Proceedings and Summary of the
Workshop on Finfish as Indicators of
Toxic Contamination
Sponsored by the :
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U.S. Environmental Protection Agency
Office of Marine and Estuarine Protection
July 27-28, 1986
Airlie, Virginia
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CONTENTS
1. Overview of the National Estuary Program and Objectives of the
Workshop on Finfish as Indicators of Toxic
Contamination. . » .3
2. Summary of the Opening Plenary
.6
Session • • •
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3. Summary of Plenary Session on Subgroup Reports. .9
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4. Summary Report from the Subgroup on Anatomic
Pathology. • ..,..,. 21
5. Report from the Subgroup on Bioaccumulation and |
•an
Enzymes • •« « • * • ° •e JU
•
6. Report from the Subgroup on Reproduction/Development,
Physiology, Behavior, and Population.....' , .38
I
7. Summary Report from Subgroup on
Immunology « - ......* 51
Appendices
A. Letter of Invitation
B. Workshop Agenda
C. List of Participants
D. Participant Workgroup Assignments
E. Workshop Background Paper
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1. OVERVIEW OF THE NATIONAL ESTUARY PROGRAM AND
OBJECTIVES OF THE WORKSHOP ON
'FINFISH AS INDICATORS OF TOXIC CONTAMINATION
In- 1985, the U.S. Environmental Protection Agency (EPA) initiated
the National Estuary Program. The program was designed to protect and
restore water quality and living resources in the nation's estuaries.
Under the authority of the Clean Water Act, the program establishes
working partnerships with other Federal agencies, state and local
governments, academic and scientific communities, industries and
businesses, public organizations, and private citizens. The goal of
these working partnerships is to address collectively the environmental
and/or management problems of estuaries. The national program, which
is administered within EPA by the Office of Marine and Estuarine
Protection (OMEP), seeks to
o increase public understanding of the nature of estuaries
and their environmental and management problems5
o provide state and local managers with the best scientific
and technical information available;
o transfer technical and management expertise and practical
experience to state and local governments;
o increase understanding of both the need for; area-wide or
basin-wide planning and its benefits;
o develop plans to control pollution sources and restore
living resources; and !
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o gain acceptance of the public and private costs of
increased pollution controls and estuarine restoration.
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Current estuary programs include the Buzzards Bay (MA), Narragansett
Bay (RI), Long Island " Sound (NY, CT), Puget Sound (WA),
Albemarle/Pamlico Sounds (NC), and San Francisco Bay (CA)-programs.
In each estuary program, the EPA regional office(s) works with
program participants to define the problems of the estuary and to reach
agreements to reduce the causes of point source pollution and the
polluting effects of other human activities contributing to these
problems. Estuary programs may establish goals to maintain currently
existing conditions, to restore a selected level of water quality or
living resources, or to maintain pristine conditions within an estuary.
Population growth and its associated increasing and conflicting demands
for water uses can cause participants of estuary programs to reexamine
and refocus their existing programs and to develop new initiatives that
adequately protect the estuary.
The principal goal of each estuary program is to produce a
comprehensive Master Environmental Plan that describes actions to
control point and non-point sources of pollution? to manage and protect
living resources; to implement sound land use practices; to control
freshwater input and removal; and/or to establish anti-degradation
policies for pristine areas. To be effective, this plan must identify
the parties responsible for these actions, and the revenue sources
necessary to do the job. The plan also must provide for monitoring of
environmental quality in the estuary, periodic program review, program
redirection in response to new problems or information, and a mechanism
to resolve conflicts among participants.
In regional estuary programs, ambient environmental quality should
be monitored primarily to set priorities for pollution control and to
verify the success of management strategies implemented under the
Master Environmental Plan. In addition, the National Estuary Program
needs to determine whether indicators of finfish health can serve as
warnings of toxic contamination, and can thus assist in setting
priorities among estuaries for inclusion in the program. To
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successfully conduct such a trend-monitoring program, indicators of
environmental quality that are both scientifically appropriate and
cost-effective must be identified and/or developed. The Workshop on
Finfish as Indicators of Toxic Contamination will partially fulfill
this need by identifying a set of appropriate indicators of toxic
contamination for assessing potential human health and ecological
concerns.
OMEP sought scientific input to assist in an jevaluation of the
many possible finfish indicators of toxic contamination to determine
which indicators may be appropriate, at present, as estuarine
monitoring tools. Therefore, the purpose of the workshop was to
systematically organize and set a priority ranking of a list of
methods, for use of fish as indicators of the health of estuaries both
emphasizing those most useful to immediate management needs and
identifing those that show the most promise for future development.
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2. SUMHARY OF THE OPENING PLENARY SESSION
Dr. Tudor Davies, Director of EPA's OMEP opened the workshop. He
reviewed the current structure of most EPA regulatory programs, many of
which are media-based (i.e., programs intended to address only specific
environmental media, such as air, land, or water). Permit limitations ,
for toxic pollutants typically are technology-based, with limited
testing of effluent toxicity and modeling of wasteload allocation.
Monitoring, therefore, is typically oriented towards assessing either
effluent pollutant loads or ambient concentrations of pollutants for
which wasteload modeling can be conducted, only recently has the need
for ambient biological monitoring received increased attention in EPA's
surface water programs. The National Estuary Program, in particular,
is seeking to develop and apply a set of scientifically appropriate and
cost-effective indicators of estuarine environmental quality as ambient
monitoring tools. This workshop's goal is to assess the extent to
which finfish indicators can serve this purpose.
Ms. Michelle Hiller, Chief of OMEP's Technical Guidance Branch,
then summarized the goals and objectives of EPA's National Estuary
Program and described how finfish indicators of toxic contamination may
be used within the program. As one potential use, EPA wants to
determine whether such indicators can assist in selecting estuaries to
be included in the national program by serving as warnings of serious
toxic contamination that either threatens or occurs in an estuary. In
addition, indicators are needed within estuaries once the estuaries
have been included in the National Program. They are needed to help
determine where the most severe biological effects are occurring; the
spatial extent of critical impacts; where possible, the specific
pollutant or pollutants most responsible for critical biological
impacts; and the success of toxic pollutant abatement strategies
implemented for an estuary.
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The workshop moderator, Dr. Gary Petrazzuolo, Technical Resources,
Inc., then described the subjects of each working group for the initial
discussion and ranking of indicators. These subgroups, based on the
participants' areas of expertise and interest, were i
o Anatomic Pathology i
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o Immunology
o Bioaccumulation and Enzymes
o Reproduction and Development,
o Physiology, Behavior, and Population. |
Each of the subgroups was to develop a list of indicators, to
consolidate and organize the list by merging closely related methods,
and to characterize each of the indicators. Indicators were
characterized by considering their usefulness for identifying effects
of toxic contamination to fish, the ecosystem, and/or human health,
along with the following set of characteristics:
o biological significance (i.e., there is a widely accepted
cause-and-effect relationship between toxic pollution and
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the indicator, or there are either few or no conflicting
plausible explanations for the observed effect other than
toxic pollution);
o cost-effectiveness;
o availability for widespread use; and
o applicability, including their use as
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early or late indicators (i.e., sensitivity — the
effect will appear following acute or chronic
exposures); !
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- pollutant-specific indicators (i.e., those that can
indicate exposure to a specific pollutant or class of
'pollutants);
species-specific (i.e., those that can be applied only
to a few or one species of fish); and
spatially-restricted indicators (i.e., those that are
only useful on small spatial scales).
The final objective of each subgroup was to rank the identified
indicators. The ranking was to be based only on the first three
criteria (biological significance, cost-effectiveness, and
availability). Information on the various aspects of applicability was
reported, but did not necessarily indicate greater or lesser usefulness
of a given indicator. Ranking was to be accomplished by asking each
subgroup to provide lists based on the four following ranking methods:
(1) identify the best indicator on the list;
(2) identify the best one-third of the indicators from
the list;
(3) rank each of the indicators in order of its
importance,and
(4) rank each indicator as either "good" or "bad."
Dr. Petrazzuolo noted that the technical background paper,
prepared for the workshop by Dr. Margaret McFaden-Carter of the
University of Delaware, provided a starting point for each subgroup's
discussion. This paper (see Appendix E) surveys the available
indicator methods. Dr. Petrazzuolo then opened the plenary session to
questions and asked whether the participants felt any major categories
of indicators had been omitted from the background paper. No such
categories were identified in the opening session. (However,
additional indicators were later identified and evaluated by the four
subgroups).
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3. SUMMARY OF THE PLENARY SESSION ON SUBGROUP REPORTS
Following the subgroup sessions, all workshop participants again
met in a plenary session to summarize and discuss each subgroup's
findings. (See the individual subgroup summaries for a more detailed
description of these discussions.) The most highly ranked indicators
from each subgroup are presented in Table 1. A glossary of the
indicator methods follows in Table 2.
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Workshop participants were asked to choose the best indicators
from those by the subgroups considered. Several participants
questioned whether the group, as a whole, was capable of making such an
evaluation. A lengthy discussion followed in which participants
presented possible scenarios for using various sets or groupings of
toxic pollution indicators. To clarify the technical questions being
asked, an EPA representative described the major stages of each estuary
program's data analysis in detail. The stages are
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o problem definition, j
o characterization, and |
o design and implementation of a monitoring program.
To screen estuaries for incorporation into the National Estuary
Program, a process analogous to the first phase of an individual
program (problem definition) is used. Then individual, regional
programs carry out all three steps to define problems, to develop
management and abatement strategies, and to monitor
recovery. Different indicators or sets of indicators could be used
during each of these phases. Workshop participants agreed to evaluate
the indicators according to their usefulness in each of these phases of
an estuary program.
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TABLE 1. SUMMARY OF SUBGROUP RANKINGS OF FINFISH INDICATORS
SUBGROUP
Anatomic Pathology
'Bioaccuraulation/Enzymes
a. Bioaccuraulation
b. Enzymes
Reproduction and Development,
Physiology, Behavior, and
Population
a. Reproduction/
Development
b. Physiology
RANK OF METHODS
1. Gross changes
2. Ordinary histological methods
3. Ultrastructural histology
1. Residue levels
2. Models
3. Metabolite profiles
1.
2.
3.
Induction (e.g., mixed function
oxygenases and metallothionein)
Inhibition
(Acetylcholinesterase
and gill ATPase)
Blood chemistry (clinical)
1.
2.
3.
1.
2.
Cytogenetics
Larval development and
viability
Embryo viability
Hematology (blood chemistry)
Swimming stamina
c. Behavior
d. Population
Immunology
1. Avoidance/attraction
2. Abnormal behavior
1. Spatial distribution profile
2. Abundance
3. Age structure profile
1. A triad of macrophage
indicators
(phagocytosis,
chemiluminescence,
and killing ability)
2. Experimental measures of
disease
resistance
3. Jerne plaque assay
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TABLE 2. GLOSSARY OF FINFISH INDICATOR METHODS
Anatomic Pathology
1. Gross changes—this category includes obvious abnormalities such
as fin erosion, skeletal deformities, tumors, etc.
2. Ordinary histological methods—observations that can be made
using standard light microscopy techniques are included in this
category.
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3. Ultrastructural histology—these techniques generally| require the
use of scanning or transmission electron microscopy.
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Bioaccumulation
1. Residue levels—this term refers to the analysis of tissues for
contaminant levels of some or all of the 129 priority pollutants,
or other contaminants of local concern.
2. Models—mathematical models can be used to predict the
bioaccumulation potential of a particular compound or group of
substances based on physical, chemical, and structural
information.
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3. Metabolite profiles—many organic compounds are metabolized by
finfish. These metabolites are not included in standard residue
analyses. Because some metabolites bind tightly to tissue
macromalecules, information on contaminant metabolites may be a
better indication of past exposure history than information on
residue levels of the parent compounds.
Enzymes
1. Induction—certain enzymes or proteins may be produced by an
animal as a result of exposure to a xenobiotic. Mixed function
oxygenases (MFOs) are enzymes that oxidize some non-polar
organic compounds to more hydrophilic forms. Metallothioneins
are proteins that bind to certain metals. Specific MFOs or metal
binding proteins may be produced as a result of exposure to a
particular contaminant.
2. Inhibition—particular enzymes or physiological processes may be
inhibited by exposure to contaminants and, therefore, such a
response may indicate a deleterious effect.
3. Blood chemistry—a number of measurements of blood chemistry are
performed routinely in clinical diagnosis, and may be adapted to
diagnosing pollutant stress in fish, They include both the
levels of certain substances (e.g., glucose, cholesterol,
triglycerides, albumin) and enzyme activities (eLg., alkaline
phosphatase, lactate dehydrogenase).
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TABLE 2. GLOSSARY OF F3MFISH INDICATOR METHODS (Continued)
Reproduction/Development , '
1. Cytogenetics—the study of the relationship between chromosomal
aberrations and pathological conditions.
2. Larval development and viability—includes such endpoints as
' growth rates, percent abnormalities and mortality.
3. Embryo viability—for many fish species this is the most
sensitive stage in their life history. The endpoints that are
commonly employed include development rate, type and extent of
developmental abnormalities, hatching success, and mortality.
Physiology
1. Hematology—the study of the blood. This includes the same types
of clinical blood measurements described above.
2. Swimming stamina—this is a measure of the general fitness of a
fish as determined by its ability to swim against a current.
Behavior
1. Avoidance/attraction—changes in these behavioral attributes are
measured by comparing the reactions of fish before and after
exposure to the test material.
2. Abnormal behavior—in addition to avoidance/attraction behavior,
other behavioral changes (e.g., erratic swimming, lethargy)
indicate specific modes of toxicity (e.g., neurological or
metabolic dysfunction).
