Proceedings of the
Integrated Research Planning Meeting for
Gulf of Mexico Estuaries
November 2-3, 1993
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida
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November 1993
PROCEEDINGS OF THE
INTEGRATED RESEARCH PLANNING MEETING FOR
GULF OF MEXICO ESTUARIES
James E. Harvey1, and Foster L. Mayer2
Avanti Corporation1
Environmental Research Laboratory
Gulf Breeze, FL 32561
U.S. Environmental Protection Agency2
Environmental Research Laboratory
Gulf Breeze, FL 32561
contract number 68-C3-0309
Work Assignment Manager
Michael A. Lewis
Project Officer
Raymond G. Wilhour
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This document has not been peer and administratively reviewed
within the EPA and is for Agency'use/distribution only. Mention of
trade names .or commercial products does not constitute endorsement
or recommendation for use.
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CONTENTS
1. Introduction (Scope and Purpose) 1
2. Research Descriptive Summaries 4
3. Aquatic Ecological Evaluation Framework 6
4. Advisory Committee Recommendations 16
5. Literature Cited 20
Appendix A, Meeting Participants 21
Appendix B, Advisory Committee Members 23
Appendix C, EPA Request for Assistance (RFA) 25
Appendix D, Cooperative Agreement Research Summaries 27
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FIGURES
Figure 1. Organizational Categories of Indicators 7
Figure 2. Candidate Selection Criteria for Indicators 9
Figure 3. Draft Site Selection Criteria Matrix 11
Figure 4. Watershed Approach to Sample Collection
and Evaluation 12
Figure 5. EMAP Sampling Strategy 13
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EXECUTIVE SUMMARY
A research planning meeting for the Aquatic Ecological
Criteria Research Program was held November • 2-3, 1993 to discuss
and coordinate the development and field validation of an
ecological diagnostic framework for Gulf of Mexico estuaries. Such
a framework is essential for developing a systems-level diagnostic
approach and developing relevant protocols to realistically and
statistically assess the condition of a give.n ecosystem, drainage
system or major watershed region. A brief review was given of this
project's relationship to the Office of "Research and Development's
Aquatic Ecologically-Based Criteria and Characterization Issue
Plan, and other EPA research initiatives: Gulf of Mexico Program,
Environmental Monitoring and Assessment Plan, etc., and it's
significance to EPA Regional and Headquarters Offices (Office of
Water). Current and near-term research was presented in the
framework of the Aquatic Ecological Criteria Research Program.
Assessments of any • missing areas of expertise in the overall
project were addressed. Criteria to identify the site(s) to be
studied were discussed. An advisory committee composed of
representatives from academia, state and federal agencies, and
industry provided suggestions relative to the selection of sites
and protocol development, assessment and validation activities,, and
will eventually provide guidance through evaluating progress by
reviewing reports and performing site visits. The meeting resulted
in a peer-reviewed conceptual approach and directives for planning
and implementation. The agreed-upon purpose of the next meeting
will be to establish a research plan addressing such specifics as
selecting a specific site, addressing temporal and seasonal
variation, identifying an appropriate stress gradient, and
delineation of responsibilities. As planning progresses, a peer
review of this research plan will be conducted in the Spring of
1994. This report summarizes research discussions and
presentations from the meeting, and also lists the Advisory
Committee recommendations.
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1. INTRODUCTION
Aquatic ecosystems are often subject to a complex and dynamic
array of physical, chemical, and -biological interactions.
Ecologists do not have a complete understanding of how toxic
chemicals and other stressors influence these interactions and how
perturbations at one level of organization are expressed at other
levels. There is a limited capability to compare or predict
effects from one species to another and only simple qualitative
approaches for dealing with population, community, or ecosystem
comparisons. Assessments from single-species approaches for site-
specific and media-specific (e.g., effluent, water column,
sediment) situations must be extended to a fundamental,
quantitative understanding of exposure-response relationships for
larger ecological units.
Regulatory actions require the best, most consistent, most
rational guidance from scientists regarding ecological
characterization and ' the choice of underlying endpoints and
indicators. The biogeographical distributions of discrete species
and populations (e.g., streams, lakes, Great Lakes, Gulf of Mexico,
North Atlantic, and Pacific coastal regions) and the influence of
seasonal factors (e.g., temperature, salinity, and nutrients, will
likely require that regulatory actions be based upon unique
characteristics of each region. Researchers and managers must
develop prudent, reliable, and cost-effective approaches to compare
and predict the effects of various stressors at all levels of
ecological organization in aquatic ecosystems of varying complexity
and size within defined biogeographical regions.
In support of these needs, a research planning meeting was
held November 2-3, 1993 to discuss and coordinate Gulf of Mexico
estuarine research within the framework of the Aquatic Ecological
Criteria Research Program. The meeting resulted in a conceptual
plan. Such a plan is essential for developing a systems-level
approach and developing protocols, since optimally, endpoints would
need to be evaluated at the same site(s) at the same times and on
the same organisms to realistically and statistically assess their
utility. Assessments of any missing areas of expertise in the
overall project were addressed. Criteria to identify the site(s)
to be studied were discussed. A presentation was assembled by the
meeting participants (Appendix A) . This information was then
presented to an advisory committee composed of representatives from
academia, state and federal agencies, and industry. The Advisory
Committee (Appendix B) provided suggestions relative to the
selection of sites and protocol development, assessment and
validation activities, and will eventually provide guidance through
evaluating progress by reviewing reports and performing site
visits. The purpose of the next meeting will be to establish a
research plan addressing such specifics as selecting the site,
addressing temporal and seasonal variation, identifying an
appropriate stress gradient, and delineation of responsibilities.
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As planning progresses, a peer review of the-overall research plan
will be conducted in the Spring of 1994.
Dr. Foster Mayer, U.S. EPA, called.the meeting to order and
articulated the agenda, introduced participants, outlined broad
goals and provided background information on the purpose of this
meeting. He called attention to the goals of the meeting and asked
participants to assist in 1) obtaining consensus purpose and goals
for this estuarine research project, 2) developing the research
program matrix, 3) reviewing the sampling site approach, and 4)
identifying and discussing deficiencies and watershed approach
needs. Dr. Mayer also related portions of this programmatic
mission to the similar goals of a 1991 workshop1 on estuarine
assessment and contaminant identification. The 1991 workshop goals
reflected the importance of criteria for selection of appropriate
endpoints and indicators that provide unambiguous information on
the condition of an ecosystem. Because of the acknowledged
importance of these goals to the EPA program and to this particular
project, the goals of that workshop warrant restatement.
1.) To define, through consensus, a set of ecological assessment
procedures to:
A) Describe, at a screening level, the condition (physical,
chemical and biological) of Gulf coast estuaries.
B) Identify and characterize ecological problems caused by
contaminants.
C) Determine the causes of observed problems, focusing on
pesticides and toxic contaminants.
2.) To establish selection criteria for demonstration sites in the
Gulf of Mexico to field test the applicability and predictive
capability of the procedures.
These goals helped to provide a basis for a subsequent Request
for Assistance (RFA) from EPA that was issued in the Spring of 1993
for collaborative research. The RFA was developed to solicit
proposals in support of development and validation of ecosystem
diagnostic techniques for Gulf of Mexico estuaries and associated
bayous. Appendix C lists the .complete RFA solicitation. After
competitive review, available resources allowed funding of three
proposals. Part of the selection criteria for these proposals was
their ability to complement current and proposed aquatic ecological
research at Gulf Breeze Environmental Research Laboratory (GBERL).
"Development and Evaluation of Diagnostic Indicators of
Ecological Condition for Gulf of Mexico Estuaries and
Associated Bayous;" Dr. Kenneth Dickson, University of North
Texas, Denton, TX (a consortium including Gulf Coast Research
Laboratory, Ocean Springs, MS - University of Southern
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Mississippi; University of Miami, FL; University of Texas at
Dallas; and ABC Laboratories, Columbia,. MO) .
"Development and Validation of Bioassays for Assessing the
Contamination of Marine Estuarine Environments;" Dr. Stephen
Safe, Texas A&M Research Foundation, College Station, TX.
"Validation of the Mummichog Fundulus heteroclitus as a
Histopathological Indicator of Pollution Along the Eastern
United States;" Dr. Wolfgang Vogelbein, Virginia Institute of
Marine Science, College of William and Mary, Gloucester Point,
VA.
Research summaries were presented from each of these funded
proposals to familiarize all the participants with the research and
collective expertise at this meeting. EPA personnel also presented
short research summaries of toxicological and histopathological
analyses performed at the GBERL for the Pensacola Bay system.
As part of EPA's Office of Research and Development Aquatic
Ecological Criteria Research Program researchers from GBERL and
extramural cooperators will share appropriate approaches, methods,
and samples to identify and develop appropriate and unambiguous
endpoints and endpoint indicators that are sensitive, accurate and
precise.' These measurements contribute to the short-term program
goal: characterize and assess a smaller aquatic ecosystem,
estuarine drainage system or bayou, and where possible, identify
diagnostic capabilities of endpoints and their suites of
indicators. The long-term program goal is to utilize an integrated
watershed approach to developing a characterization and assessment
framework that will be applicable not only to estuaries and bayous,
but to wetlands, marshes and terrestrial components of the
ecosystem. This characterization and assessment framework, once
developed and verified, would become a valuable monitoring and
regulatory tool. Future goals could include expanding the
usefulness of this framework to include an "early warning"
indicator monitoring aspect as well as predictive abilities. The
Aquatic Ecological Criteria Research Program provides fundamental
knowledge regarding the ecology of near-coastal ecosystems to
better enable formulations of risk assessment by EPA Regional
personnel, EPA headquarters regulatory personnel, and interested
state and local regulators and scientists in the Gulf of Mexico
watershed region. Advisory committee members represent this broad
regulatory and scientific diversity.
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2. RESEARCH DESCRIPTIVE SUMMARIES
Complete descriptions of objectives and approaches for each of
the three cooperative agreement.research.projects are contained in
Appendix D.
The first proposal, "Development and Evaluation of Diagnostic
Indicators of Ecological Condition for Gulf of Mexico Estuaries and
Associated Bayous" helps to identify and evaluate ecological
indicators, emphasizing those with aspects linked to diagnostic
capabilities. The approach is to catalog and reduce sets of
indicators across different spatial/temporal scales and different
levels of biological organization to produce an evaluation
framework. This evaluation framework will then be performance-
tested and verified statistically. Application of the indicators
to synthetic process-oriented simulation model output and field
data comparison will contribute to a better understanding of the
response of indicators to spatial-temporal changes in stressors.
It is important to recognize that although this project has a
modeling component, the purpose of the project is not to verify a
model using field data. The field data will be used to assess
sensitivity and reliability of indicator systems.
The second proposal, "Development and Validation of Bioassays
for Assessing the Contamination of Marine Estuarine Environments"
evaluates and validates the efficacy of a series of biomarkers to
assess exposure of marine biota to organic contaminants, more
specifically, polynuclear aromatic hydrocarbons (PAHs) and
halogenated aromatic hydrocarbons (HAHs). This project will
determine if specific biomarkers indicative of organic chemical
exposure have been induced and determine if exposure has caused
specific physiological damage, such as modification of receptor
binding and gene expression. An additional objective of this study
is to assess the relative sensitivities of biomarkers such as EROD
activity, P450IA mRNA levels, PAH metabolites in bile, and DNA
adduct formation, and compare them to arrive at an approach that
integrates the activity of contaminants in a mixture, providing a
risk-based estimate at the molecular-genetic level. Information
and experience gained from this study will be integrated into the
overall development of the evaluation framework.
The third proposal, "Validation of the Mummichog Fundulus
heteroclitus as a Histopathological Indicator of Pollution Along
the Eastern United States" seeks to determine the efficacy of
Fundulus sp. as a histopathological indicator of chemical
contamination in aquatic environments. The scope of work for this
project has been amended to include field evaluation of Fundulus
grandis in selected Gulf of Mexico estuaries. As sediments are
known to be efficient integrators of hydrophobic organic compounds,
types and concentrations of sediment contaminants (PAHs,
heterocyclic compounds, PCBs, chlorinated pesticides, trace metals,
etc.) will be correlated with liver histopathologic responses.
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These field studies will provide a method of pollution assessment
that is able to link adverse health effects in a non-migratory fish
to specific types, sources, and concentrations of environmental
contaminants. This approach can be. .implemented locally and
regionally and will help to identify specific localities where
chemical contaminants are exerting adverse biological effects in an
indigenous organism. Information and improved methods for use and
development of histopathological indicators gained from this study
will be integrated into the overall development of an aquatic
evaluation framework.
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3. AQUATIC ECOLOGICAL EVALUATION FRAMEWORK
The aquatic ecological evaluation framework will subsume many
differing measurement endpoints, indicators, and coastline
environments. This massive body of information eventually will be
organized into a database from which one can combine and retrieve
any number of biological, chemical, ecological, environmental,
geographical, hydrological, and meteorological factors to assist
in characterizing, comparing or assessing the condition of a given
estuarine drainage system. Project-specific objectives include to:
• Analyze existing performance data of candidate diagnostic
indicators gleaned from available literature and Federal
and State ecological monitoring and assessment programs
(such as EPA's EMAP).
• Develop methodology for testing and/or evaluating
indicator performance using appropriate statistical
analyses and simulation models.
• Assemble a set of habitat, exposure, stressor and effects
indicators to characterize ecological conditions for
specific spatial and temporal ranges in specific systems.
Seek causal relationships or correlations between
conditions and effects.
• Apply the candidate indicators in a known stress gradient
in a selected estuary in the Gulf of Mexico.
These objectives will be accomplished through coordinated
research by GBERL scientists and university collaborators in a
stepwise fashion. Phase 1 will consist of a comprehensive
literature search undertaken to assemble and categorize documented
ecological indicators of potential utility to assess estuarine
drainage systems at differing spatial and temporal levels. Much of
this information exists and has been captured in various workshops,
symposia and journal articles and book chapters2'3'4'5'6. This
includes molecular genetic indicators as well as physiological and
gross anatomical indicators, geochemical indicators, etc. These
indicators will then be grouped into endpoint categories:
physical/chemical level, species-level, community level, ecosystem
level, and landscape level. Rather than generate a comprehensive
list of endpoints with subsets of indicators, these endpoint
categories .were chosen to represent organizational divisions within
ecosystems (Figure 1) . Endpoint categories include both assessment
endpoints (impacts of interest) and measurement endpoints
(parameters that can be physically measured). Endpoint selection
is dependent on the particular ecosystem being examined and
potential or suspected impacts or stresses7. The indicators for
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SPECIES- AND POPULATION LEVEL ENDPOINTS - particular species of concern
because of one or more of the following characteristics:
Direct Interest
• Economic, aesthetic, recreational, nuisance, or endangered species
Indirect Interest
Interactions between species- e.g., predation, competition, parasitism
Habitat role- e.g., physically dominant species such as mangrove tree
species
Ecological role
Trophic relationships
Functional relationships
Critical species- e.g., keystone species that affect overall trophic
structure or control important ecological processes
COMMUNITY-LEVEL ENDPOINTS
• Food-web structure
• Species diversity of ecosystems
• Biotic diversity of ecosystems
ECOSYSTEM-LEVEL ENDPOINTS
• Ecologically important processes- e.g., decomposition, nutrient recycling
• Economically important processes- e.g., wastewater treatment
• Water quality
• Habitat quality
LANDSCAPE-LEVEL ENDPOINTS
• Mosiac of ecosystem types- e.g., relative coverage of plant communities
• Corridors for migration- e.g., habitat for endangered species
• Spatial and temporal patterns of habitat- e.g., timing for and location of
wetland areas necessary for bird nesting
• Feedbacks to regional and global-scale physical systems- e.g., albedo,
evapotranspiration, or sources of biogenic gases
Figure 1. Organizational Categories of Indicators.
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each endpoint will then be judged against specific selection
criteria and incorporated into a site-coupled matrix diagram.
Selection criteria for these endpoints and their respective suites
of indicators will be determined by many- factors and will largely
be driven by practicality, regulatory needs, and scientific
interpretability. Figure 2 displays some of the candidate
selection criteria that were discussed at the meeting. Many site-
coupled matrix diagrams will be constructed to provide the breadth
of applicability for different site (estuarine drainage system)
conditions. It is expected that indicators will differ among
sites, but that some will appear throughout the matrices. These
indicators will be designated as core indicators, and will allow
comparisons, at a gross level, of differing sites. It is important
to realize that core indicators should not be used as a basis for
assessing the condition of a site, but merely as a first step in
characterization. Further characterizing specific sites is
important and will identify and delineate certain processes that
drive the system under study, leading to a more complete
understanding of its resiliency or capacity for perturbation. For
example, salinity, dissolved organic carbon, dissolved oxygen, and
turbidity may be much more important parameters in a shallow,
poorly flushed bay than in a riverine system with flowing water.
Measurements of these indicators may indicate trophic relationships
that support the ecology of that system, and therefore the
potential for adverse impacts if disrupted. Examining a
perturbation that has registered across several ecosystem
organizational (trophic) levels can provide a better estimation of
the extent of an impact or potential impact.
