OSWER Directive XXXXJCX
EPA 540/R/W/XXX
PBu-uuuux
May 1997
DRAFT
SUPERFUND PROGRAM
REPRESENTATIVE SAMPLING GUIDANCE
VOLUME 3: 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 9360-4-08, EPA/540/P-91/009 (U.S. EPA 1991a). Topics covered in this compendium include: tonicity
testing; and surface water and sediment sampling.
Please note that 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 IB, MS-101
2890 Wood bridge Avenue
Edison. NJ 08837-3679
For additionaJ copies of the Superfund Program Representative Sampling Guidance, Volume 3 -- Biological, contact:
National Technical Information Services
S28S 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:
Havahartฎ - Allcock Manufacturing Co., Lititz, PA
Longworth - Longwonh Scientific Instrument Company, Ltd.. England
Museum Special - Woodstream Corporation, Lititz, PA
Sherman - HJJ. Sherman Traps, Tallahassee. FL
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CONTENTS
Notice ii
Disclaimer lii
List of Figures viii
Lost of Tables vii
Preface ix
1.0 INTRODUCTION 1
1.1 Objective and Scope 1
1.2 Risk Assessment Overview I
1.3 Conceptual Site Model 2
1.4 Data Quality Objectives 3
1.5 Technical Assistance 4
2.0 BIOLOGICAUECOLOGICAL 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 Ana Selection 6
2.2 J.2 Receptor Selection 7
2.2.3.3 Exposure-Response Relationships 8
2.2.3.4 Chemical Residue Studies 8
2.2.3.5 Population/Community Response Studies 9
2.2.3.6 Toxicity Tesung/Bioassays 9
3.0 BIOLOGICAL SAMPLING METHODS U
3.1 Chemical Residue Studies U
3.1.1 Collection Methods 11
3.1.1.1 Comparability Considerations 12
3.1.1.2 Mammals 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
3.2.2.3 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 Toxicity 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 23
4.4.1 Replicate Samples 23
4.4.2 Collocated Samples 24
4.4.3 Reference Samples 25
4.4.4 Rinsate Blank Samples 25
4.4.5 Field Blank Samples 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 Preseniauon And Analysis 27
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5.2.1 Data Presentation Techniques 27
5.2.2 Descriptive Statistics 27
5.2.3 Hypothesis Testing 27
5.3 Data Interpretation 28
5.3.1 Chemical Residue Studies 28
5.3.2 Population/Community Studies 2S
5.3.3 Toxicity Testing 28
5.3.4 Risk Calculation 28
APPENDIX A-CHECKLISTFOR ECOLOGICAL ASSESSMENT/SAMPLING 30
APPENDIX B - EXAMPLE OF FLOW DIAGRAM FOR CONCEPTUAL SITE MODEL 47
APPENDIX C- EXAMPLE SITES 50
REFERENCES 53
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List of Figures
FIGURE 1 - Conceptual Site Model 5
FIGURE 2 - Common Mammal Traps 19
FIGURE 3 - Illustrations of Sample Plots 29
List of Tables
TABLE 1 - Reference List of Standard Operating Procedures - Ecological Sampling Methods 20
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Preface
This document is third in a senes of guidance documents designed to assist Superfund Program Site Managers such as On-
Scene Coordinators (OSCs), Site Assessment Managers (SAMs), and other field staff in obtaining representative samples
at Superfund sites. It is intended to assist Superfund 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 (QAJQC) 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.
Identification and assessment of potential environmental threats are important elements for the Site Manager to understand.
These activities car be accomplished through ecological assessments such as biological sampling. This document focuses
on (he performance of ecological assessment screening approaches, more detailed ecological assessment approaches, and
biological sampling methods.
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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 the 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
ฆ 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) (งl04{a)(l)], as amended by the
Superfund Amendments and Reauthorization Act
(SARA), and the NCP [ง300.400(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 potenual.
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 sue conditions.
Representative biological sampling and ecological risk
assessment include, but are not limned to, the collection
of site information and the collection of samples for
chemical or toxicological analyses. Biological sampling
is dependent upon specific site requirements dunng
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., dunng
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 nsk.
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.
Hie 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).
1J2 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 part 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 nsk
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 10 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 Secuon 2.2 for a detailed
discussion of assessment and measurement endpoints.
