical residue studies require field collection of biota
and  subsequent  tissue  analysis.    A  representative
organism for collection and analysis is selected based on
the study  objectives and the site habitat.  Generally the
organism should be abundant, sessile (or with limited
home range), and easy to capture.  These attributes help
to provide a sufficient number of samples for analysis
thereby strengthening the linkage to the site. A number
of organism- and contaminant-specific factors should also
be considered when designing residue studies (see Philips
[1977] and [1978] for additional information).   The
subsequent  chemical  analysis may be conducted on
specific target tissues or the whole body. In most cases,
whole-body analysis is the method of choice to support
biological assessments.  This is because most prey
species are eaten in entirety by the predator.

In designing residue analysis studies, it is important to
evaluate the exposure pathway carefully.  If the organisms
analyzed  are  not  within  the site-specific  exposure
pathway, the information generated will not relate to the
environmental threat   Evaluation of the  exposure
pathway may suggest that a species other than the one of
direct  concern might provide a better evaluation of
potential  threat or bioaccumulation.

Because there are different data needs for each objective,
the study objective needs to be determined prior to the
collection of organisms.   In  these studies the actual
accumulation (dependent upon the bioavailability) of the
contaminants is  evaluated rather than assumed  from
literature values. The information collected then allows
for site-specific evaluation of the threat and reduces the
uncertainty  associated  with   the  use  of  literature
bioavailability values. These factors may be applied for
specific  areas of  uncertainty  inherent  from  the
extrapolation of available data (e.g., assumptions of 100
percent  bioaccumulation,  variations  in   sensitive
populations).

As stated previously, because  site conditions as well as
the bioavailability can change  over time, it is important
that exposure medium (soil, sediment, or water) samples
and biological samples are collected simultaneously and
analyzed for the same parameters to allow for the
comparison of environmental contaminant levels in the
tissue and the exposure medium.  This is critical in
establishing   a site-specific  linkage  that  must  be
determined on a case-by-case basis.

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 2.2.3.5   Population/Community
            Response Studies

 The fundamental approach to population or community
 response studies is  to systematically sample an area,
 documenting  the organisms of  the population  or
 community.   Individuals are typically identified and
 enumerated, and calculations are made with respect to the
 number, and species present.  These calculated values
 (e.g., indices or metrics) are used to compare sampling
 locations and reference conditions. Some population and
 community metrics include the number of individuals,
 species composition, density, diversity, and community
 structure.

 2.2.3.6   Toxicity Testing/Bioassays

 A third common assessment approach is to utilize toxicity
 tests or bioassays.  A toxicity test may be designed to
measure the effects from acute (short-term) or chronic
 (long-term) exposure to a contaminant.   An acute test
 attempts to expose the organism to a stimulus  that is
 severe enough to produce a response rapidly.  The
 duration of an acute toxicity test is short relative to the
 organism's life cycle and mortality  is the most common
 response measured.  In contrast, a chronic test attempts
 to induce  a -biological  response   of relatively slow
 progress through continuous, long-term exposure to a
 contaminant.

 In designing a toxicity test, it is critical to understand the
 fate,  transport, and  mechanisms   of toxicity  of the
 contaminants to select the test type  and conditions. The
 toxicity test must be selected to match the site and its
conditions  rather than modify the site matrix for the use
of a particular test.  Factors to consider are the test
species, physical/chemical factors  of the  contaminated
 media, acclimation  of  test  organisms,  necessity for
 laboratory versus field testing, test duration, and selection
 of test endpomis (e.g.. mortality or growth). A thorough
 understanding of the interaction of these and other factors
 is necessary to determine if a toxicity test meets the study
objectives.

The selection of the best toxicity test, including the choice
of test  organism, depends on several factors:

 •       The decisions that will be based  on the results
         of the study
•       The ecological setting of the site
•       The contaminant(s) of concern

Toxicity testing can be conducted on a variety of sample
matrices,  including water (or an aqueous effluent),
sediment, and soil. Soil and sediment toxicity tests can
be conducted on the parent material (solid-phase tests) or
on the elutriate (a water extract of the soil or sediment).
Solid-phase sediment and soil tests are currently the
preferred  tests since they  evaluate the toxicity of the
matrix of interest to the test organisms, thereby providing
more of a realistic site-specific exposure scenario.

As stated previously, one  of the most frequently used
endpoints in acute toxicity testing  is mortality (also
refaied to as lethality) hmnter ft is one of the most easily
measured parameters.

In contrast, some contaminants do not cause mortality in
            ; but rather they affect the rate or success of
reproduction or growth in test organisms. In this case,
the environmental effect of a contaminant may be that it
causes reproductive failure but does not cause mortality
in the existing population.  In either case, the population.
will either be eliminated or drastically reduced.

The use of control as well as reference groups is normally
required. Laboratory toxicity tests include a control that
evaluates die laboratory conditions, and the health and
response of the test organisms.  Laboratory controls are
required for all valid toxicity tests. A reference provides
information on how the test organisms respond to the
exposure  medium  without  the  site contaminants.
Therefore, the reference is necessary for interpretation of
the test results in the context of the site (i.e., sample data
is compared to the reference data). It is not uncommon
for conditions other than contamination to  induce a
response in a toxicity test  With proper reference and
control tests, toxicity tests can be used to establish a link
between contaminants results and adverse effects.

Within the Superfund Program, conducting toxicity tests
typically  involves collecting  field samples  (water.
sediment, soil)  and transferring  the  materials  to a
laboratory. In situ (field conducted) tests can be run if
field conditions  permit   There  are  benefits  and
limitations associated with  each approach. The most
notable benefit of laboratory testing is  that exposure
conditions are  controlled,  but  mis  leads to its most
notable limitation, a reduction of realism.  With in situ
tests, the reality of the exposure situation is increased, but
there is a reduction of test controls.  See U.S. EPA's
Compendium  of ERT Toxicity Testing Procedures,
OSWER Directive 9360.44)8, EP A/54 O/P-91/009 (U.S.
EPA 199la), for descriptions of nine common toxicity
tests and Standard Guide for Conducting Sediment
Toxicity Tests with Freshwater Invertebrates, ASTM
Standard E1383. October 1990.

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Species Seleetipn for Toxieitv Testing

Selection of the test organism is critical in designing a
study using toxicity testing.  The species selected should
be  representative relative to the assessment endpoint,
typically an organism found within the exposure pathway
expected in the field. To be useful in evaluating risk, the
test organism must respond to  the  contaminant(s) of
concern. This can be difficult to achieve since the species
and tests available are limited.  Difficult choices and
balancing of factors are frequently necessary.
                                                        10

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                        3.0  BIOLOGICAL SAMPLING METHODS
 Once a decision has been made that additional data are
 required to assess the biological threat posed by a site, an
 appropriate sampling plan must be developed.   The
 selection of ecological sampling methods and equipment
 is dependent upon  the field assessment approach, as
 discussed in Chapters 1 and 2. Thus, the selection of an
 assessment approach is the initial step in the collection
 process.  This chapter does not present step-by-step
 instructions for a particular method, nor does it present an
 exhaustive list of methods or equipment  Rather,  it
 presents specific examples of the most commonly used
 methods and associated equipment. Table 4.1 (at the end
 of this  chapter) lists some of the standard operating
 procedures   (SOPs)  used   by  the  U.S.  EPA's
 Environmental Response Team Center (ERTC).

.Because of the complex process required for selecting
 the proper assessment approach for a particular site,
 consultation with an ecologist/biologist experienced in
 conducting ecological risk  assessments is strongly
 recommended.

 3.1     CHEMICAL RESIDUE STUDIES

 Chemical residue studies are a commonly used approach
 that can address the bioavailability of contaminants in
 media (e.g., soil, sediment, water). They are often called
 tissue  residue  studies  because  they  measure  the
 contaminant body burden in site organisms.

 When collecting  organisms  for tissue analyses, it is
 critical that the measured levels  of contaminants in the
 organism  are attributable to a particular location and
 contaminant level within the site. Collection techniques
 must be evaluated for their potential to bias the generated
 data.  Collection  methods  can result in some form of
 biased data either by the size. sex. or individual health of
 the organism.  Collection techniques are chosen based on
 the  habitat present and the species of interest.  When
 representative approaches are not practical, the potential
 bias  must  be identified and considered when drawing
 conclusions  from the data.  The use of a particular
 collection technique should not be confused with the need
 to target a "class" of individuals within a population for
 collection.  For example, in a specific study it may be
desirable to collect only males of the species or to collect
fish of consumable size.
Some receptors of concern (ROCs) cannot be collected
and  analyzed  directly  because of low  numbers  of
individuals in the  study area, or other  technical  or
logistical reasons. Exposure levels for these receptors
can be estimated by collecting organisms that are preyed
upon by the  ROC.   For example, if the  ROC is a
predatory bird, the species collected for contaminant level
measurements may be one of several small mammals or
fish that the ROC is known to eat

As noted previously, it is critical to link the accumulated
contaminants both to the site and to an exposure medium.
Subsequently,   the   collection  and  analysis   of
representative soil, sediment, or water samples from the
same location are  critical.   A realistic  site-specific
Bioaccumulation Factor (BAF) or Biocohcentration
Factor (BCF) may then be calculated for use in the site
exposure models.

"Bioconcentration is usually considered to be that process
by which toxic substances enter aquatic organisms, by
gill or epithelial tissue from the water. Bioaccumulation
is a broader term in the sense that it usually includes not
only bioconcentration but  also  any uptake of toxic
substances through the consumption of one organism."
(Brungs and Mount  1978).

3.1.1  Collection Methods

It should be noted that any applicable state permits
should be acquired before any biological sampling event.
States requirements on organism,  method, sampling
location, and data usage differ widely and may change
from year to year.

The  techniques used to  collect different organisms are
specific  to  the study objectives.  All techniques are
selective to some extent for certain species, sizes, habitat,
or sexes of animals.  Therefore, the  potential biases
associated with each  technique should be determined
prior to the study. If the biases are recognized prior to
collection, the sampling may be designed to minimize
effect of the  bias.  For example,  large traps are not
effective for trapping small animals since small mammals
are not heavy enough to trigger the trap or may escape
through minute trap openings.
                                                    II

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 In determining environmental threat, the target species
 generally consist of prey species such as earthworms,
 small mammals, or fish.   Residue data from these
 organisms can be used to evaluate the risk to higher
 trophic level organisms, which may be difficult to capture
 or analyze.

 3.1.1.1   Comparability Considerations

 There are two issues that directly affect field collection.
 First, organisms such as benthic macroinvenebrates tend
 to have a patchy or non-uniform distribution in the
 environment due to micro habitats and  other factors.
 Therefore, professional evaluation in matching habitat for
 sampling, is  critical in  the  collection  of * a  truly
 representative  sample of  the  community.   Second,
 variability in sampling effort and effectiveness needs to
 be considered.

 3.1.1.2   Mammals

 Trapping is  the most common method for the collection
 of mammals. The selection of traps is determined by the
 species targeted and the habitat present Both live trap or
 kill trap methods may be acceptable for residue studies,
 but consideration of other data uses (e.g., histopathology)
 or concern for injury or death of non-target species can
 influence the use of certain trap types.
 Several trap methods are available for collecting small
 mammals.  Commonly used  traps include Museum
 Special.  Havahart  Longworth. and  Sherman  traps
 (Figure 3).  Although somewhat labor-intensive, pitfall
 trap arrays may also be established to include mammals
that are not regularly trapped using other techniques (e.g.,
shrews).

Trap  placement  is  a  key  element when collecting
samples.  Various methods of trap placement can be
utilized.  These include, but are not limited to:

 •        Sign method/Best set method
•        Paceline method
•        Grid method

When using the sign/best set method, an experienced
field technical specialist searches for fresh mammal signs
(e.g.. tacks, scat, feeding debris) to determine'where the
trap  should be positioned.   This method typically
produces higher trapping success than other methods,
however, this method is biased and is therefore generally
used to determine what species are present at the site.

The paceline method involves  placement of traps at
regular intervals  along a transect. A starting point is
selected and marked, a landmark is identified to indicate
the direction of the transect, and as the field member
walks  the. transect, the traps are placed at regular
intervals along it

The grid method is similar to the paceline method but
involves a group of evenly spaced parallel transects of
equal lengths to create a grid.  Traps are placed at each
grid node.  The size of the grid is dependent on the
species to be captured and the type of study.  Grids of
between  500  to  1,000  square meters  containing
approximately  100 traps are common.   If a grid is
established in  a  forest interior, additional  parallel
trapping lines  may be established to cover the edge
habitat

Regardless  of  the type of .trapping  used,  habitat
disturbance should be kept to a minimum to  achieve
maximum trapping success.  In most areas, a trapping
success of 10 percent is considered maximum but is
oftentimes significantly lower (e.g., 2 to 5 percent).  Part
of this reduced trapping  success is due to habitat
disturbance. Therefore, abiotic media samples (e.g., soil,
sediment, water) should be collected well in advance of
trapping efforts  or after all  trapping is completed.
Trapping success also varies with time but may increase
over time with  diminishing returns.   In other words.
extending the trapping period over several days may
produce higher trapping success by allowing mammals
that were  once  peripheral to the trapping  area  to
immigrate  into the now mammal-depauperate area.
These  immigrants would not be representative of the
trapping area. Therefore, a trapping period of 3 days is
typically used to minimize this situation.

Trapping success will also vary widely based on the
available  habitat,  targeted  species,  season,  and
geographical location of the site. When determining trap
success objectives, it is important to keep in mind the
minimum sample mass/volume requirements for chemical
residue studies.
                                                     12

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•3.1.1.3.  Fish

 Electrofishing, gill  nets, trawl nets, seine nets,  and
 minnow  traps  are  common  methods  used  for the
 collection of fish. The selection of which technique to
 use is dependent on the  species targeted for collection
 and the systerh being sampled. In addition, there are
 other available fish netting and trapping techniques that
 may be more appropriate in specific areas.   As with
 mammal trapping, disturbance in the area being sampled
 should be kept  to  a minimum to ensure collection
 success.

 Electrofishing  uses  electrical currents to gather, slow
 down, or immobilize fish for capture. An electrical field
 is created between and around two submerged electrodes
 that stuns the fish or alters their swimming within or
 around the field Depending on the electrical voltage, the
 electrical pulse frequency, and the fish species, the fish
 may swim towards one of the electrodes, swim slowly
 enough to capture, or may be stunned to the  point of
 immobilization. This technique is most effective on fish
 with swimbladders and/or shallow water since these fish
 will float to the surface for easy capture.

 Electrofishing can be done using a backpack-mounted
 electroshocker unit,  a shore-based unit or from a boat
 using either type.  Electrofishing does not work in saline
 waters and  can  be ineffective in very  soft water.
 Electrofishing is  less effective in deep water where the
 fish can avoid the current.  In turbid waters, it may be
 difficult to see the stunned fish.

 Gill netting  is  a highly effective passive collection
 technique for a wide range of habitats. Because of its low
 visibility  under  water,  a gill  net captures  fish by
 entangling their  gill plates as they attempt to swim
 through the area in which the gill net has been placed in.
 Unfortunately, this may  result  in fish to be injured or
 killed due to further entanglement, predation, or fatigue.