Population
1. Spatial- distribution profile—analysis of the spatial
distribution of a species of fish within the estuary.
Distribution may be directly related to avoidance/attraction
behavioral changes.
2. Abundance—a relative measure of population demographics.
3. Age structure profile—a skewed age structure profile may be
indicate an unstable population.
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TABLE 2. GLOSSARY OF FINFISH INDICATOR METHODS (Continued)
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Immunology '
1. A triad of macrophage indicators (phagocytosis,
chemiluminescence, and killing ability).
a. phagocytosis—a measure of the ability of macrophage
cells to engulf microorganisms, other cells, or foreign
particles.
b. chemiluminescence—the relative chemiluminescence of
macrophages may correlate with the degree of exposure to
xenobiotics. i
c. killing ability—a measure of the ability of macrophage
cells to kill microbes or tumor cells.
2. Experimental measures of disease resistance—by injecting disease
microorganisms into feral fish, the relative disease resistance
of individuals or populations can be determined.
3. Jerne plaque assay—B-lymphocyte measurement of antibody
production.
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The problem definition phase was divided into an initial
qualitative step (called "problem identification") and a later, more
quantitative process (called "screening"). Problem identification
determines initially whether an estuary should be considered for toxic
contamination screening. Problem identification may be systematic
(i.e., 'part of an on-going data collection) or anecdotal. This initial
step involves minimal new expenditures. The workshop participants
agreed that the following finfish indicators would be useful during
this phase:
o gross behavioral changes,
o anatomical changes,
o population changes, and
o tainting of commercial fish.
During the cross-estuary screening phase, scientists will
determine whether fish are significantly stressed and whether toxics
are a likely cause. The workshop generally agreed that, during this
phase, the following indicators of toxic stress would be most
appropriate, on the basis of their relatively low cost and general
applicability:
o nonpollutant-specific indicators (immunological, i.e.,
blood samples for hematocrit determinations and kidneys
for observation of macrophage phagocytes, chemotoxins, and
assays of estuarine waters with mortality as the
indicator);
o indicators of synthetic organic effects (e.g., cytochrome
P-450 and assays of liver for metabolites);
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o indicators of toxic metal effects (e.g., metallothionein
assays and/or metal residue measurements in liver
tissues);"and
o pesticide analyses (where there is reason to believe
agricultural activities are a source of pollution).
However, a few participants thought that the indicators used in
the screening phase should not be restricted to biochemical types, but
should also include indicators at the organismal and population levels
(e.g., gross pathology, abundance, and distribution). They also agreed
that it would be important during this phase of the program to archive
samples (i.e., brain, gill, liver, kidney, flesh, and spleen) for
subsequent tests or analyses. If it was determined that there may be
toxic contamination, then further histological or residue analyses
would be warranted.
As part of the characterization process for an individual estuary,
a synthesis of historical data should be performed|to further define
problems and to identify data gaps. This synthesis would include data
on pollutant loads, ambient water and sediment conditions, and any data
on biological indicators that may already exist. The indicators
selected for the characterization phase also should be useful for
distinguishing possible causes of the toxic stresses observed in the
estuary whenever possible. It was hoped that this information would
both assist in the process and perhaps help to defend the specific
pollutant abatement actions for an estuary.
^
After discussing potential characterization indicators, the
participants decided to divide characterization into two
determinations: (1) a determination of the nature, severity, and
extent of impacts on fish populations; and (2) ^ determination of
cause-and-effect relationships between specific pollutants or types of
pollutants and major observed impacts. To determine the nature,
severity, and extent of population impacts, the following indicators
were identified:
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o cytogenetics,
o egg and larval development and viability,
o histopathology (within tissue, sublethal effects),
o immunological (triad of macrophage tests), and
o enzymes (e.g., mixed function oxygenase (MFO)
system;(metallothionein).
To show cause-and-effect relationships, the workshop agreed it
would be necessary to conduct: these same tests under controlled
laboratory conditions. The techniques used during the first phase of
characterization could be modified for use in a laboratory to include
ambient water or sediment test phases and pure compounds. However,
workshop participants noted that several highly pollutant-specific
indicators have been developed under laboratory conditions and now are
ready for field testing. Other such indicators will be ready in the
next 1 to 2 years. Using such methods, it might soon be possible to
more effectively trace the causes of some impacts observed in the
field. Participants also suggested that certain ultrastructural
analyses, while expensive, could be used for evaluating
pollutant-specific causes of stress.
Monitoring ecosystem recovery, and the question of suitable
indicators for this purpose were then discussed. Participants agreed
that the indicators previously listed should be used again to compare
recovery on a site-specific basis. For a system-wide, long-term
monitoring program (i.e., to be conducted over a period of 5-20 years),
the workshop was then asked if finfish indicators should have a role
and, if so, what should it be? Participants agreed that the following
indicators would be beneficial for such a monitoring program:
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o population studies, '•
o residues,
! ''
o reproductive success (including embryo viability), and
o histological markers (sublethal effects).
The workshop generally agreed that finfish should be part of a
long-term monitoring program and that finfish indicators would be
useful for monitoring recovery.
Finally, at the plenary session, the participants were asked to
identify finfish indicators relevant to human health. They identified
tissue residues as the only unequivocal finfish indicator of human
health impacts (toxic, mutagenic, and carcinogenic). ! Metabolites were
considered a subset of residues.
Each subgroup was asked to identify the most promising methods, of
those they considered, that are presently available and those that need
further research and development. Availability was considered as (1)
immediate with widespread usage, (2) immediate with only limited
current usage (0-1 year), (3) short-term (1-2 years), and (4) long-term
(3-5 years). A summary of the subgroup conclusions follows:
o Immediately available and in widespread use
- gross pathology
- some histopathological indicators
tissue residues
- egg and larval viability and development
- population techniques
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o Immediate availability but not in widespread use (0-1
year)
- metallothionein
- cytochrome P-450 (and other mixed function oxygenase
system enzymes)
- individual macrophage triad tests
o Short-term availability, with some development and/or
field baseline data needed (1-2 years)
- macrophage triad (phagocytosis, chemiluminescence, and
killing ability)
- adducts (DNA and synthetic organics)
- bioaccuraulation models
- cytogenetics
- hematology
- ultrastruetural pathology
- combination of macrophage, T-cell, B-cell, and disease
resistance methods
o Long-term availability, with methods needing further
research and development (3-5 years)
- blood chemistry (enzymes)
- histochemistry
- development of inbred fish lines to support development
of immunological assays
- in vitro tests of several immunological indicators
PLENARY SESSION CONCLUSIONS
While trying to agree on a set of finfish indicators, workshop
participants made several recommendations and comments concerning the
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use of finfish as indicators of toxic contamination. The workshop
participants agreed that no single test was adequate. Instead, they
considered a suite of. tests necessary to characterize toxic effects.
In addition, the workshop maintained that the suite of indicators
should be field tested simultaneously in fish collected from several
study areas to compare the sensitivity of indicators across major
estuarine categories. Participants also stressed the need to study a
"pristine" reference estuary or set of estuarine areas. The purpose of
these studies would be both to provide baseline data for comparison to
• stressed systems and to determine the normal ranges of the recommended
finfish indicators under field conditions. ;
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Workshop participants also stated that finfish indicators may not
be good screening tools for cross-estuary evaluations. Participants
indicated that more easily interpreted endpoints can be evaluated to
assess whether or not a system is stressed. Also, the use of finfish
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as indicators in trend monitoring programs within! estuaries may be
limited because some key methods have not yet been field verified and
because other, less mobile organisms (e.g., certain! benthic species)
may be better suited as indicators. However, studies using less mobile
species also may be limited inasmuch as sampling heterogeneity can be
more pronounced on a local scale if pollutant distribution is patchy.
Mobile organisms, therefore, may actually be better suited at
integrating toxic contamination burdens for meso- or regional-scale
assessments. Furthermore, EPA representatives noted that there are
regulatory and programmatic needs for resource management agencies to
evaluate indicators of impacts on fish.
The workshop generally agreed that behavioral, enzymatic, and
immunological effects would appear earlier (i.e., following shorter
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toxicant exposure) than histological or gross anatomical changes.
Therefore, these effects would be better suited as early indicators of
toxic contamination. |
The workshop also pointed out that it is currently difficult to
make cross-technique assessments for those methods now used routinely
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and that it would be valuable to provide longer-term research support
to develop methods for use in the field. In addition, more detailed
information should be . obtained on both what techniques are currently
available and which institutions have the capabilities to perform theme
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4. SUMMARY REPORT FROM THE SUBGROUP ON ANATOMIC PATHOLOGY
Although death of fish has historically been used, and currently
is used often as the primary indicator of problems related to toxic
contamination, this subgroup suggested using a series of sublethal
effects occurring in a progression that may end in death to describe
and evaluate indicators of toxics in finfish (Figure 1). This
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progression includes early changes that are usually reversible, late
changes that are usually irreversible, and intermediate changes that
may or may not be reversible. It was suggested that the discussion of
indicator methods identify the stage(s) in this progression where the
methods would be applicable. Approaches to the use of pathological
indicators may be organ-specific, manifestation-specific, or systemic
(or holistic).
For the organ-specific approach, with organ defined as a
morphologically discrete and functionally organized anatomical
component, examples of changes include the following;
o Gross
Externalt discoloration, skeletal deformities, fin
rot, skin ulcerations, hyperemia, eye lesions
(opacities), neoplasia, parasites, edema !
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Internal; edema, (including ascites), organ
displacement, neoplasia, parasite
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o Histological (tissue)
Classes of changes; inflammatory, cellular alteration,
hyperplastic/neoplastic, melanomacrophage aggregation
increase, parasitic ',
o Ultrastructural (subcellular)
Classes of changes; Inclusion bodies, lysosomal,
parasitic, smooth endoplastic reticulum changes
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NORMAL
BIOCHEMICAL CHANGES
ULTRASTRUCT11RAL CHANGES
CELL DEGENERATION
1
CELL NECROSIS
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TISSUE DAMAGE
ORGAN FAILURE
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:c
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SYSTEMIC FAILURE
DEATH
Figure 1. Progression of Indicators of Toxic Stress
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o Histochemical
Enzymatic: technologies to be explored include
differential staining for glutamyl transfesrase, other
enzymes, ion-specific assays, and microprobej;.
Using the manifestation-specific approach, ! the following
manifestations have been associated with the following kinds of toxic
[
contamination and other stresses. This listing is not all-inclusive.
Additionally, in every case the effects of chemical mixtures in synergy
and opposition must be considered and may be indicated by
o Fin erosion—PAHs, PCBs, ammonia, nitrites, water-soluble
hydrocarbons, trauma
o Skin ulceration—PAHs, PCBs, parasites, trauma/injury,
sunlight (UV radiation)
o Eye disease—PAHs, phthalate esters, nutrition, parasites
o Gill disease—ammonia, nitrites, metalo-organics (tin),
organics, PAHs, metals (Cd), parasites
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o Liver neoplasms—PAHs, PCBs, chloramines, nitrosamines,
aflatoxins, metals (Cr, Cd), parasites
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o Skeletal deformities—organochlorines, herbicides:
(trifluralin), insecticides (organophosphates), metals,
nutrition
o Papillomas—viruses (from contamination?)
o Pancreatic diseases—PAHs, nitrosamines, viruses
o Kidney diseases—heavy metals (Hg, Pb, Cd), nutrition,
nitrosamines, parasites (
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o Gastrointestinal tract diseases—PCBs, petroleum
hydrocarbons, nitrosamines, viruses, bacteria, parasites.
The multiple system or holistic approach includes all parts of the
organism and all manifestations. For example, ammonia has been
associated with effects on gills, skin, gastrointestinal
tract, and aggregate macrophage changes. Industrial organics have been
associated with effects on brain and liver.
The indicators of pathology discussed by this subgroup were gross
changes, historical, • ultrastructural, and histochemical effects.
Although all the indicators were considered useful for identifying
toxic contamination in finfish, only gross changes were useful
indicators of toxic contamination at the ecosystem level. The value of
the other indicators at the ecosystem level was uncertain or unknown.
Cause-effect relationships may be implied by associating a syndrome of
effects with likely causative agents. None of the indicators was
considered valuable in assessing effects on human health. The subgroup
discussed and evaluated the methods for assessing these indicators.
Table 3 summarizes the results of this process. A brief summary of the
subgroup's discussion on indicators at all levels of pathology follows.
GROSS CHANGES
As indicators of toxic contamination, gross changes signal a very
disturbed ecosystem. The methods for assessing gross changes are
cost-effective, widely available, and easily taught. It also is of low
sensitivity. In terms of the progression shown in Figure 1, gross
pathology can identify organ failure and system failure, i.e., where
the process is usually irreversible. This methods is not
pollutant-specific, but is specific for space and time. The methods is
ready to be used with many species (i.e., it is not species-specific).
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HISTOLOGICAL EFFECTS
Histological effects may be indicators of minimally disturbed
environments. Histological methods can detect subanatomic
manifestations, at early to intermediate stages in disease progression,
i'.e., in the transition area between reversible and table 3 here
irreversible changes (See Figure 1). These methods can be performed
cost-effectively by high-production laboratories, although the
availability of such laboratories may be limited. Histological methods
also can be used in highly polluted environments, and are specific for
time and space. Certain pathological lesions can be used to identify
classes of pollutants (heavy metals, aromatic hydrocarbons,
polychlorinated compounds, and mixtures of compounds), but these
lesions are not specific for individual pollutants. The methods are
ready to be used with many species.