Except where required under other regulations, issues
such as restoration, miugation. and replacement are
i important to the program but are reserved for:
; investigations that may or may not be included in the RT
I phase. During the management decision process ofi
iselecting the preferred remedial option leading to the|
i Record of Decision (ROD), mitigation and restoration j
I issues should be addressed. Note that these issues are not |
inece^"rily 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 fust
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 part 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 (he
level(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, leachatej,
agricultural (runoff, dust, and particulates)
Potential Exposure Pathways
ingestion
waste contained in the pile on the
hazardous waste site; soil particles near
the waste pile; drum dump; or area of
agricultural activity
inhalation
dust and particulates from waste pile,
drum dump, or area of agricultural acttvuy
absorption/direct contact
soil near waste pile, drum dump, or area of
agricultural activity and surface water
downstream of sources
Potential Migration Pathways
air (particulates 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
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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
questions) 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, it is assumed that technical specialists
are available to assist Site Managers and other site
personnel m 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 (ER7C)
as well as from other sources within each region.
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FIGURE 1:
CONCEPTUAL
SITE MODEL
PRECIPITATIOI
S7AIE GAME
LANDS
WIND ROSE
PRECIPITATION
WATER PLANT
INTAKE a
LAKE fcar
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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).
Supertax) 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 art 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
houn 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
objecuves: 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 (I) the use of literature
screening values (e.g.. literature toxicity values) for
comparison to site-specific contaminant levels, (2) a
"desk-top" nsk assessment which can model existing site-
specific contaminant data to ecological receptors for
subsequent comparison to literature toxicity values, and
(3) field invesugauon/laboratory analysis that involves a
site invesugauon (which may utilize existing contaminant
da'ป for support) and laboratory analysts 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 die 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-site 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 thai upstream of the sue" 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 endpoinls. 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 minlc in this case has
been selected to be used for establishing die measurement
endpoint that is representative of piscivorous mammals.
However, it 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 mammals, 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 addiuon.
evaluation of chemical fate and transport infonnauon 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 mere 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 foundauon of
exposure-response relationships upon which the concept
of chemical residue studies, population/community
studies, and toxicity testing/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 ussue 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
durauon 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 relaovely 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 lest 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 tesung, test durauon, and selection
of test endpoints (e.g.. mortality or growth). A thorough
understanding of the inieracuon 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
re fared to as lethality) hrraircr it is one of the most easily
measured parameters.
In contrast, some contaminants do not cause mortality in
test organisms 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 comrol as well as reference groups is normally
required. Laboratory toxicity tests include a control that
evaluates the 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 (Held conducted) tests can be run if
field conditions permit. There are benefits and
limitauons associated with each approach. The most
notable benefit of laboratory testing is that exposure
conditions are controlled, but this 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 9360.4-08, EPA/540/P-91/009 (U.S.
EPA 1991a), for descriptions of nine common toxicity
tests and Standard Guide for Conducting Sediment
Toxicity Tests with Freshwater Invertebrates, ASTM
Standard El383, October 1990.
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Species Selection for Toiicitv 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 contaminants) 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.
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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 fir 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. ] (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 car 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 tn site organisms.
When collecting organisms for ussue analyses, it is
critical that the measured levels of contaminants in the
organism are attnbuiabie to a particular Iocauon and
contaminant level within the site. Collecuon 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
representauve approaches are not practical, the potential
bias must be idenufied and considered when drawing
conclusions from the data. The use of a panicular
collecuon technique should not be confused with the need
to target a "class" of individuals within a populauon 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 fROCs) 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 Bioconcentration
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
Iocauon. 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.
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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 Shennan 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 craps are placed at regular
intervals along it.
Hie 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 penod of 3 days is
typically used to minimis 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.
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3.1.1.3 Fish
Electro fishing, 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 system 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 lo 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 m the net thus creating bias
lowards a cenain sized fish. These nets are typically used
m shallow waters, but may extend to depths exceeding 50
meters. The sampling area should be free of obstructions
and floating debns. 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 ofa 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 them 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 fir applications in open water and do
not touch the bottom (Hayes 1983).
The use of minnow traps is a passive collection technique
fir 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 of fish 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 bioaccumulauve
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 pan 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 pci formed 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 ui 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 protecuon 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 m 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 resins-
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 far 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
ussue which causes effects is 1 mg/kg. the detection limit
is not artcquatr 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 Midpoints will detennine 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.
Histonatholoyical Analysis
Histopathologica! analysis can be an effective mechanism
for establishing causative relationships due to
contaminants since some contaminants can cause distinct
pathological effects. Fot 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 type(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-critical or long-term remediation-type site activiues.