The size and shape of fish captured is relative to the size
and kind of mesh used in the net thus creating  bias
 towards a certain sized fish. These nets are typically used
 in shallow waters, but may extend to depths exceeding SO
 meters. The sampling area should be free of obstructions
 and floating debris,  and provide  little to no current.
 (Hurben 1983)
Otter trawl netting is an active collection technique that
utilizes the motion of a powered boat to drag a pocket-
shaped net through a body of water. The net is secured to
the rear of a boat and pulled to gather any organisms that
are within the opening of the pocket  This pocket is kept
open through the use of underwater plates on either side
of the net that act as keels, spreading the mouth of the net
open.

Seining is another active netting technique that traps fish
by encircling mem with a long wall of netting.  The top of
the net is buoyed by floats and the bottom of the net is
weighed down by lead weights or chains. Seine nets are
effective in open or shallow waters with unobstructed
bottoms.  Beach or haul  seines are used in shallow water
situations where the net extends to the bottom.  Purse
seines are designed for applications in open water and do
not touch the bottom (Hayes 1983).

The use of minnow traps is a passive collection technique .
for minnow-sized fish. The trap itself is a metal or plastic
cage that is secured to a  stationary point and baited to
attract fish. Small funnel-shaped openings on either end
of the trap allow  fish  to swim easily  into it,  but are
difficult to locate for exit Cage "extenders" or "spacers"
that are  inserted to lengthen  the cage, allow larger
organisms such as eels,  or for a larger mass offish to be
collected.

3.1.1.4   Vegetation

Under certain  conditions, the analysis of the chemical
residue in  plants may be a highly effective  method of
assessing the impacts of a site.  The bioaccumulative
potential  of plants  varies  greatly  however, among
contaminants, contaminant species, soil/sediment texture
and chemistry, plant condition, and genetic composition
of the plant In addition to this variability,  plants can
translocate specific contaminants to different pans of the
plant.   For example,  one  contaminant may  tend to
accumulate in  the roots of a plant whereas a second
contaminant may tend  to accumulate in the fruit of the
same plant In this scenario, the collection and analysis
of a plant part that normally does not receive translocated
materials would not result in a useful sample.  Therefore.
it  is crucial to conduct a  literature review  prior to
establishing a sampling protocol.

Sampling of herbaceous plants should be  conducted
during the growing season  of the species of  interest
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 Sampling of woody plants may be conducted during the
 growing  or dormant  season, however,  most  plants
 translocate materials from the aboveground portions of
 the plant to the roots prior to dormancy.

 Collection methods and sampling specifics may be found
 in  U.S.  EPA/ERT SOP  #2037,  Terrestrial  Plant
 Community Sampling; others are provided in Table 4.1.

 3.1.2   Sample Handling and
         Preparation

 The animals or plants collected should be identified to
 species level or the lowest practical taxonomic level.
 Appropriate metrics (e.g., weight, animal body length,
 plant height) and the presence of any external anomalies,
 parasites, and external pathologies should be recorded.
 If compositing of the sample material is necessary, it
 should be performed in accordance with the study design.
Depending upon the study objectives, it may be necessary
to isolate the contaminant levels in animal tissue from the
contaminant levels in the food or abiotic matrices (e.g.,
sediment) entrained in the digestive tract of the organism.
This is an important process in that it separates the
contribution of two distinct sources of contaminants to the
next trophic  level,  thereby allowing the  data user to
recognize the relative importance of the two sources.

Clearing of the digestive tract (i.e., depuration)  of the
organism  must  then be  accomplished prior  to  the
chemical analysis. The specific depuration procedures
will vary with each type of organism  but all involve
allowing the  organism to excrete  waste products in a
manner in which the products may not be reingested,
absorbed, or deposited back onto the organism.

Biological samples should be handled with caution to
avoid personal injury, exposure to disease, parasites, or
sample contamination.   Personal  protection such  as
gloves should be worn when handling animals and traps
to reduce the transfer of scents or oils from the hand to
the trap, which could cause an avoidance reaction in the
targeted animals.

Samples collected for biological  evaluation must  be
treated in  the same manner as abiotic samples (i.e., the
same health  and  safety  guidelines, decontamination
protocols,  and  procedures  for  preventing   cross-
contamination must be adhered to). Biological samples
do  require some extra  caution in handling to avoid
personal injury and exposure to disease, parasites, and
venoms/resins. The selection of sample containers and
storage conditions (e.g., wet ice) should follow the same
protocols as abiotic samples.  Refer to Chapter 4.0 for
determination  of holding times and additional quality
assurance/quality control (QA/QC) handling procedures.

3.1.3   Analytical Methods

Chemical analytical  methods  for tissue  analysis are
similar to those for abiotic matrices (e.g., soil and water),
however, the required sample  preparation procedures
(e.g., homogenization and subsampling) of biological
samples are frequently problematic. For example, large
bones, abundant hair, or high cellulose fiber content may
result in difficult homogenization of mammals and plants.
Extra steps may be required during sample cleanup due
to high lipid (fat) levels  in animals tissue or high resin -
content in plant tissue.

Most tissue samples can be placed in a laboratory blender
with dry ice and homogenized at high speeds.  The
sample material is then  left  to  sit to allow  for the
sublimation of the dry ice.  Aliquots of the homogenate
may then be removed for the required analyses.

The requirement for split samples or other QA samples
must be determined prior to  sampling to ensure a
sufficient volume of sample is  collected.  Chapter 4.0
discusses the selection and use of QA/QC samples.

The detection limits of the analytical parameters should
be  established prior to the  collection  of samples.
Detection limits are selected  based  on the level of
analytical resolution that is needed to interpret  the data
against the study objectives. For example, if the detection
limit for a compound is 10 mg/kg but the concentration in
tissue which causes effects is 1 mg/kg, the detection limit
is not artrqiiatr  to determine if a problem exists. It should
be  noted that  standard laboratory detection limits for
abiotic matrices are often not adequate for tissue samples.
Chapter 4.0 provides details on detection limits and other
QA/QC parameters.

The tissue analysis can consist of whole body residue
analysis or analysis of specific tissues (i.e.. fish fillets).
Although less frequently used in Superfund, tissues such
as organs (e.g.. kidney or liver) may be analyzed.  The
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 study endpoints will determine whether whole body,
 fillet, or specific organ samples are to be analyzed.

 Concurrent analyses should include a determination of
 percent lipids and percent moisture.  Percent lipids may
 be used to  normalize the concentration of non-polar
 organic contaminant data. In addition, the lipid content
 of the organisms analyzed can be used to evaluate the
 organism's health.  Percent  moisture determinations
 allow the expression of contaminant levels on the basis of
 wet or dry weight Wet weight concentration data are
 frequently used in food chain accumulation models, and
 dry weight basis data  are frequently reported between
 sample location comparisons.

 Histopatholoyieal Analysis

 Histopathological analysis can be an effective mechanism
 for  establishing   causative  relationships   due  to
 contaminants since some contaminants can cause distinct
 pathological effects.   For example, cadmium causes
 visible kidney damage providing causal links between
 contaminants and  effects.   These  analyses  may  be
 performed on organisms collected for residue analysis. A
 partial  necropsy performed on the  animal tissue may
 indicate  the  presence of  internal  abnormalities  or
 parasites. The time frame and objectives of the study
 determine if histopathological analysis is warranted.

 3.2    POPULATION/COMMUNITY
        RESPONSE STUDIES

Population/community response studies are a  commonly
utilized  field assessment  approach.   The decision to
conduct a population/community response study is based
on the rype(s) of contaminants, the time available to
conduct the  study, the type of communities potentially
present at the site, and the time of year of the study.
These studies are most commonly conducted on non-
 ume-criocal or long-term remediation-type site activities.
 During  limited  time  frame  responses, however,  a
 population/community survey or screening level study
may be useful for providing information about potential
impacts associated with a site.

3.2.1   Terrestrial Vertebrate Surveys

 Methods for determining  adverse effects on terrestrial
vertebrate communities are as follows:  censusing or
population  estimates,  sex-age  ratio  determinations.
natality/mortality estimations, and diversity studies.

True or accurate censuses are usually not feasible for
most terrestrial vertebrate populations due to logistical
difficulties. Estimations can be derived by counting a
subset of organisms or counting and evaluating signs
such as burrows, nests, tracks,  feces, and carcasses.
Capture-recapture studies may  be  used to  estimate
population  size  but are  labor-intensive and  usually
require  multiple-season  sampling.    If  conducted
improperly, methods for marking captured organisms
may cause  irritation or injury or  interfere with the
species' normal activities.

Age ratios provide information on natality and rearing
success, age-specific reproductive rates, and mortality
and survival rates. Sex ratios indicate whether sexes are
present in sufficient numbers and proportions for normal
reproductive activity.

Community composition (or diversity) can be assessed by
species  frequency,  species per  unit  area,  spatial
distribution of individuals, and numerical abundance of
species (Hair 1980).

3.2.2  Benthic Macroinvertebrate
        Surveys

Benthic macroinvertebnte (BMI) population/community
evaluations in small- to medium- sized streams have been
successfully  used for  approximately  100  years to
document injury to the aquatic systems. There are many
advantages to using BMI populations to determine the
potential  ecological  impact associated with  a  site.
Sampling is relatively easy, and equipment requirements
are minimal. An evaluation of the community structure
may be used to assess overall water quality, evaluate the
integrity of watersheds, or suggest the presence of an
influence of the community structure that is independent
of water quality and habitat conditions.

Because BMIs are a primary food source for many fish
and other  organisms,  threats  beyond  the  benthic
community can be inferred from the evaluation of BMIs.
Techniques such as rapid bioassessmem protocols may
be  used as a  tool to support this type of finding and
inference. A more comprehensive discussion of general
benthological surveys may be found in U.S. EPA (1990).
                                                    15

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 3.2.2.1   Rapid Bioassessment Protocols
            for Benthic Communities

 Rapid bioassessment protocols  are  an inexpensive
 screening  tool  used  for  determining if a stream is
 supporting or not supporting a designated aquatic life
 use.  The rapid bioassessment protocols advocate an
 integrated assessment, comparing habitat and biological
 measures with empirically defined reference conditions
 (U.S.EPA1989a).
                     •\

 The three  major components of a rapid bioassessment
 essential for determining ecological impact are:

 •       Biological survey
 •       Habitat assessment
 •       Physical and chemical measurements

 As with all population/community evaluations, the habitat
.assessment is  of  particular concern with respect to
 representative sampling. Care must be taken to prevent
 bias during collection of the benthic community resulting
 from sampling dissimilar habitats. Similar habitats must
 be  sampled to make valid  comparisons  between
 locations.  In addition to habitat similarity, the sampling
 technique  and level of effort at each location must be
 uniform to achieve an accurate interpretation of results.

 In the U.S. EPA Rapid Bioassessment Protocol (RBP),
 various components of the community and habitat are
 evaluated, a numerical score is calculated, and the score
 is compared to predetermined values.  A review of the
 scores, together with habitat assessment and the physical
 and chemical data, support a determination of impact.
 U.S.  EPA Reference  (May.  1989a)  presents  the
 calculation and interpretation of scores!

 Standard  protocols,  including  the  RBP. have been
 developed  to facilitate surveying BMls  to determine
 impact rapidly. These protocols use a standard approach
 to  reduce  the  amount  of time spent collecting and
 analyzing samples.  Protocols range from a quick survey
 of  the  benthos' (Protocol  I)  to a  detailed laboratory
 classification analysis (Protocol HI).  Protocol I may be
 conducted in several hours; Protocol n is more intensive
 and focuses on major .taxonomic levels; and Protocol IH
 may require numerous hours to process each sample to a
 greater level of  taxonomic and  community assessment
 resolution.  These protocols  are  used  to determine
 community health and biological condition via tolerance
values and matrices.  They also create and amend a
historical  data base that can  be used for future site
evaluation.

3.2.22   General Benthological Surveys

Benthological surveys can be conducted with methods
other man those discussed in the RBP protocols utilizing
techniques discussed  in  the literature.   The  overall
concept is generally the same as that used in the RBP, but
the specific sampling technique changes depending on
the habitat or community sampled

3.2.2.3   Reference Stations

The use of a reference station is essential to determine
population/community effects attributable to a site. The
use of a  reference  station  within the study area is
preferable (upstream or at a nearby location otherwise
outside the area of site  influence).  In some cases this is
not possible due to regional impacts, area-wide  habitat
degradation, or lack of a similar habitat In these cases
the use of population/community studies should be re-
evaluated within the context of the site investigation. If
the choice is made to include the population/community
study, regional reference or a literature-based evaluation
of the community may be options.

3.2.2.4   Equipment for Benthic  Surveys

The  selection of the  most  appropriate sampling
equipment for a particular site is based primarily on the
habitat  being  sampled.   This subsection is  a brief
overview of the equipment available for the collection of
BNOs.  Detailed  procedures are  not discussed  in this
document  For additional information, refer to the SOPs
and methods manuals provided in Table 4.1, or consult
an ecologist/biologist experienced in this type of field
collection.

Long-handled  nets or a Surber  sampler with  a 0.5-
miliimeter (mm) size mesh are common sampling nets for
the collection of macroinvenebrates from a riffle area of
a  stream.   Samples to be collected from  deep water
gravel, sand, or soft boaom habitats such as ponds, lakes,
or rivers are more often sampled using a small Ponar or
Ekrnan dredge. Artificial substrates are used in varying
habitats when habitat  matching is problematic and/or
native substrate sampling would not be effective. The
most common types  of artificial substrate samplers are
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multiple-plate samplers or barbecue basket samplers.

The  organisms to  be  taken to the  laboratory for
identification or retained for archival purposes may be
placed in wide-mouthed plastic or glass jars (for ease in
removing contents)  and preserved  in  70 percent 2-
propanol (isopropyl alcohol) or ethyl alcohol (ethanol),
30 percent formalin, or Kahle's solution.   Refer to
methods manuals for detailed information on sample
handling and preservation.

3.2.3   Fish Biosurveys

3.2.3.1   Rapid  Bioassessment  Protocols
            for Fish Biosurveys

RBPs IV and V are two levels of fish biosurvey analyses.
Protocol IV consists of a questionnaire to be completed
with the aid of local and state fisheries experts. Protocol
V is a rigorous analysis of the fish community through
careful    species   collection,    identification,    and
enumeration.    This  level  is  comparable  to  the
macroinvenebrate Protocol ID (see Section 3.2.2.1) in
effort  Detailed information on both protocols can be
found in Rapid Bioassessments  Protocols for Use In
Streams and Rivers (U.S. EPA 1989a).

3.3  TOXICITY TESTS

Toxiciry tests evaluate the relative threat of exposure to
contaminated media  (e.g.. soil, sediment, water) in  a
controlled setting. These tests are most often conducted
in the laboratory, although they may be conducted in the
field as  well. These tests provide an estimate of the
relationship between the contaminated medium, the level
of contaminant and the severity of adverse effects under
specific lest parameters. Toxicity tests are categorized by
several parameters which  include duration of the test test
species, life stage of the organism, test end points, and
other variables.