ULTRASTRUCTURAL EFFECTS
Ultrastructural effects can be detected by electron microscopy, an
expensive method with limited availability. j The meaning of
ultra-structural changes as an indicator of toxic contamination is not
always understood, although they may offer the potential for detecting
problems early. For example, proliferation of hepatocytes tends to
reflect drug or toxicant exposure within hours of that exposure, and
may continue throughout chronic exposure. The detection of
ultrastructural changes may also be used as a confirmatory tool:
detecting changes in mitochondria, a characteristic response to
cyanide, was used to confirm cyanide as the cause of a fish kill in
Ohio.
HISTOCHEMICAL EFFECTS
The meaning of histochemical effects as an indicator is unknown,
and the methods for their detection are expensive and of limited
availability. Histochemical methods may be useful for validating
23 i
-------
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suspected toxic contamination, but they cannot currently be used for
monitoring because not enough is known about the significance of their
manifestations. The subgroup suggested that if correct enzymatic tests
are used for putative foci (e.g., the liver), alterations in metabolism
might be detected that could be associated with the development of
neoplasms. These methods may be applied to the multi-stage theory of
carcinogenesis.
DISCUSSION
I
It was emphasized that a set of tests, rather than any single
test, should be used to identify toxic contamination. Gross anatomical
examinations and histologic tests should be performed in conjunction
with chemical analyses of water and sediment and residue analyses to
determine body burdens of toxics.
Laboratory studies can establish causal relationships, but their
applicability to the field must be established. For example,
trifluralin effects (vertebral injury and hyperostosis), induced by low
concentrations in the laboratory, have been validated with the same
lesions in field-exposed, wild populations of fishes. Similarly,
PCB-induced liver damage in laboratory-exposed fish has been found in
wild, PCB-exposed fish. However, native populations may develop a
.
tolerance to the contamination, which probably would not occur in
laboratory or field testing.
The use of tumors as indicators requires evaluating their
significance in fish populations. Neoplasms in finfish, including
carcinomas, have been associated with contamination by PAHs, PCBs, and
heavy metals. The subgroup suggested that if a fish has a visible
tumor, it may contain a level of carcinogens that can pose potential
health effects for humans ingesting it. Therefore, fish with severe
fin erosion (fin rot), integumental ulcerations, and cataracts should
not be eaten. Further, an incidence of tumors that is significantly
25
-------
above expected levels in wild populations of a particular aquatic
system indicates that toxicants or carcinogens may be a major cause of
stress in the ecosystem.
RECOMMENDED INDICATORS
Based on the evaluation criteria and ranking by the four suggested
methods, the anatomic pathology indicators recommended were
o Gross changes (for highly disturbed ecosystems)
o Histological effects (for minimally disturbed
environments).
RESEARCH NEEDS
The following research needs were identified for improving the
usefulness of anatomic pathology indicators of toxic contamination in
finfish:
o Gross changes
- Conduct field tests to quantify changes across
disturbed environments to help assess significance and
commonality;
- Define impacts of fish sampling techniques on gross
lesions;
- Develop reference works on documented pathology and on
gross and microanatomy of fishes [It was noted that
development of a textbook on gross and microanatomy of
fish (identified as a critical need) is underway].
26
-------
o Histological effects >
- Improve specificity and interpretation in relation to
effects; j
- Train fish histopathologists;
- Standardize vocabulary and interpretation of lesions;
- Establish baseline data on selected species.
'[
o Ultrastructural effects |
- Conduct basic laboratory research to demonstrate
Ultrastructural effects from classes of compounds;
- Conduct research to relate laboratory studies to field
studies.
o Histochemical effects i
- Direct basic research to explore use as a monitoring
tool. !
27
-------
5. SUMMARY REPORT FROM THE SUBGROUP ON BIOACCUMULATION AND ENZYMES
The subgroup discussion began by selecting a spokesperson (Dr.
Jerry Neff, Battelle) and a recorder (Ms. Patricia Fair, NMFS). The
group was then asked to rank the major finfish indicator methods that
measure bioaccumulation/metabolism of toxicants and changes in enzyme
function due to toxic pollution. The initial indicators for each
category are ranked below and are followed by brief summaries of the
discussion for each indicator:
o Bioaccumulation
- Residue Levels
- Metabolite Profiles
- Chemical/Physical Models
- Kinetic Biology-Based Models
- Rapid Mutagenicity Tests (Ames/Lambda Prophage) on
tissues or tissue extracts.
o Enzymes
- Enzyme Induction (Particularly Mixed-Function
Oxygenases
- Enzyme Inhibition
- Blood Chemistry
- Adaptive Stress Responses
BIOACCUMULATION
Residue Concentrations/Metabolite Profiles
The subgroup ranked residue concentrations and metabolite profiles
as the best indicators, according to workshop criteria. These
indicators provide a major link to human health effects. Regarding the
use of residue levels, the subgroup noted that demersal species, which
form localized populations (i.e., have limited migratory ranges), have
historically been most useful for monitoring. The subgroup cautioned,
28
-------
however, that finfish are not always the best sentinel organisms for
many types of pollutants because even demersal species are mobile
relative to benthic invertebrates. Also finfish. metabolize many
classes of. compounds rapidly. It was stressed that residue levels in
finfish should be used in conjunction with other measures of biological
effects to establish causal relationships of particular pollutant
sources to observed field .effects.
The subgroup further noted that for some typies of pollutants,
metabolite profiles are a better indicator of past exposure than
residues. Developing metabolite profiles involves identifying the
concentrations, characteristics, and distribution of pollutant
metabolites, conjugates, and adducts in various tissues, fluids, and
subcellular sites. However, this subgroup also observed that not
enough is known about the pharmacokinetics of pollutants and their
metabolites in fish to firmly establish exposure history. Not all
this was cited as a topic for
methods are currently available!
continued research.
Chemical/Physical Models
The subgroup ranked chemical/physical models as the next most
promising method for assessing the likelihood of. stress due to
bioaccumulation. Models discussed were primarily those used to predict
distribution, environmental behavior, and fate of pollutants in marine
ecosystems. the subgroup cautioned, however, that these models cannot
be used alone. They must be used in conjunction with biological
i
models, and must be calibrated and verified under various environmental
conditions. These models are useful for providing worst-case estimates
of bioaccumulation impacts. However, models must tbe used carefully
because they do not provide the conclusive evidence that residue
monitoring can provide.
29
-------
Kinetic Biology-Based Models
These models were characterized primarily as a means to predict
and assess food chain transfer of pollutants. These models have
potential uses for both predicting ecosystem-wide effects and linking
environmental contamination to analysis of human health risk.
Rapid Hutagenicitv Tests (Ames/Lambda Prophage)
Rapid mutagenicity tests were ranked as the next most useful
indicators of the effects of bioaccumulation and metabolism. The
subgroup stated that it is often difficult to interpret these tests in
an environmental context, but they may provide a link to human health
considerations. These tests still require substantial development
because they must be modified for use with tissue/sediment extracts and
because they may yield an unacceptably high frequency of false
positives or negatives.
Table 4 summarizes the group's evaluations of these indicators
according to EPA's criteria. "Unknown" indicates that the subgroup
could not determine or agree whether the indicator met the criterion.
ENZYMES
Enzyme Induction (Particularly Mixed Function Oxygenases)
The subgroup ranked enzyme induction as the best indicator in this
category. This indicator is highly pollutant-specific for certain
classes of organic and metal pollutants. It is particularly useful for
detecting responses that are initially adaptive, but which may become
maladaptive with toxification/detoxification. These methods are
currently being refined and improved to make them more routine.
Mixed-function oxygenase system induction was singled out as a
subcategory of indicators specific to polycyclic aromatic hydrocarbons
and PCBs. Measurement of specific forms of cytochrome P450 in
30
-------
TABLE 4. BIOACCUMULATION/METABOLISM INDICATORS
Residue Levels/ ' Chemical/Physical
Criterion Metabolite Profiles Biology-Based Models
Rapid Mutagenicity
Tests
Unequivocal
meaning
Cost-
Effectiveness
Availability
Applicability
-early/late
sensitivity
Yes
Moderate
Moderate
Early
Moderate
Yes
Moderate
Early
Yes
Yes
Moderate
Unknown
-pollutant-
specific Yes Yes
Yes
-species-
specific No Can be No
-scope-
specific No No No
(spatial)
31
-------
particular is a very specific and highly sensitive indicator of
exposure to particular pollutants. Measurements of other specific
enzyme activities (e.g., aryl hydrocarbon hydroxylase, benzo[a]pyrene
hydroxylase, etc.) are subject to substantial variability, but are
still valuable indicators.
Enzyme Inhibition
The subgroup ranked this category of indicator next most useful
because many methods are readily available. However, some assays may
need to be adapted specifically for fish tissues. The subgroup also
noted that these indicators have varying degrees of pollutant
specificity, depending on the particular enzyme systems involved.
Also, there are uncertainties about the effects of natural indigenous
and exogenous factors on basal enzyme activity. Thus, the subgroup
urged caution in randomly applying enzyme inhibition; instead, it
should be used as an indication of exposure to specific classes of
chemicals. Also, baseline data are needed to establish normal ranges.
The subgroup mentioned the following examples of enzyme inhibitions
o Acetylcholinesterase
o Gill ATPase
o S-Aminolevulinate dehydratase
o DNA-polymerase
o Glucose-6-phosphate dehydrogenase.
Blood Chemistry
The subgroup ranked blood chemistries as the next most promising
set of indicators. They were mentioned as a valuable clinical
approach. However, the subgroup noted that more baseline data are
needed to further the use of this group of indicators.
32
-------
Adaptive Stress Responses
Adaptive stress responses were noted as an important set of
indicators because their meaning is fairly veil established, and they
hold promise of being cost-effective. However, the sulbgroup also noted
that these responses are non-specific, i.e., any kiind of stress will
elicit the response. Three examples of potentially useful adaptive
response indicators were specifically mentioned—stress protein
responses, adrenocortical responses, and neurochemical responses. The
subgroup felt that these methods warrant more development; infact, some
methods (particularly metallothionein assays that are specific for
metals exposure) are in the field testing stage at present.
j
The subgroup also evaluated enzyme indicators based on the
I
criteria provided by EPA. Table 5 summarizes its findings. Again,
"unknown" indicates that the group could not determine or agree whether
the indicator met the criterion.
RESEARCH AND DEVELOPMENT NEEDS ,
I
The group established the following priorities for the research
and development needs of the discussed indicators.
o Further develop and refine immunologic techniques to
quantify specific forms of cytochrome P450 (2-3 years
development time) to provide a highly specific,
inexpensive and easy-to-apply assay;
o Further evaluate the use of stress proteins, particularly
metallothioneins, and very low molecular weight proteins,
as general- and pollutant-specific indices of pollutant
stress (1-3 years); j
o Evaluate pollutant specificity of different serum enzyme
assays, and develop databases for species of interest;
33
-------
TABLE 5. ENZYME INDICATORS
Criterion
Enzyme
Induction
Enzyme Blood
Inhibition Chemistry
Adaptive
Stress Response
Unequivocal
meaning
Cost-
Effectiveness
Availability
Applicability
-early/late
sensitivity
-pollutant-
specific
-species-
specific
-scope-
specific
(spatial)
Yes
Yes
Moderate
Moderate
Moderate
Early
Yes
No
No
Yes
Yes
Unknown
Yes
No
No
Moderate
Variable
Early
Yes
No
No
No, any
stress will
elicit
response
Yes
Yes
Unknown
No, except
metallothionein
No
No
34
-------
o Develop immunological or spectrofluorometric techniques to
I
quantify pollutant-protein and pollutant-DNA adduct
relationships in blood and tissues°,
o Develop better residue-effects links using long-term,
mechanistic cause/effects studies; ' i
o Modify rapid mutagenicity tests to improve their
applicability to tissue extract assays.
The subgroup developed a number of general points relevant to all
of the indicators discussed. They are as follows:
o In using measurement of residue concentrations as an
indicator, it is vital to the proper interpretation of the
results that it be linked to effects analysis (i.e., used
in conjunction with other indicators);
o No single test is sufficient; a number of different
indicators should be used in any given field study;
o Analysis must address the combined effects of major
pollutants as this is what is found in the field;
o The mechanism of the effect must also be addressed.
Without an understanding of mechanistic links, the
indicators have little or no predictive value.
35
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6. REPORT FROM THE SUBGROUP ON
REPRODUCTION/DEVELOPMENT, PHYSIOLOGY,
BEHAVIOR, AND POPULATION
After selecting a recorder (Dr. Judith Weis, Rutgers University)
and a spokesperson (Dr. Joel O'Connor, NOAA), this subgroup listed
possible finfish indicators of toxic pollution for each of the
subcategories: reproduction and development; physiology; behavior; and
population. Initially, the subgroup was asked to identify all possible
indicators without discussing the worthiness of a particular choice.
This resulted in a long list of indicators that was then consolidated
by merging related and redundant indicators, and then deleting those
considered least useful or outside the scope of the subgroup.
During this discussion, fishery closures (i.e., closure of a
fishery by a regulatory agency due to contamination of the fish by
toxic chemicals) were considered. The subgroup questioned whether
closures should be considered as an indicator at all, and then agreed,
with reservation, to consider them as a separate category.
The subgroup also decided to consider resistance or acclimation to
pollutants when evaluating indicators. The subgroup agreed to consider
and' evaluate pollutant resistance as a separate category. Table 6
presents the final list of indicators considered for evaluation and
ranking for each category.