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 to
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
population size but are labor-intensive and usually
require multrpie-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 macroinvertebrate (BMI) population/community
evaluations in small- to medium- sized streams have been
successfully used far approximately 100 years to
document injury to the aquatic systems. There are many
advantages to using BMI populations to detennine the
potential ecological impact associated with a site.
Sampling is relatively easy, and equipment requirements
are minimal. An evaluation of the community structure
maybe 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 BMls are a primary food source for many fish
and other organisms, threats beyond the benthic
community can be inferred from the evaluation of BMls.
Techniques such as rapid bioassessment protocols may
be used as a tool to support this type of finding and
inference. A more comprehensive disotssion of general
beidhological surveys may be found in U.S. EPA (1990).
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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, compiuing habitat and biological
measures with empirically defined reference conditions
(U.S. EPA 1989a).
The three major components of a rapid bioassessment
essentia] 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 arc
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
io reduce the amount of ume spent collecung and
analyzing samples. Protocols range from a quick survey
of the benthos (Protocol I) to a detailed laboratory
classification analysis (Protocol 111). Protocol 1 may be
conducted in several hours; Protocol II is more intensive
and focuses on major taxonomic levels; and Protocol III
may require numerous hours to process each sample to a
greater level of taxonomic and community assessment
resoluuon. 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.
3.2.2.2 General Benthological Surveys
Benthological surveys can be conducted with methods
other than those rfisrussrd 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. Hiis subsection is a brief
overview of the equipment available for the collection of
BMls. 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-
miUimeter (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, re" soft bottom habitats such as ponds, lakes,
or rivers are more often sampled using a small Ponar or
Ekman 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
macroinvertebrate 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
Toxicity 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, lest 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 toxictty 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 Toxicity 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 sniH
Laboratory-raised earth worms 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 coracnuatkms in the site soil treatments may
be drawn.
Example Nn 2 (surface watcrt
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.
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3.3.2 Examples of Chronic Toxicity
Tests
Example No. 1 (surface waters
Fathead minnow larvae (PimephaUs 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 toxicological effects of the contaminant
concentrations.
Example No 1 (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 ins tar). 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
toxicological effects of the contaminant concentrations.
18
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TABLE I
Reference List of Standard Operating Procedures Ecological Sampling Methods
SOP/Method No
Source
Procedure/Method Title
Publication No.
SOP No 1820
ERTC
Tissue Homogeniiation Procedure
(In development)
SOP No 1821
ERTC
Semi-Volatiles Analysis of Tissue Samples by GC/MS
(in development)
SOP No 1822
ERTC
PeMicides/I'CB Analysis of Tissue Samples by GC/ECD
(in development)
SOP No 182}
ERTC
Microwave Digestion and Metals Analysis of Tissue Samples
(in development)
SOP No 2020
ERTC
7-Day Standard Refeience Toxicity Test Using Larval Fathead Minnows Pimtphales promtlas
OSWER EPA/54(VP 91/009
SOP No. 2021
ERTC
24-Hour Range Finding Test Using Daphnia magna or Daphnia pultx
OSWER EPA/34CVP-9I/009
SOP No. 2022
ERTC
96-Houi Acute Toiicity Test Using Larval Pimtphales promtlas
OSWER EP A/540/P91/009
SOP No 2023
ERTC
24-llour Range Finding Test Using Larval Pimtphales promttas
OSWER EPA/540/P91/009
SOP No 2024
ERTC
48-Hour Acute Toiicity Test Using Daphnia magna or Daphnia pultx
OSWER EPA/34Q/P-91/009
SOP No. 2023
ERTC,
7-Day Renewal Toxicity Test Using Ctriodaphnia dubia
OSWER EPA/340/P-91 /009
SOP No 2026
ERTC
7-Day Static Toxicity Test Using Larval Pimtphales promtlas
OSWER EPA/34Q/P-91/009
SOP No. 2027
ERTC
96-Hour Static Toxicity Test Using Selenaslrum capricomuium
OSWER EPA/54 (VP-91/009
SOP No 2028
ERTC
10-Day Chronic Toxicity Test Using Daphnia magna or Daphnia pultx
OSWER EPA/540/P-91/009
SOP No 1-001
ERTC
13-Day Solid Phase Toxicity Test Using Chironomus unions
(in development)
SOP No. 1-002
ERTC
28-Day Solid Phase Toxicity Ted Using Hyaltlla aatca
(in development)
Greene et al (1989)
14-Day Acute Toxicity Test Using adult Eistnia andrei (earthworm)
EPA 600/3-88-029
SOP No 1 003
ERTC
Field Processing of Fish
(in development)
SOP No. 2029
ERTC
Small Mammal Sampling and Processing
(in development)
SOP No. 2032
ERTC
Berth)c Sampling
(In development)
SOP No 2033
ERTC
Plant Protein DetemtinMion
(in development)
SOP No. 2034
ERTC
Plant Biomass Determination
(in development)
SOP No 2033
ERTC
Plant Peroxidase Activity Determination
(In development)
SOP No 2036
ERTC
Tree Coring and Interpretation
(In development)
SOP No. 2037
ERTC
Terrestrial Plant Community Sampling
(In development)
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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 defined two broad categories of data: screening
and defimuve.