The collection of the actual samples on  which the tests
are to  be  conducted follow the same  protocols as
collection  of  representative  samples   for  chemical
analyses. Typically, a subsample  of the media collected
for toxicity testing is submitted for chemical analyses.
The use of a concentration gradient for toxicity testing is
frequently desired to  establish a concentration gradient
within the test. This also eliminates the need to sample
all  the locations at a site.  The specific methods to be
followed for toxicity tests are described in detail in U.S.
EPA's   Compendium   of ERT  Toxiciry   Testing
Procedures, OSWER Directive 9360.4-08, EPA/540/P-
91-009 (U.S. EPA 1991a), as well as existing SOPs
listed in Table 4.1. These published procedures address
sample preservation, handling and storage, equipment
and apparatus, reagents, test procedures,  calculations,
QA/QC. and data validation.  The practical uses of
various toxicity tests, including examples  of acute and
chronic tests, are described next Each section includes
an example toxicity test

3.3.1      Examples Of  Acute  Toxicity
           Tests

Example No. 1 (solid-phase soi1>

Laboratory-raised earthworms are placed 30 per replicate
into  test chambers containing site soil. A laboratory
control and a site reference treatment are established to
provide a means for comparison of the resulting data set
Depending on the anticipated contaminant concentrations
in the  site soil,  the soil may be used in its entirety or
diluted with control  or  site reference soil.   The  test
chambers are examined daily for an exposure period of
14 days and the number dead organisms is tabulated.
When the observed mortality in  the site soil treatments is
statistically compared to control  and  site  reference
treatments, inferences regarding the toxicity of the
contaminant concentrations in the site soil treatments may
be drawn.

Example No. 2 (surface watert

Fathead minnows (Pimephales promelas)  are exposed
for 96  hours in aerated test vessels containing surface
water  from   sampling   locations   representing   a
concentration gradient. The mortality of the  organisms is
recorded at the  end of  the exposure  period   and
statistically compared to  control  and  site reference
treatments. Statistically significant differences between
treatments may be attributed to the varying contaminant
concentrations.
                                                     17

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3.3.2     Examples of Chronic Toxicfty
           Tests

Example No. 1 (surface water)

Fathead minnow  larvae (Pimcphales promelas)  are
exposed for 7 days to surface  water collected from
sampling  locations that  represent  a  concentration
gradient. Each replicate consists of 20 individuals of the
same maturity level. The test vessels are aerated and the
water is replaced  daily.  The fish,  which should have
remained  alive throughout the  exposure period,  are
harvested and measured for body length and body weight
These results represent growth rates and are statistically
compared  to the control and site reference treatments to
infer  the  lexicological effects  of  the  contaminant
concentrations.

Example No. 2 (sediment^

Midge (Chironomus sp.) larvae are exposed for 10 days
to sediment, overlain  with site  reference water, and
collected  from sampling locations  that represent a
concentration gradient  Each replicate consists of 200
individuals of the  same maturity  level (1st instar). The
test vessels are aerated and the water is replaced daily.
At the end of the exposure period, the larvae are removed
from the test vessels and measured for body length and
body weight

The organisms are then returned  to the test vessels and
allowed to mature to the adult stage. An emergence trap
is placed over the test vessel and the number of emerging
adults is recorded. These results, as well as the length
and weight results, are statistically  compared to the
control  and site  reference  treatments to infer  the
lexicological effects of the contaminant concentrations.
                                                    18

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                   Figure 2: Common Mammal Traps
                           Havahart Trap
                         Longworth live trap
(A)
                                      (B)
Folding (A) and non-folding (B) Sherman live traps
                                19

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                              TABLE I
Reference Lisl of Standard Operating Procedures -: Ecological Sampling Methods
SOP/Method No.
SOP No 1820
SOP No 1821
SOP No 1822
SOP No 1823
SOP No 2020
SOP No. 2021
SOP No 2022
SOP No 2023
SOP No. 2024
SOP No. 2025
SOP No 2026
SOP No. 2027
SOP No. 2028
SOP No 1001
SOP No. 1-002
Greene etil.( 1989)
SOP No. 1 005
SOP No. 2029
SOP No. 2032
SOP No. 2033
SOP No. 2034
SOP No. 2035
SOP No. 2036
SOP No 2037
Source
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
.
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
ERTC
Procedure/Melhod Title
Tissue Homogenizalion Procedure
Semi-Volaliles Analysis of Tissue Samples by GC/MS
Peslicides/ICB Analysis of Tissue Samples by GC/ECD
Microwave Digestion and Metals Analysis of Tissue Samples
7-Day Standard Reference Toxicily Test Using Larval Fathead Minnows Pimephalts promelat
24 Hour Range Finding Test Using Daphnia magnaot Daphnia pule*
96-Hour Acute Toxicily Test Using Larval fimephalet promelai
24-Hour Range Finding Test Using Larval PimepHalei promelai
48-Hour Acute Toxicity Test Using Daphnia magna or Daphnia pule*
7-Day Renewal Toxicily Test Using Ceriodaphnia dubia
7-Day Static Toxicily Test Using Larval FimepHalei promtlas
96- Hour Static Toxicity Test Using Stlenailrum capricomulum
10- Day Chronic Toxicity Test Using Daphnia magna'ot Daphnia pulex
15-Day Solid Phase Toxicily Test Using Chironomus lenlani
28-Day Solid Phase Toxicily Test Using Hyalella atleca
14-Day Acute Toxicily Test Using adult Eiienia andrei (earthworms)
Field Processing of Fish
Small Mammal Sampling and Processing
Benthic Sampling
Plant Protein Determination
Plant Biomass Determination
Plant Peroxidase Activity Determination
Tree Coring and Interpretation
Terrestrial Plant Community Sampling
Publication No
(In development)
(in development)
(in development)
(in development)
OSWER EPA/540/P 91/009
OSWER EPA/540/P-9 1/009
OSWER EPA/540/P-9 1/009
OSWER EPA/54(VP-9i/009
OSWER EPA/540/P-9I/009
OSWER EPA/540/P-9 1/009
OSWER EPA/540/P-9I/009
OSWER EPA/540/P-9I/009
OSWER EPA/540/P-9 1/009
(in development)
(in development)
EPA 600/3-88-029
(In development)
(in development)
(in development)
(In development)
(In development)
(In development)
(In development)
(in development)
                                 20

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                 4.0 QUALITY ASSURANCE/QUALITY CONTROL
4.1   INTRODUCTION

The goal  of representative  sampling  is  to  yield
quantitative data that accurately depict site conditions in
a given period of time. QA/QC measures specified in the
sampling procedures minimize and quantify the error
introduced into the data.

Many  QA/QC  measures  are  dependant on QA/QC
samples submitted with regular field samples. QA/QC
samples evaluate the three following types of information:
(1) the degree of site variation; (2) whether samples were
cross-contaminated during sampling and sample handling
procedures; and (3) whether a discrepancy in sample
results is attributable to field handling,  laboratory
handling, or analysis. For additional information on QA
•objectives, refer to U.S. EPA Quality Assurance/Quality
Control  (QA/QC) Guidance for Removal Activities,
EPA/540/G-90/004. April  1990.

4.2  DATA CATEGORIES

The U.S. EPA has established a process of data quality
objectives (DQOs) which establish what type, quantity.
and quality of environmental data are appropriate for
their intended application.  In  its DQO  process, U.S.
EPA has denned two broad categories of data: screening
and definitive.

Screening data are  generated by rapid, less precise
methods  of  analysis   with  less  rigorous  sample
preparation. Sample preparation steps may be restricted
to simple procedures such as dilution with a solvent,
rather than an elaborate extraction/digestion and cleanup.
At least 10 percent of the screening data are confirmed
using the analytical methods and QA/QC procedures and
criteria associated with definitive data. Screening data
without associated confirmation data are not considered
to be data of known quality. To be acceptable, screening
data must include the following:

•     chain of custody
•     initial and continuing calibration
•     analyte identification
•     analyte quantification
Streamlined  QC  requirements   are  the  defining
characteristic of screening data.

Definitive data are generated using rigorous analytical
methods (e.g., approved U.S. EPA reference methods).
These data are analyte-specific, with confirmation of
analyte identity and concentration.  Methods produce
tangible raw data (e.g., chromatograms, spectra, digital
values) in the form of hard-copy printouts or computer-
generated electronic files. Data may be generated at the
site or at an off-site location as  long as the QA/QC
requirements are satisfied. For the data to be definitive,
either analytical or  total measurement error must be
determined. QC measures for definitive data contain all
the elements associated with screening data, but  also
include trip, method, and rinsate blanks; matrix spikes;
performance evaluation samples; and replicate analyses
for error determination.

For more details on these data categories, refer to  U.S.
EPA Data Quality Objectives Process For Superfund,
EPA/540/R-93/071, Sept 1993.

4.3  SOURCES  OF ERROR

The four most common potential sources of data error in
biological sampling:

•     Sampling design
•     Sampling methodology
•     Sample heterogeneity
•     Sample analysis

4.3.1    Sampling Design

The initial selection of a habitat is a potential source of
bias  in  biological  sampling,  which might  either
exaggerate or mask the effects of hazardous substances in
the environment. In a representative sampling scheme,
habitat characteristics such as plant and animal species
composition, substrates, and degree of shading should be
similar at all locations, including the reference location.
The same individual should select both the test site and
the control and background site to minimize error in
comparing site conditions.
                                                  21

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 Standardized procedures for habitat assessment and
 selection also help minimize design error. The selection
 of an inappropriate  species may introduce an error into
 the representative sampling design.  This error can be
 minimized by selecting a species that is representative of
 the habitat and whose life-cycle is compatible with the
 timing of the study. In addition, migratory or transient
 species should be avoided.

 4.3.2      Sampling Methodology

 Sampling methodology and sample handling procedures
 may contain possible sources of error such as  unclean
 sample  containers,  improper  sample handling,  and
 improper shipment procedures. Procedures for sample
 collection and handling should be standardized to allow
 easier identification  of potential error. Follow SOPs or
 established  procedures  to  ensure that all  sampling
 techniques are performed consistently despite different
. sampling  teams,  dates,  or locations.   Use  QA/QC
 samples (Section 4.4) to evaluate errors due to improper
 sampling methodology and sample handling procedures.
 These guidelines should apply to biological as  well as
 soil, sediment, and water sampling.

 During fishing operations, the sampling crew can  prevent
 habitat disturbance by staying out of the water  body near
 the sampling locations.  The use  of any  particular
 technique  may  introduce judgment error  into the
 sampling regimen if done improperly. For all techniques,
 sampling should be conducted from the downstream
 location to the upstream location to avoid contamination
 of the  upstream stations.   Data  comparability is
 maintained by using similar collection methods and
 sampling efforts at all stations.

 Rapid bioassessments in the field should include two
 QA/QC procedures: 1) collection of replicate samples at
 stauons to check on the accuracy of the collection effort, -
 and 2) repeal a  portion (typically  10%) recount and
 ^identification for accuracy.   ,

 For tissue analyses, tools and other sampling equipment
 should  be  dedicated  to each sample,  or  must  be
 decontaminated between uses. To avoid contamination,
 sample containers must be compatible with the intended
 tissue matrix and analysis.

 4.3.3      Sample  Heterogeneity
Tissues  destined  for  chemical analysis  should be
homogenized Ideally, tissue sample homogenates should
consist of organisms  of the same species, sex, and
development stage and size since these variables all affect
chemical uptake.  There is no universal SOP for tissue
homogenization; specific procedures depend on the size
and type of the organism. For example, tissues must be
cut from fur and shell-bearing organisms as they cannot
be practically homogenized as a whole.  Homogenization
procedures  may  vary  by  site objective.    Tissue
homogenates should be stored away from light and kept
frozen at -20° C. Tissue homogenates are prepared in
the  laboratory  and  could  be  subject  to  cross-
contamination.

Refer  to  U.S.  EPA/ERT  SOP   #1820,   Tissue
Homogenizaaon Procedures for further details on tissue
homogenization procedures.

4.3.4     Sample Analysis

Analytical  procedures  may introduce  errors  from
laboratory cross-contamination,  extraction difficulties,
and inappropriate methodology. Fats naturally present in
tissues may interfere with sample analysis or extraction
and elevate detection limits. Detection limits in the tissue
samples must be the same as in  the background tissue
samples if a meaningful comparison is to be made.  To
minimize  this interference,  select an extraction  or
digestion procedure applicable to tissue samples.

Because   many   compounds   (e.g.,   chlorinated
hydrocarbons) concentrate in fatty  tissues, a percent lipid
analysis is necessary to normalize results among samples.
Lipid recoveries vary among different analytical methods;
percent lipid results for samples to be normalized and
compared must be generated by the  same analytical
method. Select a lipid analysis based on the objective of
the study (see references Heroes and Allen [1983] and
Bligh and Dyer 1959).   Sample  results may  be
normalized on a wet-weight basis. If sample results are
to be reported  on a dry-weight basis, instruct the
analytical laboratory to report  the percent moisture
content for each sample.

Appropriate   sample  preservation  prevents   loss of
compounds and decomposition of tissues before analysis.
Consult the  appropriate SOP,   analytical method, or
designated laboratory contact to confirm holding times for
tissue samples.
                                                     22

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Tissue samples destined for sorting and identification
(e.g,, benthic macroinvenebrates. voucher fish) should be
preserved in isopropyl or ethyl alcohol, formalin, or
Kahle's solution. Preservation in these solvents precludes
any chemical analysis.

4.4   QA/QC SAMPLES

QA/QC samples are collected at the site as prepared by
the laboratory. Analysis of the QA/QC samples provides
information on the variability and usability of biological
sampling data,  indicates  possible field  sampling or
laboratory error, and provides a basis for future validation
and usability of the analytical data. The most common
field QA/QC samples are field replicates, reference, and
rinsate blank samples.  The most common laboratory
QA/QC samples are performance evaluation (PE), matrix
spike (MS), and matrix spike duplicate (MSD) samples.
QA/QC results may  suggest  the need for modifying
sample collection, preparation, handling, or analytical
procedures if the resultant data do not meet site-specific
quality assurance objectives.

Refer to data validation procedures in U.S. EPA Quality
Assurance/Quality  Control (QA/QC)  Guidance for
Removal Activities, EPA/540/G-9Q/004. April 1990, for
guidelines on utilizing QA/QC samples.

4.4.1      Replicate Samples

Field Replicates

Field replicates for solid media  are samples obtained
from one sampling point that are  homogenized, divided
into separate containers, and treated as separate samples
throughout the remaining sample handling and analytical
processes.   Field replicates for  aqueous samples are
samples obtained from one location that are homogenized
and divided into separate containers. There are no "true"
field  replicates  for   biological  samples, however,
biological samples collected from the same station are
typically referred to  as replicates.  In  this case, the
biological replicates are used to determine the variability
associated  with  heterogeneity  within  a  biological
population.  Field replicates may be sent to two or more
laboratories or to the same laboratory as unique samples.

Field replicates  may be used to determine total error for
critical  samples with contaminant concentrations near the
level  that  determines  environmental  impact   To
determine error, a minimum of eight replicate samples is
recommended for valid sgtjyrirai analysis.. For total error
determination, samples should be analyzed by the same
laboratory. The higher detection limit associated with
composite samples may limit the usefulness  of error
determination.