The subgroup then tried to determine the relevance of each final
indicator to each endpoint (fishes, ecosystem, and human health). The
subgroup found that all of the indicators listed applied to the health
of individual fish and fish populations and that none applied to
ecosystem or human health effects.
After agreeing on a final list of finfish indicators, the group
evaluated each indicator based on EPA criteria. Tables 7-10 summarize
thesse findings. The presence of "unknown" indicates that the subgroup
36
-------
TABLE 6. LIST OF INDICATORS CONSIDERED FOR RANKING BY THE SUBGROUP
ON REPRODUCTION AND DEVELOPMENT/PBYSIOLOGY/BEHAVIOR/POPULATION
o Reproduction and Development
- number of eggs
- embryo viability
- cytogenetics
- larval development and viability
- growth
- fin regeneration
o Physiology
- respiration
- hematology
- endocrinology
- swimming stamina
o Behavior
- schooling
- avoidance/attraction
- reproduction
- predatory behavior
- food habits
- miscellaneous (predator avoidance/orientation/
coordination/refuge-seeking behavior)
I
o Population
- abundance
- age structure profile
- spatial distribution profile
o Fishery Closures
o Pollutant Resistance (acclimation)
37
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TABLE 7. REPRODUCTION AND DEVELOPMENT
Indicator
Criterion
Number Embryo Cytogenetics Larval
of Eggs Viability Development
Growth Fin
Regeneration
Unequivocal
Meaning
Cost-
Effectiveness
Availability
Applicability
-early/late
sensitivity
No
Yes
Yes
No
Yes
Yes
Early Early
Yes
Yes
Yes
Early
No
Yes
Yes
Early
No
Yes
Yes
Early
No
Yes
Yes
Early
-pollutant-
specific
'-species-
specific
-scope-
specific
spatial
temporal
No
No
Unknown
2-6 mo
No
No
Yes
days or
weeks
No
No
Yes
few days
to 6 mo
No
No
Yes
1 week to
2 mo
No
No
Yes3
1 week to
2 mo
No
No
Yes
1 mo to
1 yr
* Needs expertise
^ But not widely
7 .For resident species .
4 Could be a function of the female taking up toxicants and passing them on to the
embryo, in which case the time scale could be 2-6 months.
An extreme case would be up to a year
38
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TABLE 8. PHYSIOLOGY
Indicator
Criterion
Respiration Hematology Endocrinology Swimming
Stamina
Unequivocal
Meaning
Cost-
Effectiveness
Availability
No
Yes
Yes
No3
Yes
Yes
No !
Unknown
l
Unknown
No
Yes
Yes
Applicability
e
-early/late Early^
sensitivity
-Pollutant-
specific No
-Species-
specific No
-Scope-
specific
2
spatial Yes
2
temporal Yes
Early
No
No
Yes'
No
Early
No
No
Yes'
Yes
Late
No
No
Yes
No
There may be other potential indicators (e.g., scope for growth: the
amount of energy available to an organism for growth and reproduction
in excess of the energy required for maintenance)
Can be
Potential is there
3,9
-------
TABLE 9. BEHAVIOR
Criterion
Unequivocal
Meaning
Cost-
Effectiveness
Availability
Indicator
Schooling Avoidance/ Predatory Food Jisc.
S Attraction Reproduction Behavior Habits Behaviors
No No No No No No
No Yes No No Yes2 Yes4
Yes Yes Yes Yes Yes Yes
Applicability
-early/late
sensitivity
Early
Early
Early
^ Must be schooling species
2 Can be
•7 Depends -on species
^ Fairly cost-effective
Early
Late Early
-pollutant-
specific No No No
-species-
specific No Yes No
-scope-
specific
spatial Yes Yes Yes
temporal Yes Yes Yes
No No No
No No No
Yes Yes3 Yes
Yes Yes Yes
40
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TABLE 10. POPULATION
Criterion
Unequivocal
Meaning
Cost-
Effectiveness
Availability
Applicability
-early/late
sensitivity
Indicator
Abundance Age Structure Spatial Distribution
No
Yes1
Yes
Late
No
Yes1
Yes
Late
NCI
Yes
Yes
l
Early
-pollutant-
specific No
-species-
specific No
-scope-
specific
2
spatial Yes
temporal No
No No
No - No
'
Yes2 Yes
No Yes
I
For local stocks
For anadromous and resident species
41
-------
was unable to determine or agree upon the relationship between the
indicator and the criterion. The two potential finfish indicators,
pollutant resistance and fishery closures, are not directly related but
are presented together in Table 11, for convenience.
Following evaluation, each indicator was ranked within its
category on the first three criteria only (unequivocal meaning, cost
effectiveness, and availability). The ranking.within categories was
performed using the four methods specified:
o Select the best single indicator,
o Select the best few indicators,
o Give a numerical rank to each indicator, and
o Classify each indicator as "good" or "bad."
Tables 12-15 summarize the results of the rankings. Although the
indicators "pollutant resistance" and "fishery closures" were not
ranked, the group agreed to present the evaluations of these indicators
to the workshop. Although the group did not agree how to rank these
two indicators, this does not imply that they are not useful
indicators. In fact, these two indicators met the workshop's criteria
well.
42
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TABLE 11. POLLUTANT RESISTANCE AND FISHERY CLOSURE
Indicator
Criterion
Unequivocal
Meaning
Cost -
Effectiveness
Availability
Applicability
-early/late
sensitivity
-pollutant-
specific
-species-
specific
-scope-
specific
spatial
temporal
Pollutant
Resistance
Fishery
Closures
Yes
No
Yes
Late
Yes
No
Yes
No
Yes
I
Yes ;
Yes
Late
Yes
No
Yes
Yes
1
For resident species
43
-------
TABLE 12. REPRODUCTION AND DEVELOPMENT
Indicator
#1
Best
Relative
Rank
Good/Bad
Number of eggs
Embryo viability
Cytogenetics
Larval development
and viability
Growth
Fin regeneration
X
X
X
4
3
1
2
6
5
Good
Good
Good
Good
Good
Good
44
-------
TABLE 13. PHYSIOLOGY
Indicator
#1 Best
Relative
Rank
Good/Bad
Respiration
flematology
Endocrinology
Swimming Stamina
X
3 or 4
1
3 or 4
2
Good
Good
Good
Good
45
-------
TABLE 14. BEHAVIOR
Indicator
#1
Best
Relative
Rank
Good/Bad
Schooling
Avoidance/
Attraction
Reproduction
Predatory Behavior
Food Habits
Miscellaneous
Behaviors
X
X
1
5
6
4
2
Good
Good
Bad
Bad
Good
Good
46
-------
TABLE 15. POPULATION
Indicator
#1 Best
Relative
Rank
Good/Bad
Abundance
Age Structure
Profile
Spatial Distribution
Profile
X
X
2
3
1
Good
Good
Good
47
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SUMMARY REPORT FROM SUBGROUP ON IMMUNOLOGY
The field of immunology is contributing to new, sensitive, and
rapid methods for the detection and identification of microorganisms in
animals, humans, and the environment. These methods, such as
enzyme-linked immunoassays, macrophage activity assays, and passive
heraolytic assays, may be used to detect changes in animals caused by
toxic contaminants. The Immunological Indicators subgroup considered
which methods might be applied to detect changes in the immune system
of finfish caused by contaminants.
The immune system in higher animals, including finfishes, is based
on antigen exposure, resultant: antibody or cellular response, and
eventual protection (if a disease agent is involved). The subgroup
approached this problem in the chronological order of the immune
response: first considering the afferent immune response—pickup and
processing of antigen, and' then the efferent immune response—producing
the physiological result, e.g., antibody, cellular activation, etc.
Table 16 presents the inital list of immune indicators.
This subgroup assigned points to determine the relative rank of
the monitoring methods relying on the immune response, with 1 the
lowest rating, and 5 the highest rating. (See Table 17). Evaluations
are summarized below.
HACROPHAGE TESTS
One method, which included examining a combination of macrophage
phagocytosis, macrophage killing of microorganisms, and macrophage
chemoluminescence, was judged a very good method. Two committee
members considered this the best and the other two members considered
it among the top three methods.
The members also considered the use of macrophage phagocytosis
alone a good method, but not nearly as powerful as the combined
48
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TABLE 16. INITIAL LISTING OF IMMUNE INDICATORS
Macrophage indicators
phagocytosis
chemotaxis
pinocytosis (soluble particles)
killing !
microbes |
tumor cells i
chemoluminescence (a technique)
aggregates of macrophages (in kidney, for example)
melanin accumulation by macrophages
MAP (macrophage activation factor) j
MIF (macrophage inhibition factor)
Lymphocyte indicators
T-lymphocytes ;
blastogenesis
cytotoxicity i • •
lymphokine production
delayed type hypersensitivity
graft rejection (fish scale rejection)
B-lymphocytes ;
blastogenesis
antibody production (Jerne assays) |
Humoral antibody j
Direct detection of antibody in serum to specific microbes
via techniques of agglutination, precipitation, or ELISA
Disease resistance j
Directly measured by experimental animal exposure to a
specific pathogen
Assessed epidemiologically; unknown field challenge
I
Nonspecific defense mechanisms , |
CRP (C-reactive protein in blood)
natural killer cells
nonspecific antibody-like molecules
interferon
49
-------
TABLE 17. RANKING OF METHODS SUITABLE
Method
Macrophage tests
phagocytosis alone
phagocytosis,
chemoluminescence
and killing
chemo taxis
melanin &
aggregation
pinocytosis
T-lymphocyte
grafting
B-lymphocyte
Jerne assay
Humoral
Antibody produced
in injection
Antibody, non-
specific in field
Disease Resistance
Experimental
exposure
Epidemiologically
measured exposure
Natural killer cells
f" Rating scheme: 1 =
^ Tt<*«fS*4s>ts4 /M^TMT /\ne
Usefulness,
Unequivocal
Meaning
4
,
4.5
3
2
4
3
4
4
2
h
3/1°
b
3/1
2
FOR FIELD-TESTING
Cost-
Effectiveness
4
5
5
3
4
3
3
2
4
3
3
3
AT PRESENT
Availability
4
3
4
4
4'
4
5
,
2
lowest, 5 = highest
50
-------
approach described above. Adding the other tvo macrophage indicators to
a monitoring scheme was considered cost-effective. Other macrophage
tests were considered as indicators, but ranked low in comparison to
the above. I
DISEASE RESISTANCE
The second highest ranked category of toxic indicators from
immunological methods was the disease resistance area. The
experimental measurement of disease resistance in feral fish (i.e., by
injection of disease microorganisms) ranked high. However, a few
members opposed this method on the grounds that its meaning is
equivocal. The method also had a higher cost compared to others.
I
The group considered epidemiologic assessment of disease
resistance to have one especially positive feature: jit is universally
available. Again, however, the opinion was divided! as three members
felt it was useful, and one member strongly opposed it.
I
' JERNE ASSAY .! .. .
I
A final method judged to be one of the strongest immunologic
i
approaches was ,that of Jerne assay as applied to detect B-lymphocytes
as antibody-producing cells. This method received strong support from
most members and moderate support from all. Members also considered it
widely available and cost-effective.
OTHER METHODS
I
!
Other methods considered at least moderately usipful by a majority
of the members were the following:
I
o Blood levels of antibody (humoral) in feral fish injected
with microorganisms (not well tested, somewhat expensive,
•
widely available);
51
-------
o Graft rejection (T-lymphocyte involvement), not well
tried, not widely available, but promising.
The ranking of methods showed that all methods are generally
applicable to assessing toxic contaminant impacts on finfish.
Macrophage assessments were given high utility. These methods also can
be pertinent to assessing impacts on the ecosystem as well as on human
health, but are less direct measures of such impacts.
Recommended Immune Indicators for Field Testing as Monitoring Tools
Based on the evaluation criteria and ranking by the four suggested
methods, the recommended ranking of immune indicators was as follows:
(1) A combined macrophage assay consisting of
chemoluminescence, phagocytosis, and killing ability5
(2) Experimental measurement of disease resistance in feral
fish by injection of known antigens or microorganisms!
(3) Jerne plaque assays, (B-lymphocyte measurement of
antibody production);
(4) Blood levels of humoral antibody after defined ' '
injection. ;
Research Needs
The subgroup identified the following research needs for improving
the usefulness of immune indicators of toxic contamination in finfishs
(1) Immune parameters for indicating toxic assessment should
be derived from each section of the immunological system,
including
o Macrophage assays,
o T-lymphocyte assays,
52
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o B-lymphocyte assays, and
o Determination of disease resistance. I
(2) Tests in vivo should be validated with assays in vitro,
e.g., culture of immune cells or organs and demonstration
of effects of additives.
[
I
(3) To obtain better statistical information on the immune
assays, inbred lines of fish are needed.
(4) Because the endocrine system of finfish greatly affects
their immune parameters, the effects of stress on the
immune response in fish requires more investigation.
53
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Workshop
on Finfish
as Indicators
of Toxic
Contamination
Sponsored by the
U.S. Environmental
Protection Agency,
Office of Marine and
Estuarine Protection
On behalf of the U.S. Environmental Protection Agency, Office
of Marine and Estuarine Protection, I would like to invite you
to participate in a workshop on Finfish as Indicators of Toxic
Contamination in Estuaries. The workshop will be divided into
various working groups of government and academic scientists
and resource managers. The primary objective of the workshop.
is to determine preferred methodologies available for analyzing
the significance of toxic impacts on estuarine finfish.