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 dala
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
iniual and continuing calibration
analyte identification
analyte quantification
Streamlined QC requirements are the defining
characterise of screening data.
Definitive data are generated using rigorous analytical
methods (e.g., approved U.S. EPA reference methods).
These data are analyte-speciftc, 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
sue 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/D71, 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.
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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.
Dunng 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) repeat 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
homogenizalion; 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 -20ฐ C. Tissue homogenates are prepared in
the laboratory and could be subject to cross-
contamination.
Refer to U.S. EPA/ERT SOP #1820, Tissue
Homogenization 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 Herbes and Allen [1983] and
Bligh and Dyer 19S9). 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
ussue samples.
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Tissue samples destined for sotting 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 arc 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-90/004, 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 "tnie"
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 lo 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
mcnniinwMW fnr valid ซtmiซriral analyck For total CJTOr
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.
Toxicity Testing Replicates
For sediment samples, at least 3 replicate treatments
should be conducted to determine variability between
tests.;The 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 homogeneity;within the sample matrix.
Site-Specific Examples of the Use of Replicates
Example Nn 1
Two contaminant sources were identified at an active
copper smelting facility. The fust 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 m
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 tentans, 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 detetjmne the
23
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variability within the test organism population within
each treatment
The results reported mean survival for Hyalelia 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 ten tans in the contaminated
sediment (0 to 63 percent) was significantly lower than
survival in the uncontaminated reference sediment (83
percent).
Example Nn 7
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 sin 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 locauon (i.e., three "replicates" per
location) and evaluated for various quantitauve
community metrics. The purpose of these replicates were
to determine the spauai 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
funcuonal diversity varied inversely with DDT levels in
sediment and water. These results were further
substantiated by the toxicity evaluation results.
Example No. 3
Phase 1 and II Remedial Investigation and Feasibility
Studies (RIFS) 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-
cowaminated soil.
Three small mammal trapping areas were identified for
this site. Two areas were located in PCB-contaminated
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 PCB concentrations in the mammals were
as follows: on-site areas (1250 and 1340 ซg/kg. wet
weight); lefaence area (490 jig/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
ui the mammals (p<0.10). 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 rinsate blank is used to assess cross-contamination
from improper equipment decontamination procedures.
Rinsate blanks are samples obtained by running analyte-
free water over decontaminated sampling equipment
Any residual contamination should appear in the rinsate
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 fteld blank per day.
4.4.6 Trip Blank Samples
Tnp 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 I jhnratnrv Contml 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.
Tonicity 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 generauon(s) in
the present culture is viable for use in the toxicity test, or
if the culture has grown resistant or intolerant to (he
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 arc 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/MSD 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
deviauon 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 lipids, organic carbon, and particle-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
laboratsy. 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 Nauonal
Functional Guidelines (U.S. EPA 1994) and according to
US. EPA Quality Assuranee/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
Superfund 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 macroinvertebrate 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-e vide nee approach
is used to interpret the results of different studies or tests
conducted at a site.
The statistical tests and methods thai 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 stausucal procedures
require summary statistics calculated from a data set. it is
important that the summary stausiics represent the enure
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
locauon 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
IBI 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 stanstir 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 thai is applicable to vinually 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
Preiiminary screening value results are interpreted by
comparison of historical and/or new site analytical data
against literature tonicity values. This comparison will
suggest if the probability of risk exists and whether
additional evaluation is desired.
If the evaluation is pursued to an ecological risk
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.