Thus far, we have discussed the use of measurement endpoints
and indicators and their selection criteria. Certain of these
indicators can be used to diagnose the condition of a particular
ecosystem. These "diagnostic" indicators differ from other
indicators in that they are usually associated with a particular
stress; exhibit a causal relationship or correlation; are process
oriented and possess a high signal to noise ratio, which means that
few false positives would be expected. An example of a diagnostic
indicator is the use of biomarkers to assess exposure of marine
biota to polynuclear aromatic hydrocarbons (PAHs) and halogenated
aromatic hydrocarbons (HAHs) as measured by molecular biological
methods such as EROD activity, P450IA mRNA levels, PAH metabolites
in bile, and DNA adduct formation.
Another class of indicators can be termed "early warning"
indicators. Guided by experience, certain indicators may be
identified that provide very limited information about the
ecological system, but serve as a flag or trigger for additional
measurements. We must stress development of these types of
indicators are of low priority as compared to indicators which
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• Relates to an assessment endpoint
• Availability of people to run assay
• Data on laboratory and field application available
Variability - Power
Sensitivity
• Temporal practability
• Specificity/selectivity/generality
• Ease of performance/cost
• Sample requirements
• Based on fundamental principles, mechanisms understood
• Large dynamic range
• Responsive - Reflects change in ecosystem condition
• System - Wide applicability
• Unambiguous
• Integrates effects
• Low natural variability
• Interpretability
Figure 2. Candidate Selection Criteria for Indicators.
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provide site characterization information leading to an assessment
of the condition of a given system. Early warning indicators will
most likely have a rapid response time; be lower in the trophic
level; be physiological or biochemical--measurements; have a low
signal to noise ratio, therefore a large number of false positives
would be expected. As the development-of the evaluation framework
proceeds, it is expected that the screening process for indicators
will suggest assignment of certain ones having high potential as
early warning indicators.
Phase 2 of the project is to prioritize and select a field
site. Initially, the evaluation framework will focus on one site;
that site will be chosen using the criteria listed in Figure 3.
The final evaluation framework will be composed of many
ecologically diverse sites, representative of a wide range of
habitats and aquatic environments. As more sites are
characterized, they will be incorporated into appropriate site
categories of the .evaluation framework still to be decided.
Initially, sites will be chosen based on such selection criteria
as: diverse biological populations, acute exposure gradient for a
stressor, availability of a reference area, well characterized
stressors, known hydrology, close proximity to a research facility,
etc. A site selection matrix (Figure 3) shows considerations that
must be addressed to choose an appropriate site to initiate this
project.- Based on the aforementioned selection criteria and the
proximity of GBERL to the Pensacola Bay drainage basin the choices
have been narrowed to areas within Escambia Bay. To attain the
sampling density needed for such a project and to obtain meaningful
data at the appropriate spatial and temporal scales, small bays and
bayous are under consideration for the first year's effort, and may
be expanded in the following years. It was decided by meeting
participants that a small bay or bayou would be chosen and a
sampling approach selected at the next project coordination
meeting. The general concept is to sample along a stress gradient,
incorporating land-based watershed samples (streams, industrial
effluents, etc.) and extend sampling into the receiving bay,
reiterating the watershed approach of the evaluation framework
(Figure 4).
Figure 5 displays a draft sampling scheme as developed and
employed by GBERL EMAP. Note physical/chemical measurements
coupled with biological measurements designed to characterize an
area leading to an environmental assessment. Much work has gone
into developing this sampling regimen, however it should be noted
that the EMAP sampling scheme was developed for a specific spatial
and temporal scale. Thus, it can serve as a starting point to
modify according to the smaller spatial scale of this project. A
more detailed draft of a sampling scheme appears in the proposal
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SELECTION CRITERIA
Diverse Community
Acute Exposure/Gradient
Large Watershed
"Reference" Area
Historic Information
Known Stressors/Sources
Localized Stressors
"Known" Hydrology
Research Facility
/f / / /
& / / /
POTENTIAL SITES
Yes
Yes/?
Yes
Yes
Limited
Yes
Yes
Limited
Yes
Yes
Yes
Yes
Yes
Yes/Limited
Yes
Yes
Yes/Limited
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes/Limited
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes/Limited
Yes
Figure 3. Draft Site Selection Criteria Matrix.
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BLACKWATER
RIVER
MULATTO BAYOU
fi*=*y^;.
INDIAN BAYOU
GULF OF MEXICO
Figure 4. Watershed Approach to Sample Collection and Evaluation
This figure illustrates the approach- strategic sampling of a site incorporating land-
based samples, then extending into the receiving small bay or estuary along a gradient,
and ending as the gradient dissapears into the larger receiving body of water. The
sites shown have not been selected are merely illustrate the concept of the watershed
approach.
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Current
Measurement
Velocity
Direction
Nutrient
Samples
+
roc
^
1
p
' -\
COD
r l
r
TKN NH. N0,
A/0,
Benthos
Grain size
Rodox Potential Discontinuity
L
Arrive on Station
and Anchor
Record Station
Information and
Observations
Water Column Profile
at 1-Meter Intervals
Multiple Bottom
Sediment Grabs
Pull Anchor
Get Boat Under Way
Multiple Trawl Hauls
Oyster Dredge Hauls
as Directed
Microbiological
Samples
Seston Under
Development
Vibrio
E. coll
Geographical
Meteorological
General
Dissolved Oxygen -
PAR
pH
Salinity
Temperature
Transparency
Sediment Chemistry
Sediment Toxicity
Acid Volatile Sultldes
Deploy Datasonde
as Appropriate
Overnight Parameters
Population Analyses (composition / abundance)
Tissue Chemistry
Hlstopathology
Condition / Abundance
Physiological / Chemical Assessment
Figure 5. EMAP Sampling Strategy
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"Development and Evaluation of Diagnostic Indicators of Ecological
Condition for Gulf of Mexico Estuaries and Associated Bayous;" Dr.
Kenneth Dickson, University of North Texas, Denton, TX (a
consortium including Gulf Coast Research-Laboratory, Ocean Springs,
MS - University of Southern Mississippi; University of Miami, FL;
University of Texas at Dallas; and ABC Laboratories, Columbia, MO) .
Phase 3 of the project is to develop the field study design
component which will be dependent on the final selection of
indicators (phase 1) and the selection.of the estuarine site (phase
2). The field study component will be -designed to focus the best
(most appropriate) sampling approaches and methods to bear on
characterizing a particular ecosystem. The EMAP Field Operations
Manual for the Louisianian Province8 will be consulted to provide
guidance for selection of approaches and methods to be tested on a
smaller scale. The field study program does not seek to duplicate
the EMAP sampling strategy; modifications to methods and changes to
sampling strategies will be implemented to reflect appropriate
scale differences between our selected site and other sites used
originally to develop those methods. EMAP field sampling
activities usually occur from July to October to reflect the "worst
case" scenario of low dissolved oxygen. It is proposed in this
project that field activities be scheduled outside the EMAP
sampling window so that EMAP "equipment may be used. A secondary
benefit • to this approach is that data obtained using the same
equipment will provide more confidence in the measurements, and be
more comparable to the historical EMAP data. A similar strategy
was successfully employed in a demonstration project in the Back
Bay of Biloxi, MS9.
An important part of this project is to test and validate
indicators by comparing their performance to simulation models of
estuarine processes. This same approach has been suggested both as
a part of EMAP3 and at the 1992 Biloxi workshop1. The simulation
models will test the sensitivity of indicators with respect to
gradients of selected environmental conditions. This modeling
effort is not a surrogate to the statistical analyses of the field-
collected data, but will use the data to "truth" the validity and
sensitivity of an indicator to a particular system. Variables to
be included in the models will be related to the hydrodynamics,
chemistry and biology of the system. For modeling at the landscape
scale, grid cells (ca 1-10 ha) will be superimposed, encompassing
the site. The exact horizontal and vertical resolution will be
defined by the preliminary descriptions of the surrounding site.
The hydrodynamics will include patterns of horizontal and vertical
circulation, and its relationships to temperature, density and
salinity. Freshwater inflow and tidal action may be incorporated
depending on the location of the selected site. Salinity gradient
measurements will be used to calibrate the dispersion parameters.
Water quality relationships will include BOD/DO and a'sub-set of
stressors selected from the full set measured in the field. The
selected site will be more intensively examined, emphasizing
14
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chemical and biological processes at the sub-grid level.
Ecosystem, community, population, organism and biochemical
interactions will be examined at this smaller scale. Nutrient and
energy flows in trophic chains, various, biotic interactions, and
bioavailability and bioaccumulation will be addressed at this
level.
Laboratory data pertaining to the responses to toxicants at
the organism level will be used to generate population-level model
responses. Simulations of the gradual increase of pollutant
loading to the model estuary and the response time of the
indicators as applied to the model output can be used as an
estimation of the minimum detectable trend in response indicators,
and may help categorize certain indicators as being "early
warnings" of impending effects.
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4. ADVISORY COMMITTEE RECOMMENDATIONS
1. Selection of Indicators
The approach proposed for reviewing biological indicators in
ecological studies and selecting sets' of indicators for specific
purposes is a sound approach. The program should proceed with the
plan to evaluate existing ecological endpoints (assessment,
measurement) and their suites of indicators (early warning,
diagnostic) and classify their proven or potential utility for
various uses in selected habitats or community types (wetlands,
seagrasses, benthic communities, phytoplankton, etc.). A matrix
diagram developed from a master list of biological and ecological
indicators reviewed during the initial efforts for this program
would be very useful for quick review by research teams and
monitoring programs, and to provide a summary of potential
applications for these indicators.
The panel realizes that it is crucial to the ultimate success
of the program that the applications of some portion or subset of
the biological indicators be based on criteria that focus the
assessment conclusions along guidelines used for regulatory
decisions or water quality classifications. The panel recognizes
that a great deal of subtle "understanding and inference can be
built into a select suite of ecological indicators, but many
bottom-line decisions and priorities must be set within regulatory
frameworks where clear distinctions are necessary. Some
information useful for ecological research communication may be
lost through data reduction or manipulation of indices developed to
address regulatory decisions, but a goal of this program should be
development of indicators that communicate ecological conditions
consistent with classifications defined by regulatory criteria.
Thus, a means to relate selected ecological indicators to
regulatory criteria such as "fishable or swimmable waters",
"unacceptable adverse effect", "maintenance of balanced flora and
fauna", will be needed if the end products are to be widely
utilized by environmental scientists.
The panel recognizes that the proposed development of multiple
indicators, i.e., a suite of indices based on various trophic
levels or habitats, could lead to conflicting or inconclusive
assessments for specific sites. This could lead to confusion as to
the directions necessary to improve or restore habitats. Although
much of the focus of the program may be on anthropogenic chemical
stressoirs, many of the ecological indices will integrate other
stresses such. as low dissolved oxygen, habitat losses,
eutrophication, etc. These factors must be considered during the
site characterization process and evaluation of an ecological
indicators.
Early warning indicators do not have to be causally related to
specific stressors to be of value to the proposed program. They
16
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can be used as early and inexpensive indicators that alert an
investigator to potential problems, or conditions that require more
detailed assessment. Their development and application should be
followed by additional criteria that -are used to pinpoint the
nature of a problem and define ecological condition. Development
of early warning indicators should be 'a secondary goal, after the
development of suites of risk characterization and assessment
criteria for designated environments (wetlands, shallow bays, open
water, etc.).
2. Indicator Applications for Watershed Assessments or Ecological
Risk Assessments.
The potential to apply ecological indicators to enhance
ecological assessments of watersheds is an exciting development
which is pertinent to new EPA programs, arid should be evaluated in
its entirety. The panel recognizes that the development and
interpretation of ecological information relevant to site
characterization at the watershed level will be challenging, but
watershed approaches may become the basis for focusing and
prioritizing limited resources for remediation or restoration
programs. Ecological indicators that can help direct and focus
these efforts are needed.
The watershed approach under consideration is based on large
watersheds conceived at large physical scales (thousands to
millions of hectares) reflecting conditions that developed over
lengthy time scales (decades or more of various and changing land
uses). However, site characterizations and remedial actions must
be implemented at smaller spatial and temporal scales due to
resource limitations. Ecological criteria or indicators
appropriate to a number of scales of study and implementation must
be developed. The panel appreciates the challenge of addressing
problems within bays, estuaries, and watersheds, and encourages the
investigators to address this issue. Much of the initial scoping
may come from EMAP and other national, regional or local monitoring
programs, but a thorough search and evaluation of existing
ecological indicators is necessary to determine their utility for
this new characterization framework.
Because the ultimate application of these ecological
indicators may be risk-based resource management decisions, it is
important to recognize the need for appropriate baseline or
reference conditions for indicator development and application.
The research team should have clearly defined criteria for
selecting or defining reference conditions, as it is unlikely that
the research team can identify representative pristine sites for
comparison of all indicators. Historical information, ecological
analogues, models, mathematical analogues, and professional
judgement may all be necessary in development of reference
conditions or criteria.
17
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The panel encourages development of ecological indicators that
clearly discriminate between reference/bas-eline conditions and
impacted or changed ecological condition. We encourage the
research team to avoid pitfalls such as-:-
• ecological indicators that provide only tones of gray
distinctions,. without bright line differentiation between
impacted and acceptable conditions;
• the tendency for indicators to. give conflicting results,
especially as the number of inputs and assessment endpoints
increase;
• the preference of many researchers to include too many
endpoints or criteria, without focusing on critical
measurements or driving factors.
We suggest that the team start with a broad scope in reviewing
the kinds of ecological indicators that might be used for site
characterizations, but then begin to focus on those indicators that
provide reliable information for critical or priority
characteristics. A quantitative approach, with statistical and
modelling support will be necessary for scientifically defensible
products. Nevertheless, the need for definitive conclusions and
clear directions for restoration or remedial actions will require
that professional judgement enter into final interpretations and
applications of site assessment approaches.
3. Practicality/Utility of the Final Products
The committee believes that practicality of the tools and
measurements will be an important aspect of the development of
ecological indicators. The selection and weighting of indicators
should be representative of the scale of study relevant to the site
(bay, estuary, river basis), and should be relevant to the
resources common to state or local resource agencies. Highly
sophisticated or costly research approaches will have merit within
a total program, but advocation of ecological indices that require
tools or data out of the realm of day-to-day operations of state or
local agencies will result in limited real world applications.
The panel views this program as an ecological assessment
initiative, with primary emphasis on assessment, criteria and
indicators or ecological condition. The program should have a
secondary emphasis on development of diagnostic and early warning
indicators, as these tools will be relevant only when we agree on
the appropriate criteria by which a normal or healthy condition is
characterized.
Due to the location and expertise available at the Gulf Breeze
ERL, we hope that there will be close collaboration between the
cooperative agreement researchers and GBERL scientists. Selection
18
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of a study site for indicator evaluation that is near Gulf Breeze
is strongly recommended, in order to - maximize potential
participation by EPA scientists. The cooperative research program
should maximize the extent to which it can utilize information from
ongoing EPA research and monitoring programs.
4. Overall Direction
Senior management in regulatory or resource agencies often
require that information presented for decision making be free of
confusing scientific detail or caveats.- The application of these
ecological indicators for site characterization must, at some
level, be useful under these circumstances. The final indices must
incorporate various levels of data reduction and manipulation and
still reflect scientific credibility for ecological significance,
sensitivity, and reproducibility. Regulators often request a
single number for each important criterion, or some type of bright-
line distinction between impacted and unimpacted systems.
Therefore, indicators which are easily described, and which both
regulatory personnel and the lay public can relate to, are favored.
The research team should strive to develop a suite of ecological
indicators that can integrate accepted scientific approaches, stand
up to rigorous statistical and scientific review, and be defensible
in terms of regulatory criteria.
19
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5. LITERATURE CITED
1. Mayer, P., T. Duke, and W. Walker, (eds.) 1992. Estuarine
assessment and contaminant problem.identification. Summary of
a workshop held in Biloxi, Mississippi, April 23-25,1991.
NOAA Technical Memorandum NMFS-SEFSC-330, 63 p.
2. McKenzie D.H., D.E. Hyatt, V.J. McDonald (eds.) 1992.
Ecological Indicators. Vol. 1 and 2. Elsevier Applied Science.
3. Olsen, A.R. (ed.) 1992. The indicator development strategy for
the Environmental Monitoring and Assessment Program. U.S.
Environmental Protection Agency, Environmental Research
Laboratory, Corvallis, OR.
4. Huggett, R.J., R.A. Kimerle, P.M. Mehrle, Jr., and H.L.
Bergman. 1993. Biomarkers: Biochemical, physiological and
histological markers of anthropogenic stress. Lewis
Publishers, 121 S. Main Street, Chelsea, MI. 347 p.
5. Shugart, L.R., J.F. McCarthy and R. Hallbrook. 1992.
Biological markers of environmental and ecological
contamination: An overview. Risk Analysis 12(3):353-360.
6. Peakhall, D. 1992. Animal Biomarkers as pollution indicators.
Chapman & Hall, London, U.K. 29Op.
7. U.S. Environmental Protection Agency: Risk Assessment Forum.
1991. Summary report on issues in ecological risk assessment.
EPA/625/3-91/018. Washington, D.C.
8. McCauley, J.M. 1993. Near Coastal Louisianian 1993 Sampling.
Field Operations Manual, 4th Revision. ERL GB Number SR119.
U.S. Environmental Protection Agency, Office of Research and
Development, Environmental Research Laboratory, Gulf Breeze,
FL. 98 p.
9. Walker, W. 1993. Evaluating ecosystem health: A demonstration
project in the back bay of Biloxi, Mississippi. Gulf Coast
Research Laboratory Report, Ocean Springs, MS.