The workshop will be held at the Airlie House in Airlie,
Virginia, on July 28-30, 1986. Technical Resources, Inc. (TRI)
will provide technical and logistical support. Enclosed is a
registration form. Please return this form to TRI by July 11,
1986. . !
|
Also enclosed for your information is a background document,
Finfish as Indicators of Toxics in Estuaries, developed by
Margaret McFadien-Carter of the University of Delaware.
•
I hope to see you in July and look forward to your contributions
to this project.
Sincerely, ' ]
Tudor T. Davies
Director
Office of Marine and Estuarine Protection
enclosures
A-l
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WORKSHOP ON FINFISH AS INDICATORS OF TOXIC
CONTAMINATION IN ESTUARIES I
Sponsored by the U.S. Environmental Protection Agency
Office of Marine and Estuarine Protection
July 28-30, 1986
Airlie House
Airlie, Virginia i
Monday July 28
10:00"a.m. - 11:15 a.m.
11:15 a.m. - 12:15 p.m.
12:30 p.m. - 2:00 p.m.
2:00 p.m. - 3:20 p.m.
3:20 p.m. - 3:40 p.m.
3:40 p.m. - 6:00 p.m.
6:00 p.m. - 7:00 p.m.
7:00 p.m. - 8:00 p.m.
Tuesday, July 29
7:30 a.m. - 8:30 a.m.
8:30 a.m. - 10:20 a.m.
10:20 a.m. - 10:40 a.m.
10:40 a.m. - 12:00 p.m.
12:00 p.m. - 1:30 p.m.
1:30 p.m. - 3:20 p.m.
3:20 p.m. - 3:40 p.m.
3:40 p.m. - 7:00 p.m.
7:00 p.m.
Introduction:
Tudor Davies
Michelle Hi Her
Gary Petrazzuolo
Store Room
Background Paper Discussion Store Room
Lunch
Individual Subgroups
Break
Individual Subgroups
Reception - Cash Bar
Dinner
Breakfast
Subgroup Summarizing
Break
Subgroup Plenary Session
Lunch
Plenary Session
Break
Plenary Session
Cash Bar and Barbeque
B-I.
Main Dining Room
Livery, Silo House
Hitching Post, Silo House
Granary, Silo House
|West Room, Main House
South Room, Main House
Livery, Silo House
Hitching Post, Silo House
Granary, Silo House
West Room, Main House
South Room, Main House
Garden Room, Main House
Main Dining Room
Main Dining Room
Store Room
Main Dining Room
Store Room
Store Room
The Lodge
-------
neunesady, uuiy JU ' -
7:30 a.m. - 8:30 a.m. Breakfast Main House
8:30 a.m. - 10:20 a.m. Suogroup Summaries Store Room
10:20 a.m. - 10:40 a.m. Break
10:40 a.m. - 12:00 n Closing Summary Store Room
B-2
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FINFISH AS INDICATORS OF TOXIC CONTAMINATION IN ESTUARIES
July 28-30, 1986
Airlie House
Airlie, Virginia
Participant List
Dr. Douglas Anderson
National Fish Health Research Lab
U.S. Fish and Wildlife Service
P.O. Box 700
Kearneysville, WV 25430
304-725-8461
Mr. Seth Ausubel
Office of Marine and Estuarine Protection
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
202-475-7111
Dr. Michael E. Bender
Professor of Marine Science
Virginia Institute of Marine Science
College of William and Mary
201 Waller Mill Road
Williamsburg, VA 23185
804-642-7237
Dr. Richard 0. Bennett
University of Maryland
School of Medicine
Department of Pathology
10 S. Pine Street
Baltimore, MD 21201
301-528-7276
Mr. .Roland Borey
Project Biologist
Texaco Research
P.O. Box 1608
Port Arthur, TX 77641
409-989-6361
Dr. Anthony Calabrese
Laboratory Director
N.E. Fisheries Center
Milford Laboratory
National Marine Fisheries Service
212 Rogers Avenue
Milford, CT 06460
203-783-4200
C-l
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Dr. Jeffrey N. Cross
Southern California Coastal Water
Research Project
646 West Pacific Coast Highway
Long Beach, CA 90806
213-435-7071
Dr. Tudor T. Davies
. Office of Marine and Estuarine Protection
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
202-382-7166
Dr. Kim Devonald
Office of Marine and Estuarine Protection
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
202-475-7114
Dr. David W. Engel
Acting Chief
Division of Ecology
National Marine Fisheries Service
National Oceanic and Atmospheric Administration
Southeast Fisheries Center
Beaufort Laboratory
Beaufort, NC 28516-9722
919-728-8741'
Ms. Patricia Fair
National Marine Fisheries Service
National Oceanic and Atmospheric Administration
217 Ft. Johnson Road
Charleston, SC 29412
803-762-1200
Ms. Mary Jo Garreis
Chief of Division of Standards and Certification
Department of Health and Mental Hygiene.
State of Maryland
201 W. Preston Street
P.O Box 13387
Baltimore, MD 21202
•301-225-6293
Mr. Larry Goodman
Research Biologist
U.S. Environmental Protection Agency
Sabine Island
Gulf Breeze, FL 32561
904-932-5311
C-2
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Dr. William J. Hargis, Jr.
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point, VA 23062
804-642-7362
Ms. Michelle Hiller -
Office of Marine and Estuarine Protection
U.S. Environmental Protection Agency
401 M Street, SW
Washington, DC 20460
202-475-7102
Dr. Joseph B. Hunn
U.S. Fish and Wildlife Service
Columbia National Fisheries Research Laboratory
Route 1
Columbia, MO 65201
314-875-5399
Dr. Kenneth D. Jenkins
Molecular Ecology Institute
California State University, Long Beach
Long Beach, CA 90840
213-498-4906
Dr. Braulio D. Jimenez
Research Associate
Oak Ridge National Laboratory
Environmental Science Division
P.O. Box X
Oak Ridge, TN 37831
615-574-7321
Mr. Caret Lahvis
Aquatic Biologist
Great Lakes National Program Office
U.S. Environmental Protection Agency
230 South Dearborn
Chicago, IL 60604
312-353-2694
Dr. Eric B. May
Department of Pathology
University of Maryland
School of Medicine
10 S. Pine Street
Baltimore, MD 21201
301-528-7276 or 5184
Dr. Margaret McFadien-Carter
Marine Scientist
Robinson Hall
College of Marine Studies
University of Delaware
Newark, DE 19732
302-451-1194
C-3
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Mr. Victor McFarland
Aquatic Biologist
Waterways Experiment Station
U.S. Army Corps of Engineers
P.O. Box 631
Vicksburg, MS 39180
601-634-3721
Mr. Dale Miller
Northeast Utilities Environmental Laboratory
Millstone Nuclear Power Station
P.O. Box 128
Rope Ferry Road
Waterford, CT 06385
203-447-1791 x4533
Dr. Jerry Neff
Battelle New England Marine Laboratory
397 Washington Street
Duxbury, MA 02332
617-934-5682
Dr. Joel S. O'Connor
Ocean Assessments Division
National Oceanic and Atmospheric Administration
11500 Rockville Pike
Rockville,°MD 20852
301-443-8698
Dr. William A. Richkus
Director, Ecological Sciences and Analysis
Martin Marietta Environmental Systems
9200 Rurasey Road
Columbia, MD 21045
301-964-9200
Mr. Norman Rubenstein
Research Aquatic Biologist
U.S. EPA Laboratory
South Ferry Road
Narragansetft, RI 02882
401-789-1071
Mr. Jay Sauber
State of North Carolina
Division of Environmental Management
P.O Box 27687
Raleigh, NC 27611
919-733-6510
Dr. Jan Spitsbergen
Research Associate
University of Wisconsin/Madison
37 Pine Hill Road
Port Jefferson, NY 11777
516-928-7869
C-4
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Dr. John Stegeman
Associate Scientist
Bepar-tsneiYt of Biology
Woods Hole Oceanographic Institute
Woods Hole, MA 02543
617-548-1400 x2320
Dr. James P. Thomas
Estuarine Program Office F/EPO
National Oceanic and Atmospheric Administration
Universal Building, Room 625
1825 Connecticut Avenue, NW
Washington, DC 20235
202-673-5243
Dr. Beverly Anne Weeks
Associate Professor
Virginia Institute of Marine Sciences
College of William and Mary
Gloucester Point,. VA 23062
804-642-7346
Dr. Judith Weis
Professor
Department of Biological Science
Rutgers University
Newark, NJ 07102
201-648-5387
C-5
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a:a
ur IUAIU UUN i
Participant.'; by Work Group
ANATOMIC PATHOLOGY:
Eric May, Chair
Mary Jo Garreis, Recorder
William Hargis
Joseph Hunn
BIOACCUMULATION AND ENZYMES:
Jerry Neff, Chair
Patricia Fair, Recorder
Michael Bender
David Engel
Kenneth Jenkins
Braulio Jimenez
Garet Lahvis
Victor McFarland
Norman Rubenstein
John Stegemari
Dale Miller
William Richkus
James Thomas
REPRODUCTION AND DEYELOPMENT/PHYSIOLOGY/BEHAYIOR/POPULATION:
Joel 0'Conner, Chair
Judith Weis, Recorder
Roland Borey
Anthony Calabrese
Jeffery Cross
Larry Goodman
IMMUNOLOGY:
Richard Bennett, Chair
Douglas Anderson, Recorder
Margaret McFadien-Carter
Jan Spitsbergen
Beverly Anne Weeks
WORKSHOP LEADERS, FACILITATORS, AND SUPPORT STAFF
Tudor T. Davies, Director, OMEP
Michelle Hiller, Chief, Technical Guidance Branch OMEP
Seth Ausubel, OMEP
Kim Devonald, OMEP (facilitator, Immunology)
Gary Petrazzuolo, TRI (Workshop Moderator)
Joanna Fringer, TRI (facilitator, Anatomic Pathology)
Darcey Rosenblatt, TRI (facilitator, Bioaccumulation and Enzymes)
Drew Zacherle, TRI (facilitator, Reproduction etc.) !
Willie Sanderson, TRI ;
Lorraine Sickels, TRI
D-l
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1
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Background Paper
Finfish as Indicators of Toxics in Estu«iries
For EPA Cooperative Agreement CX812956-01-1
Submitted tos |
U. S. Environmental Protection Agency
Office of Marine and Estuarine Protection
Washington, DC 20460 I
Prepared by:
College of Marine Studies
University of Delaware
Newark, Delaware 19716
June, 1986
-------
1
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TABLE OF CONTENTS
1. INTRODUCTION 3
1.1 Workshop Objectives ......... e o 3
1.2 Biotic Indicators for Toxic Pollution . 3
2. OVERVIEW OF FINFISH AS INDICATORS OF TOXIC POLLUTION ... 4
2.1 Working Definitions of Toxic Pollution and Stress . 4
2.2 Historical Sources of Toxic Substances 8
2.3 Use of Finfish as Indicators of Toxic Pollution . . 12
2.1 The Problem of background Noise
3. FINFISH INDICATORS OF TOXIC POLLUTION . . . 15
3.1 Bioaccumulation/Tissue Concentrations 16
3.2 Histopathology ... 17
3.2.1 Non-Oncogenic • . 17
3*2.2 Oncogenic . . . 19
3.3 Immunology . 21
3.4 Reproduction and Development ...;.; 23
3.5 Enzymology j....... 26
3*5.1 Enzyme Induction . 26
3.5.2' Molecular Pathology ! 29
3.6 Physiology 31
3.7 Population •...;. 33
SUMMARY , . . . o 35
LITERATURE CITED ... ........... 36
TABLES i
TABLE 1 Survey of Effects of Toxic Chemicals in Finfish ... 6
TABLE 2 Abbreviated Listing of Industrial Uses of Some
Potentially Toxic Metals .. ., . 9
E- 1
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1.'INTRODUCTION
1.1 Workshop Objectives
The primary objective of this workshop is to determine the
relative usefulness of finfish indicators for toxic impacts in
estuaries. Technical specialists and resource managers will help
determine the most accurate, replicable, and cost effective metho-
dologies immediately available for analyzing toxic impacts in
estuarine finfish. Potential indicator methodologies will be
evaluated based on workshop participants'- answers to a set of
questions and criteria provided by the workshop coordinators.
These evaluations will offer estuary program managers a technical
appraisal of the strengths and weaknesses of approaches using
finfish indicators to assess toxic impacts.
1.2 Biotic Indicators for Toxic Pollution
Physical factors (e.g., hydrodynamics, sediment type, sediment
transport) and cnemical factors (eg., salinity, redox reactions,
sorption/desorption processes, and chemical reactions) affect the
fates and effects of toxic substances. Input rates.of nonconser-
vative pollutants to the environment can also influence their fate
and the expression of toxic responses (Cairns, 1986a). Organisms
can accumulate, and transport, metabolize (resulting in both
detoxification and intoxification), and physically process (e.g.,
fecal pellet formation) toxic compounds, thus affecting the physical
E-2
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and chemical factors that influence the fates of toxic substances
in the environment. Consequently, the impact of toxics in estuaries
depends on both abiotic and biotic characteristics and processes
that interact through complex relationships. Pollutant loads and
ambient concentrations may not, in themselves, offer good predic-
tions of toxic impacts. Because'of the variability of environmental
conditions, measurements of effects of toxicity often provide a more
I
meaningingful indication of environmental quality (Cairns, 1936b).
I
Field analyses of toxic responses are desirable to evaluate actual
i
impact on any given estuary. It is also particularly desirable to
I
identify members of the biota that will offer accurate early
warning systems of pollutant damage.