NOTE: A replicate biological sample may consist of
more than a single organism in those cases where the
species mass  is less  than the mass required by the
analytical procedure to attain required detection limits.
This  variability in  replicate  biological  samples  is
independent of the variability in analytical procedures.
Toxicitv Testing Replicates

For sediment samples, at least 3  replicate treatments
should be conducted to determine variability between
testsJThe function of these replicates is to determine the-
variability of the test organism population within each
treatment This assumes the sample matrix exhibits a
uniform concentration of the contaminants of concern
within each treatment Large variability may indicate a
problem with the test procedures or organisms or lack of
contaminant homogeneiry{within the sample matrix.
Site-Sr-Tic Fj
Examle No. 1
                     of the Use of Replicates
Two contaminant sources were identified at an active
copper smelting facility. The first area was a slag pile
containing high levels of copper suspected of migrating
into,  the  surrounding  surface  runoff  pathways,
subsequently leaching into the  surface water  of a
surrounding stream system.  The second area was the
contaminated creek sediment that was  present in the
drainage pathway of the slag pile.

Whole-phase sediment toxicity tests were selected to
evaluate the toxicity associated with the copper levels in
the stream sediments.  Sediment was collected at each
sampling location  (six locations  total)  to provide the
testing laboratory  with sufficient sample volume to
perform these evaluations.  Ten-day static renewal tests
using the amphipod, Hyalella azteca, and the midge,
Chironomus lentans,  were chosen.  The toxicity test
utilized  four "replicates"  per sampling  location (or
treatment), each replicate containing fifteen organisms.
The purpose of these replicates was to determine the
                                                     23

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 variability within the test organism population within
 each treatment.

 The results reported mean survival for Hyalella azteca in
 the  contaminated sediment (8  to  SO percent)  to be
 significantly lower than survival in  the uncontaminated
 reference sediment  (85 percent).   Similarly,  mean
 survival for Chironomus unions in the contaminated
 sediment (0 to 63 percent) was significantly lower than
 survival in the uncontaminated reference sediment (83
 percent).

 Example T^p, ^

 An inactive manufacturing facility had stored its stock
 compounds in unprotected piles for a number of years,
 resulting  .in DDT  contamination of  the  adjacent
 watershed.  DDT contamination in a stream located
 adjacent to  the site extended from the manufacturing
 facility to approximately 27 miles downstream.

 A field study was designed to quantitatively determine if
 the levels of DDT in  the water and sediment in this
 stream were resulting in an adverse ecological impact.
 This was accomplished  through  the  examination of
 several in situ environmental variables in conjunction
 with laboratory analyses. Water, sediment, and resident
 biota were collected and submitted for various physical
 and chemical determinations. Additional sediments were
 secured  and utilized for toxicity  testing  with three
 surrogate  species.  Finally, the benthic invertebrate
 community was sampled and the structure and function of
 this segment of the aquatic ecosystem evaluated.

Benthic invertebrates were collected from three areas at
each sampling  location  (i.e.,  three  "replicates" per
location)  and  evaluated  for  various  quantitative
community metrics. The purpose of these replicates were
to determine the spatial variability in the stream among
the   three   areas  within  each  sampling  location.
Community structure, diversity  indices,  taxonomic
evenness, an evaluation of the function feeding groups.
and statistical analyses were performed on the data set.

Qualitative and  statistical  comparison of the  results
between the contaminated areas and the uncontaminated
reference  indicated  that  the  benthic  invertebrate
community was adversely affected downstream of the site
compared to the upstream reference. Taxonomic and
functional diversity varied inversely with DDT levels in
sediment  and  water.   These  results  were  further
substantiated by the toxicity evaluation results.

Example No. 3

Phase I and D Remedial Investigation and Feasibility
Studies (RTFS) have indicated that the soils surrounding
an industrial and municipal  waste  disposal site were
contaminated with PCBs. A preliminary site survey
revealed the presence of small mammal habitat and
mammal signs in the natural areas adjacent to the site as
well as  an area that appeared to be outside-of the site's
influence (ix..  a potential reference area).   A site
investigation was subsequently conducted to determine
the levels  of PCBs accumulating into the resident
mammal community from contact with  the  PCB-
contaminated soil.

Three small mammal trapping areas were identified for
this site. Two areas were located in PCB-contanunated
areas, the third  area was a reference. Trapping grids .
were established in each area consisting of 100 traps of
various design. Six soil samples were also collected from
each trapping area to characterize the levels of PCBs
associated with the anticipated captured mammals.

A total  of  32 mammals were collected at this site.
Twelve  were collected from each on-site area and  six
were collected from the  reference area.   All captured
mammals were  submitted for whole body analysis of
PCBs. Mean PCS concentrations in the mammals were
as follows:  on-site areas (1250 and  1340 wg/kg, wet
weight); reference area (490 yg/kg, wet weight).  A one-
way analysis of variance was conducted on the data  set
treating each animal in an area as a "replicate" (i.e.. 12
replicates from each on-site area and 6 replicates from
the reference). The results of the statistical analyses
indicated that  there was a statistically  significant
difference between on-site and reference area PCB levels
in the mammals (p<0.lO).  Therefore, in this example.
there were no analytical replicates since each individual
mammal was  analyzed.   However, each mammal
represented a statistical replicate within  each trapping
area.

4.4.2      Collocated Samples

A collocated sample is collected from an area adjoining
a field sample to determine variability of the matrix and
contaminants within a  small area  of the  site.   For
example, collocated samples for chemistry analysis split
                                                    24

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 from the sample collected  for the toxicity test are
 collected about one-half to three feet away from the field
 sample location. Plants collected from within the same
 sampling plot may be considered collocated.  Collocated
 samples are appropriate for assessing variability only in
 a small area, and should not be used to assess variability
 across the entire site or for assessing error.

 4.4.3     Reference Samples

 Reference  biological  samples may  be taken from a
 reference  area  outside  the influence  of the site.
 Comparison of results from actual samples and samples
 from the reference area may indicate uptake,  body
 burden, or accumulation of chemicals on the site. The
 reference area should be close to the site. It should have
 habitats, size  and terrain similar to  the  site  under
 investigation.  The reference site need not be pristine.
 Biological  reference  samples should be of the  same
.species, sex, and developmental stage  as the field site
 sample.

 4.4.4     Rinsate Blank Samples

 A  nnsate blank is used to assess  cross-contamination
 from improper equipment decontamination procedures.
 Rinsate blanks are samples obtained by running analyte-
 frec water over  decontaminated sampling equipment.
 Any residual contamination should appear in the rihsate
 data. Analyze the rinsate blank for the same analytical
 parameters as the field samples collected that day.  When
 dedicated cutting tools or other sampling equipment are
 not used, collect one rinsate blank per device per day.

 4.4.5     Field Blank Samples

 Field blanks are samples prepared  in the  field using
 certified clean water or sand that are then submitted to the
 laboratory' for analysis. A  field blank  is used to evaluate
 contamination   or error  associated  with  sampling
 methodology,  preservation,   handling/shipping,  and
 laboratory procedures. If appropriate for the test, submit
 one field blank per day.

 4.4.6     Trip Blank Samples

 Trip blanks are samples prepared prior to going into the
 field. They consist of certified clean water or sand, and
 they are not opened until they reach the  laboratory. Use
 tnp blanks when samples are being  analyzed for volatile
organics. Handle, transport, and analyze trip blanks-in
the same manner as the other volatile organic samples
collected that day. Trip blanks are used to evaluate error
associated with sampling methodology, shipping and
handling, and analytical procedures, since any volatile
organic contamination of a trip blank would have to be
introduced during one of those procedures.

4.4.7     Performance       Evaluation
           /Laboratory Control Samples

A performance evaluation (PE) sample evaluates the
overall error from the analytical laboratory and detects
any bias in the analytical method being used.  PE samples
contain known quantities of target analytes manufactured
under strict quality control. They are usually prepared by
a third party under a U.S. EPA certification program.
The samples are usually submitted "blind" to analytical
laboratories (the sampling team knows the contents of the
samples, but  the  laboratory does not).  Laboratory
analytical error (usually bias) may be evaluated by the
percent recoveries and correct identification of the
components in  the PE sample.

4.4.8     Controls

Analytical Laboratory Control Samples

A chemical analytical laboratory control sample (LCS)
contains  quantities of target analytes known to the
laboratory  and are  used  to  monitor "controlled"
conditions.  LCSs are analyzed under the same sample
preparation, reagents, and analytical methods as the field
samples. LCS results can show bias and/or variability in
analytical results.

ToKicirv Testing Control Groups

In toxicity tests,  a laboratory reference toxicant treatment
and a control  treatment are both  typically utilized  in
addition to a site reference treatment. This test involves
exposing the test organism population to a standardized
reference toxicant at a standardized dose, then comparing
the response to historical laboratory  records  for that
culture.  The mortality results of the newly conducted
reference toxicant test should be similar to the historical
results. This is conducted to reveal if the generation(s) in
the present culture is viable for use in the toxicity test, or
if the culture has grown  resistant or  intolerant to the
toxicant over time.  Therefore, a  laboratory reference
                                                     25

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 toxicant test should be conducted prior to the testing of
 the site matrices.

 In  contrast,  a laboratory control  test is  conducted
 simultaneously with the testing of the site matrices. This
 treatment identifies mortality factors that are unrelated to
 site contaminants. This is accomplished by exposing the
 test organism population to a clean dilution water and/or
 a clean laboratory substrate.

 4.4.9    Matrix    Spike/Matrix    Spike
           Duplicate Samples   /

 Matrix spike and  matrix spike  duplicate  samples
 (MS/MSDs) are supplemental volumes of field-collected
 samples that are spiked in the laboratory with a known
 concentration  of a target analyte to determine matrix
 interference.   Matrix interference  is determined as  a
 function of the percent analyte recovery in the sample
extraction.   The percent recovery from MS/MSDs
 indicates the degree to which matrix interferences will
 affect the identification and/or quantitation of a substance.
 MS/MSDs can also be  used  to  monitor  laboratory
 performance. When two or more pairs of MS/MSDs are
 analyzed, the data obtained may also be used to evaluate
error due to laboratory bias and precision. Analyze one
MS/MS D pair to assess bias for every 10 samples, and
use the average percent recovery for the pair. To assess
precision, analyze at least eight matrix spike replicates
 from the  same sample, and determine the standard
deviation and the coefficient of variation.  See the U.S.
EPA Quality  Assurance/ Quality  Control  (QA/QC)
Guidance for Removal  Activities (April  1990) for
directions on calculating analytical error.

MS/MSDs are a  required  QA/QC  element of the
definitive data objectives.  MS/MSDs should accompany
every 10 samples.  Since the MS/MSDs are spiked field
samples, sufficient volume for three separate analyses
must be provided.  Organic analysis of tissue samples is
frequently subject to matrix interferences which causes
biased  analytical results.  Matrix spike recoveries are
often low or show poor precision in tissue samples. The
matrix  interferences will be evident in the matrix spike
results. Although metals analysis of tissue samples is
usually not subject to  these interferences. MS/MSD
samples  should  be utilized  to  monitor method  and
laboratory performance.  Some  analytical parameters
such as percent hpids. organic carbon, and panicle-size
distribution are exempt from MS/MSD analyses.
4.4.10   Laboratory Duplicate
           Samples

A laboratory duplicate  is -a  sample that  undergoes
preparation and analysis twice. The laboratory takes two
aliquots of one sample and treats them as if they were
separate samples.  Comparison of data  from the two
analyses provides a measure of analytical reproducibility
within a sample set. Discrepancies in duplicate analyses
may indicate poor homogenization in the field or other
sample preparation error, whether in the  field or in the
laboratory. However, duplicate analyses are not possible
with most tissue samples unless a homogenate of the
sample is created.

4.5  Data Evaluation

4.5.1     Evaluation of Analytical Error

Analytical error becomes significant in decision-making
as sample results approach the level of environmental
impact  The acceptable level of error is determined by
the intended use of the data and litigation  concerns.  To
be definitive, analytical  data  must  have quantitative
measurement of analytical error with PE samples and
replicates. The QA samples identified in this section can
indicate a variety of qualitative and quantitative sampling
errors.  Due to matrix interferences, causes of error may
be difficult to determine in organic  analysis of  tissue
samples.

4.5.2     Data Validation

Data  from tissue sample analysis  may  be validated
according to the Contract Laboratory Program National
Functional Guidelines (U.S. EPA 1994) and according to
US. EPA Quality Assurance/Quality Control (QA/QC)
Guidance for Removal Activities, EPA/540/G-90/004,
April  1990.  Validation of organic data may  require an
experienced chemist due to complexity of tissue analysis.
                                                    26

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                    5.0  DATA ANALYSIS AND INTERPRETATION
5.1  INTRODUCTION

The main objective of biological surveys conducted at
Supertund sites is the assessment of site-related threat or
effect. For many types of biological data (e.g., levels of
contaminants in organisms collected on site and from a
reference location), hypotheses are tested to determine
the presence or absence of an effect For some biological
tests (e.g., benthic macroinvertebratc studies, toxicity
tests), the data  analysis and interpretation process is
outlined in existing documents (U.S. EPA November
1990, U.S.  EPA  May 1996). For many Superfund
ecological assessments, a weight-of-evidence approach
is used to interpret the results of different studies or tests
conducted at a site.

The statistical tests and methods that will be employed
should be based on the objective of the data evaluation.
These components should be outlined in the Work Plan
or Sampling and Analysis Plan. This process will help
focus the study to ensure that the appropriate type and
number of samples are collected.

5.2   DATA PRESENTATION AND
       ANALYSIS

5.2.1     Data Presentation Techniques

In many cases, before descriptive statistics are calculated
from a data set. it is useful to try  various graphical
displays of the raw data. The graphical displays help
guide the choice of any necessary transformations of the
data set and  the selection  of  appropriate statistics to
summarize the data. Since most statistical procedures
require summary statistics calculated from a data set. it is
important that the summary statistics represent the entire
data set. For example,  the median may  be  a  more
appropriate measure of central tendency than the  mean
for a data set that contains outliers.  Graphical display of
a data set could indicate the need to log transform data so
that symmetry indicates a normal distribution.  Four of the
most useful graphical techniques are  described next.

A histogram  is a bar graph that displays the distribution
of a data set. and provides information regarding  the
location of the center of the sample, amount of dispersion.
extent of symmetry, and existence of outliers. Stem and
leaf plots are similar to histograms in that they provide
information on the distribution of a data set; however they
also contain information on the numeric values in the data
set Box and whisker plots can be used to compare two
or more samples of the same characteristic (e.g., stream
ffil values for two or more years).  Scatter plots are a
useful method for examining the relationship between
two sets of variables. Figure 4 illustrates the four graph
techniques described previously.

5.2.2     Descriptive Statistics

Large data sets are  often summarized using a few
descriptive statistics. Two important features of a set of
data are the central tendency and the spread. Statistics
used to describe central tendency include the arithmetic
mean, median, mode and geometric mean.  Spread or
dispersion in a data set refers to the variability in the
observations about  the  center  of the distribution.
Statistics used to describe data dispersion include range
and  standard deviation.   Methods  for  calculating
descriptive  statistics  can  be  found in  any statistics
textbook, and many software programs are available for
statistical calculations.