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Appendix A ._
Meeting Participants
Dr. Miguel Acevedo
University of North Texas
Institute of Applied Sciences
Box 13078, NT Station
Denton, TX 76203-3078
Mr. David Burke
University of Southern
Mississippi
Gulf Coast Research Laboratory
P.O. Box 7000
703 East Beach Drive
Ocean Springs, MS 39564-7000
Dr. Kenneth L. Dickson
University of North Texas
Institute of Applied Sciences -
Box 1307.8, NT Station
Denton, TX 76203-3078
Dr. William Fisher
U.S. Environmental Protection
Agency
Environmental Research Lab
1 Sabine Island Drive
Gulf Breeze, FL 32561-5299
Dr. John Fournie
U.S. Environmental Protection
Agency
Environmental Research Lab
1 Sabine Island Drive
Gulf Breeze, FL 32561-5299
Dr. Mark Harwell
RSMAS
Miami, FL
University of Miami
Mr. Mahlon C. Kennicutt, II
College of Veterinary Medicine
Texas A&M University Research
Foundation
College Station, TX 77843
Protection
Dr. Michael Lewis
U.S. Environmental
Agency
Environmental Research Lab
1 Sabine Island Drive
Gulf Breeze, FL 32561-5299
Protection
Dr. Foster Mayer
U.S. Environmental
Agency
Environmental Research Lab
1 Sabine Island Drive
Gulf Breeze, FL 32561-5299
Dr. Stephen H. Safe
College of Veterinary Medicine
Texas A&M University Research
Foundation
College Station, TX 77843
Dr. J. Kevin Summers
U.S. Environmental Protection
Agency
Environmental Research Lab
1 Sabine Island Drive
Gulf Breeze, FL 32561-5299
Dr. Michael E. Unger
College of William and Mary
Virginia Institute of Marine
Science
Gloucester Point, VA 23062
Dr. Wolfgang Vogelbein
College of William and Mary
Virginia Institute of Marine
Science
Gloucester Point, VA 23062
Dr. William W. Walker
University of S. Mississippi
Gulf Coast Research Laboratory
P.O. Box 7000
703 East Beach Drive
Ocean Springs, MS 39564-7000
21
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22
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Appendix B.
Advisory Committee Members
Dr. Don Axelrad
Florida Department of
Environmental Protection
2600 Blair Stone Road, Room 560
Tallahassee, FL 32399-2400
Dr. Thomas A. Bailey
U.S. Environmental Protection
Agency
PPTS/OPP/EFED (H7505C)
401 M Street, S.W.
Washington, DC 20460
Mr. Glen Butts
Florida Dept. of Environmental
Protection
160 Governmental Center
Suite 202
Pensacola, FL 32501-5794
Dr. James Clark
Exxon Biomedical Sciences, Inc.
Mettlers Road, CN-2350
East Millstone, NJ 08875
Dr. Carroll Cordes
U.S. Fish and Wildlife Service
National Wetlands Res. Ctr.
700 Cajundome Blvd.
Lafayette, LA 70504
Mr. Philip Crocker
U.S. Environmental Protection
Agency
Region VI
1445 Ross Ave., Suite 1200
Dallas, TX 75202-2733
Dr. Thomas Dillon
U.S. Army Corps of Engineers
Waterways Experiment Station
WESE-R, P.O. Box 631
3909 Halls Ferry Road
Vicksburg, MS 39180
Mr. Henry Folmar
Dept. of. Environmental Quality
121 Fairmont Plaza
Pearl, MS 39208
Mr. Jim Harrison
U.S. Environmental Protection
Agency
Region IV
345 Courtland Street (4WM-OPO)
Atlanta, GA 30365
Dr. Fred Kopfler
U.S. Environmental Protection
Agency
Gulf of Mexico Program,
Building 1103
Stennis Space Center, MS 39529
Dr. J. Kevin Summers
U.S. Environmental Protection
Agency
Environmental Research Lab
1 Sabine Island Drive
Gulf Breeze, FL 32561-5299
Dr. Becky Powell
Monsanto Company
800 North Lindbergh Blvd.
St. Louis, MO 63167
Dr. Maurice Zeeman
U.S. Environmental Protection
Agency
OPPTS, HERD, EEB (TS-796)
401 M Street, S.W.
Washington, D.C. 20460
23
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24
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Appendix C
EPA Request for Assistance (RFA)
Issued Spring 1993
The Aquatic Ecological Criteria Research Program - Gulf Breeze
within the Office of Research and Development of the U.S.
Environmental Protection Agency anticipates funding new RESEARCH
COOPERATIVE AGREEMENTS in FY93 for non-profit and academic
institutions. Research must relate to the development of
ecological assessment methods for estuaries in the Gulf of Mexico
region. Full proposals identifying innovative research ideas and
approaches in the research topic listed above are being sought.
Proposals should include research objectives, significance to
ecological criteria development, and experimental approach. In
addition, a time-frame for accomplishments, a budget (annual and
total), names and qualifications of investigator(s), and a
guality assurance plan should accompany the proposal.
RESEARCH TOPIC OF INTEREST IS: Development and Validation of
Ecosystem Diagnostic Techniques for Gulf of Mexico estuaries and
associated bayous.
t
The research will provide fundamental knowledge of the
ecology of near coastal ecosystems to better enable formulations
of ecological risk assessment. The research will provide tools
to assist the Agency in determining the effectiveness of
management decisions in protecting estuaries. The research will:
1. Develop procedures to measure the current health,
predict future health and to assess recovery of
estuaries. The diagnostic protocol will be field
tested to assess its applicability and predictive
capability.
2. Identify and prioritize .causes of impact on the biotic
communities, e.g., point and non-point toxics.
3. Determine the tolerance limits of major floral and
fauna communities and establish effective biocriteria
to protect the biota.
The research may include synthesis of historical data bases
and assessment of exposure in the water column and sediment.
Bioavailability may be assessed by analysis of contaminant
residues in indigenous biota. Toxicity assessment may include
sediment and water column toxicity tests, biological community
assessments, biochemical, physiological and pathological
evaluations, and productivity measurements.
25
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The anticipated funding period is up to three years.
Multiple projects (2 to 3) may be awarded. The funds available
for the research program are between $500,000-600,000 annually.
This level is subject to availability and resources may limit the
number of research projects funded. Peer review of proposals
will provide a competitive basis for selection. Proposals will
be evaluated on the basis of innovation., approach, relevance to
topics of interest, scientific staff and facilities capabilities,
and cost. Questions of a technical nature should be directed to Dr.
Michael Lewis at 904-934-9382. Application kits and assistance are
available from Ms. Janice Kurtz, 904-934-9286. Four copies of the
proposal should be sent to Ms. Kurtz (U. S. Environmental
Protection Agency, Environmental Research Laboratory, 1 Sabine
Island Drive, Gulf Breeze, Florida 32561-5299). The original
proposal and 3 copies should be sent (postmarked no later than
April 30, 1993) to:
Grants Administration Division
PM-216F
U.S. Environmental Protection Agency
499 S. Capitol Street, SW
Washington, D.C. 20460
26
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Appendix D .
Cooperative Agreement Research Summaries
"Development and Evaluation of Diagnostic Indicators of
Ecological Condition for Gulf of Mexico Estuaries and
Associated Bayous;" Dr. Kenneth Dickson, University of North
Texas, Denton, TX (a consortium including Gulf Coast Research
Laboratory, Ocean Springs, MS - University of Southern
Mississippi; University of Miami, FL; University of Texas at
Dallas; and ABC Laboratories, Columbia, MO).
"Development and Validation of Bioassays for Assessing the
Contamination of Marine Estuarine Environments;" Dr. Stephen
Safe, Texas A&M Research Foundation, College Station, TX.
"Validation of the Mummichog Fundulus heteroclitus as a
Histopathological Indicator of Pollution Along the Eastern
United States;" Dr. Wolfgang Vogelbein, Virginia Institute of
Marine Science, Gloucester Point, VA - College of William and
Mary.
27
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28
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DEVELOPMENT AND EVALUATION OF
DIAGNOSTIC INDICATORS OF
ECOLOGICAL CONDITIONS FOR
GULF OF MEXICO
ESTUARIES AND ASSOCIATED BAYOUS
A proposal for a Cooperative Research Program
Submitted to:
Aquatic Ecological Criteria Program
U.S. Environmental Protection Agency
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida
' by
Kenneth L. Dickson
Miguel Acevedo
' William Waller
James Kennedy
Institute of Applied Sciences
University of North Texas
Denton, Texas
in collaboration with
William Walker
David Burke
Gulf Coast Research Laboratory
University of Southern Mississippi
Ocean Springs, Mississippi
with consultants
Mark Harwell
RSMAS, University of Miami
Larry Ammann
University of Texas at Dallas, Richardson, Texas
Clarence Reed
ABC Laboratories, Columbus, Missouri
April 30, 1992
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OBJECTIVES
General:
Development and evaluation of diagnostic indicators capable of
estimating the ecological health conditions of Gulf of Mexico
estuaries and associated bayous and of establishing associations
between stressors and ecological responses.
Specific:
Analysis of existing field data, especially data sets collected
using EMAP methodology, for one selected estuary in the Gulf of
Mexico.
Collection of further field data using EMAP methodology
supplemented with additional diagnostic indicators in the
selected estuary in the Gulf of Mexico.
Development of a set of habitat, exposure, stressor and response
indicators to detect ecological conditions as well as to
diagnosis likely causes of these conditions.
Assure consistency of the set of indicators across different
spatial scales and different levels of biological organization.
Organize existing data on tolerance limit of major estuarine
biological organisms in the Gulf Coast populations and
communities, in order to be able to relate biological effects to
stressors.
Development of methodology for testing or evaluating indicator
performance by means of statistical analysis, using output from
process oriented simulation models and data from field
measurements.
SIGNIFICANCE TO ECOLOGICAL CRITERIA DEVELOPMENT
This project will address the issue of ecological indicators,
emphasizing those aspects related to diagnostic capabilities,
i.e. ecological conditions as well as the stressors causing that
condition. This work will contribute to the EMAP efforts by
addressing the issues of: simplification of the set of indicators
for devising cost effective strategy, maintaining consistency of
the indicators across different spatial scales and different
levels of biological organization, and of evaluating indicator
performance by means of statistical analysis. Application of the
indicators to synthetic process-oriented simulation model output
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as well as field data will contribute to a better understanding
of the response of indicators to spatial-temporal changes in the
stressors.
SUMMARY OF EXPERIMENTAL APPROACH
The work plan to achieve the above mentioned objectives is
divided into several phases;
1) select an initial group of indicators to include in a protocol
for diagnosing ecosystem health,
2) select an estuarine system in the Gulf Coast meeting adequate
criteria (existence of historical field data, EMAP-E data, and
pollution gradients),
3) evaluate indicator (from set derived in phase 1) performance
in diagnosing conditions of the selected estuarine ecosystem
(from phase 2) based on historical data and output from process
oriented models, by using statistical analysis.
4) perform data collection in the field for testing the proposed
diagnostic protocol in the selected estuary,
5) apply the indicators to the field data (from phase 4) and use
statistical analyses to assess the diagnostic capability of the
indicators,
6) comparative analysis of indicator performance from tests using
field data (from phase 5), historical data and model output (from
phase 3) .
Details on activities in these phases are given in the following
sections.
SELECTION OF INDICATORS
Key to the success of this effort will be the selection of
appropriate ecological indicators for gauging ecosystem health
and inferring causal relationships. Specific problems addressed
by the Near Coastal component of EMAP are low dissolved oxygen,
eutrophication, chemical and biological contamination, habitat
modification, and cumulative impacts. The EMAP sampling program
will provide the basic framework upon which the diagnostic
protocol will be based. A suite of chemical, biological, and
toxicological measures rather than relying on a single type of
response, will most accurately assess ecosystem health.
The main goal of this phase of the project will be to select
relevant indicators for inclusion in the diagnostic protocol. The
indicators should be exposure, stressors and habitat type as well
as response type (Knapp et al, 1990) in order to infer cause and
effect relations. This set should cover different scales of
biological organization from biomarkers to ecosystems, as well as
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different spatial scales, from sites to landscape. The list will
likely include biological markers, and diversity, biotic, and
similarity indices. While the ultimate indicators of ecological
effects are adverse changes in exposed-populations, such
population responses (e.g. occurrence, abundance, reproduction)
do not provide an indication of their cause. Inclusion of
indicators detecting and documenting exposure and environmental
stressors is fundamental (Shugart et.al, 1992).
Cairns and McCormick (1992) compiled-a list of desirable
characteristics of ecological indicators. While each indicator
cannot possess all of the desirable characteristics, reference to
such a list may aid in the selection of ecologically relevant non
redundant indicators. We will utilize these criteria in making
our initial selection of effects and diagnostic indicators. In
this phase we will not limit our evaluation to EMAP-E indicators,
but will consider a broad array of candidate diagnostic
indicators. We will examine EMAP-E results from studies
conducted at Indicator Test Evaluation Stations, as well as
candidate diagnostic indicators evaluated by other programs (i.e.
Virginia Institute of Marine Science's - comparative biomarkers
evaluation project on the Elizabeth River). Two recent reviews
of the use of biomarkers as indicators of exposure and effects
were recently published (Huggett et. al. 1993 and Peakall 1992).
These will be carefully screened to identify additional candidate
diagnostic methods.
ESTUARINE SYSTEM SELECTION
To be useful in validating the indicators, the estuarine system
should be affected by several discrete and identifiable
stressors, with gradients of impact and recovery. The presence of
reference sites is critical. Availability of an historical data
base will be an important factor in site selection. Sites for
which EMAP data are available would have distinct advantages.
Potential sites include Pensacola Bay and Mobile Bay. Personnel
at the Gulf Coast Research Laboratory have experience conducting
field studies in both of these systems and are familiar with the
historical data bases for both systems. We will coordinate with
the EPA Gulf Breeze Laboratory in selecting an appropriate
estuary to conduct the field portion of the proposed project.
FIELD ACTIVITIES
The final location and number of sampling sites for this phase
will be determined after selection of the study estuary and
examination of available data (i.e. final experimental design
will be based on examination of existing data). For scoping
purposes and for budget estimation we have assumed that
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approximately 50 sites (stations) will be studied. Proposed
field sampling approaches are outlined in Appendix A, and follow
guidelines of EMAP Field Operations Manual for the Lousianian
Province (McCauley, 1991). The .goal of--the field sampling program
is not just to duplicate EMAP-E efforts at the Province scale,
but to focus efforts to best analyze~a particular ecosystem, i.e.
Tiers 3 and 4, thereby developing a diagnostic protocol. Some
modifications to the proposed activities may be warranted after
examination of historical data and final selection of the
ecological indicators to be included-in the analysis. Thus it
should be considered as an example not as the final approach to
the field study. The final field study design will depend on the
final selection of indicators (Phase 1} and selection of the
study estuary (Phase 2)
EMAP field sampling activities usually occur from July to
October. This time frame has been selected by EMAP as an effort
to sample during the period when lowest dissolved oxygen values
are expected ("worst-case" scenario). We propose to schedule
field activities during a time period outside the EMAP sampling
window (perhaps early summer or late fall). By coordinating
effort with the ongoing EMAP program, we may be able to utilize
some of the EMAP equipment, thus substantially reducing the cost
of the project. Using the same equipment as EMAP has the second
advantage of rendering our data more comparable to the existing
data. Similar strategy was employed by the research team at the
Gulf Coast Research Laboratory in conducting a demonstration
project in the Back Bay of Biloxi, Mississippi (GCRL, 1993).
EVALUATION OF INDICATORS BY USING HISTORICAL DATA
Analysis of data collected in previous EMAP-E efforts, such as
the set of estuaries used in the Virginian Province Demonstration
Project (EPA, 1992) and the Lousianian Province Demonstration
Project (Summers, et al 1993) , will be developed at an estuary
level for the purposes of testing indicator performance.
Historical data from the selected estuary will also be used for
evaluation of indicator performance. Data along gradients of
pollution will be sought for this purpose.
EVALUATION OF INDICATORS BY MEANS OF SIMULATION MODELS
This part of the proposed research will contribute to the
development of modeling methodologies for the analysis of
indicator performance. We propose to test the indicators by
applying them to the output data resulting from simulation runs
of process models of estuarine systems. This approach has been
suggested in phase four of the EMAP methodology (Knapp et al,
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1990) as one of several potential methods to evaluate indicator
performance, and it will play an important role in Step 3, Tier 3
of the estuarine assessment methodology developed in the Biloxi
workshop (Mayer, et al, 1992) . ' ' ' •
The virtue of this technique hinges on the capability of setting
up a large number of numerical experiments that can be
statistically analyzed to evaluate the performance of the
indicators. Using the simulation models, complete control of the
environmental conditions of the simulated estuary can be achieved
in order to test the sensitivity of the indicator with respect
to gradients of these conditions. This modeling effort is not a
surrogate to the statistical analysis of the field data to be
developed during this project, but will complement and support
the relationships to be uncovered by the statistics.
For this part of the project, the data previously collected in
the field (e.g. from the Back Bay of Biloxy, GCRL, 1992) and to
be collected during the course of the project will be used for
model calibration and validation. Compilation of data from other
sources will also be necessary to complete the required data
base. The models will emphasize temporal and spatial variability
so that the response of the indicators can be evaluated with
respect to the time and space grids of the sampling design (Knapp
et al, 1990).