2. OVERVIEW OF FINFISH AS INDICATORS OF TUXIC POLLUTION
2.1 Working Definitions of Toxic Pollution and Stress
The words "toxicants" and "toxics" will be used in this
.
i
paper to mean chemicals that produce acute or chrome deleterious
l
effects on finfish and/or other estuarine biota, j Effects caused
by toxics may be systemic, teratogenic and carcinogenic as well as
any other effects that reduce reproductive fitness! or shorten the
individual's natural life.
pressure
The term "stress" may refer to any
principal that alters the natural processes (physiological
E-3
•! or forcing
al, develop-
-------
mental, or behavioral) of an organism. Stress induced by exposure
to a toxic chemical could ultimately have negative or neutral
effects.
Examples of negative stress would be toxic effects that
ultimately shorten the organism's life,' impair its reproductive
success, or result in adverse, toxic effects in organisms higher
on the food chain, through accumulation or metabolic transformations
of toxic substances.
An example of stress with neutral effects might be the
metabolism of a small amount of a toxic chemical to .a non-toxic
excretable daughter chemical, with no production of toxic, reactive
intermediates. This type of stress utilizes energy reserves of
the organism. However, the organism is not permanently impaired,
and significant effects on reproductive success are unlikely.
Beneficial results of stress are feasible. An example here
would be the stimulation of metal binding proteins by exposure to
a toxic metal that increases an organism's resistance to subsequent
exposures to toxic metals. However, these benefits generally
apply only to further toxic stresses, and often do not include
larger ecosystem effects.
. For the remainder of this paper both neutral and negative
effects will be considered. The emphasis, however, will be on the
E-4
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concept of negative stress. In summary the following classifica-
tions of "stress caused by toxic chemicals" will bei considered:
Neutral stress: pressure from toxic chemicals on finfish
I
causing altered physiology or inorphology
without permanent impairment of the orga-
nism's lifespan or reproductive success.
Negative stress; toxic pressures causing acute or chronic
l
and subiethal effects that decrease an
organism's lifespan and/or its reproductive
success„ These pressures might result in
i
Deduced survival of offspring, altered
fecundity, lowered growth rate,, suppression
of immune responses, pathological tissue
i
changes, genetic changes, or anatomical or
physiological changes deleterious to the
individual organism.
Types of effects due to toxic stress considered in this
I
paper are shown in Table 1. It is useful to distinguish among (a)
l
pathological, (b) physiological without pathological! (c) pathologi-
'
cally-induced behavioral, and (d) strictly behavioral effects of
toxic stress. The selection of stress indicators for;. this paper was
based, in part, on identification by investigators of probable
causative factors for the effects being studied. 'These are also
l
i
shown in Table 1 . While negative effects due to negative stresses
I
are emphasized, neutral stresses and their effects are also consi-
dered.
E-5
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Table 1
Survey of Effects of Toxic Chemicals in Finfish
I. Negative effects/negative stress: chronic, sublethal effects deleterious to
individual organism.
Effect
A. Bioaccumulaton/
Tissue Concentrations
Type of Effect
Documented
Causative Agents
Physiological/ PAH, PCB, Chlorinated
pathological hydrocarbons
B. Histopathological Pathological
1. Fin erosion, ulcers, cataracts
2. Neoplasms
PAH, PCB, Chlorinated
hydrocarbons
C. Immune effects
(repression/stimulation)
D. Reproductive and developmental
effects
E. Deleterious results reflected by
enzyme alteration
F. Deleterious metabolism of
chemicals (toxification)
Physiological: PAH, PCB, Metals
can lead to
pathological
Physiological: Metals, PAH, PCB,
can- lead to Chlorinated
pathological hydrocarbons
Physiological
Physiological
reflects
pathological
G. General physiological alterations Physiological/
(gill respiration, osmoregulation) pathological
PCB, PAH, Metal
PAH, PCB
PAH, PCB, Chlorinated
hydrocarbons
H. Behavioral and population alterations Pathologically PAH, Metals,
^.,« *„ off^t-.* on individuals or Chlorinated
%*•
Physiologically hydrocarbons
induced behavior
II. Neutral effects/neutral stress: chronic effects with latered physiology but
with no permanent impairment of organism.
Type of Effect Documented Stress
&f rGCt* • •" •— """
A Non-injurious enzymatic alterations Physiological/ PAH, PCB, Metals
non-pathological
B. Metabolism of toxic parent to
non-toxic daughter chemical
(detoxification)
Physiological/ PAH, PCB
non-pathological
E-6
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2.2 Historical Sources of Toxic Substances
i
The sources, fates ana possible effects of toxics in estuaries
currently are subjects of considerable concern among scientists and
environmental managers. Toxics include polyaromatic hydrocarbons
(PAHs) and other petroleum-related compounds, polychlorinated
biphenyls (PCBs), pesticides, other synthetic organics, and toxic
metals.
I
Petroleum-related sources include marinei transportation,
accidental spills, municipal wastewater discharges, refinery
wastes, industrial wastes and urban runoff. Nonpoint sources such
i
.
as urban runoff may constitute a major portion of petroleum poilu-
•
tion. There is also concern that atmospheric transport may contri-
bute more to petroleum pollution of surface waters than previously
believed or documented (National Research Council, 1985).
Primary sources of PCBs to the environment include leakage
from closed electrical systems, such as transformers and capacitors,
and losses during the manufacture and use of hydraulic fluids,
lubricants and heat transfer fluids. Other sources include adhe-
sives, plasticizers, pesticide extenders, and dyes. PCBs disposed
in landfills also may represent a significant pollution hazard.
Pesticides typically reach surface waters from nonpoint
sources, runoff and atmospheric settling which result from agricul-
E-7 !
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tural, parkland, suburban, or urban pest control efforts. Another
source of increasing concern is municipal wastewater effluents,
which contain surprisingly high pesticide concentrations as a
result of household use (Carter, 1985).
Other synthetic organic toxicants of particular concern
include: dioxins, phthalate esters, haloethers, chlorinated hydro-
carbons (other than pesticides), organoraetallic compounds, nitro-
benzenes, nitrosamines, benzidines, phenols, acrolein, acrylo-
nitrile, dichloro-5-fluoromethane, benzo(a)pyrene. These chemicals
are released from a number of industrial, domestic, and agricultural
sources.
Toxic metals includes aluminum, antimony, arsenic, barium,
beryllium, bismuth, boron, cadmium, chromium, cobalt, copper,
gold, iron, lead, lithium, manganese, mercury, molybdenum, nickel.
palladium, platinum, selenium, silicon, silver, tellurium, thallium,
tin, titanium, vanadium, zinc and zirconium. Sources of most of
these metals are primarily industrial. A partial list of industrial
metals considered toxic to humans is demonstrated in Table 2.
E-8
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Metal
Table 2
Abbreviated Listing of Industrial Uses of Some
Potentially Toxic Metals
* - Considered highly toxic
Table developed from data available in Berman (1980)
Modern Use I
Antimony:
*Arsenic:
*Barium:
Beryllium:
Bismuth:
•Cadmium:
Chromium:
Cobalt:
•Copper:
Gold:
Iron:
"Lead:
alloyed with lead, tin and copper, a flame retar-
dant in paints, enamels and lacquers: also used
in printed type.
smelting, rodenticides, insecticides, herbicides,
glass and enamel manufacture. i
electroplating, glass manufacturing, sugar refi-
ning; also in television tubes and explosives.
alloyed with copper in electrical equipment; used
in producing optical glass, nuclear reactors.
*
used in electrical fuses, facial powders and
producing artificial pearls.
used in electroplating, engraving, 'dental amalgam,
glass manufacture, ceramic glazes; Jaune brilliant
(Cadmium sulfide) used to color glass, soaps,
fireworks and textiles.
used in manufacture of stainless steel, in photo-
graphy and as corrosion inhibitor.
used in alloys and nuclear technology.
insecticides, fungicides, germicides; as pigment
in textiles and ceramics.
other than currency, used medicinally, in printed
circuits, semi-conductors and in the space industry
in glass to metal seals.
other than manufacture of steel; ferric chroraate
in pigments, ferric hydroxide in water purifica-
tion, the oxide as a polishing agent and pigment.
l
I
although reduced in paints, still high levels in
putty and plaster, glazed earthenware, in pewter,
chafing dish candles, hair dyes, color in maga-
zines.
E-9
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Metal
Table 2
Abbreviated Listing of Industrial Uses of Some
Potentially Toxic Metals
Modern Use
*Lithium:
*Molybdenum:
Nickel:
Palladium:
Platinum:
*Selenium:
*Silver:
"Thallium:
Tin:
Titanium:
Vanadium:
2inc:
Zirconium:
aerospace alloys, lubricating greases, metal
cleaners photography.
as alloy for special steel in rifle barrels,
propeller shafts, boiler plate, x-ray tubes; as
lubricant additive (toxicity has been demonstrated
to be species specific).
storage batteries, spark plugs, cooking utensils,
detergents; Raney nickel (equal parts aluminum
and nickel) used for hydrogenation of oils.
metal plating; catalyst in electrical industry;
as alloy with Ag, Au or Cu for jewelry and dentis-
try.
dentistry, jewelry, and in electrical industry.
manufacture of plastics, rubber, ceramics, ink,
glass, paint pigments, photoelectric cells.
besides tableware, jewelry, and dentistry, as
alloy with many metals. Also as steel coating;
in manufacture of solder.
amalgam with Hg; alloy in switches; in manufacture
of pigments and dyes, as rat poison.
diverse uses: food containers, electrical, radio,
automobile parts; color for china, fabric dying;
organic complexes as biocides.
for strengthening steel; useful alloy with many
metals; titanium dioxide used in creams, powders,
sun protection, in paint, plastics and leather
work, trichloride in laundering.
allows with Pb, Mn, Cr:: rust resistance, strength-
ening steel, photographic developer, dying cottons,
silks, leathers and furs.
manufacture of bronze and brass. coating on
iron, steel.
shielding in nuclear submarines and power reactors,
used in production of paints, flashbulbs and
detonators; chloride and acetate as textile water
repellents.
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2.3 Use of Finfish as Indicators of Toxic Pollution
Criteria for evaluating toxic effects in Ecosystems have
been suggested by the National Academy of Sciences (NRC, 1981).
They surveyed laboratory and field methods, including chemical
characterizations, single-species tests, multi-species tests, and
ecosystem tests. Ultimately they suggested that: |
"research should be conducted to develop test procedures
that can provide multiple sets of data. Tests snould
be designed to provide short-term results about long-
term effects."
This NRC study recommended that four classes
should be collected:
• Characterization of test substance
• Physiological responses of species
• Multi-species responses
• Ecosystem responses.
of information
Collecting all four classes of data will serve the following
purposes for testing toxic effects:
• determine partition coefficients for movement of
chemicals in the system,
• identify the toxic potential for major transformation
or degradation products,
• account for variability in natural syistems affecting
dose to biota or exposure time within a compartment,
and help distinguish natural variations ;from chemically
induced variations in ecosystems (NEC 1981).
E-ll
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By evaluating physiological responses of sensitive species
in the laboratory, discrete morphological genetic, biochemical, and
pathological effects of particular chemicals can be identified.
Subsequent ecosystem response studies can evaluate the extent to
which these processes are expressed in the field, and the ways in
which occurrence of morphological, genetic, biochemical and patho-
logical changes in single species in situ affect the abundance
and distribution of the species of concern and those that interact
with them. Identification of some single species impacts in the
field may then offer pollution abatement monitoring programs rapid
identification methods for long-term problems.
' Finfish, as a group, offer monitoring programs several bene-
fits. Analyses of physiological and pathological effects of
parent and daughter chemicals have demonstrated that finfish are
susceptible to a number of readily identifiable and some less well
understood effects of toxic stress (Couch and Harshbarger, 1985;
O'Connor et al., 1986; Malins et al., 1980, 1983, 1985; Murchelano.
and Wolke, 1985; NRC 1985; Hargis et al., 1984). They are also
capable of transforming parent chemicals into more toxic daughter
chemicals (TetraTech, 1985). They are at sufficiently high trophic
levels that they are likely to serve as early-warning indicators
because of bioconcentration effects. Altered finfish physiology
and pathology in field specimens therefore seem likely to offer
environmental managers particularly useful warning systems for
chemical pollution of estuaries.
E-12
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One potential problem with the use of finfish as indicator
i
organisms is their mobility. The benefits and disadvantages of
i
using relatively mobile organisms as toxic stress indicators will
be discussed later in this background paper.
The Problem of Background Noise
To identify pollution due to toxics, it is necessary to
distinguish between toxic effects and background noise in any
given population or ecosystem. Background noise may be defined as
i
natural variations in Measurable biotic processes that are not
i
causally related to the toxic pollutant or other disturbance that
is of concern. Differentiation between toxic effects and background
•
noise can be difficult to accomplish. Clear causal relationships
should be established in the laboratory whenever possible. It is
necessary to measure background noise in all monitoring programs
i
to obtain valid stress analyses in the field. Robert Livingston
i
has provided an excellent review of the problem of evaluating
background noise; this review is included as Appendix A of tnis
document.
E-13
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3o FINFlbH INDICATORS OF TUXIC POLLUTION
The remainder of this background document describes specific
effects of toxics on finfish as listed in Table 1. An introductory
chapter summarizing and characterizing the effects reviewed is
followed by a series of papers by specialists describing methodo-
logies for analyzing particular effects. In some cases, where a
previously published review .article or technical report presents
the necessary information on a. given method or methods, that article
or report has been included with the author's permission in lieu of
preparing original text for this report.
Effects ' are discussed under the following major headings?