28
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Figure 3 Illustrations of Sample Plots
IBI DATA
12
25
33 56
12
24
34 56
14
26
35
15
24
36
16
24
35
22
27
38
24
23
41
23
28
42
40 SO I 60
2 2334444
2 5678
3 5568
A) Histogram
B) Leaf Plot
r
o
5
c 50
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 ume 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 trained ecologisL
Checldist
The checklist has been divided into sections thai correspond to data collection methods and ecosystem types. These sections
are:
I. Site Description
1A Summary of Observations and Site Setting
H Terrestrial Habitat Checklist
HA. Wooded
IIB. 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
1. SITE DESCRIPTION
1. Site Name:
Location:
County: City: S tate:
2. Latitude: Longitude:
3. What is the approximate area of the site?
4 Is this the first site visit? ~ yes ~ no If no, attach trip report of previous site visits), if available.
Date(s) of previous site visit(s): .
5. Please attach to the checklist USGS topographic map(s) of the site, if available.
6. Are aenal or other site photographs available? ~ yes ~ 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
% Residenual
% Industrial (~ light O heavy)
% Agricultural
( Crops: )
% Recreational
(Describe; note if it is a park, etc.)
The area surrounding the site is:
mile radius
% Urban
% Rural
% Residential
% Industrial (~ light ~ heavy)
% Agricultural
(Crops: )
% Recreational
(Describe; note if it is a park, etc.)
_% Undisturbed % Undisturbed
_% Other % Other
8. Has any movement of soil taken place at the site? ~ yes ~ no. If yes, please identify the most likely cause of this
disturbance:
Agncultural Use Heavy Equipment Mining
Natural Events Erosion Other
Please describe:
32
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9. Do any potentially sensitive environmental areas exist adjacent to or in proximity to the site, e.g.. Federal and State
parks. 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?
~ Chemical ~ Manufacturing ~ Mixing ~ Waste disposal
~ 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:
~ Swales ~ Depressions ~ Drainage ditches
H Runoff ~ Windblown particulates ~ Vehicular traffic
H Other (specify^
13 If known, what is the approximate depth to the water table?
14 Is the direction of surface runoff apparent from sue observations? ~ yes ~ no If yes. to which of the following
does the surface runoff discharge? indicaie all that apply.
" Surface water j Groundwater D Sewer D Collection impoundment
15 Is there a navigable waterbody or tributary to a navigable waterbody? ~ yes ~ no
33
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16. Is there a waierbody anywhere on or in the vicinity of the site? If yes, also complete Section III: Aquatic Habitat
Checklist - Non-Flowing Systems and/or Section IV: Aquatic Habitat Checklist - Flowing Systems.
~ yes (approx. distance.
~ no
17. Is there evidence of flooding? ~ yes ~ no 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? ~ yes ~ 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 (ฎDฐF)
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
Additional Preparers.
Site Manager
Date
Affiliation
35
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II TERRESTRIAL HABITAT CHECKLIST
HA. WOODED
1. Are there any wooded areas ai the site? ~ yes ~ no If no, go to Section HB: Shrub/Scrub.
2. What percentage or area of the site is wooded? ( % acres), imam* 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 sue of the trees at the site? Use diameter at breast height
~ 0-6 in. ~ 6-12 in. ~ >12 in.
5. Specify type of understory present, if known. Provide a photograph, if available.
IIB. SHRUB/SCRUB
1. Is shrub/scrub vegetation present at the site? ~ yes ~ no If no, go to Section DC: Open Field.
2. What percentage of the site is covered by scrub/shrub vegetation? { % acres). Indicate the areas of
shnib/scrub on the site map. Please identify what information was used to determine this area.
3. What is the dominant type of scnib/shrub vegetation, if known? Provide a photograph, if available.
4. What is the approximate average height of the snub/shrub vegetation?
~ 0-2 ft. ~ 2-5 ft ~ >5fi-
36
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5. Based on site observations, how dense is the scnib/shrub vegetation?
~ Dense ~ Patchy ~ Sparse
nc. OPEN FIELD
1. Are there open (bare, barren) field areas present at the site? ~ yes ~ no If yes, please
indicate the type below:
~ Prairie/plains ~ Savannah ~ Old field ~ 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 plants)? Provide a photograph, if available.
4. What is the approximate average height of the dominant plant?
5. Describe the vegetation cover ~ Dense ~ Sparse ~ Patchy
HD. MISCELLANEOUS
1. Are other types of terrestrial habitats present at the site, other than woods, scnib/shrub, and open field? ~ yes ~ no
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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What observations, if any, were made at the site regarding the presence and/or absence of insects, fish, birds,
mammals, etc.?