The variables to be included in the models will be related to the
hydrodynamics, the chemistry and the biology of the system. For
the purpose of testing indicators across spatial scales,
consistent models at different scales will be developed. Two
different spatial scales will be selected: estuary or landscape
and site specific. The estuary or landscape models will emphasize
the transport of pollutants, and the evaluation of stressor and
habitat indicators. The site specific models will primarily deal
with the biological effects, and the evaluation of exposure and
response indicators.
Landscape or estuary model:
At the landscape scale, the estuarine system will be modeled
using grid cells of a relatively large horizontal size (ca 1-10
ha) and a few (three or four) vertical layers. The exact
horizontal and vertical resolution will be defined on the basis
of the extent of the selected estuary. The hydrodynamics will
include the patterns of horizontal and vertical circulation, and
its relationships to temperature, density and salinity.
Freshwater inflow and tidal action (if significant for the
selected estuary) will be incorporated. Salinity gradients
measurements will be used to calibrate the dispersion parameters.
Water quality relationships will include BOD/DO and a sub-set of
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pollutants selected from the full set measured in the field.
Transport of sediments and pollutants will-be the emphasis of the
models at this scale. Spatial distribution of habitats, flora and
fauna at a very limited taxonomic resolution will constitute the
biological variables at~this scale. Remote sensing and CIS
linkages of the models will provide support to this activity.
Habitat and stressor indicators will be applied and tested at
this scale
Site specific model:
Selected sites within the estuary will be subject to more
intensive modeling. For each selected site, the driving physical
variables such as temperature, salinity, etc. and pollutant
concentrations will be derived from the estuary or landscape
level model, but the chemical and biological processes at the
sub-grid level will be emphasized. Ecosystem, community,
population and organism level interactions will be included at
this scale by using a limited (but finer than the one used at the
landscape scale) taxonomic resolution. Nutrient and energy flows
in trophic chains, various biotic interactions and population
dynamics will be modeled. The kinetics of bio-availability will
be linked to the biotic interactions, and an effort will be made
to incorporate models of the kinetics of bio-accumulation.
Exposure and response indicators will be applied and tested at
this scale. Cause and effect relationships helpful for designing
indicators, can be inferred using the models. Laboratory data
pertaining to the responses to toxicants at the organism-level
will be used to generate population-level model responses.
Simulations of gradual increase of pollutant loading to the model
estuary and the response time of the indicators as applied to the
model output can be used as an estimation of the minimum
detectable trend in response indicators (Knapp et al, 1990).
STATISTICAL ANALYSIS
It is difficult to specify precisely the statistical methods to
be used before a detailed examination of the data sets. This
section describes, however, some principles that will be followed
during this project.
Within the EMAP framework, the sampling design should evaluate
the conditions of the selected estuary on the basis of sampling
only a limited number of sites in the estuary. The tiered
approach to be followed in this project requires the use of
statistical methods taking into account this characteristic.The
final experimental design will reflect measures to maximize
randomness and uniformity in sampling for evaluation of
indicators. Specific statistical evaluations to be utilized will
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be selected during the project. Appropriate statistical analyses
to describe and quantify observed differences will be performed.
Choices relative to which to use in this study will be made
cooperatively with USEPA personnel.
It is likely that the data will not satisfy the standard
distributional assumptions required of classical statistical
techniques, and hence require alternative approaches to the
analysis, such as robust statistics (Hampel et al, 1986; Ammann,
1990). Another necessary feature of the statistics would be the
flexibility to accommodate different experimental designs.
Multivariate statistical methods that incorporate the entire
range of stressful conditions as well as recognized reference
sites will also be used. Statistical analysis of biotic
integrity indicators together with exposure indicators should
provide a practical and objective means to identify ecological
conditions and causative factors. Dependent variables could
include various measures of community structure and function,
such as taxonomic or trophic categories.. Suites of environmental
factors also could be integrated into such models as independent
variables, including natural environmental parameters as well as
measures of anthropogenic impact, such as contaminant levels.
If developed carefully and systematically, output from such
refined statistical methods could be used to evaluate the
consistency of the ecological indicators. The statistics also
must discriminate among multicausal alternatives, including
noncontaminant factors and natural environmental fluctuations.
TIME FRAME
The project will be conducted over a two year period. During the
first year, emphasis will be placed upon indicator and site
selection, historical data analysis and modeling. The first set
of measurements in the field will be collected in the first year.
During the second year, emphasis will be placed upon the
evaluation of indicators by using field data and simulation
output. The field work will continue during the second year for a
second set of measurements.
QUALITY ASSURANCE
To maximize quality assurance in the protocol development, an
Advisory Committee will be formulated to provide guidance, to
review, to amend, and ultimately to approve the study protocol,
and to assess progress during the course of the study. Guidance
and requirements for quality control of measurements in each of
the stations are addressed in appropriate Standard Operating
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Procedures which presently exist for most activities specified in
this proposal. Specific guidance will be provided by the EMAP QA
Project Plan of the Lousianian Province (Heitmuller and Valente,
1992). Those activities not presently addressed will be included
in developing remaining Standard Operating Procedures. A QA plan
is included in Appendix B.
PROJECT ORGANIZATION AND RESEARCH TEAM
This project will be the collaborative effort of several
institutions and researchers, in close cooperation with
scientists of the Gulf Breeze Environmental Research Laboratory.
All institutions are located in states with estuarine systems in
the Gulf Coast: Florida, Mississippi and Texas. The research team
has expertise in all aspects related to field and laboratory work
in Gulf Coast estuaries, advanced capabilities of statistical
analysis, simulation models, and indicator development and
application. The proposed project team have prior experience
working together. Drs. Dickson and Walker have both participated
in a variety of Gulf of Mexico Program Toxics and Pesticide
Committee activities. Drs. Harwell and Acevedo have collaborated
extensively on ecological modeling projects. Drs Dickson,
Waller, Kennedy and Ammann recently collaborated on an EPA
cooperative agreement with the Duluth Environmental Research
Laboratory examining the relationships between ambient toxicity
and community response (Dickson et.al, 1992).
The Institute of Applied Sciences (IAS) of the University of
North Texas will be the organization coordinating the project and
contracting with EPA, Gulf Breeze. The IAS already has experience
in successfully developing projects for EPA, and is currently
examining estuarine data for ecological risk assessment. The
following researchers from the IAS will be part of the research
team: K. Dickson, who will be responsible for the overall
project, T.W. Waller and J. Kennedy, who will be responsible for
biological responses and eco-toxicological aspects related to the
diagnostic capabilities of indicators, and M. F. Acevedo who will
be responsible for the evaluation of performance of indicators
using simulation modeling.
The Gulf Coast Research Laboratory (GCRL) of the University of
Southern Mississippi will be in charge of the field work. This
research unit has the personnel and facilities for this activity:
including vessels and analytical laboratories. GCRL has been
actively sampling estuarine systems in the Gulf Coast, and has
recently performed a demonstration project at the Back Bay (GCRL,
1993) . The following scientists will participate in the research
team: William W. Walker, who will be responsible for the field
work aspects of the project, William D. Burke, William E.
Hawkins, Lisa S. Ortego, responsible for collection, processing
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and quality of field data, and Chet Rakocinsky, who will provide
expertise on the statistical analysis.
Additionally the following scientists will participate as
consultants: Clarence Reed (ABC Labs), who will work on the
application of the indicators to the field .data, M. Harwell
(RSMAS, University of Miami) who will provide advice on indicator
development and evaluation and L. Ammann (University of Texas at
Dallas) who will provide expertise in the statistical analysis.
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Development and Validation
of Bioassays for Assessing the Contamination of
Marine Estuarine Environments
Dr. Stephen Safe
Texas A&M Research Foundation,
College Station, TX.
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TABLE OF CONTENTS
Page
1.0 Project Description... 1
1.1 Introduction '. 1
1.2 Objectives : 2
2.0 Results and/or Benefits „: 5
3.0 Approach, Background and Rationale 6
3.1 Approach 6
3.2 Galveston Bay 7
3.3 Target Organism Selected 9
3.4 Chemical Characterization 1 1
3.5 Diagnostic in vitro Bioassays for Marine Ecological
Assessment 12
3.5.1 Background 12
3.5.2 CYP1AU) Induction Assay 14
3.5.3 Antiestrogenicity Bioassay : 16
3.6 Diagnostic in vivo Bioassays for Marine Ecological Risk
Assessment 17
3.6.1 CYP1A1 Induction Assay 17
3.7 Biliary PAH Metabolites in Fish 18
3.8 PAH-DNA Adduct Formation. 18
3.9 Epidermal Growth Factor Receptor (EGF-R) Binding and
Gene Expression 19
3.10 Timeframe of Accomplishments 20
3.11 Program Responsibilities 20
3.12 Sampling and Methods 21
4.0 General Project Information 21
5.0 Quality Assurance 22
5.1 Organic Extractions and Analyses 22
5.2 Biochemical and Molecular Analyses 24
5.3 Bile 24
6.0 References 24
7.0 Resumes 38
IX
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Stephen A. Safe 39
Susanne J. McDonald •• 41
Terry L. Wade :.: 42
Mahlon C. Kennicutt n 43
Appendix A - Experimental Methods for Biochemical and
Molecular Analyses A-1
Appendix B - Standard Operating Procedures (SOPs) for
Organic Extractions, Instrument Conditions,
and Chemical Analyses...... B-l
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1.0 PROJECT DESCRIPTION
1.1 Introduction
The increasing use of synthetic and naturally occurring chemicals by
society has resulted in widespread chronic contamination of many parts of
the global marine environment. Diverse structural classes of organic and
inorganic chemicals such as the pesticide. DDT, its metabolites and the
polychlorinated biphenyls (PCBs) have been detected in water, sediments.
invertebrates, fish and wildlife species (1-28). Many of the more persistent
halogenated pollutants; i.e.. DDT, PCBs, polychlorinated dibenzo-p-dioxins
(PCDDs) and dibenzofurans (PCDFs) are bioconcentrated as they ascend to
higher trophic levels (9.10,12,21).
Polynuclear aromatic hydrocarbon (PAHs) mixtures are another major
class of organic contaminants in the marine environment. PAHs are typically
derived from spills of crude oil and refined petroleum, runoff, and
combustion sources (29-40). The PAH are susceptible to biodegradation and
their presence in water, sediment, and invertebrate and fish tissues is
usually due to relatively recent petroleum spills and/or chronic exposure to
non-point sources.
Numerous studies have demonstrated that organic contaminants; such
as PCBs and PAHs; are probable etiologic agents in many diseases which can
result in adverse health effects in aquatic fish and wildlife populations (41-
45). Increased incidences of cancer have been observed in fish populations
which are exposed to PAHs (46,50). For example, in a non-migratory teleost
fish (mummichog, Fundulus heteroclitus) which inhabited an area
contaminated with PAH, there was a high incidence of hepatocellular
carcinomas (50). Tanabe and coworkers (41-44) have analyzed for and
assessed the potential adverse effects of halogenated aromatic hydrocarbons
(HAHs) on biota. Their studies conclude that marine species may be highly
vulnerable to the toxicity of these compounds. Recent studies by Tillitt and
coworkers (45) have correlated the hatching success of double-breasted
cormorant eggs with the relative concentrations of the "dioxin-like" PCBs in
the eggs. A "dioxin-specific" bioassay of the egg extracts (induction of
ethoxyresorufin O-deethylase, EROD activity in rat hepatoma H4IIE cells)
was used to assay for PCB's. Historically, hydrocarbon contaminant studies
measured their ambient concentrations in water, sediments and tissues
-------
using highly sensitive analytical methods to quantify these chemicals (1-40).
However, these mixtures are invariably complex and it is difficult to identify
and quantitate individual compounds. Even if the concentration of individual
components of these mixtures are obtained the extrapolation of this data in
terms of potential adverse environmental effects is not possible. The
biological significance of contaminants is difficult to assess due to the
vagaries associated with a wide diversity of exposure routes, differences in
biological availability, and individual and species differences in reactions to
toxicant exposure (Figure 1). Biomarkers and bioassays for environmental
contamination are potentially powerful alternatives for regulatory agencies
that are attempting to evaluate chemical hazards in the enviroment.
Biomarkers are sensitive and can be highly specific indicators that
demonstrate that toxicants have the potential to elicit a response at the
cellular level.
1.2 Objectives
The primary objective of the proposed study is to evaluate and field
validate the efficacy of a series of biomarkers which will assess exposure of
marine biota to organic contaminant in Calves ton Bay, Texas. This study is
designed to provide a measure of the ambient tissue and sediment
concentrations of PAHs and HAHs using a combination of analytical
(chemical) and bioassay procedures (Figure 2). The in uitro bioassay
procedures which will be utilized for sediment and tissue extracts are highly
sensitive to HAHs and PAHs and will serve to quantitate the bioactive
components that may occur in these marine extracts. Several in uiuo
markers of exposure to contaminants will also be determined for Galveston
Bay fish.
This project will (1) describe the organic contaminants to which fish
from Galveston Bay are potentially exposed, (2) determine whether specific
biomarkers of organic exposure have been induced, and (3) determine if
exposure has caused specific physiological damage as indicated by a specific
biomarker. such as modificiation of EGF-receptor binding and gene
expression. Another objective of this study is to compare the relative
sensitivities of biomarkers (EROD activity, P450IA mRNA levels, PAH
metabolites in bile, and DNA adduct formation) currently used in marine
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Effect of Toxicant
Increased mortality
Decrease in gamete or larval
viability
Disruption of physiological
processes
Reduced feeding rate
Growth inhibition
Reduced energy reserves
Reduced fecundity
POPULATION
ORGANISM
TISSUE
Alteration in cell morphology
inhibition of mitosis
neoplastic growth
Lysosomal destabilization and
autolysis
Chromosome damage
Gene mutation
Changes in conformation and
activity of enzymes
CELLULAR
rORGANELLEl-
IMOLECULAFn-
Initial Impact
of Toxicant
Homeostatic &
Adaptive Responses
Increased recruitment
Avoidance behavior
Excretion of toxicant
Change in energy allocation
Sequestration
Differential tissue growth
Hypertrophy
Hyperplasis
Lysosomal autophagy
Proliferation of ER
Sequestration in vesicles
Chromosome repair
ONA repair
Detoxication by MFO's and
metallothioneins
Enzymatic and metabolic
responses
Figure 1. The impact of a pollutant may be dissipated through successive
hierarchical organizational levels as a result of compensatory
mechanisms, this adaptive process, along with variability intrinsic
at all levels, may render such effects undetectable at the higher
levels of organization (from Bayne et al, 1985).
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Contaminant
Levels
(HAHs & PAHs)
Program Design
Mode of Uptake
Exposure Contaminant
r Levels
(HAHs & PAHs)
/^ . X S^ ^
^^ , ^ ^ — -*
k
^ ^ ^
s Extracts
)
Potential Induction Induction
(Liver Extracts) (Liver & Bile)
In Vitro In Vivo
to
(Bile)
II 1 1
CYP1A1 Antiestrogenicity CYP1A1 Biliary
Induction Bioassay Induction Metabolites
Assay I
I Product / Effect I
TCDD Toxic Equivalent
for Risk Assessment
Physiological
Alterations
(Liver)
I Vivo
1 1
Adduct EGF-R
Formation mRNA
1 Product / Effect!
DMA Damage
& Cancerous
Growths
Regulation of
Biochemical
Pathways
— *
Indicators of Current and Future Health
Figure 2. Summary of assays analyses proposed for this study.
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studies to evaluate in situ environmental contamination and compare them
to proposed in vitro and in vivo assays not currently used in marine studies.
It is proposed to collect samples from four sites within Galveston Bay.
The contaminant field will be described by measuring the concentrations of
HAHs and PAHs in sediments and fish livers. The biological effects of the
organic contaminants on fish in Galveston Bay will be evaluated by using in
vitro bioassays and in vivo markers of exposure and effect. The in vivo
biomarkers of exposure that will be measured in fish include CYP1A
(P450IA) induction as determined by hepatic EROD activity, P4501A mRNA
levels and the concentration of PAH metabolites in bile. Other biochemical
changes which will be measured in fish include levels of hepatic PAH-DNA
adduct formation using the [32PJ postlabeling assay (an indicator of PAH-
induced damage) and the levels of epidermal growth factor (EGF-R)
receptor binding and gene expression. Recent studies have confirmed
2,3,7.8-tetrachlorodibenzo-p-dioxin (TCDD) decrease EGF-R binding in both
rodents and fish and therefore the assay indicates HAH and PAH induced
changes. The relative toxicity of contaminants in tissue and sediment
extracts will also be determined using in vitro bioassays which are sensitive
to PAHs and HAHs. Previous studies in this laboratory have demonstrated
that low levels of HAHs and PAHs induced CYP1A1-dependent activity in rat
hepatoma H4IIE cells and elicit antiestrogenic activities in human MCF-7
breast cancer cells. The data generated from the in vitro bioassays can be
utilized to determine TCDD or benzo(alpyrene (BaP, for PAHs) toxic
equivalents of a mixture, which can be calculated by comparing the amount
of the mixture required to cause a specific induction response to the amount
of TCDD or BaP (the toxic reference standards) required to cause the same
response. This approach will, therefore, integrate the activity of all the
contaminants in a mixture and provide an estimate of hazard and risk of
contaminant levels in these extracts.