Bioaccumulation/Tissue concentration; Histopathology: Non-oncogenie;
Histopathology: Oncogenic; Immune Effects; Effects on Reproduction
and Development; Enzyme Alterations; Metabolism of Toxicants;
General Physiological Alterations; and Population Alterations.
Many effects fall into more than one of these categories, and it
is not intended that great significance be placed on the details
of the categorization presented here for purposes of discussion.
The objective of the workshop discussions will be to address
strengths and weaknesses of individual measurable effects as
indicators, and the merits of methodologies used for these measure-
ments. Each indicator will be evaluated on its own merits, so
that assignment to one or another general category should not
affect the evaluation.
E-14
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3.1 Bioaccumulation/Tissue Concentrations
The biological uptake of toxics through food, water, sediment
i
contacts or a combination of exposures and retention in tissues is
called bioaccumulation. A number of factors affect this process.
The bioaccumulation potential of a substance is dependent on its
chemical properties, the affected organism's mechanisms for uptake
and elimination, and the environmental factors influencing its
bioavailability. Examples of the variability of intrinsic and
- - I
extrinsic chemical processes in bioaccumulation of various toxicants
are given by trace metals. While bioaccumulation of Cd and Cu are
I
I
primarily dependent on free ion activity, bioaccuraulation and
toxicity of silver appears dependent on formation of chlorocomplexes
(Engel et al., 1981). j
.
Physical and chemical partitioning of toxics as they enter
an estuary influences exposure routes to the biota. Benthic
I
fauna and demersal fish are most likely to be affected by toxicants'
I
associated with particulates. it has been demonstrated that direct
sediment contact contributes significantly to PCB bioaccumulation
in demersal fish. Although dietary contributions of PCBs were
high, fish without direct sediment contact accumulated significantly
lower PCB residues (Rubenstein et al., 1984).
E-15
-------
Bioaccumulation is ultimately determined by the organism's
ability to metabolize, transform, and excrete a toxicant„ Clearance
ability can be related to amounts of adipose tissue as well as to
length or pattern of exposure to' the chemical. Lipid binding aue
to hydrophobia interactions, has been demonstrated by the greater
bioaccumulation of aromatic hydrocarbons over alkenes in petroleum
polluted waters (Neff, 1976). Ultimate clearance rates appear to
vary considerably among taxonomic groups of fishes and may even be
species-specific (Neff, 1976; TetraTech, 1985).
Other biological influences on ultimate tissue burden include
membrane permeability and the potential for translocation of the
chemical from the absorption site to other tissues (TetraTech,
1985). For a more detailed discussion of this topic the reader is
referred to O'Connor's review in Appendix B. Finally, it should
be noted that low tissue burdens do not necessarily indicate
insignificant effects of bioaccumulation since some metabolized
daughter compounds are toxic in extremely small amounts (TetraTech,
1985).
3.2 Histopathoiogy
3.2.1 Non-Oncogenic
Sublethal chronic exposure to certain toxics can cause
cellular damage in the skin, liver, eye lens and intestines of
E-16
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pelagic and benthic fish (Hargis et al., - 1984: Haiwkes, 1979).
Chronic exposure to petroleum causes not only direct tissue lesions
but also apparently via the lesions increases susceptibility to
parasitism, bacterial infection and viral disease indirectly via
these toxicant-induced lesions (Hodgkins et al., 1977; Sinderraann,
1979).
Synergistic actions of toxicants can increase severity of
tissue lesions. Hawkes (1979) demonstrated that while chronic
exposure to RGBs alone caused sloughing of intestinal mucosa, the
severity of the problem increased when the fish were exposed to
combined PCBs and petroleum fractions. Fin rot and cataracts have
been correlated with sublethal chronic exposure of finfish to sewage
effluents, and synergistic actions of pollutants .including toxics
.
have been identified as a possible cause of these pathological
disturbances (Hillman et al., 1986),,
i
Controlled studies have specifically correlated integumental
lesions with .certain-toxics. Finfish exposed to sediment contami-
nated with PAHs demonstrated severe lesions and uiceration within
8 days while control fish demonstrated no lesions (Hargis et al.,
.
1984). Weis and coworkers have suggested that fin deterioration
is a natural occurrence due to abrasion in demersal fish and that
lesions result from inhibition of regeneration due to toxics
i
(pers. corani. J. Weis, May, 1986). Methylmercurie chloride and
cadmium chloride have been shown to retard fin regeneration in
E-17
-------
fishes (Weis and Weis, 1978). Fin regeneration was also retarded
by DDT, malathion, carboryl, zinc, parathion and PCBs. It was not
strongly affected by quantitative diet changes or fish density
pressures (pers. cbmm. J. Weis, May, 1986)«
The possibility, of adaptation to pollution is suggested by
increased raethylmercurie tolerance in female killifish correlated
with increased fin ray count (Weis et al., 1981; Weis and Weis,
198H). A paper in preparation by J. Weis, P. weis and Zimmerer
discusses fin regeneration and its usefulness as a monitoring tool
for toxics. The correlation of exposure to toxics, especially PAHs
and PCBs, with integuraental lesions including finrot, gill deteri-
oration, and cataracts has been well documented and is reviewed
more thoroughly, with descriptions of current methodologies for
measuring the effects in Appendix C.
3.2.2 Oncogenic
Occurrences well above background levels of liver neoplasms
in finfish in polluted estuaries have been clearly demonstrated
over the last decade (Malins et al., 1980,1983; McCain et al.,
1977, 1982). Such neoplasms have also been reported in fish from
polluted fresh waters. While most estuarine and freshwater fish
studies have concentrated on the effects of PAHs and PCBs there is
evidence that heavy metals can also be correlated with carcinomas
(Couch & Harshbarger, 1985).
E-18
-------
Malins et al. (1985) have recently demonstrated field corre-
lations of increased hepatic lesions among English sole with
sediment contamination by aromatic hydrocarbons. In this case
the dietary uptake of the chemicals was documented. Baumann et
al. (1982) have similarly documented hepatomas in wild populations
of English sole and tomcod.
Contradictory evidence has been presented with regard to
cutaneous papillomas in demersal fish. While Dawe and Harshbarger
(1975) demonstrated increased occurrence of these disorders in
industrialized areas, Iwaoka et al. (1979) presented inconclu-
sive data on the relationship of toxic effects and siuc'h superficial
tumors.
Pituitary alterations can also be indicators tbf toxic stress.
Pseudocysts developed in sheepsheaa minnows' pituitary glands when
the fish were subjected to the herbicide trifluralin. These fish
were consequently functionally damaged, demonstrating bone disorders
including vertebral dysplasia (Couch, 1984). ;
In overview, there is a considerable body of literature
indicating correlation of PAHs, PCBs, and heavy metals with in-
creased occurrence of cutaneous carcinomas, liver neoplasms and
histopathological changes in freshwater and estuarine finfish
(O'Connor et al., 1986; and Malins, 1983).
E-19
-------
The body of information showing correlations of toxic chemi-
cals with lesions ana subsequent infection and with carcinomas has
lea to considerable effort towards classifying tumors and monitoring
f inf ish histopathology in polluted estuaries (Couch and Harshbarger,
1985; O'Connor et. al., 1986; Whipple, 1984; Whipple et al., 1984).
A detailed review of carcinomas is provided in Appendix D. An
important objective of the present workshop will be to discuss
strengths and weaknesses of available field monitoring method-
ologies for these effects, as well as what is known of the signifi-
cance of the effects on individual organisms,, populations and
biological communities..
3.3 Immunology
As noted above, chronic sublethal exposure to toxic chemicals
is believed to predispose finfish to parasitic, bacterial, fungal
and viral infections (Hawkes, 1979; Weeks et al., 1986a; Whipple,
1984; Whipple et al., 1984)).
Investigations comparing estuarine species from polluted
waters with controls from unpolluted areas have demonstrated that
macrophage activity in finfish is markedly affected by water (or
sediment) quality. Macrophages serve as the first line of defense
against infection by uptake or endocytosis of disease agents.
Chemotaxis (response to chemical stimulus), phagocytosis (uptake
of particulates) and pinocytosis (uptake of fluids) are processes
.E-20
-------
involved in normal endocytosis. Research has demonstrated that
each of these processes can be altered by the presence of environ-
mental pollutants, increasing finfish susceptibility to infection
i
.
and disease. However, variations are species-specific. Also,
subsequent return of fish to clean water has been found to reverse
macrophage activities to normal (Weeks et al., 1984, iy86a, 1986b).
'
Freshwater studies of the cellular immune response in finfish
have further suggested that primary and secondary responses are diet
related (Blazer et al., 1984K This would suggest the impor-
tance of toxicant uptake through ingestion.
Other studies have found that fish exposed] to enderin had
j
increased serum cortisol concentrations, contributing to the
"
repression of tne immune response (Bennett et al., 1985 a,b).
It has also been suggested that analyses of pigmented macrophages
from finfish reticulo-endothelial systems (RES) may serve as a
means of monitoring immune alterations and fish health (Wolke et
al., 1980).
A detailed review of immune effects of 'toxic chemicals
including a description of state-of-the-art methodologies has
been prepared for this report by Weeks, et al. (Appendix E)
E-21
-------
3.U Reproduction and Development
A number of studies have described reduced reproductive
capacity, fecundity and gamete viability in fish exposed to chronic
sublethal toxic stress (e.g. Engle, 1979; Whipple, 1984; Spies
1985). Surviving juveniles are often subject to abnormal hemato-
poesis (Longwell et al., 1983; Perry et al., 1984) as well as
neurological and skeletal abnormalities (Weis & Weis, 1979). Some
examples of effects of toxicants are discussed below. Detail on
these and discussion of other effects of toxic chemicals on repro-
duction and development may be furthered during the workshop.
Monocyclic aromatic hydrocarbons (MAH), zinc, DDT, PCBs and
total residual chlorine have frequently been correlated with
reproductive and larval abnormalities. Increased petrochemical
concentrations have been found correlated with egg resorption and
abnormal reproduction. Benzene has been found to cause a particu-
larly large number of effects including induction of egg resorption
and association with gill parasites in adults. Blood cell destruc-
tion and decreased serum proteins in juveniles are also associated
with benzene. When combined; with zinc, benzene affects striped
bass by severely accelerating parasitism, blood cell deterioration
and a decrease in serum proteins.
Aonormal egg development has been directly associated with
levels of DDT in striped bass ovaries (Whipple et al., 1984). DDT
E-22
-------
is also associated with neurological defects in sheepshead minnow
development (weis & Weis, 1979). PCBs caused delayed egg maturation
in striped bass (Whipple et al., 1984). Arclor 125^ (a PCB)
likewise reduced survival of embryos and fry of sheepshead minnows
(Hansen et al., "1973). Tests of toxicity of the cupric ion on
eggs of spot and Atlantic silverside found considerable differences
in sensitivity at time of hatching, with the jsilverside more
I
severely affected (Engel et al., 1979). Cadmium,! copper, nickel
i
and zinc have all been associated with reduced egg viability in
the striped bass (whipple et al., 1984). Exposure to a number of
heavy metals caused skeletal defects in developing killifish
juveniles. Mercury salts most severely affected skeletal develop-
ment while lead impairs uncurling in certain estuarine fish after
hatching (Weis & weis (1979). Kepone was found to pause scoliosis,
neurological impairment and impaired growth in juvenile sheepshead
minnows (Hansen et al., 1977).
Killifish develop cyclopia and other optic abnormalities
in response to a number of toxic chemicals. (When exposed to
carbaryl and parathion, killifish showed developmental arrest and
cardiovascular abnormalities.
Longwell has shown that the chorion of certain fishes can
become contaminated by oil-derived hydrocarbons and has suggested
l
that species differences in these contaminations: may be related
either to species spawning habits with regard to depth of water
E-23
-------
column, or to the particular developmental stage at which eggs are
exposed to the oil (Longwell, 1978). She has also shown significant
correlations between surface-layer toxics and cytologic, cytogenetic
and embryological health of mackerel eggs (Longwell & Hughs,
1980). By analyzing the yolk sac membrane in fish eggs, Longwell
and Hughs (1981 a,b) have demonstrated significant correlations
between mitotic-chroraosome irregularities and contamination with
hydrocarbons and heavy metals, this and subsequent studies have
focused on decreased egg health, defined by Longwell and Hughes
as "embryo moribundity, chromosome-mitotic abnormalities , develop-
ment rate, cell differentiation problems, gross embryo malformation
and total egg number sampled." Their studies show that poor egg
health is related to toxics, salinity and temperature (Longwell
and Hughes, 1980, 1982a,b; Longwell et al.. 1984; Cnang and
Longwell, 1984, 1985). Longwell discusses methodologies and
results of much of this work conducted on fish of the New York
Bight in Appendix F.
In summary, effects of toxic stress on reproduction and
development of estuarine f inf ish are numerous but variable according
to type of toxicant and type of fish. A manual prepared by Whipple
et al., 1984b provides methodology for the analyses of each aspect
of reproductive health of striped bass. An account of congenital
effects of toxics on a range of estuarine fish can be found in
Weis and fceis (Appendix G) while Spies discusses reproductive
impairment/success related to toxic stress in Appendix H.
E-24
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3.5 Enzymology
3.5.1 Enzyme Induction
There is a considerable body of evidence [documenting the
effects of chronic sublethal exposure to several types of toxics
I
on the mixed-function oxygenase system (MFC) in finfish*
i
The MFO system consists of an electron transport system that
oxidatively transforms nonpolar lipophilic organic compounds into
more water soluble metabolites. In fish it has been located prima-
I
rily in the liver, with lesser activity occurring in the kidney,
gills, gonads and heart. It consists of NADPH/cytochrome P-450,
reductase, cytochrome P-450 and phospholipids, which combine with
.