Review the questions in Section 1 to determine if any additional habitat checklists should be completed for this site.
38
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HL 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?
~ Natural (pond, lake)
~ Artificially created (lagoon, reservoir, canal, impoundment)
1. If known, what is the name(s) of the watertoody(ies) on or adjacent to the site?
3. If a waterbody 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? ~ yes ~ no If yes, please identify the type of vegetation present if known.
~ Emergent ~ Submergent ~ Floating
6. If known, what is the depth of the water?
7. What is the general composition of the substrate? Check all that apply.
~ Bedrock
~
Sand (coarse)
~ Muck (fine/black)
Z Boulder (>10 in.)
~
Silt (fine)
~ Debris
Z Cobble (2.5-10 in.)
~
Marl (shells)
~ Detritus
Z Gravel (0.1-2.5 in.)
~
Clay (slick)
~ Concrete
_ Other (specifyj_
8 What is the source of water in the waterbody?
Z River/Stream/Creek ~ Groundwater ~ Other (specify).
Z Industrial discharge ~ Surface runoff
39
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9. Is there a discharge from the site to the wateibody? ~ yes ~ no If yes, please describe this
discharge and its path.
] 0. Is there a discharge from the waterfoody? ~ yes ~ no If yes, and the information is available, identify from the list
below the environment into which the waterbody discharges.
~
Rivcr/Stream/Crcek
~
onsite
~
offsite
~
Groundwater
~
onsite
~
offsite
~
Wetland
~
onsite
~
offsite
~
Impoundment
~
onsite
~
offsite
Distance
Distance.
11. Identify any Held measurements and observations of water quality thai 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
Di&solved oxygen
Salinity
Turbidity (clear, slightly turbid, turbid, opaque) (Secchi disk depth.
Other (specify)
12 . Describe observed color and area of colorauon.
13. Mark the open-water, non-flowing system on the site map attached to this checklist.
40
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What observations, if any, were made at the wateibody regarding the presence and/or absence of benthic
macToinvertebrates, fish, birds, mammals, etc.?
41
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rv. 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 (arc) present at the site?
~ River ~ Stream
~ Dry wash ~ Arroyo
~ Artificially ~ Intermittent Stream
created ~ Other (specify)
(ditch, etc.)
2. If known, what is the name of the waterbody?
3. For natural systems, are there any indicators of physical alteration (e.g., channeling, debris, etc.)?
~ yes ~ no If yes, please describe indicators that were observed.
~ Creek
~ Brook
~ Channeling
4. What is the general composition of the substrate? Check all thai apply.
~
Bedrock
~ Sand (coarse)
~
Muck (fine/black)
~
Boulder (>10 in.)
~ Silt (fine)
~
Debris
~
Cobble (2.5-10 in.)
~ Marl (shells)
~
Detritus
c
Gravel (0.1-2.5 in.)
~ Clay (slick)
~
Concrete
u 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? ~ yes ~ no What information was used to make this deteimination?
42
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7. Is the flow intermittent? ~ yes ~ no If yes, please note the information that was used in making this determination.
8. Is there a discharge from the site to the waterbody? ~ yes ~ no If yes, please describe the discharge and its path.
9. Is there a discharge from the waterbody? ~ yes ~ 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? ~ yes ~ no If yes. please identify the type of vegetation present, if known.
~ Emergent ~ Submergent ~ 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
macroi n vertebrates, 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?
~ yes ~ no
Please note the sources of observations and information used (e.g., (JSCS 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 wateibody. in a floodplain) and site conditions (e.g., standing water,
dark, wet soils; mud cracks; debris line; water marks), are wetland habitats suspected?
~ yes ~ no If yes, proceed with the remainder of the wetland habitat identification checklist
3. . What type(s) of vegetation are present in the wetland?
~ Submergent ~ Emergent
~ Scrub/Shrub ~ Wooded
~ 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? ~ yes ~ no If yes, is this water ~ Fresh ~ Brackish
What is the approximate area of the water (sq. ft.)?
Please complete questions 4, II, 12 in Checklist III - Aquauc Habitat Non-Flowing Systems.
6 Is there evidence of flooding at the
Z Buttressing ~
Z Debns line ~
site? What observauons were noted?
Water marks ~ Mud cracks
Other (describe below)
45
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7.
If known, what is the source of the water in the wetland?