2.0 RESULTS AND/OR BENEFITS
The use of biomarkers to assess environmental contamination has
become increasingly important to determine the exposure of marine biota to
toxic chemicals and evaluate ecosystem health, both current and future.
However, biomarkers must be carefully evaluated before their
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implementation as routine field measurements. Consequently, the major
benefits of this study are to develop and field test in vitro bioassays, using
fish and sediment extracts, which have been "previously shown to be highly
sensitive bioindicators for HAHs and PAHs and to determine the levels of
organic contaminants in Galveston Bay sediment and fish extracts using the
in vitro bioassays. Additionally, chemical measurements which are
traditionally used to evaluate organic contamination in the marine
environment will also be compared to the results obtained from the
bioassays.
This approach will generate data that not only determines the level of
contaminants, but also determines if these contaminants have/can induce
CYPIA(I) levels/activity in both in vitro and in vivo assays, and the extent of
the associated physiological changes or damage. The products of this
proposed study are assays/analyses which (1) measure the current health of
Galveston Bay, (2) generate calculations of TCDD or BaP toxic equivalents,
which translate into a risk assessment that can be used for standard settings
of marine contaminant levels, and (3) measure specific physiological
alterations in biota which have implications for future health and/or recovery
of an ecosystem.
3.0 APPROACH. BACKGROUND AND RATIONALE
31. Approach
A series of biomarkers senstitive to PAHs and HAHs will be evaluated
to determine which are the most applicable, sensitive, and offer the greatest
potential for determining the health as well as future implications for biota
in Galveston Bay. The selection of Galveston Bay is based on several factors.
Galveston Bay is a highly industrialized estuary that supports a multi-million
dollar commercial and recreational fishery. Although, Galveston Bay is still a
relatively productive estuary it receives a wide range of environmental
contaminants, including PAHs from the petrochemical industry, petroleum
refining activities, agricultural runoff, and municipal waste water that could
have serious impacts on the fisheries and health of the Bay.
It is proposed to collect fish and sediments from Galveston Bay at four
sites twice a year. HAH and PAH concentraions will be measured for
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sediment and fish liver samples to define the environmental level of
contamination. The sites which will be selected are those that have been or
are being currently sampled in other ongoing studies which are investigating
contaminant levels in Galveston Bay (i.e., NOAA's National Status and Trends
Mussel Watch Program, EPA's Environmental Monitoring and Assessment
Program-Near Coastal, EPA's National Estuary Program, etc.). In addition,
the selected sites must contain a range of contaminant levels (from relatively
uncontaminanted to highly contaminated based on historical data) and have
sufficient numbers of target fish species. Selecting sites with a historical
and/or continuing data base will provide a temporal trend of contaminant
levels at these sites and also provide additional supporting data for
contaminant measurements made for this study. Sampling twice a year will
provide information regarding short-term variablity.
Three species of fish will be evaluated for this study. It is anticipated
only two of these species will be selected for this study. The species
selected will depend upon their availability at the sites within the Bay.
Although, we hope to collect at least one species at all sites, that may not be
possible throughout the duration of study. Therefore, the selection of two
target species should ensure continuity throughout the entire program.
The biomarkers selected for this study are specific indicators of PAH
and HAH exposure. In vitro bioassays using rodent and human cell lines will
be utilized on sediment and fish liver extracts. Traditional and innovative in
vivo assays will be determined on fish liver and fish bile will be analyzed for
PAH metabolite levels. The proposed assays/analyses range from semi-
quantitative screening methods to new quantitative methods for assessing
hydrocarbon contaminants in the marine environment. This study will
integrate and statistically evaluate the data generated from all of these
biomarkers. The products of this study will therefore provide results that
show which techniques can be best utilized for evaluating the effects of PAH
and HAH contamination in a marine environment.
3.2 Galveston Bay
Galveston Bay, with surface area of 1600 km2, is one of the largest
embayments on the U.S. coast. The Bay is very shallow, averaging only about
2 m in depth, and is isolated from the Gulf of Mexico by the Bolivar
Peninsula and Galveston Island. Tides; which average about 40 cm in height,
-------
exchange ocean and bay water primarily through the channel between these
two land barriers. Winds, rather than tidal cycles, are often the dominant
factor determining bay circulation and water exchange. Specific current
patterns in the Bay and the residence time of water in the Bay are not well
known.
For many years, Galveston Bay has been the recipient of various
pollutant inputs because of an agressivety growing urban and industrial
region. Houston, Deer Park, Bayton, Texas City, and Galveston, surrounding
Galveston Bay to the north and west, are some of the most heavily
industrialized areas in the United States. Hundreds of industrial plants,
including petrochemical complexes and refineries, bordering the Galveston
Bay estuarine system contribute significant amounts of pollutants into the
Bay. Early studies demonstrated that Galveston Bay has suffered ecological
damage related to xenobiotic inputs. Hohn (116) and Chamber and Sparks
(117) reported significant decreases in diatom species diversity and number
of invertebrates and fish in the upper Houston Ship Channel. A change in
species diversity from sciaenids to anchovy was also related to the influx of
pollutants into the Bay (118). The conclusions of these studies were that
the waters of Galveston Bay contained sublethal concentrations of pollutants
which resulted in significant changes in the estuarine community structure.
However, these contentions have never been fully assessed.
Recent studies performed by our laboratory provide an extensive high
quality database on levels of hydrocarbon contaminants that indicates
Galveston Bay provides an excellent setting for the proposed study (119).
The concentrations of PAH and pesticides/PCB have been measured in
oysters and sediments the U.S. Gulf Coast as part of the National Oceanic and
Atmospheric Administration's (NOAA) National Status and Trends "Mussel
Watch" Program. This program was designed to monitor the current status
and long-term trends of selected environmental organic and trace metal
contaminants, e.g., chlorinated pesticides, PCBs and PAHs along the
Atlantic. Pacific and Gulf coasts of the U.S.A. Several overviews of the
concentrations and distributions of PAHs and chlorinated hydrocarbons have
already been reported (120-124).
Higher PAH and pesticide/PCB concentrations were measured in
oysters from the upper portion of the Galveston Bay, e.g., Ship Channel and
Yacht Club, and near the city of Galveston. e.g.. Confederate Reef and Offatts
8
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Bayou. Oyster samples from areas further away from urban centers, e.g.,
Hanna Reef, had average concentrations one to two orders of magnitude
lower. In general, these concentrations are "in good agreement with those
previously encountered during temporal studies in Galveston Bay (125;
Sericano, unpublished data).
Most contaminant concentrations determined for the NS&T Program
are normaly distributed on a logrithmic scale (126). From frequenty
distribution plots the median concentration for the Gulf Coast can be
determined. The concentrations that are plus one sigma or minus one
sigma from the mean can then be termed high or low as compared to the
mean (unpublished data). This type of distribution for total DDTs is
provided in Figure 3. The Galveston Bay NS&T sites are indicated and it is
apparent that they cover the range of the distribution. Therefore a sufficient
contaminant gradient exists within Galveston Bay for the purpose of this
study. In addition, Galveston Bay is representative of all U.S. coastal NS&T
sites, which will make the results of this study applicable to this expanded
geographic area.
3.3 Target Organism Selected
Fish have been proposed as the target organism for this study because
numberous labatory and field studies have shown that levels of the
cytochrome P4501A and associated enzyme activities are increased following
exposure to HAHs and PAHs. The fish cytochrome P450 system is similar to
the mammalian system, although levels/activities are typically lower (60-76).
Fish and other laboratory animals express the Ah receptor (51-58) and
exposure to various HAHs and PAHs result in the induction of CYPlA(l) gene
expression in these species (Table 1). Therefore the induction of the
CYP1A1-dependent EROD and AHH activities has been historically used as
biomarkers of fish exposure to aromatic hydrocarbons (64). In recent years
multiple cytochrome P450 proteins have been identified in several species
of fish. One form purified from fish livers has been identified as a P450
induced by HAHs and PAHs and is apparently a functional analogue of rat
P450IAI (127-129). Although PAHs are Ah receptor agonists and induce
CYP1A, these compounds are also metabolized by the induced P450 to form
metabolites which may be highly toxic or carcinogenic (47-49). Some of
these highly reactive PAH metabolic intermediates are known to alkylate
9
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NS&T Mussel Watch
1986-1988 Mean
Mean = 44 ppb
Md + 1s = 140 ppb
100
DDTs (ppb)
1000
10000
Figure 3. Cumulative frequency distribution of total DDT in bivalves from Galveston Bay and other US
coastal sites. (•-Summary of US Coastal NS&T sites; •- GaJveston Bay
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cellular macromolecules such as protein, RNA and DNA in fish and these
interactions have been associated with hepatotoxicity and the development
of hepatic cancer (47-49). For this reason.'this proposal will incorporate
bioassays which will also assess the metabolism of PAHs and their
interaction with cellular DNA in fish.
Table 1. Selected aromatic and chlorinated hydrocarbons tested as inducers of
microsomal monooxygenase activity, in fish.
Active inducers Inactive as inducers
Benzo[ajpyrene DDT
Dibenzanthracene 2,2'.4,4'-Tetrachlorobiphenyl
Methylcholanthrene 2.2,4,41,5,5'-Hexachlorobiphenyl
3.3',4.4'-Tetrachlorobiphenyl
2,3,7,8-Tetrachlorodibenzodioxin
b-Naphthoflavone
(from 64)
Three species of fish will be examined to determine which is (are)
most appropriate for this study. The species proposed are Micropogonias
undulatus (Atlantic croaker), Arms felis (sea catfish), and Paralichthys
lethostigma (southern flounder) because they are commonly found in
Galveston Bay during the warmer months and are associated with or feed on
the bottom. Additionally, the croaker and flounder are important catches in
commercial and sport fisheries. The Atlantic croaker has been sampled in
Galveston Bay since 1984 for contaminant studies for the National Benthic
Surveillance Project of NOAA's National Status and Trends Program.
Additionally, the sea catfish has been sampled by local regulatory agencies as
part of their monitoring studies. It is proposed to use two fish species in
this study. However, which species or combination of species used in this
study will depend upon availability at the proposed sites. To avoid spawning
activities the best sampling times will most likely be during April-May and
September-October.
3.4 Chemical Characteriziation
Three objectives will be addressed by the chemical characterization
portion of this study. First contaminant concentrations within fish tissues
will be determined to document the level of uptake. In particular fish livers
will be analyzed. Secondly the level of contaminants in the environment will
be documented by measuring analytes in the sediments. Third, extracts of
sediments and fish will be used to estimate potential lexicological
1 1
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contaminants contained in them. These data will provide a framework from
which to describe potential exposure, expressed in body burden of
chemicals, and potential inducible biological 'effects. In conjuction with the
in vivo toxicology measurements a more complete understanding of
exposure will be possible.
As a first step in a contaminant study design, ambient concentrations
of specific analytes are measured. While the ambient concentrations cannot
easily be directly extrapolated to a biological effect it does at least define
what contaminants are present and at what levels. The expected or
predicted impact on the associated flora and fauna is difficult to assess. The
presence of contaminants does not infer effect or impact and various
mitigating factors such as bioavailability and metabolism factors may in fact
ultimately control exposure. However contaminant concentrations are
essential to provide a freamework within which the observed ecological,
physiological, and biochemical varitions can be interpreted.
The analytes of interest will include PAHs and HAHs. including AHH
active congeners. The methods will be identical to those of EPA EMAP-NC
and NOAA Status and Trends Program. Standard operating proceedures are
provided in Appendix B. The quality assurance for chemical contaminant
analyses will be equivalent to the EPA EMAP/NC Program.
3.5 Diagnostic in vitro Bioassays for Marine Ecological Assessment
3.5.1 Background
The most toxic member of the HAH class of environmental
contaminants is TCDD which is formed as an industrial by-product and has
been identified in aquatic ecosystems (11, 14-28). Several studies have
reported that 2.3.7.8-TCDD and related toxic halogenated aromatics elicit a
number of common toxic responses which include body weight loss, thymic
atrophy, impairment of immune responses, hepatotoxicity and porphyria.
chloracne and related dermal lesions, tissue-specific hypo- and hyperplastic
responses, carcinogenesis, teratogenicity and reproductive toxicity (51-56).
One of the hallmarks of exposure to HAHs and PAHs is their induction
of both phase I and phase II drug-metabolizing enzymes in laboratory
animals and mammalian cell cultures (34.35, 48-56. 59-76). 2.3,7.8-TCDD
and related toxic halogenated aromatics induce the cytochrome P4501A1
12
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(CYP1A1) and cytochrome P4501A2 (CYP1A2) hemoproteins and their
associated microsomal monooxygenases, which include aryl hydrocarbon
hydroxylase (AHH) and EROD activities. These latter enzyme activities have
been extensively used in fish as biomarkers of exposure to the TCDD-like
HAHs and PAHs. In addition, several PCB congeners induce other forms of
cytochrome P450 (in the rat) and these include cytochromes P4502B1
(cytochrome P450b), P4502B2 (cytochrome P450e), P4502A1 (cytochrome
P450a) and P4503A1 (cytochrome P450p) and their associated
monooxygenase enzyme activities. However, these enzyme are not inducible
in fish and cannot be utilized as biomarkers of, exposure to these
compounds. Toxic aromatic and halogenated hydrocarbons also induce the
phase II drug-metabolizing enzymes, glucuronosyl transferases, and
glutathione S-transferases. HAHs also induce many other biochemical
responses and these include the induction of diverse enzymes, the
modulation of hormone receptor binding activities, the alteration of thyroid
hormone and vitamin A levels, and enzymes which metabolize steroid
hormones. Many of these induced responses are potentially useful as
biomarkers of fish exposure to PAH and HAH; however, only minimal
characterization of HAH-induced hormone/hormone receptor responses has
been reported in fish and marine biota.
The mechanism of action of HAHs and PAHs have been extensively
investigated (51-57. 59) and the results of genetic and molecular biology
studies are consistent with the role of the aryl hydrocarbon (Ah) receptor as
the initial cellular target for the HAHs (Figure 4). The Ah receptor has been
identified in diverse species/organs (57) including seven species of teleost
and elasmobranch fish (scup, winter flounder, killifish, rainbow trout, brown
trout and dogfish) (58). The molecular biology of Ah receptor-mediated
effects have primarily been derived from studies on the induction of CYP1A1
gene expression by TCDD and 3-methylcholanthrene (a PAH) (51). Initial
binding of the toxin to the cytosolic Ah receptor is followed by an activation
or transformation step and the subsequent accumulation of occupied nuclear
receptor complexes. These nuclear complexes then interact with specific
DNA sequences or dioxin regulatory elements (DREs) which are located in
the S'-upstream region from the CYP1A1 gene. These interactions lead to
the enhancement of CYP1A1 gene expression. It is assumed that many of
the toxic effects elicited by halogenated and aromatic aryl hydrocarbons are
13
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NUCLEUS
Nuclear Binding Sites
CYTOPLASM
Cytochrome
P-4501A1
mRNA
Cytochrome P-4501A1
Induction (AHH and other
monooxygenases)
Nv"lnduced Proteins"X
Pleiotropic Resoponses
* 2.3.7,8-TCDD and
related PAH isomers
Figure 4. Proposed model for the mechanism of action of 2,3,7.8-TCDD and
related toxic halogenated aromatic hydrocarbons. The initial
formation of a cytosolic receptor complex is followed by an
activation step, the formation of nuclear receptor complexes, and
their interaction with specific nuclear-binding sites. This
interaction contributes to an increase or induction of a 2,3,7,8-
TCDD-inducible gene such as CYP1A1.
also the result of altered receptor-mediated gene expression, however, the
molecular mechanisms of these responses are currently unknown. One of
the hallmarks of the toxicity of HAHs are their remarkable structure-activity
relationships which is determined, in part, by their parallel structure-
dependent affinities for the Ah receptor. This induced activity has been
adapted for determining the in vivo exposure of aquatic and marine biota to
HAHs and PAHs and will be extensively utilized in this proposal.
3.5.2 CYPlAfDInduction Assay
Current analytical techniques to measure PAHs and HAHs are costly
and require sophisticated equipment and trained personnel. Therefore, the
14
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development of bioassays for these compounds would be advantageous since
the bioassay-derived toxicity is an integrated assessment of the potential
biological effects of a complex mixture. Several groups have developed
mechanism-based bioassays for HAHs and PAHs (77-81). Bradlaw and
coworkers (82,83) first reported that HAHs readily induced AHH activity in
rat hepatoma H4IIE cells in culture demonstrated the utility of this assay
system for quantifying toxic halogenated aromatics in diverse matrices,
including fish tissue extracts, PCB/PCDF-contaminated rice oil, diverse food
extracts (including gelatin samples containing pentachlorophenol) and trace
levels of higher chlorinated PCDDs. Research in our laboratory has focused
on validating the utility of the P450 induction bioassay for predicting the
toxicity of individual PAH and HAH congeners and HAH mixtures derived
from diverse sources including fish tissue extracts (59, 85-94)
(59,77.84-92). From these results, the TCDD or toxic equivalents of a
mixture can be calculated by comparing the amount of the mixture required
to cause a specific induction response to the amount of TCDD (the toxic
reference standard) required to cause the same response (e.g. EC50 - half-
maximal induction). Based on the dilutions of the extract, the toxic 2.3.7,8-
tetrachlorobenzo-p-dioxin (TCDD or dioxin) equivalents (TEQs) or
benzo(a]pyrene (BaP) for this extract can be calculated (Figure 5). This
bioassay has been validated by comparing the "bioassay-derived" TEQs for a
mixture to the in vivo toxicity of the same mixture and there was a good
correlation between the results of both assays (92). Moreover, recent
laboratory studies have demonstrated that in vitro the AHH or EROD
induction bioassay can be used for extracts of aquatic samples (45, 95-97)
(45.93-95) and the results have been utilized to implicate HAHs as etiologic
agents in wildlife toxicity (45).