5.2.3     Hypothesis Testing

Biological studies  are conducted at Superfund sites to
determine adverse effects due to site-related factors. For
many types of biological data, hypothesis testing is the
statistical procedure used to evaluate data.  Hypothesis
testing involves statistically evaluating a parameter of
concern, such as the mean or median, at  a specified
probability  for  incorrectly interpreting the   analysis
results.  In conventional statistical analysis, hypothesis
testing for a trend or effect is based on a null  hypothesis.
Typically, the null hypothesis is presumed when there is
no trend or effect present.  To test this hypothesis, data
are collected to estimate an effect The data are used to
provide a sample estimate of a test statistic, and a table
for the test statistic is consulted to determine how  unlikely
the observed value of the statistic is if the null hypothesis
is true.  If the observed value of  the test statistic is
unlikely, the null hypothesis is rejected. In ecological risk
assessment a  hypothesis  is  a question  about the
relationship among assessment endpoints and  their
                                                   27

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 predicted responses when exposed to contaminants. The
 most basic hypothesis that is applicable to virtually all
 Superfund sites is that site-related contaminants are
 causing adverse effects of the assessment endpoint(s).

 5.3   DATA INTERPRETATION

 5.3.1      Chemical Residue Studies

 Chemical  residue data may be evaluated in two ways.
 First, the contaminant  concentrations by themselves
 provide evidence of bioaccumulation and probable food
 chain transfer of the contaminants, and an overall picture
 of the distribution of contaminants in the biological
 community. Second, the residue data may be evaluated
 against literature residue values that are known to cause
 no effect or an adverse effect in the organism.

 5.3.2      Population/Community
            Studies

 The  interpretation of  population/community data is
 extensive, therefore, the reader is referred to a detailed
 treatment  in U.S. EPA (November 1990), U.S.  EPA
 (1989a), Karr et al. (1986). and other literature.

 5.3.3     Toxicity Testing

 Measurement endpoints obtained in toxicity tests are
.generally compared to results from a laboratory control
 and a reference location sample to determine whether
 statistically significant differences exist.  If significant
 effects (e.g.. mortality, decreased reproduction) are
 observed,  additional statistical analyses can be run to
 determine  whether observed  effects correlate  with
 measured contaminant levels. The reader is referred to a
 detailed treatment in  ASTM (1992).  U.S. EPA (May
 1988). U.S. EPA (March 1989b).

 5.3.4      Risk Calculation

 Preliminary screening value results are interpreted by
 comparison of histohcal and/or new site analytical data
 against literature toxicity values.  This comparison will
 suggest if the probability of risk exists and whether
 additional  evaluation is desired.
assessment, mathematical models, such as the Hazard
Quotient method, are used to  evaluate the  site  data
against literature toxicity values. Based on the type of
model used, the results can be extrapolated to suggest the
presence of ecological risk.
If the evaluation is pursued to an  ecological  risk
                                                   28

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                Figure 3 Illustrations of Sample Plots
                            IBI DATA

                         12 25  33 56
                         12 24  34 SB
                         14 26  35
                         15 24  36
                         16 24  35
                         22 27  3B
                         24 23  41
                         23 28  42
 A) Histogram
B) Leaf Plot
                                                     250   300   350    '
                                                       Sediment Zinc (mg/Kg)
C) Whisker Plot                                    D) Scatter Plot

                               29

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                              APPENDIX A - CHECKLIST FOR ECOLOGICAL
                                         ASSESSMENT/SAMPLING
Introduction

The checklist that follows provides guidance in making observations for an ecological assessment  It is not intended for
limited or emergency response actions (e.g., removal of a few drums) or for purely industrial settings with no discharges.
The checklist is a screening tool for preliminary site evaluation and may also be useful in planning more extensive site
investigations. It must be completed as thoroughly as time allows. The results of the checklist will serve as a starting point
for the collection of appropriate biological data to be used in developing a response action.  It is recognized that certain
questions in  this checklist are not universally applicable and that site-specific conditions will influence interpretation.
Therefore, a site synopsis is requested to facilitate final review of the checklist by a mined ecologist


Checklist

The checklist has been divided into sections that correspond to data collection methods and ecosystem types. These sections
are:

I.   Site Description

    1A.   Summary of Observations and Site Setting

IL  Terrestrial Habitat Checklist

    HA.  Wooded
    Iffi.  Shrub/Scrub
    EC.  Open Field                                                                  .
    ED.  Miscellaneous

m. Aquatic Habitat Checklist - Non-Flowing Systems

IV. Aquatic Habitat Checklist - Flowing Systems

V.  Wetlands Habitat Checklist
                                                     30

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                 Checklist for Ecological Assessment/Sampling
I. SITE DESCRIPTION
1.  Site Name:.

    Location: _
    County:	City:	State:.
2.  Latitude: 	,	                Longitude:
3.   What is the approximate area of the site?.
4.   Is this the first site visit? D yes D no If no, attach trip report of previous site visit(s), if available.

    Date(s) of previous site visitfs):



5.   Please attach to the checklist USGS topographic map(s) of the site, if available.
6.   Are aerial or other site photographs available? D yes D no If yes, please attach any available photo(s) to the site
    map at the conclusion of this section.
                                                31

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7.  The land use on the site is:


    	% Urban

    	_% Rural

    	% Residential

    	% Industrial  (D light D heavy)

    	% Agricultural

    (Crops:	
    	% Recreational

    (Describe; note if it is a park, etc.)
         _%  Undisturbed

         _%  Other
                   The area surrounding the site is:
                   	mile radius

                   	% Urban

                   	% Rural

                   _,	% Residential

                   	% Industrial (D light D heavy)

                   	% Agricultural

                   (Crops:	;	
                   Recreational

                   (Describe; note if it is a park, etc.)
                        _% Undisturbed

                        _% Other
8.   Has any movement of soil taken place at the site? D yes D no. If yes, please identify the most likely cause of this
    disturbance:
    	Agricultural Use

    	Natural Events

    Please describe:
. Heavy Equipment

 Erosion
. Mining

.Other
                                                    32

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9.  Do any potentially sensitive environmental anus exist adjacent to or in proximity to the site, e.g., Federal and State
    paries. National and State monuments, wetlands, prairie potholes? Remember, flood plains and wetlands are not
    always obvious; do not answer "no" without confirming information.
    Please provide the source(s) of information used to identify these sensitive areas, and indicate their general location
    on the site map.
10. What type of facility is located at the site?

    D Chemical          O  Manufacturing D Mixing        D Waste disposal

    D Other (specify)	
11.  What are the suspected contaminants of concern at the site? If known, what are the maximum concentration levels?
12. Check any potential routes of off-site migration of contaminants observed at the site:

    O Swales                    D Depressions                   D  Drainage ditches

    " Runoff                    O Windblown paniculate* D  Vehicular traffic

    [3 Other (specify)	
13.  If known, what is the approximate depth to the water table?.
14.  Is the direction of surface runoff apparent from site observations? D yes D  no If yes, to which of the following
    does the surface runoff discharge?  Indicate all that apply.

    Z  Surface water      U Groundwater          D Sewer        O Collection impoundment


IS.  Is there a navigable waterbody or tributary to a navigable waterbody?        D yes O no
                                                     33

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 16.  Is there a wateibody anywhere on or in the vicinity of the site? If yes, also complete Section ffl: Aquatic Habitat
     Checklist - Non-Flowing Systems and/or Section IV: Aquatic Habitat Checklist - Flowing Systems.

     D yes (approx. distance	)        D no


 17.  Is there evidence of flooding? OyesDno Wetlands and flood plains are not always obvious; do not answer "no"
     without confirming information. If yes, complete Section V: Wetland Habitat Checklist


 18.  If a field guide was used to aid any of the identifications, please provide a reference. Also, estimate the time spent
     identifying fauna.  [Use a blank sheet if additional space is needed for text]
19. Are any threatened and/or endangered species (plant or animal) known to inhabit the area of the site? D yes D no
    If yes. you are required to verify this information with the U.S. Fish and Wildlife Service.  If species' identities are
    known, please list them next
20. Record weather conditions at the time this checklist was prepared:

    DATE:__	'

    	Temperature (°C/CF)          	Normal daily high temperature

    	Wind (direction/speed)        	Precipitation (rain, snow)

            	Cloud cover
                                                     34

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 1A. SUMMARY OF OBSERVATIONS AND SITE SETTING
Completed by	i	 Affiliation.
Additional Preparers.




Site Manager	
Date	




                                           35

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IL TERRESTRIAL HABITAT CHECKLIST

DA.      WOODED


1.  Are there any wooded areas at the site? Dyes D no If no, go to Section HE: Shnib/Scnib.

2.  What percentage or area of the site is wooded? (	_%	acres). Indicate the wooded area on the site map
    which is attached to a copy of this checklist. Please identify what information was used to determine the wooded
    area of the site.
3.  What is the dominant type of vegetation in the wooded area? (Circle one; Evergreen/Deciduous/ Mixed) Provide a
    photograph, if available.
    Dominant plant, if known:.
4.  What is the predominant size of the trees at the site? Use diameter at breast height

     D  0-6 in.           D 6-12 in.      D>12in.


5.  Specify type of understory present, if known. Provide a photograph, if available.
ITB.     SHRUB/SCRUB


1.   Is shrub/scrub vegetation present at the site? D yes D no If no, go to Section EC: Open Field.


2.   What percentage of the site is covered by scrub/shrub vegetation? (	%	acres). Indicate the areas of
    shrub/scrub on the site map. Please identify what information was used to determine this area.
3.   What is the dominant type of scrub/shrub vegetation, if known? Provide a photograph, if available.



4.   What is the approximate average height of the scrub/shrub vegetation?

    D 0-2 ft.            D 2-5 ft.        D > 5 ft.

                                                   36

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S.  Based on site observations, how dense is the scrub/shrub vegetation?

     D Dense            D Patchy       D Sparse


IIC      OPEN FIELD

1.  Are there open (bare, barren) field areas present at the site? Dyes Ono If yes, please
    indicate the type below:
    D Prairie/plains      D Savannah     D Old field      D Other (specify).
2.  What percentage of the site is open field? (	% ___ acres). Indicate the open fields on the site map.

3.  What is/are the dominant plant(s)? Provide a photograph, if available.
4.  What is the approximate average height of the dominant plant?.
5.  Describe the vegetation cover  D Dense        D  Sparse        D Patchy


ITO.      MISCELLANEOUS

1.  Are other types of terrestrial habitats present at the site, other than woods, scrub/shrub, and open field?  Dyes Dno
    If yes. identify and describe them below.
2.   Describe the terrestrial miscellaneous habitat(s) and identify these area(s) on the site map.
                                                     37

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3.  What observations, if any, were made at the site regarding the presence and/or absence of insects, fish, birds,
    mammals, etc.?
4.  Review the questions in Section I to determine if any additional habitat checklists should be completed for this site.
                                                     38

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    AQUATIC HABITAT CHECKLIST - NON-FLOWING SYSTEMS

Note:    Aquatic systems are often associated with wetland habitats. Please refer to Section V, Wetland Habitat
         Checklist.

1.  What type of open-water, non-flowing system is present at the site?

    O Natural (pond, lake)
    D Artificially created (lagoon, reservoir, canal, impoundment)


2.  If known, what is the iiame(s) of the waterbody(ies) on or adjacent to the site?
3.  If a waterbpdy is present, what are its known uses (e.g.: recreation, navigation, etc.)?
                                 *'
4.  What is the approximate size of the waterbody(ies)?  	acre(s).


5.  Is any aquatic vegetation present?  O yes D no If yes. please identify the type of vegetation present if known.

          O Emergent             D Submergent           O Floating


6.  If known, what is the depth of the water?	
7.  What is the general composition of the substrate? Check all that apply.

    Z Bedrock                  G Sand (coarse)         D Muck (fine/black)

    Z Boulder (>10 in.)   '       D Silt (fine)             D Debris

    Z Cobble (2.5-10 in.)        C Marl (shells)          D Detritus

    Z Gravel (0.1-2.5 in.)        D Clay (slick)           D Concrete

    Z Other (specify)	'
8.   What is the source of water in the waterbody?

    Z  River/Stream/Creek                D Groundwater          D Other (specify).

    Z  Industrial discharge                D Surface runoff
                                                   39

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9.  Is there a discharge from the site to the waterbody? Dyes Dno  If yes, please describe this
    discharge and its path.
10. Is there a discharge from the waterbody? D yes O no  If yes. and the information is available, identify from the list
    below the environment into which the waterbody discharges.
    D River/Stream/Creek        D onsite        D offsite        Distance	

    D Groundwater              D onsite        D offsite

    D Wetland                  D onsite        D offsite        Distance	

    D Impoundment              D onsite        D offsite


11.  Identify any field measurements and observations of water quality that were made. For those parameters for which
    data were collected provide the measurement and the units of measure below:
                         Area

                         Depth (average)

                         Temperature (depth of the water at which the reading was taken)	

                         pH

                         Dissolved oxygen

                         Salinity

                         Turbidity (clear, slightly turbid, turbid, opaque) (Secchi disk depth.

                         Other (specify)
12.  Describe observed color and area of coloration.
13.  Mark the open-water, non-flowing system on the site map attached to this checklist
                                                     40

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14. What observations, if any, were made at the waterbody regarding the presence and/or absence of benthic
    rnacroinvertebrates, fish, birds, mammals, etc.?
                                                     41

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 IV. AQUATIC HABITAT CHECKLIST - FLOWING SYSTEMS

 Note:     Aquatic systems are often associated with wetland habitats. Please refer to Section V. Wetland Habitat
          Checklist.

 1.   What type(s) of flowing water system(s) is (are) present at the site?

          D  River                D  Stream                       D Creek
          D  Dry wash            D  Arroyo                       D Brook
          D  Artificially           D  Intermittent Stream             D Channeling
            created               D  Other (specify)	;	
            (ditch, etc.)

 2.   If known, what is the name of the waterbodv?
3.  For natural systems, are there any indicators of physical alteration (e.g., channeling, debris, etc.)?
    D yes   D no If yes, please describe indicators that were observed.
4.  What is the general composition of the substrate? Check all that apply.

    D Bedrock                  D Sand (coarse)          D Muck (fine/black)

    D Boulder (>10 in.)          D Silt (fine)             D Debris

    D Cobble (2.5-10 in.)        D Marl (shells)          D Detritus

    C Gravel (0.1-2.5 in.)        D Clay (slick)            D Concrete

    C Other (specify)	.


5.   What is the condition of the bank (e.g., height, slope, extent of vegetative cover)?
6.   Is the system influenced by tides? O yes D no  What information was used to make this determination?
                                                    42

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7.  Is the flow intermittent? Dyes Dno If yes, please note the information that was used in making this determination.
8.  Is there a discharge from the site to the waterbody? Dyes Dno If yes. please describe the discharge and its path.
9.  Is there a discharge from the waterbody? D yes D no  If yes. and the information is available, please identify what
    the waterbody discharges to and whether the discharge is on site or off site.
10. Identify any field measurements and observations of water quality that were made. For those parameters for which
    data were collected, provide the measurement and the units of measure in the appropriate space below:

    	               Width (ft.)