NADPH, oxygen and a substrate. The substrate may' be transformed
into highly reactive toxic intermediates, and to more or less toxic
I
daughter compounds prior to excretion. This has lead some investi-
gators to refer to the MFO system as a toxification /detoxifica-
tion system (Neff, 1984). The activity of the hepatic MFO system
'
is induced (increased) in finfish exposed to petroleum and PAHs
(Lech et al., 1982; Neff, 1976; Neff 19b4; Stegeman, 1981). PCBs,
i
dioxins and heavy metals may also induce MFU activity (Lech et
al., 1982; NRC, 1985). Natural environmental factors and intrinsic
i
biological chemicals (e.g. testosterone) may also increase or
i
decrease MFO activity, suggesting that it be used jwith caution as
an indicator of environmental pollution (Neff, 1984; NRC, 1985).
E-25 !
-------
However, induced MFO activity can indicate rather specific exposure
to several organic pollutants (Neff, 1984). This fact combined
with its potential to increase the toxic load in the organism
through production of carcinogenic metabolites encourages continued
research on the system. Stegeman has provided a review of toxic
effects on enzyme systems, particularly the MFO systems in Appendix
I.
The components and function of the MFO system, sometimes
referred to as the toxification/detoxification system, were intro-
duced above. MFO enzymes metabolize environmental xenobiotics and
endogenous steroids (Spies et al.,-1982). The resulting molecules
(e.g. sulfates) are small and polar, thus easily excreted. While
this prevents accumulation of PAHs and certain other toxics in
tissues, their oxidation can produce carcinogenic .and mutagenic
daughter compounds (Kurelec et al., 1977; Spies, 1982; Varans -et
al., 1980). The system responds in a similar fashion to PCBs
(Gruger et al., 1977, Spies et al., 1982).
Ingested PCBs and PAHs increase aryl hydrocarbon hydroxylase
(AHH) and microsomal proteins in some flatfish (Spies, 1982).
As a result of these studies, it has been the suggested that
activity of the MFO system be used as an indicator of at least
petroleum pollution (Payne and Penrose, 1975; Stegeman, 1978,1980)=
Toward this end research has demonstrated that, unlike mammalian
systems where there are specific inducers, the finfish show similar
E-26
-------
qualitative responses to petroleum, PCBs and a range of other
xenobiotics (Spies et al., 1982). However, DDT and DDE do not
I
induce MFO activity in flatfish (Addison et al(i, 1977; Spies,
1982), but do induce MFO activity in white croaker« (Brown et al.,
1982). Thus MFO induction by chlorinated hydrocarbons may not be
universal in finfish.
While PCBs from sewage effluents seem poorly metabolized by
flatfish, chronic sublethal exposure to PCBs jjeems to hinder
I
reproductive success in starry flounder. This suggests a possible
indirect toxic effect of PCB metabolic intermediates via the MFO
system (see Spies's Appendix H).
.
Metallothioneins are components of an intracellular mechanism
for sequestering and detoxifying trace metals. Metallothioneins
are metal binding, low molecular weight, cysteine rich proteins
that lack aromatic amino acids. Metallothionein systems have been
studied in fish and other animals, including mammals. While the
metallothionein system is believed to sequester metal-s that might
bind to sensitive cellular sites, it has also been found to be
part of the normal metabolism of copper and zinc in mammals (England
Houseyidi pers. • comra.; Jenkins et al., 1982). |Metallothionein
|
synthesis is induced by low levels of a number of metals resulting
.
in highly stable metal-thiol bonds which allow tihe organism to
tolerate increasing amounts of the trace metals. Metallothioneins
thus serve as an excellent specific indicator for metals, but
their activity has caused difficulties in analyses of potential
E-27
-------
toxicity of these metals. Cytosolic metal distribution studies
offer a possible approach to resolving such difficulties,, as well
as a method for clarifying avenues of metal uptake and synergistic
activities of metals with other pollutants.
A survey of the research on metallothionein systems in parti-
cular and current methods for assessing toxicant metabolic activi-
ties in general can be found in Jenkins1 review in Appendix J.
3.5.2 Molecular Pathology
Blood enzymes have long been used for clinical diagnosis in
mammals. Changes in concentrations of tissue-specific enzymes are
routinely used in the diagnosis of liver and heart disease or
damage, for instance. Activity in fish tissue-specific enzymes
may also be useful for diagnosing pollution caused cellular damage
and pathological conditions. For example, delta amino levulinic
acid dehydratase (ALA-D) is an enzyme involved in the synthesis of
hemoglobin and other porphyrin-proteins. Lead inhibits ALA-D
activity in finfish erythrocytes (Beritid et al., 1977).
Two other blood enzymes of use in analyzing pollutant damage
to fish are glutamate oxaloacetate transaminase (GOT) and gluta-
mate-pyruvate transaminase (GPT). Activity changes in these
enzymes primarily reflect liver damage although GOT can also
reflect damage to heart tissue. GOT activity decreases with
E-28
-------
exposure of certain finfish to lead but increases with exposure to
I
phenol, PCBs and municipal sewage effluent with synthetic organics
(Neff 1984). GPT increases with exposure to all of these pollutants
i
(lead, phenol, PCBs, municipal sewage effluent). • Otner blood
enzymes found to vary in activity when fish are exposed to pollu-
tants are LDH (lactate dehydrogenase), alkaline phosphatase, glucu-
ronidase, amino levulinic acid dehydratase and isocyfcric dehydro-
I
genase. However, several of these enzymes, as well as creatine
phosphokinase, show less change in the plasma with CC1,. exposure
than do GOT and GPT (Neff, 1984). At present then, ALA-D, GPT, and
GUT appear the most promising blood enzyme indicators of pollutant
mediated damage. Further investigations of pollutant effects on
I
enzyme systems, and of non pollutant effects on blood enzyme
activity may expand this list.
Tissue enzyme analyses have included in vitro cissays and in
vivo studies. The results of the two forms of ansilysis are often
quite different. Also, it is not always clear whether pollutant
mediated enzyme changes will result in significant changes in
metabolic processes in the fish (Neff, 1980).
Relationships have been demonstrated between toxicant expo-
i
sures and altered activity of some tissue enzymes. The gill
epithelia contain several adenosine triphospatases (ATPases).
These are important in osmotic and ionic regulation. Direct
i
correlations between chronic sublethal exposure to; pollutants and
E-29
-------
changes in activity of these enzymes have been documented (Miller
and Kinter, 1977; Neff, 1984). Likewise altered acetylcholines-
terase (AChE) activity in the brain, gill and muscles of fish
clearly correlates with exposure to chlorinated hydrocarbons,
carbamate and organophosphates (Neff, 1981).
As with blood enzymes, further research would be needed to
determine what relationships exist between tissue enzyme activities
in finfish and most toxicant stresses, and between any altered
enzyme activities and ultimate biological effects.
3.6 Physiology
Alterations of physiological functions such as osmoregulation
and respiration can also indicate toxic stress in fish. However,
distinguishing physiological effects from other effects is sometimes
difficult. Coho salmon sraolt demonstrate osmoregulatory failure
under toxic burdens, for example, but it has been suggested that
this failure is secondary and symptomatic of other negative stres-
ses. The investigators found no evidence of toluene or naphthalene
altering osmoregulatory ability in the smolt as a function of
salinity (Stickle et al.t 1982).
While respiratory rates have been examined as indicators of
sublethal stress by toxics (esp. petroleum), results are again
. E-30
-------
inconsistent ' because of the numerous other factors that affect
respiration. It has also been shown that responses vary conside-
rably among animal classes. Oxygen-consumption rates are depressed
in silver stressed cunners and mud- snails, but- elevated in all
bivalves studied (Goulde et al., 1977). Severely ulcerated gills
of finfish caused by PAH exposure do clearly demonstrate impaired
respiration (R. Huggett pers. comm., April 1986). A potential
answer to the problems of separating individual physiological
i
effects from other possible primary causes is the use of an energy
budget to evaluate toxic stress effects on respiration and other
physiological parameters (Bayne et al., 1976,, J1979). Such a
method is valuable because it avoids a need to separate intrinsic
and extrinsic effects on respiration and offers comparisons within
and among species on integrative stress indices such as growth
rate (NRC, 1985). " -
Bioenergetics methods have been effectively used as toxic
stress indicators by several investigators (Gilfillan et al.,
1985; NRC, 1985). Estimations of the catabolic energy of proteins
i
and amino acids, for example, can be determined by determining the
i
ratio of oxygen used to nitrogen excreted (0:N ratio)«,
r
Another way of assessing physiological effects of pollutants
is through correlation of altered enzyme activity, as discussed
above, with altered physiological function. ATPases may be either
.
stimulated or inhibited by pollutants including chlorinated hydro-
E-31 !
-------
carbons, metals and crude oil (Poston et al., 1979; Lorz et al.s
1978; Miller et al., 1977). These ATPases are found in finfish
gills and control ionic and osmotic regulation.
Other biochemical changes "induced by toxicants have also
been shown to correlate with specific physiological dysfunctions
and toxic stress. Changes in the adenylate energy charge (AEC)
for instance, reflect the metabolic energy available from the
adenine nucleotide pool. Stresses altering this pool then alter
available metabolic energy (Neff, 1984). Declines in liver glycogen
have been shown to coincide with hyperglycemia (Neff, 1984),,
Decreased growth rates and fecundity as well as altered energy
metabolisms have been correlated with toxicant induced depression
in liver glycogen and hypoglycemia (Conan, 1982; Neff, 1984).
Thus physiological dysfunctions may serve as relatively
easily observed symptoms of some biochemical and enzyme impairments
due to toxic stress.
3.7 Population
Population effects can include those caused by changed
reproductive rates, or changed distribution and migration patterns.
Effects of suppressed reproductive rates on population density have
been clearly demonstrated in the diminishing population of the
striped bass in San Francisco Bay. The suppressed reproductive
. E-32
-------
rates have been shown by these authors to correlate with toxic
concentrations in the Bay (Whipple et al., 1984).
i
Several laboratory studies have demonstrated that certain
fish can avoid toxics including DOT, enderin, and Duroban. There
is, however, no proof that fish avoid toxicants in the field
(Hansen et al.t 1972, 1974; Hansen, 1969). However, if they
do, the potential for beneficial effect on natural populations is
considerable. If the behavior is genetically controlled, it would
I
tend to increase through selection in environments with "hot
spots" of pollution. The hotspots would be increasingly avoided
by such species and the population would suffer less exposure to
toxic effects.
Observations of the entire life cycle of individuals under
toxic stress can serve as a first level of research in effects of
toxics on whole populations. Such laboratory studies have suggested
severe effects on reproductive efficiency in sh^epshead minnows
i
(Hansen and Parrish, 1977; Hansen et al., 1977).
One of the greatest problems in analyzing effects of toxics
I
on populations is separating direct toxic effects^from natural or
non-pollution related variations including overfishing. Appendix
K provides a discussion of this problem.
E-33
-------
SUMMARY
The effects of toxic stress on finfish may be deleterious as
in the case of neoplasms or neutral as in the cases of raetallothi-
onein binding of metals or metabolism and excretion of the pollu-
tants. The effects may be histopathological, biochemical, physio-
logical, behavioral or combinations of these. It is necessary
for pollution assessment and abatement programs to identify which
toxic effects on biota can be of immediate value as indicators
of toxic contamination in estuaries. There is an especially
pressing need for indicators that will give early warning of long
term problems. The choice of effects to use at present must
center around those that already have well developed methodologies.
The effects should be easily observed and measured at affordable
cost in order to serve as a screening system for pollution in the
field. There are often a number of methodologies available for
measuring the same or similar effects. Each of these should be
considered and the most suitable for immediate use in pollution
monitoring and abatement programs be given highest priority for
refinement and field implementation at this time. The most promi-
sing methods for future use should be recommended to research and
development arms of management agencies for continued support.
E-34
-------
LITERATURE CITED !
Addison, R. F. M. E. Zinck and D. E. Willis. 1977. Mixed function
oxidase enzymes in trout (Salvelinus fontinalis) liver:
absence of induction following feeding of P.P-DDT or P,P,
DDE. Comp. Biochem. Physiol. 57C: 39-13.
Baumann, P. C., W. D. Smith and M. Ribick. 1982. Hepatic tumor
rates and polynuclear aromatic hydrocarbon levels in two
populations of Brown bullhead (Ictalurus nebulosus) In
Polynuclear Aromatic Hydrocarbons: Sixth International
Symposium on Physical and Biological Chemistry. M. W.
Cooke, A. J. Dennis and G. L. Fisher (Eds.). Battelle Press,
Columbus, OH. pp. 93-102.
i
Bayne, B. L.., D. R. Livingston, M. N. Moore and J. Widdows.
197b. A cytocheraical and biochemical index of stress in
My til us edulis. L. Marine Pollution Bulletin.. 7:221-224.
Bayne, B. L., M. N. Moore, J. Widdows, D. R. Livingston and P.
Salkheld. 1979. Measurement of the responses of individuals
to environmental stress and pollution: studies with bivalve
mollusks.. In The Assessment of Sublethal Eiffects of Pollu-
tants. in the Sea. .The Royal Society. London, pp 165-180.
Bennett, R. 0. and R. E. Wolke. 1985. The effect of sublethal
endrin exposure on rainbow trout, Salmo gairdneri. Richardson
I Evaluation of Serum Cortisol Concentrations and Immune
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