This project proposes to utilize the rat hepatoma H4IIE cells as a
bioassay for HAHs and PAHs and the responses which will be measured
includes a CYP1A1-dependent enzyme activity (EROD). This bioassay is one
of the most sensitive and widely utilized procedures for determining TEQs
and will be used as a quantitative bioassay of extracts from field samples.
15
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100
% Maximal
Response 50
Test Mixture (TM)
frig extract/g sample)
Dioxin or Toxic Equivalents Calculation:
ECsofTMJztCsofTCDOJrX
(Y u,g extract/g sample)
.-. Y \ig extract/g sample =
X (ECso cone, for TCDD or BaP)
Concentration
Figure 5. Calculation of TEQs from in vitro bioassays.
3.5.3 Antiestrogenicitv Bioassav
Recent studies have demonstrated that HAHs and PAHs exhibit
antiestrogenic activity in the female rat and in human breast cancer cell
lines (98,99). In MCF-7 human breast cancer cells, TCDD and related HAHs
inhibit several estrogen-induced responses including cell proliferation and
the secretion of the 52-, 34- and 160-kDa proteins (98-102) and the results
are consistent with an Ah receptor-mediated response. These same
compounds also induce CYP1A1 gene expression; however, this induction
response in MCF-7 cells is less sensitive than that observed in rat hepatoma
H4IIE cells. Recent studies in this laboratory have shown that an
electrophoresis assay coupled with double-staining of the resulting gels with
an ISS ProBlue and silver staining solution can be used for determining the
estrogen-induced secretion of the 52-kDa protein, and the antiestrogenic
effects of TCDD and related HAHs of 2.3,7,8-TCDD (101). The results of
these studies (see Table 2) show that for several classes of HAHs their
antiestrogenic potencies were similar to those observed for other Ah
receptor-mediated responses (102), including the induction of CYP1A1.
The sensitivity of the antiestrogenicity assay was comparable to the CYP1A1
induction bioassay. The results of recent studies showed that for PAHs
(103). the antiestrogenicity assay was more sensitive to these compounds
16
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than CYP1A1 induction. For this reason, the antiestrogenicity bioassay will
also be evaluated for sensitivity and specifically for contaminants extracted
from marine samples.
Table 2. £€50 values for selected PCB, PCDD, and PCDF congeners as inhibitors of l?p-
estradiol-induced secretion of the 52-kDa protein from MCF-7 cellsa.
Congener /
2.3.7.8-TCDD
2.3,4.7.8-PeCDF
2.3,7.8-TCDF
1,2.3.7.8-PeCDD
1.2.3.7.9-PeCDF
1.3.6.8-TDDF
3.3'.4,4'-TCBP
3.3'.4.4'.5-PCBP
3.3'.4.4'.5.5'-HCBP
2.3.3'.4.4',5'-HCBP
2.3.3'.4.4'-PCBP
2.3.4,4',5-PCBP
Approximate £€50
value (nM)
8.6
63
31
69
680
1900
170
30
180
>1000
>1000
>1000
Relative Potency
Antiestrogenicity
1.0
0.14
0.28
0.12
0.02
0.004
0.05
0.29
0.05
<0.009
<0.009
<0.009
Relative Potency
Other Assays
1.0
0.8-0. 1 1
0.43-0.006
0.64-0.007
0.009-0.0008
0.3-0.0006.
0.1-0.0012
<0-001
<0.001
<0.001
aThe values for the relative potencies of these congeners compared to TCDD as Ah receptor
agonists were recently reviewed (Safe. 1990)
3.6 Diagnostic In Vivo Bioassays for Marine Ecological Risk Assessment
3.6.1 CYP1A1 Induction Assay
The uptake and contamination of PAHs and HAHs by fish living in
marine or aquatic environments has been extensively investigated utilizing
the induction of hepatic CYPlA(l) in the exposed animals (50, 60-76).
Numerous studies have correlated the magnitude of the induction response
in fish with the levels of PAH/HAH contamination and, thus, the induction of
CYP1A in fish is a quantitative in vivo bioassay which integrates the effects of
all TCDD-like" contaminants in these species. Both the in vivo and in uitro
induction bioassays provide induction responses which give the total TEQs
associated with a contaminated species or extract, This integrated value
represents a quantitative assessment of levels or exposure to HAHs and
PAHs. Thus, the induction of EROD activity and CYP1A mRNA levels will be
utilized in this study as a measure of in uiuo exposure of fish to pollutants
derived from estuarine environments. Measuring both catalytic enzyme
activity and mRNA levels can be advantageous. It has been documented that
some inducers also inhibit the catalytic activity of P450 induced. Thus
17
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measuring only catalytic activity may not represent the total induction
response whereas induced mRNA levels will be a more consistent indicator
of CYP1A induction.
3.7 Biliary PAH Metabolites in Fish
Statham et al., (130) first proposed assessing hydrocarbon pollution by
analyzing the bile of fish for hydrocarbon metabolites. A number of PAH
metabolites formed in the livers of fish are excreted into bile. The gall
bladder is the storage site of PAH metabolites and represents an important
pathway for excretion (131). A nonradiometric technique employing high
performance liquid chromatography (HPLC/fluorescene. detection) was
developed to estimate biliary PAH metabolites in fish (132,133).
Studies have shown that fish captured in areas with contaminated
sediments have higher biliary concentrations of PAH metabolites than fish
from less contaminated areas (134-136). Our studies on several species of
fish from Galveston Bay. Texas showed that the concentrations of PAH
metabolites in bile was variable between sites and was related to the degree
of hydrocarbon contamination (unpublished data). Studies have also shown
that fish exposed to spilled oil exhibit elevated concentrations of PAH
metabolites in their bile (137-139). Additionally studies have correlated
high biliary concentrations of benzo[a]pyrene equivalent metabolites in fish
with idiopathic liver lesions (132,133), reduced ovarian maturation (140)
and elevated AHH activity (141). Bile metabolites will be analyzed to
demonstrate uptake and induced biochemical reaction to exposure.
This method has been useful as a semi-quantitative indicator of PAH
exposure and complements the more quantitative P4501A1 bioassay and
compound specific PAH-adduct assay which will be used in this study.
3.8 PAH-DNA Adduct Formation
Although PAHs can function as Ah receptor agonists and induce
CYPlA(l) gene expression in fish, these compounds also elicit both toxic
and carcinogenic responses via Ah receptor-independent pathways. The
metabolism of benzo[a]pyrene and other PAHs to both non-toxic and highly
reactive carcinogenic intermediates has been extensively investigated in
diverse species including fish (34.35, 46-49, 102-104). The conversion of
benzo(a|pyrene into the 7,8-epoxy and 7,8-dihydrodiol metabolites followed
18
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by P450-dependent oxidation into the 7,8-diol-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene results in the formation of a highly reactive
alkylating agent which forms covalent DNA adducts which are believed to be
markers of PAH-initiated cancer in target tissue.
The metabolic activation of PAHs into carcinogenic metabolites can be
monitored in exposed fish by measuring DNA adduct formation (102-104)
using the [32P]-postlabeling procedure described by Randerath, Gupta and
coworkers (105,106). This bioassay quantitates all PAH adducts and thus
serves to integrate overall exposure of the fish to those PAHs which are
activated and form DNA adducts with the more carcinogenic PAHs. Thus,
this assay will be evaluated in field collected fish as an in vivo biomarker of
exposure to PAHs and can also serve as an indicator of DNA damage and risk
assessor of future development of cancerous lesions.
3.9 Epidermal Growth Factor Receptor fEGF-R) Binding and Gene
Expression
Recent studies in this laboratory demonstrated that relatively low
doses of TCDD (<0.016 n.mol/kg) inhibited constitutive and estrogen-
induced EGF-R binding in uterine and hepatic membranes and EGF-R mRNA
levels in the uterus and liver of female rats (107). In laboratory studies,
Newsted and Giesy also reported that TCDD caused a dose-response
decrease in EGF-R binding in hepatic plasma membrane in rainbow trout
and structure-activity studies suggested that this effect was Ah receptor
mediated (108). Moreover, it was demonstrated that the TCDD-induced
decrease in EGF-R binding was nearly two-orders of magnitude more
sensitive than the CYP1A1 induction response in rainbow trout. This
proposed study will, therefore, evaluate the EGF-R binding and EGF-R
mRNA levels as in vivo bioassays for determining the impact of PAHs and
HAHs on fish. The results will be compared with the P4501A1 induction
data from the same fish samples and serve to validate the utility of the EGF-
R binding and EGF-R mRNA levels in fish as bioindicators of estuarine
pollution by HAHs and PAHs. Since the EGF-R is involved in the regulation
of diverse biochemical pathways, the validation of this response in marine
fish will provide a physiologically-relevant bioindicator of exposure to
contaminants which may also play a role in contaminant-mediated adverse
effects in these species.
19
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3.10 Timeframe of Accomplishments
Year I
Year II
Task
0-6 6-12 0-6 6-12
Months Months Months Months
Responsibility
Establish Study Sites
Select Target Sites X
Standardize in vitro assays X
for sediments/fish
Standardize in vivo assays X
for fish livers
Sample study sites X
Extract sediment and fish X
samples
Analyze sediment and fish X
livers for PAHs/HAHs
Conduct in vitro assays X
Analyze bile for PAH X
metabolites
Conduct in vivo assays for X
fish livers
Integrate and evaluate data
X
X
X
X
X
X
X
X
X
Safe/McDonald/
Wade/Kennicutt
Safe/McDonald/
Wade/Kennicutt
Safe
Safe/McDonald
X McDonald
X Wade
X Wade/Kennicutt
X Safe
X McDonald
X Safe/McDonald
X Safe/McDonald/
Wade/Kennicutt
3.11 Program Responsibilities
Dr. Stephen Safe will have overall management, supervisory and data
synthesis responsibilities for the project. Dr. Safe will also be responsible
for the development and implementation of in vitro bioassays and the
following in vivo assays using fish livers, CYPIA mRNA levels, DNA adduct
formation and EGF-R binding and gene expression. Dr. Safe will be assisted
by two graduate students. Dr. Susanne McDonald will be reponsible for the
field sampling, biliary metabolite analyses, in uiuo EROD activities in hepatic
fish samples as well as assist Dr. Safe. Dr. McDonald will be assited by a half-
time technician. Dr. Terry Wade will be responsible for the extraction of ail
20
-------
samples for contaminant concentrations as well as in uitro bioassays and the
analysis and interpretation of HAH analytical data. Dr. Wade will be assisted
by a half-time technician. Dr. Mahlon C. Kehnicutt II will be responsible for
the analysis and interpretation of PAH analytical data.
All field and laboratory equipment and/or instrumentation are
currently available at Texas A&M University for completion of this project.
3.12 Sampling and Methods
Sediment, water, and fish samples from Galveston Bay will be
collected twice a year during April-May and September-October. Fish will
be collected by trawling, gill nets, or hook and line. Fish will be processed
immediately upon capture. Livers will be excised and subaliquoted. Liver
aliquots to be used for in vivo assays will be frozen and stored in liquid
nitrogen until analysis. Liver samples used to measure contaminant levels
and in in uitro bioassays will be stored on ice in the field and upon arrival at
the laboratory frozen and stored at -20°C until analysis. Bile will be collected
using vacutainers, stored on ice in the field and upon arrival in the
laboratory stored at -20°C until analysis.
Sediments will be collected using a boxcore. The top 2cm of the
boxcore will be sampled for contaminant analysis. Samples will be stored on
ice in the field and frozen at -20°C until analysis in the laboratory.
Details of analytical methods are in Appendices A and B.
4.0 GENERAL PROJECT INFORMATION
The amounts and composition of HAHs and PAHs wil be measured in
sediments and the livers of two species of fish collected at four sites in
Galveston Bay to assess the levels of these classes of contaminants. These
samples will be extracted using EPA approved methods (see Appendix B).
PAHs will be identified and quantified using gas chromatography/mass
spectrometry. HAHs will be identified and quantified using gas
chromatography/ECD detection. Duplicate extractions, excluding surrogates
and internal standards, will be conducted on all sediment and fish liver
samples for use in the in uitro bioassays.
Fish livers will be further analyzed to assess CYPIA induction. EROD
activity is a measure of the catalytic rate of a CYPIA-associated
21
-------
monooxygenase and determining the levels of CYPLA mRNA evaluates the
induction of CYP1A gene transcription. Measuring 'the levels of DNA adducts
is an in situ biomarker of environmental geno-toxicity since the DNA damage
can be expressed in mutations and disease. Additionally, levels of DNA
adducts in ferrel fish is proported to be representative of cumulative
exposure. The levels of EGF-R binding and gene expression are also in situ
measurements of PAH/HAH contaminant exposure in which physiologically
important growth factor-mediated signal transduction pathway is modulated.
Based on the results of preliminary studies in rainbow trout (108) this assay
should prove to be a highly sensitive indicator of (fish) estuarine exposure to
toxic pollutants. As such this technique is a measure of both exposure and
effect following exposure to genotoxic pollutants.
The levels of compounds fluorescing at naphthalene, phenanthrene
and BaP excititation/emission wavelengths will be estimated using HPLC and
fluorescence detection. The types of PAHs identified in bile can be useful in
describing the type(s) of PAH to which fish are exposed. Anthropogenic-
derived PAHs typically contain high concentrations of BaP, which can be a
bi-product of combustion.
All samples will be collected in triplicate and all asssays/analyses will
be performed in triplicate. Triplicate analyses will facilitate the use of
statistical methods to evaluate the products of this project. This project will
not use human subjects or research animals.
5.0 QUALITY ASSURANCE
5.1 Organic Extractions and Analyses
The quality assurance for this study is two-fold. First the availability of
environmental test cases is especially helpful to attaining project goals. Only
test cases with EPA approved quality assurance work plans or programs of
comparable high quality will be chosen. QA routinely includes replication,
system blanks, standard reference materials, matrix spikes, calibration
checks, surrogates, and internal standards (see Appendix B-QAMP, QAPP.
SOPs).
The Pi's are highly experienced in trace contaminant analysis and are
fully aware of the critical need for rigorous quality control to ensure
program results and success. The principal investigators in this proposal
22
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have had senior positions in a number of major environmental studies
conducted for NOAA, the Minerals Management Service (MMS), U.S. Fish
and Wildlife Service (USFWS), the Environmental Protection Agency (EPA),
the Office of Naval Research (ONR), the National Science Foundation (NSF)
and the Department of Energy (DOE). For example, GERG currently
conducts the following high quality environmental programs that involve
field sampling, design and analyses:
• NOAA's National Status and Trends Mussel Watch Project for the Gulf of
Mexico. We have for the last 7 years conducted the Gulf of Mexico
portion of this program. We have a long-standing record of
intercalibration using the NIST/NOAA methods for a wide array of trace
metal and trace organic analytes.
• EPA's Environmental Monitoring and Assessment Program - Near Coastal
(EMAP-NC) in the Gulf of Mexico and Virginian Province. We are
conducting the sediment analyses for the EMAP-NC program in the Gulf
of Mexico (Louisianan Province) and tissue analyses for the Virginian
Province (EPA's Narragansett Office).
• Exxon Valdez Damage Assessment Studies (NOAA, USFWS and the State
of Alaska). We are conducting most of the analytical hydrocarbon
analyses for the Exxon Valdez Trustee's as part of the oil spill damage
assessment effort. We have also provided Exxon's contractors with
hydrocarbon analyses as part of Exxon's Oil Spill Litigation studies.
• U.S. FWS Contract Laboratory. GERG is one of the U.S. Fish and Wildlife
Service's two and four contract laboratories for trace organic and trace
metal contaminant analyses, respectively.
• EPA's National Estuary Programs (Galveston, Boston and Casco Bays).
GERG has conducted several of EPA's National Estuary Program analytical
and sampling efforts in Galveston Bay, Boston Harbor and Casco Bay.
• NSFs Bahia Paraiso Antarctic Oil Spill Project. GERG was chosen by the
National Science Foundation to provide the hydrocarbon component of
the Bahia Paraiso oil spill rapid response in Antarctic in 1989.
• IOC's International Mussel Watch. GERG has been selected by the IOC to
participate in International Mussel Watch as the only U.S. laboratory.
• Cooperative Environmental Research Consortium for the Wider
Caribbean. GERG has been selected by EPA to participate in this Puerto
Rico based Institute to work with government and industry in the wider
Caribbean region on environmental issues and studies.
23
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All contaminant analyses will be made by methods equivalent to those
used in the above programs. QA will be equivalent to these programs.
5.2 Biochemical and Molecular Analyses
The proposed in vivo and in vitro bioassays are all currently ongoing in
this laboratory. For the induction of EROD activity, CYP1A1 mRNA levels,
EGF-R binding and gene expression and PAH adduct formation, the assays
will be standardized using fish which have been treated witeither TCDD or
BaP at a maximal or near maximal dose. The appropriate subcellular
fractions are frozen and stored at -80°C and will be used in each assay as
standards to ensure that assay procedures vary by less than 10%. The in
vitro induction and antiestrogenicity assays will also be standardized for each
run by determining dose response curves for TCDD and BaP.