    	               Depth (ft.)

    	               Velocity (specify units):
                         Temperature (depth of the water at which the reading was taken.

                         pH

                         Dissolved oxygen

                         Salinity               ^  ,

                         Turbidity (clear, slightly turbid, turbid, opaque)
                         (Secchi disk depth        '  	)

                         Other (specify)	
                                                      43

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11. Describe observed color and area of coloration.
12. Is any aquatic vegetation present? Dyes Ono If yes, please identify the type of vegetation present if known.

    D Emergent                 D Submergent           D Floating


13. Mark the flowing water system on the attached site map.
14.  What observations were made at the waterbody regarding the presence and/or absence of benthic
    macroinvenebrates, fish, birds, mammals, etc.?
                                                     44

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V.  WETLAND HABITAT CHECKLIST

1.  Based on observations and/or available information, are designated or known wetlands definitely present at the site?
    Dyes Dno

    Please note the sources of observations and information used (e.g., USGS Topographic Maps, National Wetland
    Inventory. Federal or State Agency, etc.) to make this determination.
2.  Based on the location of the site (e.g., along a waterbody, in a floodplain) and site conditions (e.g., standing water.
    dark, wet soils; mud cracks; debris line; water marks), are wetland habitats suspected?
    Dyes D no If yes, proceed with the remainder of the wetland habitat identification checklist


3.  What type(s) of vegetation are present in the wetland?

    D Submergent                       D  Emergent
    D Scrub/Shrub                       D  Wooded

    D Other (specify)	
4.  Provide a general description of the vegetation present in and around the wetland (height, color, etc.).  Provide a
    photograph of the known or suspected wetlands, if available.
5.  Is standing water present? Dyes O no If yes, is this water O Fresh D Brackish
    What is the approximate area of the water (sq. ft)?	;	
    Please complete questions 4,11,12 in Checklist ID • Aquatic Habitat - Non-Flowing Systems.
6   Is there evidence of flooding at the site? What observations were noted?

    ~ Buttressing                D Water marks          D Mud cracks

    Z Debris line                D Other (describe below)
                                                    45

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 7.  If known, what is the source of the water in the wetland?

     D StreanVRiver/Creek/Lake/Pond               D  Groundwater

     D Flooding                                   D  Surface Runoff
 8.   Is there a discharge from the site to a known or suspected wetland? D yes D no  If yes, please describe.
. 9.   Is there a discharge from the wetland? D yes D no. If yes, to what waterbody is discharge released?

     G  Surface Stream/River       D Groundwater  D Lake/Pond            D Marine
 10.  If a soil sample was collected, describe the appearance of the soil in the wetland area. Circle or write in the best
     response.

     Color (blue/gray, brown, black, mottled)                                          '
     Water content (dry. wet. saturated/unsaturated).
 11. Mark the observed wetland area(s) on the attached site map.
                                                      46

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          APPENDIX B - Example of Flow Diagram For Conceptual Site Model
                               Figure B-1
    Migration  Routes of  a Gas Contaminant
                from Origin to  Receptor
 Original atal
of contaminant
 of concern*
Pathway
 from
 origin
                 condenaatlon
      **
             Air
                 solidification
Change of
contaminant
atate In
pathway
n
Liquid —
_ **
f^*»*»
UaS
Solid
\j\jt\\j
n
Final
pathway
to receptor
,- > SO
1— > SW
1 — » so
•. AT
- • • > Ml
. QW
* OVY
a. QO
r oU
_> SW
r O VV
!
Human
_G'D
G,D
I,D
I,D
G,D
G,D
G,D
Receptor
Ecological Threat
Terrestrial
G,p
G,D
I,D
I,D
I,D
G,D
G,D
Aquatic
N/A
G,D
N/A
N/A
G,D
N/A
G.D
          *  M_
          ** Inc
    e a transformation product
    IBS vapors
                                        Receptor Key
                                        N/A
• Dermal Contact
- Inhalation
   ittton
   Applicable
                              • lnfl0$Uon
                              - Not Appld
Pathway Key

so - Son
SW - Surface Water
(Including aedlmenta)
QW • Ground Water
                                  47

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                                   Figure B-2
       Migration  Routes of a  Liquid Contaminant
                     from  Origin to  Receptor
    Original slate
   of contaminant
     of e neern*
Liquid
Pathway
 from
 origin
 Change of
contaminant
  state In
  pathway
  Final
 pathway
to receptor
               SW
                   crystallization
 Liquid 	>

  Gas"  -[^

 Solid  	»
               SO
                     laachate,
                     Infiltration
            Liquid
                AI
  *  May be a transformation product
  ** Includes vapors
              Gas
                                    **
    SW

    AI

    SW

    SW


    SO

    SW

    GW
             >   SO

             •>   AI

             ->   sw
                    Pathway Key

                 AI . AIT
                 30-Sod
                 SW • Surtae* Witw
                    (kiehidlng ledlmtnti)
                 QW - Qround Water
                                                          Receptor
Human


 G,D

 b*>
 G^D

 G,D
  Ecological Threat

Terrestrial  Aquatic

 G,D    G,p

         N/A

        J3,A

         G,D
                                         G,J)

                                         G,D
G,D
oTo
G,D
G,D
G,D
N/A
N/A
Q,D
N/A
G,D
I,D
G,D
G,D
I,D
G,D
N/A
N/A
G,D
                              Recaptor K»y

                             D • Dwnui ConMel
                             I - tnhilitlon
Q                                Im---•!-.—
                               • mgvnrun
                             N/A - Not Appncabl*
                                      48

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                             Figure B-3
       Migration  Routes of  a Solid  Contaminant
                    from Origin  to  Receptor
 Original atate
of contaminant
  of concern*
Solid
 Pathway
  from
  origin
 Change of
contaminant
 •tale In
 pathway
              AI
                  partlculatea/
                    dual
              Solid
SW
»  Solid

>  Liquid

        **
               so
                            Liquid
  *  May be a transformation product
  ** Includes vapors
                     jjeceptor Key

                    0 - (Mmd ContMl
                    I - Inhilrion
                    0 • (ng»«"on
                    N/A - Not ApplkaMi
                Final
               pathway
              to receptor
                  AI
                  sw
                  so
                              _
                              Gas    —
              Solid
                  SO

                  AI

                  SW

                  SO

                  SO

                  GW

                  SW
                      Pathway Key
                     • Air
        AI
        SO
        BW - Surfie* Wilw
           flndudkiB Mdhntnti)
        OW - Ground Wittr
Receptor
Human
1,0
Q,D
G,D
Ecological Threat
Terrestrial
1,0
G.D
G,D
Aquatic
N/A
6,0
N/A
                                                         Q,D
,0    G,D
                                                  G,D
                                      G,D
G,D
1,0
G.D
G,D
G,D
G,D
G,D
G,D
1,0
G.D
G,D
G,D
N/A
G,D
N/A
N/A
G,D
N/A
N/A
N/A
G,D

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                                 APPENDIX C - EXAMPLE SITES

Example sites are presented in this document to demonstrate how information from the checklist for ecological
assessment/sampling is used in conjunction with representative biological sampling to meet the study objectives. A
genera] history for each site is presented first, then additional preliminary information

I.   SITE HISTORIES

Site A — Copper Site

This is a former municipal landfill located in an upland area of the mid-Atlantic plain. Residential, commercial, and
industrial refuse were disposed at the site from 1961 to 1980.  Large amounts of copper wire were also disposed at this
site. Minimal grass cover has been placed over the fill. Terrestrial ecosystems in the vicinity of the landfill include
upland forest, successional fields, agricultural land, and residential and commercial areas. The surface of the landfill has
deteriorated in several locations. Leachate seeps have been noted on the slope of the landfill, several of which discharge
to a 5-acrc pond down-gradient of the site.

Site B - Stream DDT Site

This is a former chemical production facility located adjacent to a stream.  The facility manufactured and packaged
dichlorodiphenyltrichloroethane (DDT).  Due to poor storage practices, several DDT spills have occurred.

Site C — Terrestrial PCB Site

This site is a former waste oil recycling facility located in a remote area. Oils contaminated with polychlorinated
biphenyl compounds (PCBs) were disposed in a lagoon. The lagoon is not lined and the substrate is composed mostly of
sand.  Oils contaminated with PCBs have migrated through the soil and contaminated a wide area adjacent to the site.

n.  USE OF THE CHECKLIST FOR ECOLOGICAL ASSESSMENT/SAMPLING

Site A - Copper Site

A preliminary site visit was conducted, and the checklist indicated the following:  1) the pond has an organic substrate,
2) emergent vegetation including cattail and Phragmites occurs along the shore near the leachate seeps, and 3) the pond
reaches a depth of five feet toward the middle.  Several species of sunfish. minnows, and carp were observed. A diverse
benthic macroinvertebrate community also has been noted in the pond. The pond appears to function as a valuable
habitat for fish and other wildlife.

Preliminary sampling indicated elevated copper levels in the seep as well as elevated base cations, total organic carbon
(TOO. and depressed pH levels (pH 5.7).

Copper can cause toxic effects in both aquatic plants and invertebrates at relatively low water concentrations, thereby
affecting the pond s ability to support macroinvertebrate and fish communities, as well as the wildlife that feed at the
pond  Terrestrial ecosystems do not need to be evaluated because the overland flow of the seeps is limited to short
gullies.  Thus, the area of concern has been identified as the 5-acre pond and the associated leachate seeps.

A review of the literature on the ecotoxicity of copper to aquatic biota and plants, both algae and vascular, was
conducted. In general it was found that young organisms are more sensitive to copper with decreasing sensitivity  as
body weight increases. The toxiciry of copper in water is influenced by water hardness, alkalinity, and pH.


                                                    50

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Site B - Stream DDT Site

The ecological checklist was completed as part of the preliminary site visit The information gathered indicates that
surface water drainage from the site flows through several drainage swales toward a small unnamed creek. This creek is
a second order stream containing riffle-run areas and small pools. The stream substrate is composed of sand and gravel
in the pools with some small depositional areas in the backwater areas, and primarily cobble in the riffles. Previous
sampling efforts have indicated the presence of DDT and its metabolites in the stream sediments at a concentration of
230 milligrams per kilogram (mg/kg). A variety of wildlife, especially piscivorous birds, utilize this area for feeding.
Many species of minnow have been noted in this stream.  DDT is well known for its tendency to bioaccumulate and
biomagnify in food chains, and available evidence indicates that it can cause reproductive failure in birds due to eggshell
thinning.

In freshwater systems, DDT can have direct effects on animals, particularly insects. A literature review of the aquatic
toxicity of DDT was conducted, and a no observed adverse effects level (NOAEL) was identified for aquatic insects.
Aquatic plants are not affected by DDT. Additional information on the effects of DDT on birds identified decreased
reproductive success due to eggshell thinning.

Site C — Terrestrial PCB Site

During a preliminary site visit, the ecological checklist was completed.  Most of the habitat is upland forest, old field,   -
and successions] terrestrial areas. Biological surveys at this site have noted a variety of small mammals, and red-tailed
hawks were also observed. The area of concern has been identified as the 10-acre area surrounding the site. PCBs have
been shown to reduce reproductive success in mammals or target liver functions. PCBs are not highly volatile, so
inhalation of PCBs would not be an important exposure pathway. However, PCBs have been shown to biomagnify
indicating that the ingestion exposure route needs evaluation. Shrews and/or voles would be appropriate mammalian
receptors to evaluate for this exposure route. Potential reproductive effects on predators that feed on small mammals
would also  be important to evaluate. The literature has indicated that exposure to PCBs through the food chain can
cause chronic toxicity to predatory birds.

Limited information was available oh the effects of PCBs to red-tailed hawks. Studies on comparable species have
indicated decreased sperm concentration that may affect reproductive success.

ID. CONCEPTUAL SITE MODEL FORMULATION

Site A -- Copper Site

The assessment endpoint for this site was identified as the maintenance of pond  fish and invertebrate community
composition similar to that of other ponds in the area of similar size and characteristics. Benthic macroinvertebrate
community studies may be relatively labor-intensive and potentially an insensitive measure in this type of system.
Measuring the fish community would also be unsuitable due to the limited size of the pond and the expected low
diversity of fish species. In addition, copper is not strongly food-chain transferrable. Therefore, direct toxicity testing
was selected as an appropriate measurement endpoint. Toxicity was defined as  a statistically significant decrease in
survival or juvenile growth rates in a population exposed to water or sediments, as compared to a population from the
reference sites.

One toxicity test selected was a 10-day solid-phase sediment toxicity test using early life-stage Hyalella azteca. The
measurement endpomis for the test are mortality and growth rates (measured as length and weight changes). Two water-
column toxicity tests were selected: a 7-day test using the alga Selenastrum capricornutum (growth test) and a 7-day
larval fish test using Pimephales promelas (mortality and growth endpoints).
                                                    51

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Five sediment samples were collected from the pond bottom at intervals along an identified concentration gradient
Reference sediment was also collected. A laboratory control was utilized in addition to the reference sediment in this
toxiciry test The study design specified that sediment for the toxicity tests was collected from the leachate seeps known
to be at the pond edge, and from four additional locations transecting the pond at equidistance locations. A pre-sampling
visit was required to confirm that the seep was flowing due to the intermittent nature of leachate seeps.

Site B — Stream DDT Site

A conceptual model was developed to evaluate the environmental pathways for DDT that could result in ecological
impacts. DDT in the sediments can be released to the water column during natural resuspension and redistribution of
the sediments. Some diffusion of DDT to the water column from the sediment surface may also occur. The benthic
macroinvertebrate community would be an initial receptor for Vie DDT in sediments. Fish that feed on the benthic
macroin vertebrates could be exposed to the DDT both in the water column and in their food. Piscivorous birds would
be exposed to the DDT that has accumulated in the fish.  For example, belted kingfishers are known to feed in the
stream. Given the natural history of this species, it is possible that they forage entirely in the contaminated area. From
this information, the assessment endpoint was identified to be the protection of piscivorous birds from eggshell thinning
due to DDT exposure. From this assessment endpoint eggshell thinning in the belted kingfisher was selected as the
measurement endpoint.                                                                                    .

Existing information identified a DDT gradient in the stream sediments. Forage fish (e.g., creek chub) were selected to ,
measure exposure levels for kingfishers. The study design for measuring DDT residue levels specified that 10 creek
chub of the same size and sex will be collected at each location for chemical residue analysis.  Although analytical data
for the stream sediment exists, new co-located sediment samples were specified to be collected to provide a stronger
link between the present state of contamination in the sediment and in the fish.