~ Strcam/River/Creek/Lake/Pond ~ Groundwater
~ Flooding ~ Surface Runoff
8. Is there a discharge from the site to a known or suspected wetland? ~ yes ~ no If yes, please describe.
9. Is there a discharge from the wetland? ~ yes ~ no. If yes. to what waterbody is discharge released?
~ Surface Stream/River ~ Groundwater ~ Lake/Pond ~ 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 itilt
of contaminant
of concern*
Pathway
from
origin
Change of
contaminant
state In
pathway
condensation
-~ Liquid
**
Gas ~ Air
Gas
**
~ Solid
solidification
Final
pathway
to receptor
~ SO
~ sw
> so
> AI
> SW
> SO
~ sw
i
Human
J3'0
G,D
Receptor
Ecological Threat
Terrestrial
G,D
G,D
Aquatic
N/A
G,D
I.D
I,D
N/A
I.D
I .D
N/A
G,D
I,D
G,D
G,D
G,D
N/A
GtD
G,D
G,D
* May be a transformation product
* Includes vapors
Receptor Key
D ป Darmal Contact
I - Inhalation
Q Inowtlon
N/A - Not AppHcabla
Pathway Key
AI - Ah
SO - Son
SW ป Surtaca Watar
(Including aadimanti)
GW - Ground Watar
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
~ sw
Change of
Pathway contaminant
from ซtate In
origin pathway
* Liquid
Gas**
~ Solid
crystallization
Final
pathway
to receptor
-u
~ so
leachate.
Infiltration
~ Liquid
~ AI
* May be a transformation product
** Includes vapors
Gas
*
sw
AI
SW
sw
Pathway Kay
M . Mr
SO - Soil
8W Suriaca WMซ
(Indudlna tadhnanti)
QW - Oround Watar
Human
h9
G, D
G,D
Receptor
Ecological Threat
Terrestrial
G J)
IiD_
G,J)
G,D
Aquatic
G,D
N/A
J3,A
G,D
~
so
G,D
G,D
N/A
~
sw
G, D
G,D
G,D
~
GW
G,D
N/A
N/A
~
so
G,D
G,D
N/A
~
AI
I.D
I, D
N/A
p.
SW
G,D
G,D
GfD
Receptor Key
0 - Darmal Contact
1 - Inhalation
O - Ingettlon
M/A m Not AppOcabla
48
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Figure B-3
Migration Routes of a Solid Contaminant
from Origin to Receptor
Original state
of contaminant
ol concern*
Solid
Pathway
from
origin
Change ot
contaminant
atate In
pathway
AI
~ SW
partlculatea/
duet
~ SO
Solid
> Solid
~ Liquid
Gas
Solid
Liquid
* May be a transformation product
** Includes vapors
Receptor Key
0 Darniat Contact
1 - Inhalation
0 . higatfon
NM Not Appneabte
Final
pathway
to receptor
AI
SW
SO
SW
SW
**
Receptor
Ecological Threat
Human
Terrestrial
Aquatic
IปD
IปD
N/A
G,D
G,D
G,D
G,D
G,D
N/A
G, D
G,D
~G,D
G,0
G,D
G,D
~ so
G, D
G,D
N/A
~ AI
I ปD
I,D
N/A
~ SW
G,D
G,D
G,D
~ so
G,D
G,D
N/A
~ so
G,D
G,D
N/A
~ GW
G,D
N/A
N/A
~ SW
G,D
G,D
G,D
Pathway Key
AI /Ur
SO - 8ol
SW - Surface Witw
(Including MdtmcnU)
OW - Ground Water
49
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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
general 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, succession^ 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-acre 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
A preliminary site visit was conducted, and the checklist indicated the following: I) 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
benihic macroinvenebrate community also has been noted in the pond. The pond appears to function as a valuable
habitat for fish and otheT wildlife.
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).
Copper can cause toxic effects in both aquatic plants and invertebrates at relatively low water concentrations, thereby
affecting the pond & ability to support macroinvenebrate 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 toxicity 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 pan of the preliminary site visit. The information gathered indicates that
surface water drainage from the site flows through several drainage swales toward a small 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 (hat it can cause reproductive failure in buds 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 successional 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 on the effects of PCBs to red-tailed hawks. Studies on comparable species have
indicated decreased sperm concentration that may affect reproductive success.
m. 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 macroinvenebrate
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 transferable. 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 Hyalelia azieca. The
measurement endpoints 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 capricormuum (growth test) and a 7-day
larval fish test using Pimephales promelas (mortality and growth endpoints).