5.3 Bile
Blanks, calibration standards, reference bile, and duplicates are
analyzed with each run. Details of quality assurance are provided in the
Standard Operating Procedure (SOP-9009) for the HPLC analysis of bile in
Appendix B.
-------
A PROPOSAL
Submitted to:
The United States Environmental Protection Agency
by
The College of William and Mary
. School of Marine Science
Virginia Institute of Marine Science
Gloucester Point, VA 23062
VALIDATION OF THE MUMMICHOG, FUNDULUS HETEROCLJTUS AS A
HISTOPATHOLOGICAL INDICATOR OF POLLUTION
ALONG THE EASTERN UNITED STATES
Project duration: 2 Years
Wol&^fK.jVoga#ein, PI
Assistant-Research Scientist
(§0^)642-7261,
' Robert J. JWfcgett,
Dept. of Environmental
Science
Craig L. Smith, Co-Pi
Associate Research Scientist
Michael AUnger, Co-Pi
Assistant Research Scientist
fU I'// •^Hrvv^x
. Byrne, Assofc.
of Research'
ne A. Lopez,
Sponsored Pi
David E. Zweraer, Co-Pi
Marine Scientist
-------
INTRODUCTION
Laboratory and field studies indicate that exposure to toxic chemical agents can cause the development
of characteristic pathologic disorders in aquatic organisms. In fishes, lesions can develop in almost every organ
system, however, the liver appears to be the most common target organ. An important function of the
vertebrate liver is the metabolic transformation of endogenous and exogenous chemicals, often resulting in the
formation of reactive and potentially cytotoxic, mutagenic, or carcinogenic metabolites. The liver is therefore a
prime target for the damaging effects of many xenobiotic chemical contaminants. For example, a spectrum of
liver lesions including cancer develops in .both laboratory rodents and fishes following exposure to potent
hepatotoxic and hepatocarcinogenic agents (eg. Frith and Ward, 1980; Maronpot et al., 1986; Hawkins et al.,
1985; Hendricks et al., 1985; Couch & Courtney, 1987). Similarly, fishes from waterways adjacent to urban
areas exhibit a spectrum of pathological conditions, including liver neoplasms, associated with exposure to
xenobiotic chemical contaminants (eg. Smith et al., 1979; Murchelano & Wolke. 1985, 1991; Malins et al..
1988; Vogelbeinet al., 1990; Myers et al.. 1993).
The chemical compounds most frequently implicated as causative agents of liver neoplasia in feral
fishes are the polycyclic aromatic hydrocarbons (PAH). The U.S. EPA has identified 16 unsubstituted PAH as
priority pollutants. Eight of these chemicals, benz[a]anthracene, chrysene. benzo[b]fluoranthene,
benzo[k]nuorantheae. benzo[a]pyrene, indeno[l,2,3,-cd]pyrene, dibenz(a.h]anthracene, and benzo[ghi]perylene,
exhibit possible or probable carcinogenic activity in rodents (Menzie et al., 1992). Although significant
concentrations of these eight compounds are often present in polluted aquatic environments, only benzo[a]pyrene
has been examined for its carcinogenic activity in fishes (Hendricks et al., 1985; Hawkins et al., 1989).
Further evidence of PAH carcinogenesis in fishes is provided by field studies (Malins et al., 1984, 1985a,
1985b; Baumann et al., 1989; Vogelbein et al., 1990). English sole, Paroohrvs yemlus from urban areas of
Puget Sound exhibit a spectrum of liver lesions associated with 1) high PAH concentrations in surficial
sediments and stomach contents of fish (Malins et al., 1984; Myers et al., 1987; 1991; 1993), 2) induction of
-------
cytochrome P450 mediated xenobiotic metabolizing enzymes in liver (Collier et al., 1992), and 3) elevation of
PAH metabolites in the bile (Krahn et al.. 1984). Identification of bile metabolites in this species indicates
exposure to aromatic hydrocarbons and verifies that these compounds are bioavailable. Myers et al. (1993)
suggest that because of their mobility, bemhic fishes are more reflective of exposure to chemical contaminants
over a broad geographic range than are measurements of contaminant concentrations in sediments or in the more
sedentary organisms. Thus, bemhic fishes such as the English sole can act as "integrators" of contaminants
from a variety' of sources within specific geographic regions, and are therefore useful as indicators of
environmental contamination. Nevenheless, a need exists for the development of new indicator species.
especially ones having a broad geographic .distribution and a non-migratory life style. These species would
reflect environmental conditions on a local rather than regional level and could be very useful in evaluating the
adverse biological effects, for example, in waters adjacent to specific point source discharges.
Because the effects of exposure to toxic chemical agents are often expressed as characteristic tissue
lesions, histopathologic methods arc gaining acceptance in pollution monitoring programs. The U.S.
Environmental Protection Agency (EPA) has recognized the value of histopathological endpoims as indicators of
pollution (U.S. EPA. 1986) and has selected fish liver histopathology as one of several indicators of biological
impacts for selected marine dischargers holding 301(h)-modified NPDES permits (Becker and Grieb, 1987).
Both the EPA's Environmental Management and Assessment Program (EMAP) (Foumie, pers. commun.) and
NOAA's National Bemhic Surveillance Project (Varanasi et al., 1989; McCain et al., 1989; Myers et al.. 1993)
currently use fish liver histopathology in their assessments of environmental pollution in coastal habitats.
Histopathologic evaluations of contaminant effects offer several advantages to agencies responsible for
implementing pollution monitoring programs: 1) tissue processing procedures are routine, quick, and relatively
inexpensive, 2) effects in multiple target organs are easily and rapidly evaluated. 3) acute as well as chronic
effects can be distinguished. 4) mechanisms of action can sometimes be inferred, and S) correlations with
potential higher order effects (ie. at the organismal & population levels) are sometimes possible.
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PRELIMINARY DATA
We recently reported high prevalences of hepatic neoplasms in mummichog (Funduliis heteroclitus)
inhabiting a creosote- contaminated site in the Elizabeth River, Virginia (Vogelbein et al., 1990). In addition to
the liver neoplasms, these fish also exhibited a broad spectrum of preneoplastic and non-neoplastic liver lesions,
as well as elevated prevalences of extrahepatic neoplasms including rumors of the exocrine pancreas, bile ducts,
vascular system, kidney, and lymphoid tissues. To date, we have seen neoplastic changes in five different
organ systems and eight specific cell types in mummichog inhabiting this particular site (our unpublished data).
For the past 2 years we have been funded by the U.S. EPA to conduct morphological, field, and
laboratory studies of this new rumor epizootic. To evaluate the mummichog's potential as an indicator of
chemical contaminants in urbanized coastal environments, we conducted a histological survey of fish from eight
estuarine habitats in Virginia. This study has identified a gradient of pathological responses ascribable to
toxicant exposure in this species. Prevalences of hepatic and extra-hepatic prolifcrative lesions arc illustrated
for the eight estuarine habitats in Fig. la (putatively preneoplastic altered hepatic foci), Fig. Ib (hepatic
adenomas (HA), hepatoccllular carcinomas (HCC), total altered foci (AF(T]), total hepatic neoplasms (HN[T]).
and Fig. Ic (biliary prolifcrative lesions, (BP), vascular proliferative lesions (VP), pancreatic neoplasms (PN).
Highest liver lesion prevalences were found in mummichog from the creosote-contaminated site (AW) in the
Elizabeth River. VA, with 86.7 % of the fish exhibiting putatively pre-neoplastic altered hepatocellular foci
(AF), and 50 % having hepatic neoplasms (HN) (Fig. Ib). Lowest lesion prevalences were found in fish from a
relatively uncontaminated reference site (CI) in the lower York River, VA. A single individual from this
locality exhibited an AF, however, no other lesions ascribable to toxicant exposure were observed in this
population. In contrast, mummichog from six moderately contaminated sites, five of them in the Elizabeth
River and one in the York River, exhibited intermediate lesion prevalences (Figs, la, b, c). Fish from two of
these sites (SC & CTF) exhibited only AF (Fig. Ib), whereas fish from 4 localities other than the AW site
(ERW, ASP. CSY. and RF-SB) exhibited both AF and HN (Fig. Ib). Extra-hepatic proliferative lesions
-------
FIGURE 1
1UU
80
60
40
20
0
a
"E'F
DBF
B AF ..---.
0CF
DVF
n
1 1
II
1 1
4^ F :-
tr A n
C BBP g
SVP ^
DPN ^
1
1
1
1 1
. ill
|
\
N
S
v
\
s
s
V
>
N
S
N
S
S
N
S
s
V
V
s
s
\
•a
SC CTF ERW ASP CSV RF-SB AW
o A
i i M
-------
occurred in mummichog from 5 of the 8 localities examined (Fig. Ic). A broad spectrum of degenerative and
regenerative liver lesions also occurs in fish from all stations except the reference site. Morpholoeical studies,
characterizing the non-neoplastic liver lesions are in progress. Recent studies indicate that some of these lesions
may be equally or even more useful than the pre-neoplastic and neoplastic lesions as indicators of toxicant
exposure in feral fishes (Hinton et al., 1992; Myers et al., 1993).
Figure 2a illustrates the positive relationship between total aromatic hydrocarbon concentrations in
sediments from the various localities and liver lesion prevalences in resident mummichog. This relationship is
even more apparent when the high molecular weight PAH benzo(a]pyrehe (BaP) is selected for comparisons
(Fig. 2b). We are not implying that the observed liver lesions are attributed entirely to exposure of fish to BaP
in the sediments. Obviously, these fish are exposed to extremely complex mixtures of chemicals, the
interactions of which are presently not understood. In fact, investigations with rodents indicate that the
contribution of BaP to the carcinogenic potency of emission condensates from gasoline and diesel engines, coal
combustion processes, and sidestrcam cigarette smoke is minor and accounts for only 0.17^ to 4.0^ of the
total carcinogenicity (Grimmer et al.. 1991). However, our data do indicate that the mummichog is responsive
to toxicant exposure and exhibits great potential as an indicator species. Logistic regression analyses are in
progress to identify statistically significant differences in lesion prevalences among the different sites.
To aid in validation of this species as a field indicator and to develop methods that will allow us to
evaluate toxicogenic and carcinogenic activities of hydrophobic organic compounds, we exposed larval
laboratory-reared mummichog to aqueous and paniculate fractions of a potent carcinogenic PAH. 7,12-
dimeihylbenz[a]anthracene (DMBA). We recently examined a 6 mo post-exposure sub-sample from this study
and observed a spectrum of toxicopathic liver lesions in the high doses including degenerative and inflammatory
changes, nuclear arypia, and putatively preneoplastic altered hepatocellular foci. The majority of the fish
comprising this study remain in grow-out and are scheduled to be processed at the end of 1 year (Aug. 1993).
These data suggest that the mummichog is responsive to the toxicogenic and carcinogenic effects of DMBA and
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LU
o
2
LLJ
LLJ
9.
CO
LU
100
80
60
40
20
> 1 oc
..2,500
100
40
20
EJAF(T)
EDHN(T)
• AH(T)
C!
SC CTF ERW ASP CSY RF-SB AW
SAF(T)
QHN(T)
• BAP(T)
CI
SC CTF
ASP CSY RF-SB AW
SAMPLING SITES
FIGURE 2
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may therefore be an effective new model for laboratory studies of chemical hepatocarcinogenesis, especially
evaluations of environmentally relevant compounds such as PAH.
RATIONALE FOR CHOICE OF EXPERIMENTAL ANIMAL
Several attributes suggest that the mummichog may be an effective indicator of chemical pollution in
estuarme environments of the Eastern Seaboard of the U.S.. This small fish has a wide geographic distribution.
inhabiting coastal marshes from Nova Scotia to Florida (Bigelow and Schroeder, 1953). It is one of the most
abundant intenidal marsh fishes along the-east coast of the U.S: and thus, relatively easy and inexpensive to
collect. A very important attribute is that local sub-populations are non-migratory, exhibiting a summer home
range on the order of 30-40 m (Lotrich. 1975) and greatly restricted winter movements (Fritz et a]., 1975).
This, coupled with the demonstrated responsiveness to chemical toxicants (Vogelbein et al., 1990, our
unpublished data), suggests that native mummichog integrate a broad range of environmental insults and can
thus be used to evaluate potential adverse biological effects in waters in close proximity to point source
discharges. The mummichog is extremely hardy and will tolerate broad fluctuations in environmental variables
such as temperature, salinity, dissolved oxygen, etc. This has created the widely held view that this fish is not
effective for environmental monitoring and toxicity testing. THIS VIEW IS NOT SUPPORTED BY OUR
RECENT FINDINGS. We suggest that hardiness and sensitivity to chemical toxicants are unrelated. In fact,
several closely related cyprinodontid fishes have been recommended as 'sensitive* carcinogen assay organisms
(Counney and Couch, 1985; Koenig and Chasar, 1985). The hardy nature of this fish is, in our opinion, a
positive attribute in the context of pollution monitoring. Because of its adaptability and hardiness, we would
expect to find mummichog in even the most heavily contaminated environments (as already demonstrated for the
creosote-contaminated AW site). Less hardy, more "sensitive* species would probably not survive long-term
exposures and might disappear entirely from many chemically contaminated habitats.
The mummichog is currently the only known species exhibiting neoplasia in polluted environments that
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also is amenable to laboratory culture and experimental manipulation. It is therefore the best available model
system to verify the putative cause-effect relationship between contaminant exposure and the development of
liver pathology in feral fishes.
OBJECTIVES
Based on results of our previous field and laboratory studies, we hypothesize that the mummichoa.
Fundulus heieroclirus is an effective histopaihological indicator of chemical contamination in coastal embavmems
along the Eastern Seaboard of the U.S. f urther, we hypothesize that the high prevalences of idiopathic liver
lesions occurring in populations from industrialized environments are caused by chronic exposure of the fish to
xenobiotic chemical contaminants, in particular a suite of high molecular weight polycyclic aromatic
hydrocarbons, including those identified by EPA as priority pollutants. The objective of the proposed study is
to conduct field and laboratory studies that will test these hypotheses and validate this small estuarine teleost as
an effective field sentinel and laboratory' model.
EXPERIMENTAL DESIGN
FIELD STUDIES
We propose a liver histopathological analysis of mummichog from ten different localities on (he Eastern
Seaboard of the United States including areas in the Hudson River (i.e. New York Harbor. Arthur Kill, and
Passaic River), Delaware Bay, Chesapeake Bay, and the Savannah River in Georgia. Specifics of site selection
will follow discussion with EPA scientists and will reflect available knowledge of chemical contamination in
these waterways, however, we would like to include several EMAP sampling stations in our design. Each of
the broad geographical regions that we plan to sample will also include at least one relatively uncontaminated
/•
reference site. Our general goal is to select habitats that are contaminated with major classes of hydrophobic
chemicals originating from different industrial processes. Because sediments are known to be efficient
integrators of hydrophobic contaminants, we further propose to correlate results of the sediment chemical
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analyses with liver histopathologic responses in mummichog native to these localities. Analyses will identify
specific classes and concentrations of hydrophobic organics such as PAH, PCB, polar hydrocarbons, pesticides
- • _'
and trace metals. In addition, we propose wide spectrum sediment chemical analyses that will flag high
concentrations of unknown compounds. We will attempt to identify these unknowns by mass spectrometry.
LABORATORY STUDIES
We propose to conduct four chronic (1 year duration) sediment and dietary exposures with
mummichog in order to evaluate toxicogenic and carcinogenic activities of complex mixtures of environmentally
relevant hydrophobic organic chemicals. We are proposing a stepwise series of laboratory exposures to an
increasingly narrow set of chemicals extracted from contaminated sediments in order to evaluate the role of the
higher molecular weight PAH fraction in the etiology of liver pathology. Fish in treatment A (Fig. 3) will be
exposed to a diluted Elizabeth River sediment, and fed a diet amended with a high molecular-weight aromatic
fraction of the Elizabeth River sediment extract. Whole sediment extract is not appropriate for inclusion in the
diet, because of large concentrations of-low molecular-weight PAH such as naphthalene and its alkylated
derivatives which are not as likely to accumulate in food organisms and may also cause acute toxicity at high
dietary concentrations. This treatment will provide exposure via gills, skin, and alimentary tract. Treatment B
will use the same high molecular-weight extract in both sediment and in the diet. Treatment C will consist of
dietary exposure to the high molecular-weight extract alone, and treatment D will serve as a control, with fish
maintained over an uncontaminated reference sediment and fed a unfortified control diet.
METHODS
Fish Collection: Because our recent studies in Virginia indicate that liver lesion prevalences are influenced by
age and season, and that highest prevalences occur during the fall and in the largest fish, we will collect the
field samples during September and October 1994. Adult fish (TL >75 mm) will be collected using standard
10
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FIGURE 3. EXPERIMENTAL DESIGN FOR LABORATORY EXPOSURES
FOOD WITH
HIGH M.W.
PAH ADDED
/ FOOD WITH
HIGH M.W.
V PAH ADDED
'!,CONTROL SEDI MEN
' •' '••l'<;{':^^A^f^fiSMM
c
/FOODWITH\
HIGH M.W.
VPAH ADDED/
t-
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baited minnow traps. Sixty fish (30 maJes and 30 females) will be obtained from each of the 10 sampline sites
Fish will be shipped live by overnight mail to VIMS, where they will be necropsied and processed for
histopaihologic evaluation within 5 days of capture.