Site C  - Terrestrial PCB Site

A conceptual model was prepared to determine the  exposure pathways by which predatory birds could be exposed to
PCBs originating in the soil at the site. The prey of red-tailed hawks includes voles, deer mice, and various insects.
Voles are  herbivorous and prevalent at the site. However. PCBs do not strongly accumulate in plants, thus voles may
not represent a strong exposure pathway to hawks.  Deer mice are omnivorous and may be more likely than voles to be
exposed to PCBs. The assessment endpoint for this site was identified to be the protection of reproductive success in
high trophic level species exposed to PCBs via diet

Initially, a sampling feasibility study was conducted to confirm sufficient numbers of the deer mice. Two survey lines of
10 live traps were set for deer mice in the area believed to contain the desired concentration gradient for the study
design. Previous information  indicated a gradient of decreasing PCB concentration with increasing distance from the
unlined lagoon. Three locations were selected along this gradient to measure PCB concentrations in prey. Co-located
soil and water samples were also collected. The analytical results of these matrices were utilized as variables in a food
chain accumulation model which predicted the amount of contaminant in the environment that may travel through the
food chain, ultimately to the red-tailed hawk.   .                                   .
                                                      52

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                                         REFERENCES
ASTM. 1992. Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. American Society for
Testing and Materials. £1241-92.

Bligh. E.G., WJ. Dyer. 1959. Ltpid Extraction and Purification. Canadian Journal of Biochemistry and Physiology. Vol
37. pp. 912-917

Brungs, W.A. and D.I. Mount 1978. Introduction to a Discussion of the Use of Aquatic Toxiciry Tests for Evaluation
of the Effects of Toxic Substances. Cairns, J. Jr., ILL. Dickson and A.W. Makei (eds.) Estimating the Hazard of
Chemical Substances to Aquatic Life.  ASTM 657. Amer. Soc. Test Materials, Philadelphia, PA. p. 1526.

Green, J.C., CL. Baucis, WJ. Warren-Hicks, B.R. Parkhurst, GJ-. Under, S.A. Peterson, and W£. Meiller.  1989.
Protocol for Short Term Toxiciry Screening of Hazardous Waste. VS. Environmental Protection Agency,
Environmental Research Laboratory, Corvallis, OR. EPA 600/3-88/029.

Hair, JD. 1980. Measurement of ecological diversity, in S.D. Schemnitz, ed. Wildlife Management Techniques
Manual. Fourth Edition. The Wildlife Society, Washington, D.C. pp269-275.

Hayes, MX. 1983. Active Fish Capture Methods, Chapter 7 in Fisheries Techniques. American Fisheries Society, pp.
123-145.                                                                                   .

Heroes, S.E. and C.P. Allen. 1983. Lipid Quantification of Freshwater Invertebrates: Method Modification for
Microquanritation. Canadian Journal of Fisheries and Aquatic Sciences. 40(8). pp. 1315-1317.

Hurben, W.A.  1983. Passive Capture Methods, Chapter 6 in Fisheries Techniques. American Fisheries Society, pp. 95-
122.05

Karr, J.R., K.D. Fausch, PI.. Angermeier. P.R. Yam, and U. Schlosser. 1986. Assessing Biological Integrity in
Running Waters: A Method and Its Rationale. Special Publication 5. Illinois Natural History Survey.

Philips. DJ.H.  1977. The Use of Biological Indicator Organisms to Monitor Trace Metal Pollution In Marine and
Estuarine Environments-A Review. Environmental Poll  13, pp. 281-317.

Philips. DJ.H.  1978. Use of Biological Indicator Organisms to Quantitate Organochlorine Pollutants in Aquatic
Environments-A Review. Environmental Poll. 16, pp. 167-229.

Timbrell. J.A. 1989. Introduction to Toxicology. Taylor and Francis, London. 155p.

U.S. EPA (Environmental Protection Agency). 1997. Ecological Risk Assessment Guidance for Superfund: Process for
Designing and Conducting Ecological Risk Assessments. Office of Solid Waste and Emergency Response. EPA 540-R-
97/006.

U.S. EPA (Environmental Protection Agency). 1994. CLP National Functional Guidelines for Inorganic Data .
Review Office  of Solid Waste and Emergency Response. Publication 9240.1-05

U.S. EPA (Environmental Protection Agency). January 1991. Compendium ofERTToxicity Testing Procedures.
OSWER Directive 9360.4-08.
                                                   53

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 U.S. EPA (Environmental Protection Agency). 1992. Framework for Ecological Risk Assessment. EPA/630/R-92/001.


 U.S. EPA (Environmental Protection Agency).  December 1991b. ECO Update. Volume 1, Number 2, Publication
 9345.0-051.  Office of Emergency and Remedial Response, Hazardous Site Evaluation Division (OS-230).

 U.S. EPA (Environmental Protection Agency). .April 1990. Quality Assurance/Quality Control (QA/QC) Guidance
for Removal Activities. Sampling QA/QC Plan and Data Validation Procedures. EPA/540/G-90/004.

 U.S. EPA (Environmental Protection Agency).  November 1990. Macroinvertebrate Field and Laboratory Methods
for Evaluating the Biological Integrity of Surface Waters.. Aquatic Biology Branch and Development and Evaluation
 Branch, Quality Assurance Research Division, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio,
 EPA/600/4-90/030.

 U.S. EPA (Environmental Protection Agency). March 1989b. Short-Term Methods for Estimating the Chronic Toxicity
 of Effluents and Receiving Waters to Freshwater Organisms. EPA/600/4-89/001.

 U.S. Environmental Protection Agency. May I989a. Rapid Bioassessment Protocols For Use In Streams And Rivers:
Benthic Macroinvertebrates and Fish. EPA/444/4-89-001.

U.S. Environmental Protection Agency. May 1988. Short-Term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Marine and Estuarine Organisms. EPA/600/4-87/028.
                                                   54

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                APPENDIX C



SUPPLEMENTAL GUIDANCE ON LITERATURE SEARCH

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                                   APPENDIX C
         SUPPLEMENTAL GUIDANCE ON  LITERATURE SEARCH
       A literature search is conducted to obtain information on contaminants of concern,
 their potential ecological effects, and species of concern.  This appendix is separated into two
 sections; Section C-l describes the information necessary for the literature review portion of
 an ecological risk assessment.  Topics include information for exposure profiles,
 bioavailability or bioconcentration factors for various compounds, life-history information for
 the species of concern or the surrogate species, and an ecological effects profile. Section C-2
 lists information sources and techniques for a literature search and review. Topics include  a
 discussion of how to select key words on which to base a search and various sources of
 information (i.e., databases, scientific abstracts, literature reviews, journal articles, and
 government documents).  Threatened and endangered species are discussed separately due to
 the unique databases and information sources available for these species.

       Prior to conducting a literature search, it is important to determine what information is
 needed for the ecological risk assessment.  The questions raised in Section D-l must be
 thoroughly reviewed, the information necessary to complete the assessment must be
 determined, and the purpose of the assessment must be clearly defined. Once these activities
 are completed, the actual literature search can begin.  These activities will assist in focusing
 and streamlining the search.
C-1    LITERATURE REVIEW FOR AN ECOLOGICAL RISK ASSESSMENT

       Specific information.  During problem formulation, the risk assessor must
determine what information is needed for the risk assessment. For example, if the risk
assessment will estimate the effects of lead contamination of soils  on terrestrial vertebrates,
then literature information on the effects of dissolved lead to fish would not be relevant. The
type and form of the contaminant and the biological species of concern often can focus the
literature search.  For example, the toxicity of organometallic compounds is quite different
from the comparable inorganic forms.  Different isomers of organic compounds also can have
different toxic effects.

       Reports of toxicity tests should be reviewed critically to ensure that the study was
scientifically sound.  For example, a report should specify the exposure routes, measures of
effect and exposure, and the full study design. Moreover, whether the investigator used
accepted scientific techniques should be determined.

       The exposure route used in the study should also be comparable to the exposure route
in the risk assessment.  Data reported for studies where exposure is by injection or gavage are
not  directly comparable to dietary exposure studies. Therefore, an uncertainty factor might
                                          C-l

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 need to be- included in the risk assessment study design, or the toxicity report should not be
 used in the risk assessment.

       To use some data reported in the literature, dose conversions are necessary to estimate
 toxicity levels for species other than those tested. Doses for many laboratory studies are
 reported in terms of mg contaminant/kg diet, sometimes on a wet-weight basis and sometimes
 on a dry-weight basis. That expression should be converted  to mg contaminant/kg wet
 bodyweight/day, so that estimates of an equivalent dose in another species can be scaled
 appropriately. Average ingestion rate and wet body weight for a species often are reported in
 the original toxicity study. If not, estimates of those data can be obtained  from other
 literature sources to make the  dose conversion:

   Dose  = (mg contaminant/kg diet) x ingestion rate (kg/day) x (I/wet body weight (kg)).

 If the contaminant concentration is expressed as  mg contaminant/kg dry diet, the  ingestion
 rate  should also be in terms of kg of dry diet ingested per day.

       Exposure profile.  Once contaminants of concern are selected for the ecological risk
 assessment, a general overview of the contaminants' physical and chemical properties is
 needed.  The fate and transport of contaminants in the environment determines how biota are
 likely to be exposed.  Many contaminants undergo degradation (e.g., hydrolysis, photolysis,
 microbial) after release into the environment. Degradation can affect toxicity, persistence,
 and fate and transport of compounds. Developing an exposure profile for a contaminant
requires information regarding inherent properties of the contaminant that can affect fate and
transport Or bioavailability.

       Bioavailability.  Of particular importance in an ecological risk assessment is the
bioavailability of site contaminants in the environment.  Bioavailability influences exposure
levels for the biota.  Some factors that affect bioavailability of contaminants in soil and
sediment include  the proportion of the medium composed of organic matter, grain size of the
medium, and its pH.  The aerobic state of sediments is important because it often affects the
chemical form of contaminants.  Those physical  properties of the media can change the
chemical form of a contaminant to a form that is more or less toxic than the original
contaminant.  Many contaminants adsorb to organic matter, which can make them less
bioavailable.              •

       Environmental factors that influence the bioavailability of a contaminant in water are
important to aquatic risk assessments.  Factors including pH, hardness, or aerobic status can
determine both the chemical form and uptake of contaminants by biota.  Other environmental
factors can influence how organisms process contaminants. For example, as water
temperatures  rise, metabolism of fish and aquatic invertebrates increases, and the rate of
uptake of a contaminant from  water can increase.
                                           C-2

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       If the literature search on the contaminants of concern reveals information on the
bioavailability of a contaminant, then appropriate bioaccumulation or bioconcentration factors
(BAFs or BCFs) for the contaminants should be determined.  If not readily available in the
literature, BAF or BCF values can be estimated from studies that report contaminant
concentrations in both the environmental exposure medium (e.g., sediments) and in the
exposed biota (e.g., benthic macroinvertebrates). Caution is necessary, however, when
extrapolating BAF or BCF values estimated for one ecosystem to another ecosystem.

       Life history.  Because it is impossible and unnecessary to model an entire ecosystem,
the selection of assessment endpoints and associated species of concern, and measurement
endpoints (including those for a surrogate species if necessary) are fundamental to a
successful risk assessment. This process is described in Steps 3 and 4.  Once assessment and
measurement endpoints are agreed to by the risk assessor and risk manager, life history
information for the species of concern or the surrogate species should be collected. Patterns
of activity and feeding habits of a species affect their potential for exposure to a contaminant
(e.g., grooming activities of small mammals, egestion of bone and hide by owls).  Other
important exposure factors include food and water ingestion rates, composition of the diet,
average body weight, home range size, and seasonal activities such as migration.

       Ecological effects profile.  Once contaminants and species of concern are selected
during problem formulation, a general overview of toxicity and toxic mechanisms is needed.
The distinction between the species of concern representing an assessment endpoint and a
surrogate species representing a measurement endpoint is important. The species of concern
is the species that might be threatened by contaminants at the site.  A surrogate species  is
used when it is not appropriate or possible to measure attributes of  the species of concern.  A
surrogate for a species of concern should be sufficiently similar biologically to allow
inferences on likely effects in the species of concern.

       The ecological effects profile should include toxicity information from the literature
for each possible exposure route. A lowest-observed-adverse-effect level  (LOAEL) and the
no-observed-adverse-effect level (NOAEL)  for the species of concern or its surrogate should
be obtained.  Unfortunately, LOAELs are available for few wildlife species and contaminants.
If used with caution, toxicity data from a closely related species can be used to estimate a
LOAEL and a NOAEL for a receptor species.
C-2   INFORMATION SOURCES

       This section describes information sources that can be examined to find the
information described in Section 3-1. A logical and focused literature search will reduce the
time, spent searching for pertinent information.
                                           C-3

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       A first step in a literature search is to develop a search strategy, including a list of key
words.  The next step is to review computerized databases, either on-line or CD-ROM-based
information systems.  These systems can be searched based on a number of parameters.

       Scientific abstracts that contain up-to-date listings of current, published information
also are useful information sources. Most abstracts are indexed  by author or subject.
Toxicity studies and information on wildlife life-histories often are summarized in literature
reviews published in books or peer-reviewed journals. Original  reports of toxiciry studies can
be identified in the  literature section of published documents.  The original article in which
data are reported must be reviewed before the data are cited in a risk assessment.

       Key words.  Once the risk assessor has prepared a list of the specific information
needed for the risk  assessment, a list of key words can be developed. Card catalogs,
abstracts, on-line databases, and other reference materials usually are indexed on a limited set
of key words. Therefore, the key words used to search for information must be considered
carefully.

       Useful key words include the contaminant of concern, the biological species of
concern, the type of toxicity information wanted, or other associated words. In addition,
related subjects can be used as key words. However, it usually is necessary to limit
peripheral aspects of the subject in order to narrow the search.  For example, if the risk
assessor needs information on the toxicity  of lead  in soils to moles, then requiring that both
"lead" and "mole" are among the key words can focus the literature search.  If the risk
assessor needs information on a given plant or animal species  (or group of species), key
words should  include both the scientific name (e.g., genus and species names or order or
family names) and an accepted common name(s).  The projected use of the data in the risk
assessment helps determine which  key words are most appropriate.

       If someone outside of the risk assessment team will conduct the literature search, it is
important that they  understand both the  key words and the  study objectives for the data.

       Databases. Databases are usually on-line or CD-ROM-based information systems.
These systems can be searched using a number of parameters. Prior to searching databases,
the risk assessor should determine  which database(s) is  most likely to provide  the information
needed for the risk  assessment. For example, U.S. Environmental Protection Agency's
(EPA's) AQUIRE database (AQUatic Information REtrieval database) provides information
specifically on the toxicity of chemicals to aquatic plants and animals.   PHYTOTOX includes
data on the toxicity of contaminants to terrestrial and aquatic plants, and TERRETOX
includes data on toxicity to terrestrial animals.  U.S. EPA's IRIS (Integrated Risk Information
System) provides information on human health risks (e.g., references to original toxicity
studies) and regulatory information (e.g., reference doses and cancer potency factors) for a
variety of chemicals.  Other useful databases include the National Library of Medicine's
HSDB (Hazardous Substances Data Bank) and the National Center for Environmental
Assessment's  HEAST Tables (Health Effects Assessment Summary Tables).  Commercially

                                          C-4

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 available databases include BIOSIS (Biosciences Information Services) and ENVIROLINE.
 Another database, the U.S. Public Health Service's Registry of Toxic Effects of Chemical
 Substances (RTECS) is a compilation of toxicity data extracted from the scientific literature
 and is also available online.