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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
toxicity 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
macroinvenebrate community would be an initial receptor for tfie DDT in sediments. Fish that feed on the benthic
macroinveitebrates 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 prepaied 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.
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REFERENCES
ASTM. 1992. Standard Guide for Conducting Early Ufe-Stage Toxicity Teas with Fishes. American Society for
Testing and Materials. El 241-92.
Bligh, E.G., WJ. Dyer. 1959. Lipid Extraction and Purification, fanarfian Journal of Biochemistry and Physiology. Vol
37. pp. 912-917
B rungs. WA. and D.I- Mount. 1978. Introduction to a Discussion of the Use of Aquatic Toxicity Tests for Evaluation
of the Effects of Toxic Substances. Cairns, J. Jr., KX. Dickson and A.W. Makei (cds.) Estimating the Hazard of
Chemical Substances lo Aquatic Life. ASTM 657. Amer. Soc. Test Materials, Philadelphia. PA. p. 1526.
Green. 3.C., CJ_. Battels, WJ. Warren-Hicks, B.R. Parichurst, Gi..Linder, SA. Peterson, and"W.E.Meiller. 1989.
Protocol for Short Term Toxicity Screening of Hazardous Waste. U.S. 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, Ml.. 1983. Active Fish Capture Methods, Chapter 7 in Fisheries Techniques. American Fisheries Society, pp.
123-145.
Herbes, S.E. and C.P. Alien. 1983. Lipid Quantification of Freshwater Invertebrates: Method Modification for
Microquantitation. Canadian Journal of Fisheries and Aquatic Sciences. 40(8). pp. 1315-1317.
Hurbert, W.A. 1983. Passive Capture Methods, Chapter 6 in Fishtries Techniques. American Fisheries Society, pp. 95-
122.05
Kan, J.R., K.D. Fausch. Pi.. Angermeier. P.R. Yarn, and LJ. Schlosscr. 1986. Assessing Biological Integrity in
Running Waters: A Method and Its Rationale. Special Publication 5. Illinois Natural History Survey.
Philips. D.J.H. 1977 The Use of Biological Indicator Organisms to Monitor Trace Metal Pollution In Marine and
Estuanne Environments-A Review. Environmental PoU. 13, pp. 281-317.
Philips. D.J.H. 1978. Use of Biological indicator Organisms to Quantitate Organochlonne Pollutants in Aquatic
Environments-A Review. Environmental Poll 16, pp. 167-229.
Timbrel). 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
Destining and Conducting Ecological Risk Assessments. Office of Solid Waste and Emergency Response. EPA 540-R-
97/006.
U.S. EPA f 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 of ERTToxicity Testing Procedures.
OSWER Directive 9360.4-08.
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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 1989a. 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.
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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-I 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
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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 (1/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.
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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.
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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 toxicity 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-iine 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
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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 toxicological 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
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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 not 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/OMA-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 l 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
D-1
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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 (Type II error) = p
However, if one tries to evaluate the probability of Type H 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 II 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).
1 With a specified alternative hypothesis, once a and the sample size (N) are set, is determined.
D-2
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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 1 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 II 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 - P) 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
powerful one-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:
D-3
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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.
D-4
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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 and Data Analysis. Boca Raton, FL: CRC Press,
Inc., Lewis Publishers.
Siegel, S. 1956. Non-parametric Statistics. New York, NY: McGraw-Hill.
D-5
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EXHIBIT 1-2
Eight-step Ecological Risk Assessment Process for Superfund
STEP 1: SCREENING-LEVEL:
Site Visit
Problem Formulation
Toxicity Evaluation
Risk Assessor
and Risk Manager
Agreement
STEP 2: SCREENING-LEVEL:
Exposure Estimate
Risk Calculation
SMDP
STEP 3: PROBLEM FORMULATION
Toxicity Evaluation
7
Assessment
Endpoints
J
I
Conceptual Model
Exposure Pathways
I
c
o
o
"o
O
ta
re
Q
Questions/Hypotheses
SMDP
STEP 4: STUDY DESIGN AND DQO PROCESS
ฆ Lines of Evidence
Measurement Endpoints
Work Plan and Sampling and Analysis Plan
SMDP
STEP 5: VERIFICATION OF FIELD
SAMPLING DESIGN
SMDP
STEP 6: SITE INVESTIGATION AND
DATA ANALYSIS
[SMDP]
STEP 7: RISK CHARACTERIZATION
STEP 8: RISK MANAGEMENT
SMDP
1-9
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