Animal Necropsv/ Histology Methods: Fish will be anesthetized with tricane methanesulphonate (MS-222)
sexed, measured to the nearest mm (SL and TL), and weighed to the nearest 0.001 g. Visceral organs will be
removed and carefully inspected. The liver will be separated from other viscera, weighed (0.001 g), and
photographed. Gross morphological characteristics of the liver (i.e. color, texture, size, shape, lesions) will be
recorded. Livers will be cut with a razor, blade into 5 slices and placed in Bouin's fluid. Selected liver tissues
and lesions will be sub-sampled and processed by routine methods for electron microscopic analysis (Hayat,
1989) because morphologic studies of mummichog lesions, although not emphasized in this scope of work, will
continue in collaboration with EPA investigator Dr. J.W. Fournie. Otoliths will be removed and stored for
future aging studies. The remaining viscera, head, and section of body containing the kidney will be fi.xed and
archived for future studies. Livers will be processed by routine methods for paraffin histology (Luna, 1968).
Briefly, after 48 h of fixation, tissues will be rinsed to remove all traces of fixative, dehydrated, and embedded
in paraffin. Sections 5^m in thickness will be cut on a rotary microtome, adhered to glass slides, and stained
with hematoxylin and eosin. All five liver slices will be embedded in the same block so that five different
levels of the liver can be evaluated simultaneously. Diagnoses will be based on lesion nomenclature! systems
developed in our prior EPA study (CR 818165-01-0).
Laboratory Studies: Chronic exposures of mummichog will require a series of range-finding tests to determine
levels of sediment and dietary contaminants that will preclude acute morbidity and mortality. These tests are
planned for summer 1993. To further reduce occurrence of potential acute toxicity during the definitive study.
we propose to use 10 day old F, larvae of fish obtained at the Atlantic Wood site in Virginia's Elizabeth River.
These fish appear to be resistant to the acute toxicity of the sediment contaminants (C. Horton and W.
Vogelbein . unpublished results). Continuous flow-through exposures will be conducted in glass aquaria on a
12
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wet table supplied with filtered, temperature controlled (25'C) York River water. Our experimental design is
outlined in Fig. 3. We will evaluate several different diets to minimize potential problems with palatability of
food amended with high molecular weight PAH. Fish will be sampled intermittently for histopathologic
evaluation according to the schedule outlined in Fig. 4. This approach will maximize the information available
to us regarding potential etiology and histogenesis of developing toxicopathic liver lesions.
FIGURE 4. SAMPLING STRATEGY FOR LABORATORY EXPOSURES
TIME 0 Iwk ,2wk 4wk 8wk I2wk 6mo I2mo
TAVIV- CT7C
FISH PER
TREATMENT
SAMPLE
TAKEN
Statistical analyses :
.•)(] oal
280 240
20 20
Statistical significance
<« oil
— 2j gaj
70 gal
220 200 180 150 120 60
20 20 20 30 60 60
of differences in lesion prevalences in feral fish fr<
test sites and laboratory fish dosed by different treatments will be evaluated by logistic regression analyses.
This approach permits examination of the influence of multiple risk factors on the probability of disease
occurrence. It has been applied to retrospective epidemiological studies (Breslow and Day, 1980) and has
recently also been used to examine the epizootiology of liver and kidney lesions occurring in feral English sole,
flathead sole, starry flounder, hornyhead turbot, white croaker, and black croaker in chemically contaminated
west coast sites (eg. Myers et al.t 1993).
Field Collection and Preparation of Chemistry Samples: A single sampling effort at each of selected field sites
will be used to collect surface sediment grab samples. Probable sites will be selected, though considerable
latitude will be exercised during the actual collection, so that unusually sandy or otherwise anomalous samples
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may be avoided. Sediment samples will be taken with a pre-cleaned Ponar grab. The grab sampler will be
washed with copious amounts of water and pesticide grade methaflal between stations to prevent contamination
of the samples. The upper 2 cm of the intact grab will be removed from five grabs at each station location and
mixed in a solvent rinsed stainless steel bucket to produce a composite sample. This composite sample will be
homogenized and placed in three precleaned quart glass jars with teflon lined lids, one for organics analysis, the
second for metals. TOC, and grain size, and a third for duplication in case of sample loss. Sample storage jars
will be washed, acid rinsed, baked and solvent rinsed prior to use. Samples will be stored on ice in the field
and immediately frozen when returned to the laboratory. One site sample will be collected in sufficient amount
to permit sub-sampling, and divided into three well-mixed replicates, each of which will be separately analyzed
to enable the estimation of variance due to analysis.
Chemical Analvsis-Orsanics: Frozen sediment samples will be thawed, placed in clean stainless steel trays
and lyophilized in a freeze-dryer fitted with a nitrogen bleed valve to prevent contaminant backstreamine during
vacuum operation and venting. All labwarc which contacts the samples will be scrupulously pre-cleaned and
baked. Weighed dry samples of about 100 g will be spiked with internal reference standards and
Soxhlet-extracted with methylene chloride for 48 hours. The extract will be concentrated by rotary vacuum
evaporation and cleaned up by gel permeation chromatography to remove large biogenic molecules. The
resulting aliquot will be separated by polarity into aliphatic, aromatic, and polar fractions by micro-liquid
chromatography on silica gel by gradient elution with hexane, methylene chloride, and methanol. The efficacy
of the aliphatic/aromatic hydrocarbon separation is routinely monitored with standards and flow parameters
adjusted as required. These separation techniques will also be used to produce fractionated extracts from
sediments collected at the Atlantic Wood site in the Elizabeth River, Virginia. Composition of these extracts
will be verified by gas chromatographic analysis prior to use in laboratory exposure experiments.
Liquid-chromatographic fractions will have the solvent exchanged to hexane-toluene and be
concentrated for gas chromatographic separation with fused silica capillary columns coated with DBS or
14
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equivalent crosslinked silicone polymer stationary phase. Standard analytical conditions use helium carrier gas
at constant pressure, a splitless injection technique, and oven temperature programming at 6° C per min from
75° C to 300° C. Flame lonization Detectors (FID) will be used" to detect hydrocarbons, and Electrolytic
Conductivity Detectors (ELCD) will be used for analysis of halogenated compounds. Detector responses are
digitized and stored for further analysis using a HP3350 Laboratory Data System. Standard integration
algorithms are used for peak integrations, but Relative Retention Index programs are used for peak identification
and quantification using internal standard methods from raw retention time and peak area data. Daily injections
of standard mixtures are monitored to ensure instrument performance and precision. Aromatic fractions will be
further analyzed by electron impact and tiegative chemical- ionization mass spectroscopy to confirm the identity
of suspected aromatics and halogenated species and to attempt the identification of selected unknowns. Routine
sediment parameters will be measured for comparative purposes, including grain size, total organic carbon
(TOC), and moisture content.
Metals Analysis: Aliquots of the sediment homogenaics will be analyzed for chromium, copper, manganese.
zinc, aluminum, arsenic, cadmium, nickel, lead and mercury by atomic absorption spectroscopy. Sediments
will be digested by the same techniques used when analyzing sediments in previous Chesapeake Bay Monitoring
Programs. Each sediment homogenate sample will be analyzed in triplicate and appropriate blanks and a
standard reference sediment (SRM 1646) will be analyzed with the environmental samples. All glassware will
be thoroughly detergent washed and rinsed with deionized water, followed by successive washings with 1:1 HC1
and 1:1 HNO,.. Final rinsings will be performed with semiconductor grade deionized water. All glassware and
other materials used for processing samples will be capped or covered prior to use to prevent contamination
from laboratory paniculates. All sediment teachings and standard solutions will be prepared with 'Trace Metal
Grade" HCI and/or HNO}. Acid blanks will be periodically .checked for contamination. Standard solutions of
each element will be prepared from sequential dilutions of commercially available AAS standards (Fisher) and
stored in polyethylene bottles. Estuarine sediment SRM 1646 from the NIST will be used for reference values
to check recoveries in the leaching procedure. A Perkin-Elmer Model 1100B Atomic Absorption
15
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Spectrophotometer will be used for all determinations via name (Cr, Cu, Mn, and Zn), graphite furnace (Al,
As, Cd, Ni, and Pb), and cold vapor reduction (Hg). Calibration curves will be generated from prepared
standards for every instrument run. Instrument drift will be checked after every 10-15 samples during each run
by measuring the concentration of a standard solution used to prepare the curve. New working curves will be
prepared if the standard concentration reported is not within 10% of the expected value.
Sediment samples will be digested in accordance with procedures followed previously when analyzing
samples for EPA sponsored monitoring of the Chesapeake Bay. Each sample will be placed on a large
watchglass and inspected to remove foreign materials (twigs, etc.). Samples will be dried overnight at 102 °C
or until a constant weight is obtained. Dried samples will be thoroughly ground in mortars to a homogeneous
consistency and transferred to glass vials for archival storage.
Samples on the order of 1 g will be transferred to 150 mL polyethylene beakers, after which 1 mL of
dcionizcd water and then 10 mL of aqua regia will be added. The beakers will then be covered with
waichglasscs and refluxed on hot plates for two hours, with two additional 5 mL portions of aqua regia added to
prevent evaporation to dryness. After cooling. 15-20 mL of water will be added, and the samples quantitatively
transferred to 50 mL polyethylene centrifuge tubes for centrifugation. The resulting supernatant solution is then
quantitatively transferred into 100 mL volumetric flasks. The residual sediment will be washed an additional
two times and the resulting wash added to the leachate solution. After dilution, the solutions will be transferred
to polyethylene bottles for storage prior to analysis. Acid blanks and SRM 1646 will be processed in an
identical manner. Each environmental sample will be processed in triplicate.
% Moisture Analysis: Sediment samples will be thoroughly thawed and mixed to provide as homogeneous a
consistency as possible. Samples on the order of 1 g will be placed on acid washed and oven dried watch
glasses and weighed to the nearest O.I mg. Samples will be dried at 110° C overnight (18 hr) and reweighed.
A second weighing after an additional 4 hr drying will be made to insure that all moisture has been removed.
16
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Again, samples will be analyzed in triplicate and the mean concentration and variance reported.
Grain Size Analysis: The technique used for separating sediment .samples into gravel, sand, silt and clay
fractions will follow the procedure of Carver (1971). A 50 g aliquot of each homogenized sediment sample will
be wet sieved to separate the sample into a coarse and fine fraction. The coarse fraction will be further
separated into gravel and sand fractions by dry sieve separation. The fine fraction is then separated into silt and
clay by pipette analysis. Select replicate samples of the. sediment homogenates will be analyzed blind by the
analyst to assure that good precision is maintained throughout the procedure.
Sediment Total Organic Carbon Analysis: Aliquots of the sediment homogenates will be analyzed for total
organic carbon (TOO by a high temperature combustion technique using a Carlo Erba NA1500 analyzer. To
prepare sediment samples for TOC analysis, 10% HCL will be added to a small portion of the dried sample. If
effervescence is observed, another aliquot of the sample will be weighed into a tared dish. Acid will be added
to this sample in small amounts until no effervescence is seen. The sample will then be dried and analyzed for
carbon using the Carlo Erba Analyzer.
OA/OC Procedures:
Sampling Procedures: Drs. Vogelbein and Unger will conduct all environmental sampling for this project to
assure uniform procedures are used at all locations. Sediment samples will be labeled at time of collection, and
remain in the custody of project personnel throughout analysis. Aliquots of environmental sediment samples will
be retained frozen until the project final report is approved by EPA.
Calibration Procedures: The VIMS Analytical Protocol is an internal standards method, relying on addition of
a known amount of surrogate standards to samples prior to extraction. These surrogate standards are prepared
in concentrated form from reputable sources each year, and aliquots are diluted for working standards on a
regular basis. Standard solutions are stored in the dark at -20°C. The VIMS Analytical Protocol also relies on
17
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a relative retention index (AJU) system of seven marker compounds for qualitative identifications. A pre-
weighed known mixture of these compounds are analyzed by GC-FID each day to confirm constancy of
response factors, retention times, and peak shape.
Data Reduction, Validation etc.: Chemical data will be collected using an HP 3350 Lab Automation Svstem.
This system will be used to integrate samples and further process the raw data using custom computer programs
for qualitative and quantitative analysis. Data collected for this project will be backed up and stored on
magnetic tape. Additional hard copies of chemical data will be retained by both W.K. Vogelbein and M.A.
Unger.
Internal QC Checks: In addition to doily analysis of the marker standard mixture, reagent blanks will be
analyzed with each group of samples to ensure that no compounds that are derived from the laboratory
procedure interfere with, or are reported as pan of, the sample. In addition, the NIST reference sediment
#1941 will be analyzed for PCBs and PAHs to verify the accuracy of the methodology. If unsuitable
contamination is discovered in a blank sample, then analysis of environmental samples will stop until the
laboratory contamination is rectified. Routine preventive maintenance is preformed on all analytical equipment
by Institute personnel and logs are maintained for each instrument. Daily analysis of GC standards assures
proper performance of equipment. If any step of the analytical procedure is found to be out of expected limits,
ie. retention times are off, contamination in procedural blanks, low recovery of surrogate standard, then analysis
of environmental samples will be stopped until the problem is rectified. Additional aliquots of all environmental
samples will be kept frozen and available for reanalysis in the event of sample loss during the analytical
procedure.
RESULTS AND BENEFITS EXPECTED
The proposed field studies will provide managers with a cost-effective method, applicable on both a local as
18
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well as a regional level, thai can be used to link adverse health effects in.a native fish species with sediment
contamination. It can also be used to identify specific localities wiihin shallow-water, near shore habitats that
are heavily impacted and that may require remediation. Laboratory exposures will provide direct evidence that
liver pathology observed in feral fishes from chemically contaminated habitats is caused by chronic exposure to
xenobiotic chemical contaminants. They will also provide a detailed understanding of the histogenesis of
toxicant-induced liver lesions in a feral species and important information regarding developmental relationships
between the different types of lesions. These studies will provide critical information required for validation of
this species as a field sentinel and form the methodologic basis for future studies examining potential
synergistic, antagonistic, and passive promotional effects between individual as well as broad classes of
hydrophobic organic contaminants.
RELATIONSHIP OF PROPOSED STUDIES TO OTHER WORK
The proposed scope of work is a logical extension of EPA Contract 818165-01-0 in which we have
characterized the liver and exocrine pancreatic proliferativc lesions occurring in feral mummichog. conducted an
epizootiological survey of mummichog inhabiting chemically contaminated Virginia environments, and elicited
toxicopathic liver lesions in fish exposed to the potent carcinogen DMBA. The proposed studies are critical in
our stepwise approach to validating this species as a field sentinel and laboratory model and are related to
studies that have been conducted with English sole, winter flounder, and Atlantic tomcod. However, unlike to
these species, the mummichog is amenable to lab culture and experimental manipulation. This suggests that
where prior studies have been unsuccessful or only marginally successful in examining cause and effect '
relationships in the laboratory, we have the highest likelihood of being able to demonstrate these relationships.
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ILL NO .8U4-b42-fl«b ' U6C ^U.yd lt>:bU No . UU4 K . 06
ADDENDUM TO EPA PROPOSAL: Validation of the Mummichog, Fundulua
heteroclitua as a Histopathological indicator of Pollution along
the Eastern United States by Vogelbein et al. Virginia Institute
of Marine Science, Gloucester Point, VA -23062.
The Gulf killifish, Fundulus grandis inhabits coastal marshes of
the southern U.S., ranging from Florida into Mexico. It replaces
the mumraichog, Fundulus heteroclitus in northern Florida and ia
very similar to this species with respect to taxonomy, habitat
preference, life history, and physiology. Despite a lack of
specific data regarding its' migratory habits, it is highly
probable that the Gulf killifish, like the muranichog, has a
restricted home range. It therefore may serve as an effective
indicator of chemical contamination in the Gulf of Mexico. In
order to expand the original scope of work to include a
histopathological examination of a shallow-water estuarine
teleost native to the Gulf of Mexico, we recommend the following
modifications to our proposal;
1. That the proposed field study sites in New York (Hudson
River) and Delaware (Delaware Bay) be eliminated and replaced by
a liver hietopalhological evaluation of feral Gulf killifish,
Pundulua crrandie from 4 sites in the northern Gulf of Mexico (3
chemically contaminated sites including i creosote-polluted site
if possible, and 1 uncontaminated site) . Specific sites will be
selected following evaluation of available sediment contaminant
data and discussion with EPA scientists.
2. That experimental exposure studies be modified to include an
evaluation of the Gulf killifish without compromising existing
experimental design or cost. We suggest that:
a. sample size of the original mummichog exposure study be
reduced from 280 to 150 fish/treatment and that only the six
and twelve month sampling times be retained. This will
essentially eliminate the information obtained on the
histogenesis of the developing liver lesions but allow us to
retain the original experimental design.
b. a parallel study using the identical experimental design
be conducted with F. orandis, and that experimental animals
and sediments for this study be obtained, if possible, from
a creosote- or PAH-contaminated Gulf coast environment. For
the original study we have proposed to expose offsping of
AW mummichog because they are resistant to the acute
toxicity of the AW sediments, and therefore more likely to
survive long term sediment exposures. Ideally, we would
want to use a resistant population of Gulf killifish as
well.
c. monies saved by reducing the scope of the field studies
will be allocated to conduct chemical analyses required for
the extended laboratory exposures.
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