       Several states have Fish and Wildlife History Databases or Academy of Science
 databases, which often provide useful information  on the life-histories of plants and animals
 in the state.  State databases are particularly useful for obtaining information on endemic
 organisms or geographically distinct habitats.

       Databases searches can yield a large amount of information in a short period of time.
 Thus, if the key words do not accurately describe the information needed, database searches
 can provide a large amount of irrelevant information. Access fees and on-line fees can apply;
 therefore, the selection of relevant key words and an organized approach to the search will
 reduce the time and expense of on-line literature searches.

       Abstracts.  Published abstract compilations (e.g., Biological Abstracts, Chemical
 Abstracts, Applied Ecology Abstracts) contain up-to-date listings of current, published
 information.  Most abstracts are indexed by author or subject. Authors and key words can be
 cross-referenced to identify additional publications. Abstract compilations also include, for
 each citation, a copy of its abstract from the journal or book in which it was published.
 Reviewing the abstracts of individual citations is a relatively quick way to determine whether
 an article is applicable to the risk assessment.   As with computerized database searches, it is
 important to  determine which  abstract compilations are most suitable for the risk assessor's
 information needs.

       Published abstract compilations that are indexed by author are particularly useful.  If
 an author is known to conduct a specific type  of research, their name would be referenced in
 the abstract for other articles on similar subjects.  If the risk assessor considers an abstract
 pertinent to the assessment, the original article must be retrieved and reviewed before it can
 be cited in the risk assessment.  Otherwise, the results of the risk assessment could be based
 on incorrect and incomplete information  about a study.

       Abstracts usually must be searched manually, which can be a very time consuming.
The judicious use of key words can help to reduce the  amount of time needed to search
through these volumes.

       Literature review publications.  Published literature reviews often cover toxicity
or wildlife information of value to an ecological risk assessment.  For example, the U.S. Fish
and Wildlife  Services (U.S. FWS) has published several contaminant-specific documents that
list lexicological data on terrestrial, aquatic, and avian studies (e.g., Eisler, 1988).  The U.S.
EPA publishes ambient  water  quality criteria documents (e.g., U.S. EPA, 1985) that list all
the data used to calculate those values.  Some literature reviews critically evaluate the original
studies (e.g., toxicity data reviewed by NOAA, 1990).  The Wildlife Exposure Factors

                                           C-5

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 Handbook (U.S. EPA, 1993a,b) provides pertinent information on exposure factors (e.g., body
 weights, food ingestion rates, dietary composition, home range size) for 34 selected wildlife
 species.

        Literature reviews can provide an extensive amount of information. However, the risk
 assessor must obtain a copy of the original of any studies identified in a literature review that
 will be used in the risk assessment. The original study must be reviewed and evaluated
 before it can be used in the risk assessment.  Otherwise, the results of the risk assessment
 could be based on incorrect and incomplete information about a study.

        References cited in previous studies.  Pertinent studies can be identified in the
 literature cited section of published documents that are relevant to the risk assessment, and
 one often can identify several investigators who work on related studies.  Searching for
 references in the literature cited section of published documents, however,  takes time and
 might hot be very effective. However, this is probably the most common approach to
 identifying relevant literature.  If this approach is selected, the best place to start is a review
 article.  Many journals do not list the title of a citation for an article, however, limiting the
 usefulness of this technique.  Also, it can be difficult to retrieve literature cited in obscure or
 foreign journals or in unpublished masters' theses or doctoral dissertations. Although this
 approach tends to  be more time consuming than the other literature search approaches
 described above, it probably is the most common approach used to locate information for a
 risk assessment.

       Journal articles, books, government documents. There are a variety of
journals, books, and government documents that contain information useful to risk
 assessments. The  same requirement for retrieving the original reports for any information
 used in the risk assessment described for other information sources applies to these sources.

       Threatened and endangered species.  Threatened and endangered species are of
 concern to both federal and state governments. When conducting an ecological risk
 assessment, it often is necessary to determine or estimate the effects of site contaminants to
 federal  threatened  or endangered species.  In addition, other special-status species (e.g.,
 species listed by a state as endangered or threatened within the state) also can be the focus of
 the assessment.  During the problem formulation step, the  U.S. FWS or state Natural Heritage
 programs should be contacted to determine if these species are present or might be present on
 or near a Superfund site.

       Once the presence of a special-status species is confirmed or considered likely,
 information on this species, as well as on surrogate  species, should be included in the
 literature search.  There are specific federal and state programs that deal with issues related to
 special-status species, and often there is more information  available for these than for non-
 special-status species  used as surrogates for an ecological risk assessment. Nonetheless, the
 use of surrogate species usually is necessary when an assessment endpoint is a special-status
 species.

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REFERENCES

Eisler, R.  1988. Lead Hazards to Fish, Wildlife, and Invertebrates: A Synoptic Review.
       U.S. Fish and Wildlife Service Patuxent Wildlife Research Center, Laurel MD: U.S.
       Department of the Interior; Biological Report 85(1.14), Contaminant Hazard Reviews
       Rep. No. 14.

National Oceanic and Atmospheric Administration (NOAA).  1990.  The Potential for
       Biological Effects of Sediment-Sorbed Contaminants Tested in the National Status and
       Trends Program.  Seattle, WA: Office of Oceanography and Marine Assessment.
       NOAA/TM/NOS/6MA-52. Technical memorandum by Long, E.R. and Morgan, L.G.

U.S. Environmental Protection Agency (U.S. EPA).  1993a.  Wildlife Exposure Factors
       Handbook Volume I. Washington, DC: Office of Research and Development;
       EPA/600/R-93/187a.

U.S. Environmental Protection Agency (U.S. EPA).  1993b.  Wildlife Exposure Factors
       Handbook Volume II:  Appendix.  Washington, DC: Office of Research and
       Development; EPA/600/R-93/187b.

U.S. Environmental Protection Agency (U.S. EPA).  1985.  Ambient Water Quality Criteria
      for Copper-1984.  Washington, DC:  Office of Water, Regulations and Standards,
       Criteria and Standards Division.  EPA/440/5-84-031.  PB85-227023.
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        APPENDIX D



STATISTICAL CONSIDERATIONS

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                                    APPENDIX D
                        STATISTICAL CONSIDERATIONS
       In the biological sciences, statistical tests often are needed to support decisions based
 on alternative hypotheses because of the natural variability in the systems under investigation.
 A statistical test examines a set of sample data, and, based on an expected distribution of the
 data, leads to a decision on whether to accept the hypothesis underlying the expected
 distribution or whether to reject that hypothesis and accept an alternative one.  The null
 hypothesis is a hypothesis of no differences. It usually is formulated for the express purpose
 of being rejected.  The alternative or test hypothesis is an operational statement of the
 investigator's research hypothesis.  An example of a null hypothesis for toxicity testing would
 be that mortality of water fleas exposed to water from a contaminated area is no different
 than mortality of water fleas exposed to water from an otherwise similar, but uncontaminated
 area. An example of the test hypothesis is that mortality of water fleas exposed to water
 from the contaminated area is higher than mortality of water fleas exposed to uncontaminated
 water.

 D-1   TYPE I AND TYPE II ERROR

       There are two types of correct decisions for hypothesis testing: (1) accepting a true
 null hypothesis, and  (2) rejecting a false null hypothesis.  There also are two types of
 incorrect decisions:  rejecting a true null hypothesis, called Type I error; and accepting a false
 null hypothesis, called Type II error.

       When designing a test  of a hypothesis, one should decide what magnitude of Type I
error (rejection of a true null  hypothesis)  is acceptable.  Even when sampling from a
population of known parameters, there are always some sample  sets which, by chance, differ
 markedly.  If one allows 5 percent of samples to lead to a Type I error, then one would on
average reject a true null hypothesis for 5 out of every 100 samples taken. In other words,
we would be confident that, 95 times out of 100, one would  not reject the null hypothesis of
no difference "by mistake" (because chance alone produced such deviant results). When the
probability of Type I error (commonly symbolized by a) is set at 0.05, this is called a
significance level of 5 percent. Setting a significance level of 5 percent is a widely accepted
convention in most experimental sciences, but it is just that, a convention.  One can demand
more confidence (e.g., a = 0.01) or less confidence (e.g., a = 0.10) that the hypothesis of no
difference is not rejected by mistake.

       If one requires more confidence for a given sample size  that the null hypothesis is not
rejected by mistake (e.g., a =  0.01), the chances of Type II error increase. In. other words,
the chance increases that one will mistakenly accept a false null hypothesis (e.g., mistakenly
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believe that the contaminated water from the site has no effect on mortality of water fleas).
The probability of Type II error is commonly denoted by p.  Thus:

       p (Type I error) = a                                                            -
       p (Typell error) = p
                               v
However, if one tries to evaluate the probability of Type n error (accepting a false hypothesis
of no difference), there is a problem.  If the null hypothesis is false, then some other
hypothesis must be true, but unless one can specify a second hypothesis, one can't determine
the probability of Type n error.  This leads to another important statistical consideration,
which is the power of a study design and the  statistical  test used to evaluate the results.

D-2   STATISTICAL POWER

       The power of a statistical test is equal  to (1 - P) and is equal to the probability of
rejecting the null hypothesis (no difference) when it should be rejected (i.e., it is false) and
the specified alternative hypothesis is true.  Obviously,  for any given test (e.g., a toxicity test
at a Superfund site), one would like the quantity (1 -  P) to be as large  as possible  (and P to
be as small as possible). Because one generally cannot specify a given alternative hypothesis
(e.g., mortality should be 40 percent in the exposed population), the power of a  test is
generally evaluated on the basis of a continuum of possible alternative hypotheses.
                                                                                •
       Ideally, one would specify both a and P before an experiment or  test of the hypothesis
is conducted.  In practice, it is  usual to specify a (e.g.,  0.05) and the sample size because the
exact alternative hypothesis cannot be  specified.1  Given the inverse relationship between the
likelihood of making Type I and Type II errors, a decrease in a will increase P  for any given
sample size.

       To improve the statistical power of a test (i.e., reduce p), while keeping a constant,
one can either increase the sample size (N) or change the nature of the statistical test.  Some
statistical tests  are more powerful than others, but it is important that the assumptions
required by the test (e.g., normality of the underlying distribution) are  met for the  test results
to be valid. In general, the more powerful tests rely on more assumptions about the data (see
Section D-3).

       Alternative study designs  sometimes can improve statistical power (e.g., stratified
random sampling compared with random sampling if something is known about the history
and location of contaminant release).  A discussion of different statistical sampling designs is
beyond the scope of this guidance, however.  Several references provide guidance on
statistical sampling design, sampling techniques, and  statistical analyses appropriate for
hazardous waste sites (e.g., see Cochran, 1977; Green,  1979; Gilbert, 1987; Ott, 1995).
   '   With a specified alternative hypothesis, once a and the sample size (N) are set, P is determined.

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        One also can improve the power of a statistical test if the test hypothesis is more
 specific than  "two populations are different," and, instead, predicts the direction of a
 difference (e.g., mortality in the exposed group is .higher than mortality in the control group).
 When one can  predict the direction of a difference between groups, one uses a one-tailed
 statistical test; otherwise, one must use the less powerful two-tailed version of the test.
                                        Highlight D-2
          Key Points About Statistical Significance, Power, and Sample Size

     (1)    The significance level for a statistical test, a, is the probability that a statistical test will
            'yield a value under which the null hypothesis will be rejected when it is in fact true.
            In other words, a defines the probability of committing Type I error (e.g., concluding
            that the site medium is toxic when it is in fact not toxic to the test organisms).

     (2)    The value of p is the probability that a statistical test will yield a value under which the
            null hypothesis is accepted when it is in  fact  false.  Thus, P defines the probability of
            committing Type n error (e.g., concluding that the site medium is not toxic when it is
            in fact toxic to the test organisms).

     (3)    The power of  a statistical test (i.e., 1 -  f3) indicates the probability of rejecting the null
            hypotheses when it is false (and therefore  should be rejected).   Thus, one wants the
            power of a statistical test to be as high as possible.

     (4)    Power is related to the nature of the statistical test chosen.  A one-tailed test is more
            powerful than  a two-tailed test.  If the alternative to the null  hypothesis can state the
            expected direction of a difference between a test and control group, one can use the more
            powerrulone-tailed test.

     (5)    The power of  any statistical test increases with increasing sample size.
D-3   STATISTICAL MODEL

       Associated with every statistical test is a model and a measurement requirement.  Each
statistical test is valid only under certain conditions.  Sometimes, it is possible to test whether
the conditions of a particular statistical model are met, but more often, one has to assume that
they are or are  not met based on an understanding of the underlying population and sampling
design.  The conditions that  must be met for a statistical test to be valid often are referred to
as the assumptions of the test.

       The most powerful statistical tests (see previous section) are those  with the most
extensive assumptions.  In general, parametric statistical tests (e.g., t test, F test) are the most
powerful tests,  but also have the most exacting assumptions to be met:

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       (1)    The "observations" must be independent;

       (2)    The "observations" must be drawn from a population that is normally
              distributed;

       (3)    The populations must have the same variance (or in special cases, a known
              ratio of variances); and

       (4)    The variables must have been measured at least on an interval scale so that it is
              possible to use arithmetic operations (e.g., addition, multiplication) on the
              measured values (Siegel, 1956).

The second and third assumptions are the ones most often violated by the types of data
associated with biological hypothesis testing.  Often, distributions are positively skewed (i.e.,
longer upper than lower tail of the distribution).  Sometimes, it is possible to transform data
from positively skewed distributions to normal distributions using a mathematical function.
For example, many biological parameters turn out to be log-normally distributed (i.e., if one
takes the log of all  measures, the resulting values are normally distributed).  Sometimes,
however, the underlying shape of the distribution cannot be normalized  (e.g., it is bimodal).

       When the assumptions required for parametric tests are not met,  one must use
nonparametric statistics (e.g., median test, chi-squared test). Nonparametric tests are in
general less powerful than parametric  tests because less is known or assumed about the shape
of the underlying distributions.  However, the loss in power can be compensated for by an
increase in sample size, which is the concept  behind measures of power-efficiency.

       Power-efficiency reflects  the increase in sample size necessary to make test B (e.g., a
nonparametric test) as efficient or powerful as test A (e.g., a parametric test). A power-
efficiency of 80 percent means that in order for test B to be as powerful as test A, one must
make 10 observations for test B  for every 8 observations for test A.

       For further information .on statistical tests, consult references  on  the topic (e.g., see
references below).
REFERENCES

Cochran, W. G. 1977.  Sampling Techniques. Third edition.  New York, NY: John Wiley
       and Sons, Inc.
                                                           ^

Gilbert, R.O.  1987. Statistical Methods for Environmental Pollution Monitoring.  New York,
       NY: Reinhold.
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Green, R. H.  1979. Sampling Design and Statistical Methods for Environmental Biologists.
       New York, NY: Wiley.

Ott, W.R.  1995. Environmental Statistics arid Data Analysis.  Boca Raton, FL:  CRC Press,
       Inc., Lewis Publishers.

Siegel, S. 1956. Non-parametric Statistics.  New York, NY: McGraw-Hill.
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